RU2777988C2 - New enzymes and crispr systems - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to a method for the modification of a target locus of interest, including the delivery to the specified locus of a composition not found in nature or constructed, including monomolecular effector protein CRISPR-Cas containing three catalytic domains of RuvC nuclease, but not containing HNH domain, and one or more components, which are nucleic acids including a heterologous directing sequence and tracr-RNA sequence. A composition not found in nature or constructed, and a system for the modification of a target sequence of interest, as well as a delivery system containing it, and a vector system are also disclosed. The invention also relates to a particle, a cell, a cell line containing it, as well as to a method for the production of a plant, and a method for the inhibition of cell growth.

EFFECT: invention is effective for the modification of a target locus of interest.

61 cl, 53 dwg, 12 tbl, 5 ex

Description

RELATED APPLICATIONS AND INCLUSION AS A REFERENCE

[001] This application claims priority to U.S. Provisional Application No. 62/181663 filed June 18, 2015 and 62/245264 filed October 22, 2015.

[002] All documents cited or referred to in the documents cited in this application, together with any manufacturer's instructions, descriptions, specifications and product flow sheet, for any of the products mentioned in this description or in any document included in this description as links are included in the present description by reference and can be used in the implementation of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document were specifically and individually indicated as being incorporated by reference.

STATEMENT FOR FEDERAL RESEARCH SPONSORSHIP

[003] The present invention was made with the support of government grant numbers MH100706, MH110049, DK097768, GM10407, awarded by the National Institutes of Health. The government has certain rights to the invention.

FIELD OF THE INVENTION

[004] The present invention relates to systems, methods and compositions used to control gene expression, including targeting sequences, such as altering gene transcription or editing nucleic acids, which can use vector systems related to short palindromic repeats regularly arranged in clusters. (CRISPR), and their components.

STATE OF THE ART TO which THE INVENTION RELATES

[005] Recent advances in genome sequencing techniques and analysis of the resulting sequences have greatly increased the speed of cataloging and mapping genetic factors associated with a wide range of biological functions and diseases. Technologies for targeted modification of the genome are necessary for the systematic search for genetic variations that are the causes of diseases, as well as for the development of synthetic biology, applications in biotechnology and medicine. Despite the existence of various genome editing techniques, such as engineered zinc fingers, transcription activator-like effectors (TALE), homing meganucleases, there remains a need for new genome engineering technologies that use the latest strategies and molecular mechanisms, and that are affordable, easy to install. , scalable and capable of targeting multiple positions within the eukaryotic genome. This may provide a major resource for new approaches in genetic engineering and biotechnology.

[006] The adaptive immunity systems of bacteria and archaea CRISPR-Cas exhibit a huge diversity in the protein composition and architecture of genomic loci. The loci of the CRISPR-Cas system include more than 50 gene families, and there are no strictly universal genes, which indicates rapid evolution and extreme diversity of loci architecture. To date, approximately 395 profiles of 93 Cas proteins have been exhaustively identified as cas genes using an integrated approach. To classify CRISPR-Cas systems, gene profiles and characteristic features of loci architecture are used. In the latest proposed classification, CRISPR-Cas systems are most generally divided into two classes: class 1 systems consist of multi-subunit effector complexes, and in class 2 systems, the effector module consists of a single protein, for example, the Cas9 protein (Fig. 1A and 1B). New effector proteins associated with CRISPR-Cas class 2 systems can serve as powerful tools for genomic engineering, and the prediction of putative new effector proteins and their design and optimization are important.

[007] Citation or reference to any document in this application is not an admission that such document is available as prior art for the present invention.

SUMMARY OF THE INVENTION

[008] There is an urgent need to develop alternative and reliable systems and techniques for targeting nucleic acids and polynucleotides (eg, DNA or RNA, and any hybrids and derivatives thereof) with a wide range of practical applications. The present invention addresses this problem and has a number of associated advantages. Enrichment of the existing repertoire of technologies for targeting a DNA or RNA sequence of interest (targeting) in the genome and epigenome with the new systems presented in the present description can lead to transformations not only in the study, but also in changing or editing certain target regions through direct detection, analysis and modification. In order to effectively use the genomic or epigenomic DNA or RNA targeting systems contemplated herein without adverse consequences, it is important to understand the basic principles of engineering and optimizing these DNA or RNA targeting tools.

[009] The present invention relates to a method for modifying sequences associated with or within a target locus, the method comprising delivering to said locus a non-naturally occurring or engineered composition comprising an effector protein of the CRISPR-Cas type V loci and one or more nucleic acid components, wherein the effector protein forms a complex with one or more nucleic acid components, and upon binding said complex to the locus of interest, the effector protein induces modification of sequences associated with or within the locus of interest. In a preferred embodiment, the modification is the introduction of a chain break. In a preferred embodiment, the sequences at the target locus, associated with it or located directly in it, include DNA and the effector protein is encoded by CRISPR-Cas subtype V-A loci, or CRISPR-Cas subtype V-B loci, or CRISPR-Cas subtype V-C loci.

[0010] It will be understood that the terms "Cas enzyme", "CRISPR enzyme", "CRISPR protein", "Cas protein", and "CRISPR Cas" are used generally interchangeably and in each case when they are mentioned in the present description, denote new effector CRISPR proteins described hereinafter, unless otherwise indicated by the context, in particular when Cas9 is specifically mentioned. The CRISPR effector proteins described herein are preferably C2c1 or C2c3 effector proteins.

[0011] The invention relates to a method for modifying sequences associated with or at a target locus of interest, the method comprising delivering to said sequences associated with or located at the locus a non-naturally occurring or engineered composition comprising an effector protein of the C2c1 loci or C2c3 and one or more nucleic acid components, wherein the C2c1 or C2c3 effector protein forms a complex with one or more nucleic acid components, and upon binding of said complex to the locus of interest, the effector protein induces modification of sequences associated with or located in the target locus of interest. In a preferred embodiment, the modification is the introduction of a chain break. In a preferred embodiment, the C2c1 or C2c3 effector protein is complexed with one β-nucleic acid component, preferably an engineered or non-naturally occurring nucleic acid component. The induction of modification of sequences associated with or at the target locus of interest can be directed by a C2c1 effector protein or C2c3 nucleic acid complex. In a preferred embodiment of the invention, said nucleic acid component is CRISPR RNA (cr-RNA). In a preferred embodiment, the nucleic acid component is a mature crRNA or guide RNA, wherein the mature crRNA or guide RNA contains a spacer sequence (or guide sequence) and a sequence containing direct repeats, or derivatives thereof. In a preferred embodiment, the spacer sequence, or a derivative thereof, comprises a primer sequence, the primer sequence being critical for recognition and/or hybridization to the target locus sequence. In a preferred embodiment, the sequences associated with or located at the target locus of interest comprise linear or supercoiled DNA.

[0012] Aspects of the invention relate to C2c1 or C2c3 effector protein complexes comprising one or more non-naturally occurring or engineered or modified or optimized nucleic acids. In a preferred embodiment of the invention, the nucleic acid component of the complex may comprise a targeting sequence linked to a direct repeat containing sequence, wherein the direct repeat containing sequence contains one or more hairpin structures or optimized secondary structures. In certain embodiments of the invention, direct repeats are at least 16 nucleotides in length, as well as one hairpin structure. In further embodiments of the invention, direct repeats may be greater than 16 nucleotides in length, preferably 17 nucleotides, as well as one or more hairpin structures or optimized secondary structures. In a preferred embodiment of the invention, a direct repeat may be modified to contain one or more protein-binding RNA aptamers. In a preferred embodiment of the invention, one or more aptamers may be included as part of an optimized secondary structure. Such aptamers may be capable of binding bacteriophage coat protein. The bacteriophage coat protein can be selected from the group consisting of Qβ, F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕСb5, ϕСb8r, ϕСb12r, ϕСb23r, 7s and PRR1. In a preferred embodiment of the invention, bacteriophage MS2 coat protein is used. The invention also relates to the components of the above complexes, which are nucleic acids with a length of 30 or more, 40 or more, and 50 or more nucleotides.

[0013] The invention relates to methods for editing a genome, the method comprising two or more steps of targeting a C2c1 or C2c3 effector protein and cleavage. In certain embodiments of the invention, the first step includes cleavage with the C2c1 or C2c3 effector protein of sequences associated with the target locus located at a large distance from the primer sequence, while the second step includes cleavage with the C2c1 or C2c3 effector protein of sequences at the target locus. In preferred embodiments, the first step of targeting the C2c1 or C2c3 effector protein results in an insertion-deletion, while the second step of targeting the C2c1 or C2c3 effector protein can be repaired by homology-guided repair (HDR). In the most preferred embodiment of the invention, one or more of the steps of targeting the C2c1 or C2c3 effector protein results in a sticky-ended rupture that can be repaired by inserting a repair matrix.

[0014] The invention relates to methods for editing or modifying a genome associated with or carried out directly at a target locus of interest, wherein the method comprises introducing a C2c1 or C2c3 effector protein complex into any desired cell type, prokaryotic or eukaryotic cells, resulting in the complex effector protein C2c1 or C2c3 effectively functions by inserting a DNA insert into the genome of a eukaryotic or prokaryotic cell. In preferred embodiments, the cell is a eukaryotic cell and the genome is a mammalian genome. In preferred embodiments, insertion of the DNA insert is facilitated by non-homologous end-joining (HCK-NHEJ) gene insertion mechanisms. In preferred embodiments, the DNA insert is an exogenously introduced DNA template or repair template. In one preferred embodiment, the exogenously introduced DNA or repair template is delivered with a C2c1 or C2c3 effector protein complex or a single component or polynucleotide vector to express any component of the complex. Even more preferably, the eukaryotic cell is a non-dividing cell (eg, a non-dividing cell in which genome editing by homologous repair (HDR) is particularly difficult). In preferred genome editing techniques in human cells, C2c1 or C2c3 effector proteins may include, but are not limited to, the specific types of C2c1 or C2c3 effector proteins described herein.

[0015] The invention also relates to a method for modifying a target locus of interest, the method comprising delivering to said locus a non-naturally occurring or engineered composition comprising an effector protein of the C2c1 loci and one or more nucleic acid components, wherein the effector protein C2cl forms a complex with one or more nucleic acid components, and upon binding of said complex to the locus of interest, the effector protein induces modification of the target locus of interest. In a preferred embodiment of the invention, such a modification is a chain break.

[0016] The invention also relates to a method for modifying a target locus of interest, the method comprising delivering to said locus a non-naturally occurring or engineered composition comprising an effector protein of the C2c3 loci and one or more nucleic acid components, wherein the effector protein C2c3 forms a complex with one or more nucleic acid components, and upon binding of said complex to the locus of interest, the effector protein induces modification of the target locus of interest. In a preferred embodiment of the invention, such a modification is a chain break.

[0017] In such methods, the target locus of interest may be part of a DNA molecule. In such methods, the target locus of interest may be part of an in vitro DNA molecule.

[0018] In such methods, the target locus of interest may be part of a DNA molecule in a cell. Such a cell can be either prokaryotic or eukaryotic. Such a cell may be a mammalian cell. Such a mammalian cell may be a non-human primate cell, a bovine, porcine, rodent or mouse cell. Such a cage may be a poultry, fish or shrimp cage. Such a cell may be a plant cell derived from plants such as cassava, corn, sorghum, wheat, or rice. Such a plant cell may also be an algal, tree or vegetable cell. Modifications made to such a cell in accordance with the present invention can be made in such a way that such a cell and its descendants are modified to improve the production of biological products such as antibodies, starch, alcohol, or other desired product of the cell. Such modifications made to a cell in accordance with the present invention may be capable of causing the cell or its daughter cells to produce an altered biological product produced.

[0019] The mammalian cell can be a non-human mammalian cell, e.g., a primate cell, a cow, a sheep, a pig, a dog, a rodent, a member of the Leporidae family, a mammalian cell such as a monkey, a cell of a cow, a sheep, a pig, a dog, a rabbit, a rat or mice. The cell may be a eukaryotic cell of a non-mammalian animal such as poultry (eg chicken), vertebrate fish (eg salmon), or invertebrate (eg oyster, bivalve, lobster, shrimp). Such a cell may also be a plant cell. Such a plant cell can be obtained from a monocot plant or dicotyledonous plant, as well as an agricultural plant or cereal such as cassava, corn, sorghum, soybean, wheat, oats or rice. Such a plant cell may also be a cell of an algae, tree or producer plant, fruit or vegetable (for example, a tree such as citrus trees such as orange, grapefruit or lemon; peach or nectarine; apple and pear; nut tree such as almond , walnut or pistachio; nightshade plant; plants of the genus Brassica; plants of the genus Lactuca; plants of the genus Spinacia; plants of the genus Capsicum; cotton, tobacco, asparagus, carrots, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberries, blueberries, raspberries, blackberries, grapes, coffee, cocoa, etc.).

[0020] The invention also relates to an engineered CRISPR protein, complex, composition, system, vector, cell or cell line of the invention for use in modifying a locus of interest in a cell. Said modification preferably includes bringing the cell into contact with any of the compositions described above or any of the systems described above. The invention also relates to the use of an engineered CRISPR protein, complex, composition, system, vector, cell or cell line of the invention for the manufacture of a medicament for modifying a locus of interest in a cell.

[0021] The invention also relates to an engineered CRISPR protein, complex, composition, system, vector, cell, or cell line of the invention for use in modifying a locus of interest in a cell. The invention also relates to the use of an engineered CRISPR protein, complex, composition, system, vector, cell or cell line of the invention in the preparation of a drug for modifying a locus of interest in a cell. Said modification preferably includes bringing the cell into contact with any of the compositions described above or any of the systems described above.

[0022] The invention relates to a method for modifying a target locus of interest, the method comprising delivering to said locus a non-naturally occurring or engineered composition comprising an effector protein of type IV CRISPR-Cas loci and one or more nucleic acid components, wherein the effector protein forms a complex with one or more nucleic acid components, wherein upon binding said complex to the locus of interest, the effector protein induces modification of the target locus of interest. In a preferred embodiment of the invention, such a modification is a chain break.

[0023] In a preferred embodiment, the target locus of interest is DNA.

[0024] In such methods, the target locus of interest may be part of a DNA molecule or an RNA molecule. In a preferred embodiment, the target locus of interest is RNA.

[0025] In such methods, the target locus of interest may be within a DNA molecule within a cell. The cell may be a prokaryotic or eukaryotic cell. The cell may be a mammalian cell. Such a mammalian cell may be a non-human mammalian cell, such as a primate, cow, sheep, pig, dog, rodent, Leporidae, mammalian cell such as a monkey, cow, sheep, pig, dog, rabbit, rat, or mouse cell. . Such a cell may be a eukaryotic cell of a non-mammalian animal such as poultry (eg chicken), vertebrate fish (eg salmon) or invertebrate (eg oyster, bivalve, lobster, shrimp). Such a cell may also be a plant cell. Such a plant cell can be obtained from a monocot plant or dicotyledonous plant, as well as an agricultural plant or cereal such as cassava, corn, sorghum, soybean, wheat, oats or rice. Such a plant cell may also be a cell of an algae, tree or producer plant, fruit or vegetable (e.g. a tree such as citrus trees such as orange, grapefruit or lemon; peach or nectarine; apple or pear; nut tree such as almond , walnut or pistachio; nightshade plant; plants of the genus Brassica ; plants of the genus Lactuca ; plants of the genus Spinacia ; plants of the genus Capsicum ; cotton, tobacco, asparagus, carrots, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberries, blueberries, raspberries, blackberries, grapes, coffee, cocoa, etc.)

[0026] In any of the methods described, the target locus of interest may be a genomic or epigenomic locus. In any of the methods described, said complex may be delivered by multiple targeting molecules for multiplex application. More than one protein(s) may be used in any of the methods described.

[0027] In preferred embodiments of the invention, biochemical or in vitro or in vivo cleavage of sequences associated with or directly located at the target locus of interest occurs without the putative transactivating cr-RNA (tracr RNA) sequence, for example, by cleavage with an effector protein C2c1 or C2c3. In other embodiments, cleavage may occur with the putative transactivating cr-RNA (tracr RNA) sequence, for example, through cleavage with other effector proteins of the CRISPR family.

[0028] In any of the methods described, the effector protein (e.g., C2c1 or C2c3) and nucleic acid components can be provided by one or more polynucleotide molecules encoding the protein and/or nucleic acid component(s), wherein one or more polynucleotide molecules are operably adapted to express the protein and/or nucleic acid component(s). One or more polynucleotide molecules may contain one or more regulatory elements operably adapted to express the protein and/or nucleic acid component(s). One or more polynucleotide molecules may be in one or more vectors. The invention covers such polynucleotide molecules, for example, polynucleotide molecules, functionally adapted for the expression of protein and/or component(s), which is a nucleic acid, as well as the specified vector(s).

[0029] The invention also relates to an engineered CRISPR protein, complex, composition, system, vector, cell, or cell line of the invention for use in modifying a target locus of interest in a cell. Said modification preferably includes bringing the cell into contact with any of the compositions described above or any of the systems described above. The invention also relates to the use of an engineered CRISPR protein, complex, composition, system, vector, cell or cell line of the invention for the preparation of a drug for modifying a target locus in a cell.

[0030] The invention also relates to an engineered CRISPR protein, complex, composition, system, vector, cell, or cell line of the invention for use in modifying a target locus of interest in a cell. The invention also relates to the use of an engineered CRISPR protein, complex, composition, system, vector, cell or cell line of the invention for the preparation of a drug for modifying a target locus of interest. Said modification preferably includes bringing the cell into contact with any of the compositions described above or any of the systems described above.

[0031] The invention also relates to a method for modifying a target locus of interest, said method comprising delivering to said locus a non-naturally occurring or engineered composition comprising an effector protein of the C2c2 locus and one or more nucleic acid components, wherein the effector the C2c2 protein forms a complex with one or more nucleic acid components, and upon binding of said complex to the locus of interest, the effector protein modifies the target locus of interest. In a preferred embodiment, the modification is the introduction of a single strand break.

[0032] In such methods, the target locus of interest may be contained within the DNA molecule in vitro . In such methods, the target locus of interest may be contained within a DNA molecule within a cell. Preferably, in such methods, the target locus of interest may be incorporated into the RNA molecule in vitro . It is also preferred that in such methods the target locus of interest can be incorporated into an RNA molecule within the cell. The cell may be a prokaryotic or eukaryotic cell. The cell may be a mammalian cell. The cell may be a rodent cell. the cell may be a mouse cell.

[0033] The invention also relates to an engineered CRISPR protein, complex, composition, system, vector, cell or cell line of the invention for use in modifying a locus of interest in a cell. Said modification preferably includes bringing the cell into contact with any of the compositions described above or any of the systems described above. The invention also provides for the use of an engineered CRISPR protein, complex, composition, system, vector, cell or cell line according to the invention in the manufacture of a drug for modifying a locus of interest in a cell.

[0034] The invention also relates to an engineered CRISPR protein, complex, composition, system, vector, cell, or cell line for use in modifying a locus of interest in a cell according to the invention. The invention also relates to the use of an engineered CRISPR protein, complex, composition, system, vector, cell or cell line in the preparation of a drug of the invention to modify a locus of interest in a cell. Said modification preferably includes bringing the cell into contact with any of the compositions described above or any of the systems described above.

[0035] In any of the methods described, the target locus of interest can be a genomic or epigenomic locus of interest. In any of the methods described, the complex can be delivered using multiple targeting molecules for multiplex application. More than one protein(s) may be used in any of the methods described.

[0036] In other aspects of the invention, the nucleic acid components may comprise a putative CRISPR RNA (crRNA) sequence and/or a putative cr-RNA transactivating sequence (tracr-RNA). In some embodiments, cleavage, such as biochemical or in vitro cleavage, or cleavage in cells, may occur without the putative transactivating cr-RNA (tracr RNA) sequence. In other embodiments, cleavage, such as biochemical or in vitro cleavage, or cleavage in cells, may occur with the putative transactivating cr-RNA (tracr-RNA) sequence;

[0037] In some embodiments, where the effector protein is a type V CRISPR-Cas loci effector protein, such as a CRISPR-Cas loci C2c1 effector protein or a CRISPR-Cas C2c3 loci effector protein, preferably a CRISPR-Cas C2c1 effector protein, the components , which are nucleic acids, may contain a putative CRISPR RNA sequence (cr-RNA) and a putative trans-activating cr-RNA sequence (tracr RNA).

[0038] In further aspects of the invention, the nucleic acid components may comprise a putative CRISPR RNA (cr-RNA) sequence and not include any putative trans-activating cr-RNA (tracr-RNA) sequence. As a non-limiting example, Applicants contemplate that in such cases the pre-crRNA may contain a secondary structure sufficient for processing to produce mature crRNA as well as loading the effector protein with the crRNA. As a non-limiting example, such a secondary structure may comprise, essentially consist of, or consist of a hairpin within a pre-crRNA, more specifically within a direct repeat.

[0039] In some embodiments, where the effector protein is a type VI CRISPR-Cas loci effector protein, such as the C2c2 effector protein, the nucleic acid components may contain a putative CRISPR type sequence (cr-RNA) and not include any or putative trans-activating cr-RNA sequence (tracr RNA).

[0040] In any of the methods described, the effector protein and nucleic acid components can be delivered by one or more polynucleotide molecules encoding the protein and/or nucleic acid components, and wherein the one or more polynucleotide molecules are operably arranged to express the protein and/or component(s) which is a nucleic acid. One or more polynucleotide molecules may contain one or more regulatory elements operably arranged for the expression of a protein and/or a nucleic acid component. One or more polynucleotide molecules may be included in one or more vectors. In any of the methods described, the target locus of interest may be a genomic or epigenomic locus. In any of the methods described, the complex can be delivered via multiple targeting molecules for multiplex application. More than one protein(s) may be used in any of the methods described.

[0041] In any of the methods described, the chain break can be a single-strand break or a double-strand break.

[0042] Regulatory elements may include inducible promoters. Polynucleotides and/or vector systems may include inducible systems.

[0043] In any of the methods described, one or more polynucleotide molecules may be included in the delivery system, or one or more vectors may be included in the delivery system.

[0044] In any of the methods described, such a non-naturally occurring or engineered composition can be delivered using liposomes and particles, including nanoparticles, exosomes, microvesicles, gene guns, or one or more viral vectors, for example, using a nucleic acid molecule or viral vector.

[0045] The invention also relates to a non-naturally occurring or engineered composition, which is a composition having the characteristics described in the present description or defined in any of the methods described in the present description.

[0046] The invention also relates to an engineered CRISPR protein, complex, composition, system, vector, cell or cell line of the invention for use in therapy.

[0047] In certain embodiments, the invention thus relates to a non-naturally occurring or engineered composition, such as, in particular, a composition capable of or adapted to modify a target locus of interest, said composition comprising an effector protein a type V CRISPR-Cas locus and one or more nucleic acid components, wherein the effector protein forms a complex with one or more nucleic acid components, and upon binding of said complex to a locus of interest, the effector protein causes a modification of the target locus of interest. In certain embodiments, the effector protein may be encoded by the V-A subtype of the CRISPR-Cas loci, or the V-B subtype of the CRISPR-Cas loci, or the V-C subtype of the CRISPR-Cas loci. In certain embodiments, the effector protein may be a C2c1 effector protein or a C2c3 effector protein.

[0048] Thus, in some embodiments, the invention relates to a naturally occurring or engineered composition, in particular, a composition capable of or adapted to modify a target locus of interest, said composition comprising an effector protein of CRISPR-Cas type VI loci and one or more nucleic acid components, wherein such effector protein forms a complex with one or more nucleic acid components, and upon binding of said complex to a locus of interest, such effector protein induces modification of the target locus of interest. In certain embodiments of the invention, such an effector protein may be an effector protein of the C2c2 loci.

[0049] Also in a further aspect, the present invention relates to a non-naturally occurring or engineered composition, in particular a composition capable of or adapted to modify a target locus of interest, said composition comprising: (a) a guide RNA molecule (or a combination of guide RNA molecules, such as a first guide RNA molecule or a second guide RNA molecule) or a nucleic acid encoding a guide RNA molecule (or one or more nucleic acids encoding a combination of guide RNA molecules); (b) an effector protein of type V CRISPR-Cas loci or a nucleic acid encoding an effector protein of type V CRISPR-Cas loci. CRISPR Cas. In certain embodiments, the effector protein can be C2c1 effector protein of CRISPR Cas loci or C2c3 effector protein of CRISPR Cas loci.

[0050] Another aspect of the invention also relates to a non-naturally occurring or engineered composition, such as, in particular, a composition capable of or adapted to modify a target locus of interest, said composition comprising: (a) a guide molecule RNA (or a combination of guide RNA molecules, for example, a first guide RNA molecule and a second guide RNA molecule) or a nucleic acid encoding a combination of guide RNA molecules; (b) an effector protein of type VI CRISPR-Cas loci or a nucleic acid encoding an effector protein of type VI CRISPR Cas loci. In some embodiments, the effector protein may be a C2c2 effector protein.

[0051] In a further aspect, the invention also relates to a non-naturally occurring or engineered composition comprising: (i) one or more CRISPR-Cas system polynucleotide sequences comprising (a) a guide sequence capable of hybridizing to a target sequence in polynucleotide locus, (b) a tracr partner sequence, (c) a tracr RNA sequence, and (II) a second polynucleotide sequence encoding an effector protein of type V CRISPR-Cas loci, wherein, upon transcription, the tracr partner sequence hybridizes to a tracr RNA sequence and the targeting sequence directs the sequence-specific binding of the CRISPR complex to the target sequence, and wherein the CRISPR complex comprises an effector protein of CRISPR-Cas type V loci complexed with (1) a targeting sequence that hybridizes to the target sequence and (2) a tracr partner sequence , to which hybridizes with the tracr-RNA sequence. In certain embodiments, the effector protein may be encoded by the V-A subtype of the CRISPR-Cas loci, or the V-B subtype of the CRISPR-Cas loci, or the V-C subtype of the CRISPR-Cas loci. In certain embodiments, the effector protein can be C2c1 effector protein of CRISPR-Cas loci or C2c3 effector protein of CRISPR-Cas loci.

[0052] In a further aspect, the invention also relates to a non-naturally occurring or engineered composition comprising: (i) one or more CRISPR-Cas system polynucleotide sequences comprising (a) a guide sequence capable of hybridizing to a target sequence in polynucleotide locus, (b) a tracr partner sequence, (c) a tracr RNA sequence, and (ii) a second polynucleotide sequence encoding an effector protein of type VI CRISPR Cas loci, wherein, upon transcription, the tracr satellite sequence hybridizes to the tracr RNA sequence and guide the sequence directs the sequence-specific binding of the CRISPR complex to the target sequence, and wherein the CRISPR complex comprises an effector protein of CRISPR Cas type VI loci complexed with (1) a targeting sequence that hybridizes to the target sequence and (2) a tracr partner sequence, which hybridizes to the tracr-RNA sequence. In certain embodiments, the effector protein may be encoded by the V-A subtype of the CRISPR-Cas loci, or the V-B subtype of the CRISPR-Cas loci, or the V-C subtype of the CRISPR-Cas loci. In some embodiments, the effector protein may be the C2c2 effector protein of the CRISPR-Cas loci.

[0053] In certain embodiments, tracr-RNA may not be required. Therefore, in some embodiments, the invention also relates to a non-naturally occurring or engineered composition comprising: (i) one or more polynucleotide sequences of the CRISPR-Cas system, containing (a) a guide sequence capable of hybridizing to a target sequence in polynucleotide locus, and (b) a direct repeat sequence, and (ii) a second polynucleotide sequence encoding an effector protein of the type V or type VI CRISPR-Cas loci, wherein during transcription the guide sequence controls the sequence-specific binding of the CRISPR complex to the target sequence, and wherein the CRISPR complex comprises an effector protein of the CRISPR-Cas type V or type VI loci complexed with (i) a guide sequence that is hybridized to the target sequence and (2) a direct repeat sequence. In certain embodiments, the type V effector protein may be encoded by the V-A subtypes of the CRISPR-Cas loci, or the V-B subtypes of the CRISPR-Cas loci, or the subtypes V-C of the CRISPR-Cas loci. In certain embodiments, the effector protein can be C2c1 effector protein of CRISPR-Cas loci or C2c3 effector protein of CRISPR-Cas loci. Preferably, the effector protein is an effector protein of type VI CRISPR-Cas loci. More preferably, the effector protein is the C2c2 effector protein of the CRISPR-Cas loci. Without imposing restrictions, Applicants contemplate that in such cases the direct repeat sequence may contain a secondary structure sufficient to load the effector protein with crRNA. As a non-limiting example, such a secondary structure may include, essentially consist of, or consist of a hairpin within a direct repeat.

[0054] The invention also relates to a vector system comprising one or more vectors, wherein one or more vectors contain one or more polynucleotide molecules encoding components of a non-naturally occurring or engineered composition, which is a composition having the characteristics as defined in any of the ways described in the present description.

[0055] The invention also relates to a delivery system comprising one or more vectors or one or more polynucleotide molecules, wherein the one or more vectors or polynucleotide molecules comprise one or more polynucleotide molecules encoding components of a non-naturally occurring or engineered composition that is a composition having the characteristics described in the present description or defined in any of the described methods.

[0056] The invention also relates to a non-naturally occurring or engineered composition, or one or more polynucleotides encoding components of said composition, or a vector or delivery system containing one or more polynucleotides encoding components of said composition, for use in therapeutic method of treatment. Such a therapeutic method of treatment may include gene or genome editing or gene therapy.

[0057] The invention also relates to an engineered CRISPR protein, complex, composition, system, vector, cell, or cell line of the invention for use in the therapy of a disease, disorder, or infectious disease in an individual in need thereof. The disease, disorder, or infection may include a viral infection. The viral infection may be hepatitis B (HBV). The invention also relates to the use of an engineered CRISPR protein, complex, composition, system, vector, cell or cell line of the invention for the manufacture of a medicament for the treatment of a disease, disorder or infection in an individual in need thereof. The disease, disorder, or infection may include a viral infection.

[0058] The invention also relates to computational (bioinformatics) methods and algorithms for predicting new CRISPR-Cas Class 2 systems and identifying their components.

[0059] The invention also relates to methods and compositions in which one or more amino acid residues of an effector protein can be modified, for example, in an engineered or non-naturally occurring effector protein, or C2c1 or C2c3. In one of the embodiments of the invention, the modification may include a mutation of one or more amino acid residues of the effector protein. One or more mutations may be located in one or more catalytic domains of the effector protein. The nuclease activity of such an effector protein may be reduced or absent compared to an effector protein lacking one or more of the mutations. Such an effector protein may not be able to direct cleavage of the DNA or RNA strand at the target locus of interest. A preferred embodiment includes two such mutations. In a preferred embodiment of the invention, such one or more amino acid residues are modified in a C2c1 or C2c3 effector protein, such as an engineered or non-naturally occurring effector protein, or C2c1 or C2c3.

[0060] The invention also relates to one or more mutations, or two or more mutations in the catalytically active domain of the effector protein containing the RuvC domain. In some embodiments, such a RuvC domain may be one of the RuvCI, RuvCII, or RuvCIII domains, or a catalytically active domain, homologous to the RuvCI, RuvCII, and RuvCIII domains, etc., or any corresponding domain as described in any of those described herein. ways. In some embodiments, such one or more mutations can be located in the catalytically active domain of the effector protein, which is the HEPN domain, or the catalytically active domain, homologous to the HEPN domain. Such an effector protein may have one or more nuclear localization signal (NLS) domains. Such one or more heterologous functional domains may contain at least two or more NLS domains. Such one or more NLS domains may be located at or near the end of the effector protein sequence (for example, C2c1 or C2c3), and in the case of two or more NLSs, each of these two may be located near the end of the effector protein (for example, C2c1 or C2c3) . Such one or more heterologous functional domains may be one or more transcriptional activation domains. In a preferred embodiment, the transcriptional activation domain may include VP64. Such one or more functional domains may be one or more transcriptional repression domains. In a preferred embodiment, such a transcriptional repression domain is a KRAB domain or a SID domain (eg, SID4X). Such one or more heterologous functional domains may be one or more nuclease domains. In a preferred embodiment, the nuclease domain is a Fok1 domain.

[0061] The invention also relates to one or more heterologous functional domains of the following types of activity: methylase activity, demethylase activity, transcription activation, transcriptional repression, transcription termination factor activity, histone modification, nuclease activity, single-stranded RNA cleavage, double-stranded RNA cleavage, single-stranded RNA cleavage DNA, double-stranded DNA cleavage and nucleic acid binding. At least one or more heterologous functional domains may be located at or near the N-terminus of the effector protein, while also at least one or more heterologous functional domains may be located at or near the C-terminus of the effector protein. Such one or more heterologous functional domains may be fused to such an effector protein. Such one or more heterologous functional domains may be attached to such an effector protein. Such one or more heterologous functional domains may be linked to such an effector protein via a linking group.

[0062] The invention also relates to an effector protein comprising an effector protein from an organism belonging to one of the following genera: Streptococcus , Campylobacter , Nitratifractor , Staphylococcus , Parvibaculum , Roseburia , Neisseria , Gluconacetobacter , Azospirillum , Sphaerochaeta , Lactobacillus , Eubacterium , Corynnoebacter , , Rhodobacter , Listeria , Paludibacter , Clostridium , Lachnospiraceae , Clostridiaridium , Leptotrichia , Francisella , Legionella , Alicyclobacillus , Methanomethyophilus , Porphyromonas , Prevotella , Bacteroidetes , Helcococcus , Letospira , Desulfovibrio , Desulfonatronum , Opitutaceae , Tuberibacillus , Bacillus , Brevibacilus , Methylobacterium или Acidaminococcus . Such an effector protein may be a chimeric effector protein, the first fragment of which is derived from the first effector protein orthologue and the second fragment from the second effector protein orthologue, the first and second effector protein orthologues being different. At least one of the first and second effector protein orthologs may be an effector protein from one of the following organisms: Streptococcus , Campylobacter , Nitratifractor , Staphylococcus , Parvibaculum , Roseburia , Neisseria , Gluconacetobacter , Azospirillum , Sphaerochaeta , Lactobacillus , Eubacterium , Corynebacter , Carnobacterium , Listeria , Paludibacter , Clostridium , Lachnospiraceae , Clostridiaridium , Leptotrichia , Francisella , Legionella , Alicyclobacillus , Methanomethyophilus , Porphyromonas , Prevotella , Bacteroidetes , Helcococcus , Letospira , Desulfovibrio , Desulfonatronum , Opitutaceae , Tuberibacillus , Bacillus , Brevibacillus , Methylobacterium или Acidaminococcus .

[0063] In some embodiments, such an effector protein, in particular a type V loci effector protein, and more particularly a VB loci effector protein, in particular C2c1p, can be derived from, be derived from, or be derived from bacterial metabolism related to such taxa such as Bacilli, Verrucomicrobia, alpha proteobacteria or delta proteobacteria. In some embodiments, such an effector protein, in particular a type V loci effector protein, more specifically a type VB loci effector protein, in particular C2c1p, may be derived from, isolated from, or derived from the metabolism of bacteria belonging to one of the following genera: Alicyclobacillus , Desulfovibrio , Desulfonatronum , Opitutaceae , Tuberibacillus , Bacillus , Brevibacillus , Desulfatirhabdium , Citrobacter , and Methylobacterium . In some embodiments, such an effector protein, particularly a type V loci effector protein, more specifically a VB loci effector protein, in particular C2c1p, may be derived from, isolated from, or derived from the metabolism of the following bacteria: Alicyclobacillus acidoterrestris (e.g., ATCC 49025 ), Alicyclobacillus scontaminans (e.g. DSM 17975), Desulfovibr ioinopinatus (e.g. DSM 10711), Desulfonatronum thiodismutans (e.g. strain MLF-1), Opitutaceae TAV5 bacteria, Tuberibacillus calidus (e.g. DSM 17572), Bacillus thermoamylovorans (e.g. strain B4166), Brevibacillus sp. CF112, Bacillus sp. NSP2.1, Desulfatirhabdium butyrativorans (eg DSM 18734), Alicyclobacillus herbarius (eg DSM 13609), Citrobacter freundii (eg ATCC 8090), Brevibacillus agri (eg BAB-2500), Methylobacterium nodulans (eg ORS 2060). In some embodiments, such an effector protein, in particular a type V loci effector protein, more specifically a VB type loci effector protein, in particular C2c1p, can be derived from, be isolated from, be derived from the metabolism of bacteria from the list tabulated in FIG. 41A -B.

[0064] In some embodiments, such an effector protein, namely a type V loci protein, more specifically an effector protein of type V-B loci, in particular C2c1p, may comprise, consist essentially of, or consist of an amino acid sequence selected from a list including amino acid sequences, shown in multiple sequence alignments in FIGS. 13D-H.

[0065] In some embodiments, a V-B type locus, as contemplated herein, may encode for a Cas1-Cas4 fusion, Cas2, and a C2c1p effector protein. In some embodiments, a V-B type locus, as contemplated herein, may be adjacent to a CRISPR sequence. The characteristic organization of V-B type loci is illustrated in Fig. 9 and Fig. 41A-B.

[0066] In some embodiments, a Cas1 protein encoded by a type V-B locus, as contemplated herein, may cluster with a type I-U system. 10A and 10B and 10C-V illustrate the Cas1 tree, including Cas1 encoded by representative loci of the V-B type.

[0067] In certain embodiments, the effector protein, specifically the V-type loci effector protein, more specifically the V-B type loci effector protein, and in particular C2c1p, such as native C2c1p, may have a length of from about 1100 to about 1500 amino acids, for example, from about 1100 to about 1200, or about 1200 to about 1300 amino acids, or about 1300 to about 1400 amino acids, or about 1400 to about 1500 amino acids, e.g., about 1100, about 1200, about 1300, about 1400, or about 1500 amino acids.

[0068] In some embodiments, the effector protein, specifically the type V loci effector protein, more specifically the V-B loci effector protein, in particular C2c1p, and preferably the C-terminal portion of said effector protein, contains three RuvC-like nuclease catalytic motifs ( i.e. RuvCI, RuvCII and RuvCIII). In some embodiments, said effector protein, and preferably the C-terminal portion of said effector protein, may further comprise a region corresponding to a bridging helix (also known as an arginine-rich cassette) that in the Cas9 protein is involved in cr-RNA binding. In some embodiments of the invention, said effector protein, and preferably the C-terminal portion of said protein, may further include a zinc finger containing domain, which may be inactive (i.e., which does not bind zinc, e.g., in which Zn-binding cysteine residues are absent) . In some embodiments, said effector protein, and preferably the C-terminal portion of said effector protein, may comprise three RuvC-like nuclease catalytic motifs (i.e., RuvCI, RuvCII, and RuvCIII), a bridging helix region, a zinc finger domain, preferably in the following order, from N- to C-terminus: RuvCI-bridged helix-RuvCII-zinc finger-RuvCIII See FIGS. 11, 12 and FIGS. 13A and 13C for an illustration of the domain architecture of representatives of V-B type effector proteins.

[0069] In some embodiments, V-B type loci as described herein may contain CRISPR repeats from 30 to 40 bp in length, more typically from 34 to 38 bp, even more typically from 36 bp. up to 37 bp, for example, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 bp

[0070] In some embodiments, the effector protein, specifically the type V loci effector protein, more specifically the V-C type loci effector protein, in particular C2c3p, may originate from, be isolated from, or derived from a bacterial metagenome selected from the bacterial metagenomes listed in Table on Fig.43 A-B.

[0071] In some embodiments, an effector protein, specifically an effector protein of type V loci, more specifically an effector protein of type V-C loci, in particular C2c3p, may include, consist essentially of, or consist only of an amino acid sequence selected from the group of amino acid sequences shown in the multiple sequence alignment in FIG.

[0072] In some embodiments, the V-C type locus, as described herein, may encode for the effector protein Cas1 and C2c3p. See Fig.14 and Fig.43A-B to illustrate the organization of characteristic loci type V-C.

[0073] In some embodiments, a Cas1 protein encoded by a type V-C locus as described herein can cluster with a type I-B system. See FIGS. 10A and 10B and FIGS. 10C-W illustrating the Cas protein tree including the Cas1 protein encoded by characteristic V-C type loci.

[0074] In some embodiments, the effector protein, namely the type V loci protein, more specifically the V-C loci effector protein, even more specifically C2c3p, in particular C2c3p, may be from about 1100 to about 1500 amino acids in length, e.g. , from about 1100 to about 1200 amino acids, or from about 1200 to about 1300 amino acids, or from about 1300 to about 1400 amino acids, or from about 1400 to about 1500 amino acids, for example, about 1100, about 1200, about 1300, about 1400 or about 1500 amino acids, or at least about 1100, at least about 1200, at least about 1300, at least about 1400, or at least about 1500 amino acids.

[0075] In some embodiments, the effector protein, namely the type V loci effector protein, more specifically the V-C type loci effector protein, in particular C2c3p, preferably the C-terminal portion of said effector protein, comprises three RuvC-like catalytic motifs. nucleases (i.e., RuvCI, RuvCII and RuvCIII). In some embodiments, said effector protein, preferably the C-terminal portion of said effector protein, may contain a region corresponding to a bridging helix (also known as an arginine-rich cassette) that in the Cas9 protein is involved in crRNA binding. In some embodiments, said effector protein, preferably the C-terminal portion of said effector protein, may further include a zinc finger domain. It is preferable to retain the Zn-binding cysteine residues in C2c3p. In some embodiments, said effector protein, preferably the C-terminal portion of said effector protein, may include three RuvC-like nuclease catalytic motifs (i.e., RuvCI, RuvCII, and RuvCIII), a bridging helix region, and a zinc finger domain. , preferably in the following order from N to C end: RuvCI-bridged helix-RuvCII-zinc finger-RuvCIII. See FIGS. 13A and 13C for an illustration of the characteristic domain architecture of V-C type effector proteins.

[0076] In some embodiments, V-C type loci, as contemplated herein, may contain CRISPR repeats 20 to 30 bp in length, more typically 22 to 27 bp in length. long, and even more typically 25 bp. length, for example, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 b.p.

[0077] In some embodiments of the invention, the effector protein, namely the effector protein of type VI loci, in particular C2c2p, can originate from, be isolated from or obtained from bacteria belonging to the taxa of alpha-proteobacteria, Bacilli, Clostridia, Fusobacteria and Bacteroidetes. In some embodiments, the effector protein, more specifically the type VI loci effector protein, in particular C2c2p, may originate from, be isolated from, or be derived from bacteria belonging to one of the following genera: Lachnospiraceae , Clostridium , Carnobacterium , Paludibacter , Listeria , Leptotrichia , and Rhodobacter . In some embodiments of the invention, the effector protein, namely the effector protein of type VI loci, in particular C2c2p, may originate from, be isolated from or obtained from the following bacterial species: Lachnospiraceae bacterium MA2020, Lachnospiraceae bacterium NK4A179, Clostridium aminophylum (e.g., DSM 10710), Lachnospiraceae bacterium NK4A144, Carnobacteriumgallinarum (eg DSM 4847 strain MT44), Paludibacterpropionicigenes (eg WB4), Listeriaseeligeri (eg serovar ½b strain SLCC3954), Listeriaweihenstephanensis (eg FSL R9-0317 c4), Listerianewyorkensis (eg FSL strain M6-0635) , Leptotrichiawadei (eg F0279), Leptotrichiabuccalis (eg DSM 1135), Leptotrichia sp. Oraltaxon 225 (eg str. F0581), Leptotrichia sp. Oraltaxon 879 (eg strain F0557), Leptotrichiashahii (eg DSM.19757), Rhodobactercapsulatus (eg SB 1003, R121, or DE442). In some embodiments, the effector protein, more specifically the type V loci effector protein, even more specifically C2c2p, can be derived from, isolated from, or derived from the bacterial species listed in the table in FIGS. 42A-B.

[0078] In some embodiments of the invention, the effector protein, namely the effector protein of type VI loci, in particular - C2c2p, may contain, consist only of, or include an amino acid sequence selected from the sequences shown in the multiple alignment in Fig.13J-N .

[0079] In some embodiments, the type VI locus, as contemplated by the present invention, may encode the Cas1, Cas2, and C2c2p effector proteins. In some embodiments of the invention, the type V-C locus, as contemplated by the present invention, may contain a CRISPR sequence. In some embodiments, the V-C type locus, as contemplated by the present invention, may contain the c2c2 gene and the CRISPR sequence, and not contain the cas1 and cas2 genes . See Fig. 15 and Fig. 42A-B for an illustration of the characteristic organization of type VI loci.

[0080] In some embodiments, the Cas1 protein encoded by the type VI locus, as contemplated by the present invention, can form clusters with a type II subtree, including a small type III-A branch, or within a type III-A system. See Figures 10A and 10B and Figures 10C-W for a Cas1 tree including the Cas1 protein encoded by typical type VI loci.

[0081] In some embodiments, the effector protein, specifically the type VI loci effector protein, in particular C2c2p, e.g. natural C2c2p, may be from about 1000 to about 1500 amino acid residues in length, such as from about 1100 to about 1400 amino acids in length, for example , about 1000 to about 1100 amino acids long, about 1100 to about 1200 amino acids long, or about 1200 to about 1300 amino acids long, or about 1300 to about 1400 amino acids long, or about 1400 to about 1500 amino acids long, e.g. , about 1000, about 1100, about 1200, about 1300, about 1400, or about 1500 amino acids long.

[0082] In some embodiments, the effector protein, namely the effector protein of type VI loci, and in particular C2c2p, contains at least one, or preferably at least two, most preferably exactly two, conserved RxxxxH motifs. Catalytic RxxxxH motifs are characteristic of HEPN domains (a DNA binding domain present in eukaryotes and prokaryotes). Therefore, in some embodiments of the invention, the effector protein, namely the effector protein of type VI loci, in particular C2c2p, contains at least one, or preferably at least two, most preferably exactly two, HEPN domains. See Fig. 11 and Fig. 13B for an illustration of the characteristic domain architecture of type VI effector proteins. In some embodiments, the HEPN domains may have RNase activity. In other embodiments, the HEPN domains may have DNase activity.

[0083] In some embodiments of the invention, type VI loci, as contemplated by the present invention, may contain CRISPR repeats 30 to 40 bp in length, more typically 35 to 39 bp in length, for example, 30 , 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 b.p.

[0084] In some embodiments, a protospacer adjacent sequence (PAM) or PAM-like motif directs the binding of an effector protein complex to a target locus of interest, as described herein. In some embodiments, the PAM may be 5'-PAM (ie, located upstream of the 5' end of the protospacer). In other embodiments, the PAM may be 3'-PAM (ie, downstream of the 5' end of the protospacer).

[0085] In a preferred embodiment, an effector protein, specifically a type V loci effector protein, more specifically a VB type loci effector protein, even more specifically C2c1p, can recognize 5'-PAM. In some embodiments, an effector protein, specifically a type V loci effector protein, more specifically a VB type loci effector protein, even more specifically a C2c1p, can recognize a 5'-PAM that is 5'-TTN- or 5'-ATTN- sequence where N is A, C, G, or T. In some preferred embodiments, the effector protein may be the C2c1p protein of the bacterium Alicyclobacillus acidoterrestris , more preferably the ATCC 49025 C2c1p protein of the bacterium Alicyclobacillus acidoterrestris , in which the 5'-PAM is represented by the 5' - sequence TTN, where N is A, C, G or T, more preferably where N is A, G or T. In other best embodiments of the invention, the effector protein is the C2c1p protein of the bacterium Bacillus thermoamylovorans , more preferably the C2c1p protein of strain B4166 the bacterium Bacillusthermo amylovorans , in which the 5'-PAM is represented by the 5'-sequence ATTN, where N is A, C, G, or T.

[0086] In some embodiments, the CRISPR enzyme is engineered and may contain one or more mutations that reduce or completely eliminate nuclease activity.

[0087] Mutations can also be introduced in adjacent residues, for example, in amino acids close to those mentioned above, which are involved in nuclease activity. In some embodiments, only the RuvC domain is inactivated, and in other embodiments, the other putative nuclease domain is inactivated, with the effector protein complex functioning as a nikase and cleaving only one strand of DNA. In some embodiments, two C2c1 or C2c3 variants (each a different nikase) are used to increase specificity, two nikase variants are used to cleave DNA on targets (where both nikases cleave the DNA strand, minimizing or completely eliminating off-target modifications, where only one the DNA strand is cleaved and then repaired). In preferred embodiments, the C2c1 or C2c3 effector protein cleaves sequences associated with or directly at the target locus of interest as a homodimer containing two C2c1 or C2c3 effector protein molecules. In a preferred embodiment, the homodimer may contain two C2c1 effector protein molecules, or two C2c3 effector protein molecules, or a mixture of C2c1 and C2c3 containing various mutations in the respective RuvC domains.

[0088] The invention relates to methods for using two or more nikazas, in particular, to a method involving the use of a double or double niqazah. In some aspects and embodiments, one type of C2c1 or C2c3 nickase can be delivered, for example, a modified C2c1 or C2c3 or a modified C2c1 or C2c3 nickase, as described herein. This results in the target DNA being bound by two C2c1 or two C2c3 nickases, or a mixture of C2c1 and C2c3 nickases. In addition, it is also contemplated that different orthologues can be used, for example, a C2c1 or C2c3 nickase on the same strand (eg, coding strand) of DNA and an orthologue on a non-coding or opposite DNA strand. An ortholog may be, but is not limited to, a Cas9 nickase, such as a SaCas9 nickase or an SpCas9 nickase. It may be preferable to use two different orthologues that require different PAMs and may also have different requirements for guide sequences, allowing for a greater degree of control when using the invention. In some embodiments, DNA cleavage will include at least four types of nicases, with each type targeting a different nucleotide sequence of the target DNA, with each pair of nicases introducing a first strand break on one DNA strand and a second pair introducing a strand break on a second DNA strand. . In such methods, at least two pairs of nicks are introduced into the target DNA, where the introduction of the first and second pair of nicks in the helix excise target sequences between the first and second pair of nicks. In some embodiments, one or both of the orthologs are controlled, ie. induced.

[0089] In some embodiments, the guide RNA or mature crRNA contains, consists primarily of, or consists only of a straight repeat sequence and a guide sequence or spacer. In some embodiments, the guide RNA or mature crRNA consists of a 19 nucleotide partial direct repeat followed by a 23-25 nucleotide guide or spacer. In some embodiments, the effector protein is a C2c1 or C2c3 effector protein and requires a target sequence of at least 16 nucleotides in length to achieve detectable DNA cleavage and a target sequence of at least 17 nucleotides in length to achieve efficient DNA cleavage in vitro . In some embodiments, the direct repeat sequence is located upstream (ie, at the 5' end) of the guide sequence or spacer. In a preferred embodiment, the primer sequence (i.e., the sequence required for recognition and/or hybridization to the target locus sequence) of the C2c1 or C2c3 guide RNA is approximately within the first 5 nucleotides of the 5' end of the guide sequence or spacer.

[0090] In preferred embodiments, the mature crRNA contains a hairpin structure or an optimized hairpin structure or any optimized secondary structure. In preferred embodiments, the mature crRNA contains a hairpin structure or an optimized hairpin structure in a direct repeat sequence, where the hairpin structure or optimized hairpin structure is important for cleavage. In some embodiments, the mature crRNA preferably includes a single hairpin structure. In some embodiments, the direct repeat sequence preferably includes a single hairpin structure. In some embodiments, the cleavage activity of the effector protein complex can be modified by introducing mutations that affect the structure of the hairpin RNA duplex. In preferred embodiments, mutations can be introduced to maintain the hairpin RNA duplex structure, thereby retaining the enzymatic activity provided by the effector protein complex. In other preferred embodiments, mutations that disrupt the structure of the hairpin RNA duplex can be introduced to completely remove the enzymatic activity carried out by the effector protein complex.

[0091] The invention also relates to a nucleotide sequence encoding an effector protein that is codon-optimized for expression in a eukaryotic organism or eukaryotic cell by any of the methods or compositions described herein. In one embodiment, the codon-optimized effector protein is any C2c1 or C2c3 discussed herein and has been codon-optimized for ease of use in a eukaryotic cell or organism, such as such a cell or organism as described elsewhere herein. for example, but not limited to, in a yeast cell, or a mammalian cell or organism, including a mouse cell, a rat cell, and a cell of a human or non-human eukaryotic organism, such as a plant.

[0092] In some embodiments, at least one nuclear localization signal (NLS) is attached to nucleic acid sequences encoding C2c1 or C2c3 effector proteins. In preferred embodiments of the invention, at least one or more C-terminal or N-terminal NLS sequences are attached (hence, nucleic acid molecules encoding a C2c1 or C2c3 effector protein may include code for NLS sequences so that the expressed product has an attached or linked sequence NLS). In a preferred embodiment, the C-terminal NLSs are fused for optimal expression and nuclear targeting of eukaryotic cells, preferably human cells. In a preferred embodiment, the codon-optimized effector protein is C2c1 or C2c3 and the guide RNA spacer is 15 to 35 nucleotides long. In some embodiments, the length of the guide RNA spacer is at least 16 nucleotides, such as at least 17 nucleotides. In some embodiments, the spacer length is 15 to 17 nucleotides, 17 to 20 nucleotides, 20 to 24 nucleotides, such as 20, 21, 22, 23, or 24 nucleotides, 23 to 25 nucleotides, such as 23, 24 or 25 nucleotides, 24 to 27 nucleotides, 27 to 30 nucleotides, 30 to 35 nucleotides, or 35 nucleotides or longer. In some embodiments, the codon-optimized effector protein is C2c1 or C2c3 and the direct repeat of the guide RNA is at least 16 nucleotides in length. In some embodiments, the codon-optimized effector protein is C2c1 or C2c3 and the direct repeat of the guide RNA is 16 to 20 nucleotides in length, such as 16, 17, 18, 19, or 20 nucleotides. In certain preferred embodiments of the invention, the direct repeat of the guide RNA is 19 nucleotides in length.

[0093] The present invention also encompasses methods for delivering multiple nucleic acid components, wherein each nucleic acid component is specific to a different target loci of interest to alter the multiple target loci of interest. The nucleic acid component of the complex may include one or more protein-binding RNA aptamers. One or more aptamers may be capable of binding to a bacteriophage coat protein. The bacteriophage coat protein can be selected from the group consisting of: Qβ, F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95 , TW19, AP205, ϕСb5, ϕСb8r, ϕCb12r, ϕСb23r, 7s and PRR1. In a preferred embodiment of the invention, the bacteriophage coat protein is the MS2 protein. The invention also provides that the nucleic acid component of the complex is 30 or more, 40 or more, or 50 or more nucleotides in length.

[0094] The invention also relates to cells, components and/or systems of the present invention, containing a small amount of cations present in the cells, components and/or systems. In general, this cation is magnesium, such as Mg ²⁺ . The cation may be present in trace amounts. The preferred concentration range for the Mg ²⁺ cation is from about 1 mM to about 15 mM. The preferred concentration may be about 1 mM for human-based cells, components and/or systems, and from about 10 mM to 15 mM for bacteria-based cells, components and/or systems. See, for example, Gasiunas et al., PNAS, posted online on September 4, 2012, www.pnas.org/cgi/doi/10.1073/pnas. 1208507109.

[0095] Accordingly, it is not the object of the invention to cover within its scope any previously known product, process for producing a product, or method of use, thus, the applicants reserve the right and hereby acknowledge the disclaimer of any previously known product, process, or method . It is further noted that the invention does not intend to cover within its scope any product, manufacturing process, or method of using a product that does not comply with the written description and permissive conditions of the USPTO (35 U.S.C. 112, paragraph one) or EPO (Article 83 EPC), so Applicants reserve the right to and hereby waive any previously described product, process, or method of using a product. The practice of the invention shall be governed by Article 53(c) of the EPC and Rule 28(b) and (c) of the EPC. Nothing in this description should be construed as a promise.

[0096] It is noted that in this application and especially in the claims and/or paragraphs, terms such as "comprise", "contained", "comprising", etc., may have the meaning assigned to them in the US Patent Law; for example, they may mean "includes", "included", "including", etc.; terms such as "essentially consisting of" and "essentially consisting of" have the meaning assigned to them in US Patent Law, for example, they include elements not expressly stated, but exclude elements that are covered in prior applications or that affect main or new features of the invention.

[0097] In a further aspect, the invention relates to a eukaryotic cell comprising a modified target locus of interest, wherein the target locus of interest is modified according to any of the methods described herein. Another aspect of the invention relates to the creation of a cell line of the specified cell. The next aspect of the invention provides for the creation of a multicellular organism, including one or more of these cells.

[0098] In some embodiments, modification of the target locus of interest may result in a eukaryotic cell with altered expression of at least one gene product; eukaryotic cells with altered expression of the product of at least one gene, and the expression of this product is increased; a eukaryotic cell with an altered expression of the product of at least one gene, and the expression of this product is reduced; or a eukaryotic cell containing an edited genome.

[0099] In some embodiments, the eukaryotic cell may be a mammalian or human cell.

[00100] In further embodiments, non-naturally occurring or engineered compositions, vector systems, or delivery systems as described herein can be used for site-specific gene knockout; site-specific genome editing; specific interference of the DNA sequence; or multiplex genome engineering.

[00101] The invention also relates to the production of a gene product from a cell, cell line, or organism as described herein. In some embodiments, the amount of the expressed gene product may be greater or less than the amount of the gene product in a cell with no altered expression or edited genome. In some embodiments, the gene product may be changed from a gene product from a cell without altered expression or an edited genome.

[00102] These and other embodiments of the invention are disclosed in, evident from, or covered by the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

[00103] New features of the invention are detailed in the appended claims. A better understanding of the features and advantages of this invention will be obtained by reading the following detailed description, which includes materials illustrating the principles of the invention, and the accompanying Figures thereto:

[00104] Figures 1A and 1B show a new classification of CRISPR-Cas systems. Class 1 includes multi-subunit cr-RNA effector complexes (Cascade), and Class 2 includes single-subunit cr-RNA effector complexes (Cas9-like). FIG. 1B shows another description of the new classification of CRISPR-Cas systems.

[00105] Figure 2 depicts the molecular organization of CRISPR-Cas systems.

[00106] Figure 3 depicts the structures of type I and III effector complexes: a common architecture/common ancestry is traceable despite extensive sequence divergence.

[00107] Figure 4 depicts CRISPR-Cas as a system based on RNA recognition motifs (RRM).

[00108] Figure 5 depicts the phylogeny of Cas1, showing a major aspect of the evolution of the CRISPR-Cas system, the recombination of cr-RNA effector modules for adaptation.

[00109] Figure 6 is a listing of CRISPR-Cas systems, namely the distribution of types/subtypes of CRISPR-Cas systems among archaea and bacteria.

[00110] FIG. 7 depicts an algorithm for identifying Cas candidates.

[00111] FIGS. 8A and 8B depict the organization of the complete loci of class 2 CRISPR-Cas systems. Three subtypes of type II and subtypes VA, VB and VC and type VI are designated. Subfamilies based on Cas1 phylogeny are also labeled. The scheme only includes the common genes present in each subtype; additional genes present in some variants are omitted. The red rectangle denotes a degenerate repeat. Gray arrows indicate the direction of transcription of the CRISPR sequence. PreFran, Prevotella-Francisella . Figure 8B shows another description of the organization of complete loci of several Class 2 CRISPR-Cas systems.

[00112] FIG. 9 shows neighborhoods of C2c1, i. genomic architecture of CRISPR-Cas C2c1 loci. The number of repeats in CRISPR sequences is indicated. For each genomic contig, a numeric Genebank ID and locus coordinates are indicated.

[00113] FIGS. 10A and 10B depict representations of the Cas1 protein sequence tree. The tree in FIG. 10B was constructed from a multiple alignment of 1498 Cas1 sequences that contained 304 phylogenetically informative positions. Branches corresponding to class 2 systems are underlined: blue - type II; orange - subtype V-A; red - subtype V-B; brown - subtype V-C; purple - type VI. Insets show extended branches of new (sub)types. Bootstrap support values are given as a percentage and are only shown for some of the relevant branches.

[00114] Figures 10C-10W show the complete Cas1 tree, which is schematically shown in Figure 10B in Newick format with species names and bootstrap support values. The tree was built using the FastTree program (options "-gamma -wag"). Multiple alignment of Cas1 sequences was filtered with a uniformity threshold of 0.1 and a gap occurrence threshold of 0.5 prior to tree reconstruction.

[00115] Figure 11 shows the domain organization of class 2 families.

[00116] Figure 12 depicts TnpB homology regions of class 2 proteins.

[00117] Figures 13A-13N are another depiction of the domain architecture and conserved motifs of class 2 effector proteins. Figure 13A illustrates types II and V: TnpB-derived nucleases. The top row of the figure shows RuvC nucleases from Thermos thermophilus (PDB ID: 4EP5) with catalytic amino acid residue designations. The architecture of each domain is shown below, with an alignment of conserved motifs for individual members of the respective protein families (the only sequence for RuvC). Catalytic residues are shown in white letters on a black background, conserved hydrophobic residues are shown in yellow; conserved small amino acid residues are highlighted in green; in the helical bridge alignment, the positively charged residues are highlighted in red. The predicted secondary structure is shown below the aligned sequences: H denotes α-helices, E denotes a straightened conformation (β-structure). Less conserved spacer sequences between alignment blocks are indicated by numbers. 13B illustrates type VI: the proteins contain 2 HEPN domains that can show RNase activity. The top alignment blocks contain individual HEPN domains described earlier, the bottom blocks contain catalytic motifs from type VI effector proteins. Designations as in Fig. 13A. Fig. 13C illustrates the closest homologues of new type V effector proteins among transposon-encoded proteins: non-overlapping sets of homologues. 13D-H illustrates the multiple alignment of the C2c1 protein family. This alignment was made using the MUSCLE program and modified manually based on local pairwise alignments obtained using PSI-BLAST. For each sequence, a GenBank Identifier (GI) number and the systematic name of the organism are given. The secondary structure is predicted using Jpred and is shown below the sequence used as the query sequence (notations: H - alpha helix, E - beta structure). CONSENSUS is calculated for each alignment column by comparing the sum of the paired weights of each column for homogeneous columns (matching residue in all aligned sequences) and a random column with a homogeneity threshold of 0.8. The active site motifs of the RuvC-like domain are shown below the alignment. 13I shows a multiple alignment for the C2c3 protein family. This alignment was performed using the MUSCLE program. For each sequence, the internal number assigned to it and the GenBank identifier (GI) number of the metagenomic contig encoding the corresponding C2c3 protein are indicated. The secondary structure is predicted by Jpred and shown below the alignment (notations: H - alpha helix, E - beta structure). CONSENSUS is calculated for each alignment column by comparing the sum of the pairwise weights of each column for homogeneous columns (matching residuals in all aligned sequences) and a random column with a homogeneity threshold of 0.8. The active site motifs of the RuvC-like domain are shown below the alignment of the C-terminal domain. 13J-N illustrates the multiple alignment of the C2c2 protein family. The alignment was done using the MUSCLE program and changed manually based on local alignments using PSI-BLAST. Each sequence is labeled with a GenBank Identifier (GI) number and the systematic name of the organism. The secondary structure is predicted using Jpred and is shown below the sequence used as the query sequence (notations: H - alpha helix, E - beta structure). CONSENSUS is calculated for each alignment column by comparing the sum of the pairwise weights of each column for homogeneous columns (matching residuals in all aligned sequences) and a random column with a homogeneity threshold of 0.8. The active site motifs of the HEPN domain are shown below the alignment.

[00118] FIG. 14 shows neighborhoods of C2c3, i. genomic architecture of C2c3 loci of the CRISPR-Cas system. The number of repeats in CRISPR sequences is indicated. For each genomic contig, the GenBank identifier number and locus coordinates are indicated.

[00119] FIG. 15 shows neighborhoods of C2c2, i. genomic architecture of C2c2 loci of the CRISPR-Cas system. The number of repeats in CRISPR sequences is indicated. For each genomic contig, the GenBank identifier number and locus coordinates are indicated.

[00120] Figure 16 depicts the RxxxxH motif of the HEPN domain of the C2c2 family.

[00121] Figure 17 depicts C2C1: 1. Alicyclobacillus acidoterrestris ATCC 49025.

[00122] Figure 18 depicts C2C1: 4. Desulfonatronum thiodismutans strain MLF-1.

[00123] Figure 19 depicts C2C1: 5. Opitutaceae TAV5 bacteria.

[00124] Figure 20 depicts C2C1: 7. Bacillus thermoamylovorans strain B4166.

[00125] Figure 21 depicts C2C1: 9. Bacillus sp. NSP2.1.

[00126] Figure 22 depicts C2C2: 1. Lachnospiraceae MA2020 bacteria.

[00127] Figure 23 depicts C2C2: bacteria 2. Lachnospiraceae NK4A179.

[00128] Figure 24 depicts C2C2: 3. [Clostridium] aminophilum DSM 10710.

[00129] Figure 25 depicts C2C2: 4. bacteria Lachnospiraceae NK4A144.

[00130] Figure 26 depicts C2C2: 5. Carnobacterium gallinarum DSM 4847.

[00131] Figure 27 depicts C2C2: 6. Carnobacterium gallinarum DSM 4847

[00132] Figure 28 depicts C2C2: 7. Paludibacter propionicigenes WB4.

[00133] Figure 29 depicts C2C2: 8. Listeria seeligeri serovar 1/2b.

[00134] Figure 30 depicts C2C2: 9. Listeria weihenstephanensis FSL R9-0317.

[00135] Figure 31 depicts C2C2: 10. Listeria bacterium FSL M6-0635.

[00136] Figure 32 shows C2C2: 11. Leptotrichia wadei F0279.

[00137] Figure 33 shows C2C2: 12. Leptotrichia wadei F0279.

[00138] Figure 34 shows C2C2: 14. Leptotrichia shahii DSM 19757.

[00139] Figure 35 shows C2C2: 15. Rhodobacter capsulatus SB 1003.

[00140] Figure 36 depicts C2C2: 16. Rhodobacter capsulatus R121.

[00141] Figure 37 depicts C2C2: 17. Rhodobacter capsulatus DE442.

[00142] FIG. 38 shows a tree of DR sequences.

[00143] Figure 39 depicts the C2c2 protein tree.

[00144] FIGS. 40A-40D contain a table including descriptions of 63 large protein-coding genes near cas1 genes identified using the computational method described herein. Described in the present description, representatives of the new subtypes (VB, VC, VI) are highlighted in color. Protein sequences for representatives of type VB and type IV encoding AUXO014641567.1, AUXO011689375.1, AUXO011689375.1, AIJXO011277409.1, AUXO014986615.1 have not been analyzed, since these sequences cannot be species-specific.

[00145] Fig.41A-41M contain a table showing the results of the analysis of loci V-B type (coding protein C2c1). * cas1cas4 - gene containing cas4 and cas1 domains; CRISPR - CRISPR repeats; SOS - SOS response genes; unk - hypothetical protein; > - the direction of the coding sequence of the gene; [D] - degenerate repetition (defined where possible); [T] - tracr-RNA. 41C-J illustrates the analysis of CRISPR sequences for type VB loci (coding for C2c1 proteins) as described herein (the CRISPR section is part of the core output of pilercr (see the description of the output on the pilercr site: http://www. drive5.com/pHercr/); repeats were folded using mfold (see the description of the result on the mfold website: http://mfold.ma.albany.edu/?q=mfQld/DNA-Folding-FGrm); folding results repeats and analysis of CRISPR sequences are placed after the detailed description of each case, the location of CRISPR, see the reference in the table in Fig. 41 A-B. description using CRISPRmap (details see http://rna.informatik.uni-freiburg.de/CRISPRmap/Input.jsp) Figure 41L illustrates degenerate repeats of type VB loci (coding for C2c1 protein) found as described herein , using the CRISPR-sequence search algorithm values (http://crispr.u-psud.fr/Server/). The column of normal repeats contains a normal repeat, the column of spacers contains the last spacer, the downstream column contains the region of underlying sequences, starting with a degenerate repeat (250 bp); the sequence number corresponds to the CRISPR sequence number in the corresponding locus (see Table in Fig.41 A-B); the area highlighted in yellow has a full match between the normal repeat and the degenerate repeat (if it does not match with another part of the degenerate repeat). Fig.41M illustrates the predicted structures of tracr-RNA, forming base pairs with these repeats. Tracr-RNA for Alicyclobacillus acidoterrestrial was identified by RNA sequencing. For the remaining loci, putative tracr-RNAs were identified based on the presence of an anti-direct repeat (DR) sequence (Anti-DR). Anti-forward repeat sequences were identified by Geneious (www.geneious.com) by searching for sequences within each relevant CRISPR locus with high homology to DR. The 5' and 3' ends of each putative tracr RNA were determined by computational prediction of bacterial transcription start and termination sites using BPROM (www.softberry.com) and ARNOLD (rna.ig-mors.u-psud.fr) respectively. /toolbox/amold/). Cofolding predictions from Geneious, 5' ends in blue, 3' ends in orange.

[00146] Fig.42A-42N contains a table showing the results of the analysis of loci type VI (coding protein C2c2). *CRISPR - CRISPR repeats; unk - hypothetical protein; > - the direction of the coding sequence of the gene; [D] - degenerate repetition (defined where possible); [T] - tracr-RNA, fig.42C-I show the results of the analysis of CRISPR sequences of the type VI loci (coding for the C2c2 protein) as described in the present description (the CRISPR section is part of the basic result of the pilercr work (see the description of the result on the site pilercr : http://www.drive5.com/pilercr/); repeats were folded using mfold (see the description of the result on the mfold website: http://mfold.rna.albany.edu/?q=mfQld/DNA- Folding-Form); results of repeat folding and analysis of CRISPR sequences are placed after the detailed description of each case; see the location of CRISPR by reference in the table in Fig.42 A-B. Fig.42J illustrates the classification of CRISPRmap CRISPR repeats of type VI loci (coding for protein C2c2) using as described in the present CRISPRmap description (for details see http://rna.informatik.uni-freiburg.de/CRISPRmap/Input.jsp). C2c2) as described in the present description, detected using al CRISPR sequence search algorithm (http://crispr.u-psud.fr/Server/). The column of normal repeats contains a normal repeat, the column of spacers contains the last spacer, the downstream column contains the region of underlying sequences, starting with a degenerate repeat (250 bp); the sequence number corresponds to the numerical value of the CRISPR sequence in the corresponding locus (see table in Fig.42 A-B); the area highlighted in yellow has a full match between the normal repeat and the degenerate repeat (if it does not match with another part of the degenerate repeat). 42M-N illustrates the predicted structures of tracr-RNA base pairing with these repeats. Putative tracr-RNAs were identified based on the presence of an anti-direct repeat (DR) (Anti-DR) sequence. Anti-forward repeat sequences were identified by Geneious (www.geneious.com) by searching for sequences within each relevant CRISPR locus with high homology to DR. The 5' and 3' ends of each putative tracr RNA were determined by computational prediction of bacterial transcription start and termination sites using BPROM (www.softberry.com) and ARNOLD (rna.ig-mors.u-psud.fr/ toolbox/amold/) respectively. Cofolding predictions from Geneious, 5' ends in blue, 3' ends in orange.

[00147] Fig.43A-43F contains a table showing the results of the analysis of loci type V-C (coding protein C2c3). *CRISPR - CRISPR repeats; unk - hypothetical protein; > - the direction of the coding sequence of the gene; [D] - degenerate repeat (defined where possible). Fig.43C - show the results of the analysis of CRISPR sequences of V-C loci (coding for the C2c3 protein) as described in the present description (the CRISPR section is part of the core output of the pilercr work (see the description of the work on the site pilercr: http://www. drive5.com/pilercr/); repeats were folded using mfold (see the description of the result on the mfold website: http://mfold.ma.albany.edu/?q=mfQld/DNA-Folding-Form); folding results repeats and analysis of CRISPR sequences are placed after the detailed description of each case, see the location of CRISPR at the link in the table in Fig. 43 A-B. Statistically significant BLAST matches for the spacer with prokaryotes and their viruses are shown. Fig. 43E illustrates the classification of CRISPRmap CRISPR repeats loci of the V-C type (coding for the C2c3 protein) as described herein using CRISPRmap (details see http://rna.informatik.uni-freiburg.de/CRISPRmap/Input.jsp) Figure 43F illustrates the degenerate repeats of the loci type V-C (coding ie C2c3 protein) as described herein, detected using the CRISPR sequence search algorithm (http://crispr.u-psud.fr/Server/). The normal repeat column contains the normal repeat, the spacer column contains the last spacer, the downstream column contains the area of underlying sequences, starting from the degenerate repeat (250 bp); the sequence number corresponds to the number of the CRISPR sequence in the corresponding locus (see table in Fig.43 A-B); the area highlighted in yellow has a full match between the normal repeat and the degenerate repeat (if it does not match with another part of the degenerate repeat).

[00148] FIGS. 44A-44E show a complete list of CRISPR-Cas loci in genomes where C2c1 or C2c2 proteins have been found. The genes for the C2c1 and C2c2 proteins are highlighted in yellow.

[00149] Figures 45A-45C illustrate the alignment of Listeria loci encoding the putative type VI CRISPR-Cas system. Aligned synthetic region corresponding to contig AODJ01000004.1 Listeria weihenstephanensis FSL R9-0317 at coordinates 42281-46274 and contig JNFB01000012.1 Listeria newyorkensis strain FSL M6-0635 at coordinates 169489-173541. Legend: C2c2 gene in blue, CRISPR repeats in magenta, degenerate repeats in magenta, spacers in bold.

[00150] Figures 46A-46D show functional confirmation of the C2c1 locus of Alicyclobacillus acideoterrestris . 46A: RNA sequencing demonstrates high expression of the C2c1 locus of Alicyclobacillus acideoterrestris with a truncated crRNA including a 14 nucleotide long direct repeat at the 5' end and a 20 nucleotide spacer. The putative tracr-RNA, 79 nucleotides long, is stably expressed in the same direction as the cas gene cluster. 46B: Northern blot analysis of RNA expressed from the endogenous locus (M) and minimal first spacer sequence (S) show processed crRNA with a direct repeat at the 5' end and the presence of a small putative tracr RNA. The arrows indicate the positions of the probes and their direction. Fig.46C: Co-folding direct repeat of cr-RNA and putative tracr-RNA in silico shows a stable secondary structure and complementarity between the two RNA. 5' bases are colored blue and 3' bases are colored orange. Fig.46D: Heterologous expression of the Alicyclobacillus acideoterrestris C2c1 locus in pACYC-184 transformed into E. coli gives identical results with the expression observed in the endogenous strain (Fig.46A). Processed crRNAs have a 14 nt long direct DR repeat at the 5' end and a 20 nt long spacer, and the putative 79 nt tracr RNA is expressed stably.

[00151] Figures 47A-47C show the identification of a protospacer adjacent motif (PAM) for the C2c1 enzyme from Alicyclobacillus acideoterrestris. 47A: Screening scheme for PAM. 47B: Depletion at the left 5'end of the PAM library indicates the presence of TTM PAM at the 5'end. Attrition is defined as a base 2 logarithm, with the amount of PAM above a threshold of 3.5 being used to calculate an entropy estimate for each position. Fig.47C: PAM confirmation for C2c1 from Alicycclobacillus acideoterrestris by measuring interference with eight different PAM. PAMs corresponding to the TTN motif demonstrate the presence of depletion as measured by the colony forming unit (CFU) method.

[00152] Figures 48A-F provide a characterization of the conditions under which C2c1 from Alicyclobacillus acideoterrestris cleaves in vitro . 48A: In vitro cleavage of the EMX1 target by a human cell lysate expressing C2c1 from Alicyclobacillus acideoterrestris shows that in vitro targeting of Alicyclobacillus acideoterrestris C2c1 is reliable and dependent on tracr-RNA. Non-targeting cr-RNA. (cr-RNA 2) cannot cleave the EMX1 target, while cr-RNA 1 targeting EMX1 provides strong cleavage in the presence of Mg ⁺⁺ and weak cleavage in the absence of Mg ⁺⁺ . 48B: In vitro cleavage of the EMX1 target in the presence of various length tracr RNAs identifies 78 bp types as the minimum length of tracr RNA, and increased cleavage efficiency for the 91 bp form is also noted. Fig.48C: Analysis of the temperature dependence of the cleavage of the target EMX1 in vitro shows that the optimal temperature range for cleavage of AacC2c1 is from 40 to 55°C. 48D: In vitro validation of the PAM requirements of the AacC2c1 protein with four different PAMs. Those PAMs that were complementary to the TTN motif were cleaved efficiently. Fig. 48E: In silico folding of the chimeric sg-RNA AacC2c1 shows the formation of a stable structure with a direct repeat: reverse connection of segments derived from tracr-RNA (marked in red) and cr-RNA (marked in black). 48F: Comparison of in vitro target cleavage in the presence of cr-RNA-tracr-RNA AacC2c1 and sg-RNA shows similar cleavage efficiency.

[00153] Figures 49A-49C show functional confirmation of the C2c1 locus of Bacillus thermoamylovorans. 49A: Heterologous expression of the Bacillus thermoamylovorans C2c1 locus in E. coli . The putative tracr-RNA is highly expressed and processed down to 91 nucleotides. Processed crRNAs are also present and have a 14 nt long direct repeat and a 19 nt spacer at the 5' end. Fig. 49B: In silico cofolding of a crRNA direct repeat and putative tracr RNA shows the formation of a stable secondary structure and complementarity between the two RNAs. The 5' ends are colored blue and the 3' ends are colored orange. Fig.49C: Depletion at the 5'end of the left PAM library shows the presence of ATTN PAM at the 5'end. Attrition is measured as a base 2 logarithm, and the amount of PAM above a threshold of 3.5 is used to calculate an entropy score for each position.

[00154] FIG. 50 shows all reads for the C2C1 locus of Bacillus sp .

[00155] Figure 51 shows filtered reads from 0 to 55 base pairs at the C2C1 locus of Bacillus sp .

[00156] Figure 52 shows the cofolding of the direct repeat sequence and tracr RNA corresponding to the B. sp C2C1 locus.

[00157] FIG. 53 illustrates an evolutionary scenario for CRISPR-Cas systems. It is assumed that the Cas8 protein arose through the inactivation of Cas10 (denoted by a white X), which was accompanied by a significant acceleration of evolution. Abbreviations: TR, terminal repeats; TS, end sequences, HD, HD endonuclease family; HNH, HNH endonuclease family, RuvC, RuvC endonuclease family; HEPN, a putative endoribonuclease of the HEPN superfamily. The regions of genes and proteins shown in gray represent sequences that were encoded in the corresponding transposable elements but were lost during the evolution of CRISPR-Cas systems.

[00158] The drawings herein are for illustrative purposes only and are not necessarily drawn to scale.

DETAILED DESCRIPTION OF THE INVENTION

[00159] Before the methods of the present invention are described, it should be understood that the present invention is not limited to the individual methods, components, products, or combinations thereof described herein, due to the fact that such methods, products, and combinations, of course, can, undoubtedly vary. It is also to be understood that the terminology used herein is not intended to be limiting, as the scope of the present invention is limited only by the appended claims.

[00160] In general, the term "CRISPR-Cas or CRISPR system", also used in prior documents such as W0 2014/093622 (PCT/US2013/074667), is used to collectively refer to transcripts and other components involved in expression or control activity of CRISPR-associated ("Cas") genes, including sequences encoding the Cas gene, tracr sequence (transactivating CRISPR) (e.g., tracr RNA or active partial tracr RNA), tracr helper sequence (including "forward repeat" and processable tracr-RNA partial direct repeat within the endogenous CRISPR system), a targeting sequence (also referred to as a "spacer" within the endogenous CRISPR system), or "RNA molecule(s)" as used herein (e.g., molecule(s) Cas guide RNA such as Cas9, e.g. CRISPR RNA and transactivating (tracr) RNA or single guide RNA (sgRNA) (chimeric RNA)) or other sequences and trans anscripts of the CRISPR locus. In general, the CRISPR system is characterized by components that facilitate the formation of the CRISPR complex at the site of the target sequence (also referred to as the protospacer within the endogenous CRISPR system). In the context of CRISPR complex formation, "target sequence" means a sequence for which a complementary target sequence has been purposefully designed, wherein hybridization between the target sequence and the target sequence promotes the formation of the CRISPR complex. The target sequence may include any polynucleotide, including RNA or DNA polynucleotides. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell. In some embodiments, direct repeats can be identified in silico by looking for repeat motifs that meet any or all of the following criteria: 1. found within a 2 kb region of the genomic sequence flanking type II CRISPR loci; 2. length from 20 to 50 bp; and 3. gaps separating them 20-50 bp long. In some embodiments, any 2 of these criteria may be used, such as 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all 3 criteria may be used.

[00161] In embodiments of the invention, concepts such as mature crRNA, single guide RNA, guide sequence, and guide RNA, i.e. RNAs capable of directing Cas to a target locus of the genome are used interchangeably in prior cited documents such as W0 2014/093622 (PCT/US2013/074667). In general, a guide sequence is any polynucleotide sequence having sufficient complementarity to the target polynucleotide to hybridize to the target sequence and direct the sequence to direct specific binding of the CRISPR complex to the target sequence. In some embodiments, the level of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using an appropriate alignment algorithm, is close to or greater than 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or higher. The optimal alignment can be determined using any appropriate sequence alignment algorithm including but not limited to: Smith-Waterman algorithm, Needleman-Wunsch algorithm, Burrows-Wheeler transform based algorithms (e.g. Burrows Wheeler Aligner), ClustalW, Clustal X , BLAST, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA, USA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge .net). In some embodiments, the guide sequence has a length approximately equal to or greater than 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 , 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides. In some embodiments, the guide sequence is less than 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides in length. The preferred length of the guide sequence is 10-30 nucleotides. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence can be assessed by any suitable technique. For example, components of a CRISPR system sufficient to form a CRISPR complex, including a verifiable targeting sequence, can be delivered into a host cell containing the appropriate target sequence, including by transfection using vectors encoding components of the CRISPR sequence, followed by quantitative assessment of the location of the preferred target. splitting the target sequence, in particular, using the analysis, as described in the present description. Similarly, cleavage of a target nucleotide sequence can be assessed in vitro using the target sequence, components of the CRISPR complex, including a target target sequence and a control target sequence other than the target target sequence when comparing the level or rate of target sequence cleavage between reactions involving test and control guide sequences. Other methods of analysis are also acceptable and can be chosen by a person skilled in the art.

[00162] In classical CRISPR-Cas systems, the level of complementarity between a guide molecule sequence and its corresponding target sequence can be approximately equal to or greater than 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97, 5%, 99% or 100%; the guide molecule or RNA or sgRNA can be about or greater than 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 in length , 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides; or the guide molecule or RNA or sgRNA may be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides in length; and preferably tracr-RNA is 30 or 50 nucleotides long. However, in one of its aspects, the invention is intended to reduce off-target interactions, for example, to reduce the interaction of a guide molecule with a target molecule with low complementarity. Indeed, the examples show that the invention involves mutations that render the CRISPR-Cas system capable of distinguishing between target and non-target sequences with complementarity greater than 80% to about 95%, e.g., 83%-84% complementarity, 88 -89% or 94-95% (for example, distinguishing a target molecule having 18 nucleotides of a non-target molecule 18 nucleotides long with 1, 2 or 3 complementarity violations). Accordingly, in the context of the present invention, the level of complementarity between the guide sequence and its corresponding target molecule exceeds 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.9% or 100%. For a non-target molecule, it is less than 100%, 99.9%, 99.5%, 99%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95%, 94.5%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83 %, 82%, 81%, or 80% complementarity between its sequence and the guide molecule, with the non-target molecule preferably having sequence complementarity of 100%, 99.9%, 99.5%, 99%, 99%, 98, 5%, 98%, 97.5%, 97%, 96.5%, 96%, 95.5%, 95% or 94.5% with targeting molecule.

[00163] In particularly preferred embodiments of the present invention, a guide RNA (capable of directing Cas to a target locus) may include (1) a guide sequence capable of hybridizing to a genomic target locus in a eukaryotic cell; (2) tracr sequence; and (3) partner sequence tracr. Components (1)-(3) can be located on the same RNA, i.e. sg-RNA (located in the direction from 5' to 3'-end), or tracr-RNA can be RNA other than RNA containing the guide sequence and the tracr sequence. Tracr hybridizes to the tracr partner sequence and directs the CRISPR/Cas complex to the target sequence.

[00164] Aspects of the invention relate to the identification and engineering of novel effector proteins associated with CRISPR-Cas class 2 systems. In a preferred embodiment, the effector protein comprises a single subunit effector module. In a future embodiment, the effector protein is functional in prokaryotic or eukaryotic cells for in vitro , in vivo or ex vivo use. One aspect of the invention encompasses computational methods and algorithms for predicting new Class 2 CRISPR-Cas systems and determining their constituent components.

[00165] In one embodiment, a computational method for identifying novel loci of the CRISPR-Cas class 2 system includes the following steps: detecting all contigs encoding the Cas1 protein; identification of all predicted protein-coding genes within 20 kb. from the cas1 gene; comparison of identified genes with specific profiles of Cas proteins and prediction of CRISPR sequences by selecting unclassified candidate CRISPR-Cas loci containing proteins with sequences greater than 500 amino acids (>500 a.k.); analysis of selected candidates using PSI-BLAST and HHPred assays to isolate and identify new CRISPR-Cas class 2 loci. In addition to the above steps, possible candidates can be further analyzed by searching for homologues in metagenomic databases.

[00166] One aspect of finding all contigs encoding the Cas1 protein is to use the GenemarkS program to predict genes, as described in more detail in the article "GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions," John Besemer, Alexandre Lomsadze and Mark Borodovsky, Nucleic Acids Research (2001) 29, pp 2607-2618, incorporated herein by reference.

[00167] In one aspect, all predicted protein-coding genes are identified by comparing the identified genes to specific Cas protein profiles and annotating them according to the NCBI Conservative Domains Database (CDD), which consists of a collection of well-annotated multiple sequence alignment models for ancient domains and full-length proteins. They are available as positional weighting matrices (PSSM) for the rapid identification of conserved domains in protein sequences through RPS-BLAST analysis. CDD includes domains curated by NCBI that use 3D structure information to accurately define domain boundaries and provide insight into sequence/structure/function relationships, as well as domain models imported from many external databases (Pfam, SMART, COG, PRK , TIGRFAM). In the following aspect, PILER-CR, which is a publicly available CRISPR repeat search software, was used to predict CRISPR sequences, as described in "PILER-CR: fast and accurate identification of CRISPR repeats", Edgar, R.C., BMC Bioinformatics, Jan 20:8:18 (2007), incorporated herein by reference.

[00168] In the following aspect, an individual analysis of each case is carried out using PSI-BLAST (Position Based Basic Local Alignment Finder). PSI-BLAST analysis uses a positional weight matrix (PSSM) or multiple alignment profile of sequences found above a predetermined weight threshold in protein-protein BLAST analysis. This PSSM matrix is used to further search for new matches in the database and is updated for subsequent iterations with these newly discovered sequences. Thus, PSI-BLAST is a way to detect distant relationships between proteins.

[00169] In another aspect, individual analysis of each case involves the use of HHpred, a sequence database search and structure prediction method that is as easy to use as BLAST or PSI-BLAST, and at the same time much more sensitive in finding distant homologues. In fact, the sensitivity of the HHpred method is competitive with the most powerful structure prediction servers currently available. HHpred is the first server based on pairwise comparison of Hidden Markov Model (HMM) profiles. While most conventional sequence search methods search sequence databases such as UniProt or NR, HHpred searches alignment databases such as Pfam or SMART. This makes it much easier to obtain a list of matches with many families of sequences instead of disparate single sequences. All major public profile and alignment databases are available through HHpred. HHpred uses a sequence or multiple alignment as a single search query. Within minutes, HHpred presents search results in an easy-to-read PSI-BLAST-like format. Search options include local or global alignment and secondary structure weight similarity. HHpred can produce pairwise search and template sequence alignments, combined multiple search and template alignments (eg for transitive searches), and 3D structure models computed by the MODELLER software based on HHpred alignments.

[00170] The term "nucleic acid targeting system", where the nucleic acid is DNA or RNA, and in some aspects may also refer to RNA-DNA hybrids or derivatives thereof, refers collectively to transcripts and other elements involved in expression or targeting DNA or RNA targeting activities of genes associated with the CRISPR system ("Cas"), which may contain sequences encoding Cas proteins, targeting DNA or RNA, and targeting RNA, targeting DNA or RNA, including the sequence cr-RNA (cr-RNA ) and (in the CRISPR-Cas9 system, but not all systems) an RNA sequence that trans-activates the CRISPR/Cas system (tracr-RNA), or other sequences and transcripts of the CRISPR locus that target DNA or RNA. In general, the RNA targeting system is characterized by elements that facilitate the formation of an RNA targeting complex at the site of the target RNA sequence. In the context of forming a DNA or RNA targeting complex, the term "target sequence" refers to a DNA or RNA sequence that is complementary to a DNA or RNA targeting guide RNA, where hybridization between the target sequence and the RNA targeting guide RNA promotes formation targeting the RNA complex. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell.

[00171] In one embodiment, the novel DNA-targeted systems, also referred to as DNA-targeted CRISPR-Cas or CRISPR-Cas-DNA-targeted systems in the context of this application, are based on identified Cas type V proteins (e.g., subtype V-A and subtype V-B), which do not require the production of specialized proteins for specific RNA sequences, but contain only one effector protein or enzyme that can be programmed by an RNA molecule to recognize a specific DNA target; in other words, the protein can be attracted to a specific DNA target with the help of the specified RNA molecule. Aspects of the invention relate in particular to DNA-targeted RNA-directed CRISPR C2c1 or C2c3 systems.

[00172] In one aspect of the invention, the novel RNA targeting systems, also referred to as RNA or RNA-targeted CRISPR/Cas or the RNA targeting system with the CRISPR-Cas system, described herein, are based on identified type VI Cas proteins, not requiring the creation of individual proteins to target specific RNA sequences, in addition to a single enzyme that can be programmed by an RNA molecule to recognize a specific target DNA, in other words, the enzyme can be designed to work with a specific target DNA using said RNA molecule.

[00173] As used herein, a Cas protein or CRISPR enzyme refers to any of the proteins present in the new classification of CRISPR-Cas systems. In a preferred embodiment, the present invention relates to effector proteins identified at CRISPR-Cas type V loci, for example, the Cpf1-encoding loci referred to as the V-A subtype. Currently, V-A subtype loci include cas1, cas2, a single gene designated cpf1, and the CRISPR cassette. Cpf1 (CRISPR-associated Cpf1 protein, PREFRAN subtype) is a large protein (approximately 1300 amino acids) containing a RuvC-like nuclease domain homologous to the corresponding Cas9 domain, along with the corresponding arginine-rich Cas9 cassette. However, Cpf1 does not have the HNH nuclease domain present in all Cas9 proteins, and the RuvC-like domain is contiguous in the Cpf1 sequence, in contrast to Cas9, where it contains long insertions including the HNH domain. Accordingly, in certain embodiments, the CRISPR-Cas enzyme only includes a RuvC-like nuclease domain.

[00174] In a preferred embodiment, the present invention relates to compositions and systems containing effector proteins identified at the C2c1 loci, referred to as the V-B subtype. In the present description, C2c1 refers to class 2 candidate 1. All C2c1 loci encode for a fusion of Cas1-Cas4, Cas2, and a large protein, which Applicants have designated C2c1p, and is usually adjacent to the CRISPR cassette.

[00175] In a preferred embodiment, the present invention relates to an effector protein identified at CRISPR-Cas type VI loci, eg C2c2 loci. In the present description, C2c2 means 2 class 2 candidates. The C2c2 loci include the cas1 and cas2 genes, along with a large protein designated by the Applicants as C2c2p and the CRISPR cassette; however, unlike C2c1p, C2c2p often encodes the sequence following the CRISPR cassette, but not cas1-cas2.

[00176] In a preferred embodiment, the present invention relates to compositions and systems containing effector proteins identified at the C2c3 loci, designated as the V-C subtype. All C2c3 loci encode a fusion of Cas1-Cas4, Cas2 and a large protein designated by the Applicants as C2c3p.

[00177] Aspects of the present invention also include methods and uses of the compositions and systems described herein for genomic engineering, for example, to modify or direct the expression of one or more genes or one or more gene products in prokaryotic or eukaryotic cells in vitro, in vivo or ex vivo .

[00178] The nucleic acid targeting systems, vector systems, vectors and compositions described herein can be used in the practice of nucleic acid targeting, alteration or modification of the synthesis of gene products such as proteins, nucleic acid cleavage, nucleic acid editing, splicing nucleic acids, target nucleic acid transfer, target nucleic acid tracking, target nucleic acid isolation, target nucleic acid imaging, etc.

[00179] Aspects of the invention also cover methods and uses of the compositions and systems described herein in genetic engineering, for example, to alter or manipulate the expression of one or more genes or one or more gene products, in prokaryotic or eukaryotic cells, in vitro , in vivo or ex vivo .

[00180] The methods of the present invention described herein involve introducing one or more mutations into a eukaryotic cell ( in vitro , i.e., in an isolated eukaryotic cell) as described herein, including delivery to the cell of a vector as described in this description. Mutations may include the insertion, deletion or substitution of one or more nucleotides in each target sequence of the cell/cells with guide/their RNA or sgRNA molecule/molecules. Mutations may include insertion, deletion or substitution of 1-75 nucleotides in each/s of the specified/th cell/s with guide/their RNA or sg-RNA molecule/molecules. Mutations may include insertion, deletion, or substitution 1, 5, 10, 11, 12, 13, 14, 15, 16, 1/, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30, 35, 40, 45, 50 or 75 nucleotides in each/s of the indicated/s cell/s using guide/their RNA or sg-RNA molecule/molecules. Mutations may include insertion, deletion or substitution 10, 11, 12, 13, 14, 15, 16, 17, F 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35 , 40, 45, 50 or 75 nucleotides in each/s of the indicated/s cell/s using guide/their RNA or sg-RNA molecule/molecules. Mutations may include insertion, deletion, or substitution of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or 75 nucleotides in each cell(s) indicated using guide/their RNA or sg-RNA molecule/molecules. Mutations can include insertion, deletion or substitution of 40, 45, 50, 75, 100, 200, 300, 400 or 500 nucleotides in each specified cell/s with guide RNA/s or sgRNA molecule/molecules.

[00181] To minimize toxicity and reduce the possibility of off-target effects, it is important to control the concentration of delivered Cas mRNA and guide RNA. Optimal concentrations of Cas mRNA and guide RNA can be determined by testing different concentrations in cellular or non-human eukaryotic animal models and using deep sequencing techniques to analyze the degree of modification at potential non-target genomic loci. Another alternative way to minimize the level of toxicity and off-target effects is to deliver Cas nickase mRNA (eg Cas9 of S. pyogenes with D10A mutation) with two guide RNAs targeted at the target site. Guide sequences and strategies to minimize toxicity and off-target effects can be used as described in WO 2014/093622 (PCT/US 2013/074667) or by mutation as described herein.

[00182] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence complexed with one or more Cas proteins) results in cleavage of one or both strands near the target sequence (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from it) or in itself. Without wishing to be bound by theory, a tracr sequence that may contain or consist of a portion of the wild-type tracr sequence (e.g., about or more than 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of the wild-type tracr sequence ) may also form part of the CRISPR complex, in particular by hybridization of at least a portion of the tracr sequence with the entire tracr partner sequence or a portion thereof operably linked to the targeting sequence.

[00183] Preferably, the nucleic acid molecule encoding Cas presents a glitch codon-optimized sequence to Cas codons. An example of a codon-optimized sequence here is a sequence optimized for expression in a eukaryote, eg, a human (i.e., optimized for expression in a human) or other eukaryote, animal, or mammal, as discussed herein; see for example the human codon-optimized SaCas9 sequence in WO 2014/093622 (PCT/US2013/074667). While this is preferred, it is understood that other examples and known codon optimization for a non-human host or codon optimization for specific organs are possible. In some embodiments, the enzyme coding sequence encodes Cas and is codon-optimized for expression in specific cells, such as eukaryotic cells. Such eukaryotic cells may belong to or be isolated from a particular organism, in particular a mammal, including, but not limited to: a human, non-human eukaryote, animal or mammal as described herein, for example, mice, rats, rabbits, dogs, livestock or non-human mammal or primate. In some embodiments, methods for modifying the genetic makeup of a human germline and/or methods for modifying the genetic makeup of animals that are likely to cause suffering without significant medical benefit to humans or animals, as well as the animals resulting from such methods, may be excluded. In general, codon optimization refers to the process of changing a sequence to increase expression in cells of a given organism by changing at least one codon (e.g., about or more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence into codons that are more frequently or most frequently used in the genes of those of cells of the target organism while maintaining the original amino acid sequence. Different species show a specific shift in the frequency of occurrence of individual codons for individual amino acids. Codon bias (difference in codon usage between different organisms) often correlates with messenger RNA (mRNA) translation efficiency, which in turn is thought to depend, among other things, on the properties of the codons being translated and the availability of individual transfer RNA (tRNA) molecules. The predominance of specific tRNAs in a cell usually corresponds to those codons that are used most often in peptide synthesis. Accordingly, genes can be designed to optimize codons to optimize gene expression in an organism. Tables of codon usage are available, for example, from the "Codon Usage Database" available at www.kazusa.orjp/codon/, and such tables can be adapted in a number of ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000). Computer algorithms for optimizing codons for a specific sequence and expression in a specific host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA, USA). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a Cas coding sequence correspond to the most commonly used codons for a particular amino acids.

[00184] In certain embodiments, the methods as described herein may include providing a Cas transgenic cell in which one or more nucleic acids encoding one or more guide RNA molecules are provided or introduced operably linked in the cell to a regulatory an element containing the promoter of one or more genes of interest. The term "Cas transgenic cell", as used herein, refers to a cell, in particular a eukaryotic cell, into the genome of which the Cas gene is inserted. The nature, type or origin of such a cell is not particularly limited in accordance with the present invention. Also, the method of introducing the Cas transgene into a cell may vary and be any of the methods available in the art. In some embodiments, a transgenic Cas cell is obtained by introducing a Cas transgene into an isolated cell. In some other embodiments, a transgenic Cas cell can be obtained by isolating cells from a transgenic Cas organism. As a non-limiting example, a transgenic Cas cell as used herein can be derived from a Cas transgenic eukaryotic organism, such as a eukaryotic organism with an inserted Cas. See WO 2014/093622 (PCT/US13/74667), incorporated herein by reference. Methods according to US Pat. No. 8,771,985 issued to Sangamo BioSciences, Inc. and Sigma-Aldrich Co. LLC and US Patent Publication No. 20110265198 assigned to Sangamo BioSciences, Inc. aimed at targeting the Rosa locus may be modified to use the CRISPR Cas system of the present invention. The methods of US Pat. No. 20130236946 assigned to Cellectis to target the Rosa locus can also be modified to use the CRISPR-Cas system of the present invention. For the following example, a reference is made to an article by Platt et. al. (Cell; 159(2):440-455 (2014)) describing a mouse with a Cas9 mouse insert, incorporated herein by reference. The Cas transgene can also include a Lox-Stop-polyA-Lox(LSL) cassette, which makes Cas expression driven by the Cre recombinase. Alternatively, a Cas transgenic cell can be obtained by introducing a Cas transgene into an isolated cell. Transgene delivery systems are well known in the art. An example would be the delivery of a Cas transgene, eg, into a eukaryotic cell via a vector (eg, AAV, adenovirus, lentivirus) and/or a particle and/or nanoparticle, as also described elsewhere herein.

[00185] It will be understood by the skilled artisan that a cell, such as a Cas transgenic cell as used herein, may contain further genomic changes in addition to having an inserted Cas gene or mutations resulting from sequence-specific action of Cas in complex with RNA capable of directing Cas to the target locus, such as, for example, one or more oncogenic mutations, as described by way of non-limiting example in Platt et al. (2014), Chen et al., (2014), or Kumar et al. (2009).

[00186] In some embodiments, the Cas sequence is fused to one or more nuclear localization signal (NLS) sequences, such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs. In some embodiments, the implementation of the Cas contains about or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLS at or near the N-terminus, about or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at the C-terminus, or a combination thereof (e.g., zero or at least one or more NLSs at the N-terminus and zero or at least one or more NLSs at the C-terminus end). If more than one NLS is present, each of them may be selected independently of the others, in particular, a single NLS may be represented by more than one copy and/or in combination with another one or more NLS represented by one or more copies. In a preferred embodiment, Cas contains no more than 6 NLS. In some embodiments, an NLS is considered N- or C-terminal if the nearest amino acid of the NLS is 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more amino acids away from the polypeptide chain. from the N- or C-terminus. Non-limiting examples of NLSs include NLS sequences derived from: SV40 large T antigen NLS having the amino acid sequence PKKKRKV (SEQ ID NO: X); NLS from nucleoplasmin (eg, two-part nucleoplasman NLS with the sequence KRPAATKKAGQAKKKK) (SEQ ID NO: X); c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: X) or RQRRNELKRSP (SEQ ID NO: X); NLS hRNPA1 M9 having the sequence NQ S SNFGPMKGGNFGGRS SGPYGGGGQYFAKPRNQGGY (SEQ ID NO: X); sequences RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: X) from the importin-alpha domain; VSRKRPRP (SEQ ID NO: X) and PPKKARED (SEQ ID NO: X) myoma T-protein sequences; sequence POPKKKPL (SEQ ID NO: X) human p53; mouse c-abi IV sequences SALIKKKKKMAP (SEQ ID NO: X), DRLRR (SEQ ID NO: X) and PKQKKRK (SEQ ID NO: X) sequences of influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: X) of the hepatitis delta antigen; sequence REKKKFLKRR (SEQ ID NO: X) of the mouse Mx1 protein; sequences KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: X) of human poly(ADP-ribose) polymerase; and sequences RKCLQAGMNLEARKTKK (SEQ ID NO: X) of glucocorticoid steroid hormone receptors (human). In general, such one or more NLSs are strong enough to cause accumulation of Cas in appreciable amounts in the nucleus of a eukaryotic cell. In general, the strength of nuclear localization activity can be determined by the number of NLSs in the Cas proteins, the particular NLS(s) used, or a combination of these factors. For example, a detectable label can be associated with Cas in such a way as to visualize localization in the cell, in particular, through a combination of methods for determining the localization of the nucleus (for example, a specific nuclear dye, such as DAPI). Cell nuclei can also be isolated from cells, the contents of which can then be analyzed using any suitable protein detection method, such as immunohistochemistry, Western blot, or enzyme activity analysis. Accumulation in the nucleus can be determined indirectly, in particular by analyzing the effect of CRISPR complexing (for example, analyzing DNA cleavage or mutations in the target sequence, or analyzing changes in gene expression due to CRISPR complexing and/or Cas enzyme activity), in comparison with a control not exposed to Cas or complex, or exposed to Cas lacking one or more NLSs.

[00187] In some aspects, the invention includes such vectors, for example, for the delivery or introduction into the cell of Cas and / or RNA capable of directing Cas to the target locus (i.e., guide RNA), as well as for reproducing these components (for example, in prokaryotic cells). As used herein, a "vector" is a tool that allows or facilitates the transfer of an object from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another piece of DNA can be inserted in such a way as to cause the inserted piece to replicate. In general, the vector is capable of replication when associated with the appropriate controls. In general, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is associated. Non-limiting examples of vectors are nucleic acid molecules that are single stranded, double stranded, or partially double stranded; nucleic acid molecules having one or more free ends that do not have free ends (eg, circular); nucleic acid molecules, including DNA, RNA, or both DNA and RNA, and other polynucleotide varieties known in the art. One type of vector is a "plasmid", which is a circular double-stranded loop of DNA into which additional DNA segments can be inserted, including using standard cloning methods. Another type of vector is a viral vector containing a sequence derived from viral DNA or RNA necessary for packaging into a virus (eg, retroviruses, replication-defective retroviruses, adenoviruses, adenoviruses, retroviruses, and replication-defective adeno-associated viruses (AAV, AAV)). Viral vectors also include a polynucleotide inserted into a virus for transfection into a host cell. Some vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) integrate into the genome of a host cell when introduced into the host cell and thereby replicate along with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are directly associated. Such vectors are referred to herein as "expression vectors". Vectors that result in expression in a eukaryotic cell may be referred to herein as "eukaryotic expression vectors". A common form of expression vectors in recombinant DNA methods is often plasmids.

[00188] Recombinant expression vectors may include a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements that can be selected based on host cells that will be used for expression, i.e. operably linked to a nucleic acid sequence to be expressed. In a recombinant expression vector, "operably linked" should mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced). into the host cell). With regard to recombination and cloning methods, reference is made to US Patent Application No. 10/815730, published September 2, 2004 as US 2004-0171156 A1, the contents of which are incorporated herein by reference in their entirety.

[00189] Vector(s) may include regulatory element(s), such as promoter(s). The vector(s) may include sequences encoding Cas proteins and/or a single RNA guide sequence, but at least 3 or 8 or 16 or 32 or 48 or 50 sequences encoding RNA guide sequences are also possible (e.g., sg-RNA), e.g. 1-2, 1-3, 1-4 1-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-8, 3-16, 3 -30, 3-32, 3-48, 3-50 RNA (e.g. sg-RNA). A single vector may contain a promoter for each RNA (e.g. sgRNA), preferably up to 16 RNA molecules (sgRNA) and if a single vector provides more than 16 RNAs (e.g. sgRNA), one or more promoters can express more than one RNA (e.g. sgRNA), e.g., in the presence of 32 RNAs (e.g., sgRNA), each promoter can express two RNAs (e.g., sgRNA) and in the presence of 48 RNAs (e.g., sg-RNA), each promoter can provide the expression of three RNAs (for example, sg-RNA). Based on simple arithmetic and well established cloning protocols and the concepts of the present invention, one skilled in the art can easily apply the present invention to RNA molecule(s) (e.g. sgRNA) for a suitable commonly used vector such as AAV, and a suitable promoter, in particular a U6 promoter, eg U6-sgRNA. For example, the maximum possible packing volume in AAV is ~4.7 kb. The length of U6-sg-RNA alone (including restriction sites for cloning) is 361 bp. Therefore, one skilled in the art can easily place about 12-16, eg 13, U6-sgRNA cassettes into one vector. It can be assembled using any suitable method, in particular the "golden gate strategy" for TALE assembly (http://www.genome-engineering.org/taleffectors/). The skilled artisan may also use a tandem guide strategy to increase the amount of U6-sgRNA by about 1.5 times, eg from 12-16, eg 13, to 18-24, eg, about 19 U6-sgRNA. Therefore, one skilled in the art can easily make about 18-24, eg about 19 promoter-RNA constructs, eg U6-sg-RNA, in a single vector, eg AAV vector. Other means of increasing the number of promoters and RNA are given in WO 2016/205749 PCT/US2016/038238, e.g., sgRNA molecule(s) in a vector to use a single promoter (e.g., U6) to express RNA sequences, e.g., sgRNA, separated by cleavable sequences. Further methods for increasing the number of promoter-RNA constructs, eg sgRNA in a vector, are to express a promoter-RNA sequence, eg sgRNA in a vector separated by cleavable sequences within an intron of a coding sequence or gene; in this case it is preferable to use a polymerase II promoter which can enhance expression and allow transcription of long RNA tissue type specific (see e.g. http://nar.oxfordjournals.org/content/34/7/e53.short, http ://www.nature.com/mt/journal/vl6/n 9/abs/mt20081 44a.html). In a preferred embodiment, the AAV vector can be packaged with a U6-sgRNA tandem targeting up to about 50 genes. Accordingly, based on the knowledge in the art and the guidance in this application, a qualified person can create and use vector(s), for example, a single vector expressing multiple RNAs or other targeting molecules under control, operably or functionally associated with one or more promoters, especially for the number of RNA or other guide molecules discussed in the present description, without any undue experimentation.

[00190] Sequences encoding guide RNAs, such as sgRNAs, and/or sequences encoding Cas proteins can be operably or directly linked to the regulatory element(s) and therefore the regulatory element(s) directs their expression. The promoter(s) may be a constitutive promoter(s) and/or a transient promoter(s) and/or an inducible promoter(s) and/or a tissue-specific promoter(s). The promoter may be selected from the group consisting of RNA polymerases: pol I, pol II, pol III, T7, U6, H1, Rous sarcoma virus (RSV) retroviral LTR promoter (LTR), cytomegalovirus (CMV) promoter, virus promoter SV40, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerate kinase (PGK) promoter and EF1α promoter. The preferred promoter is the U6 promoter.

[00191] In general, the CRISPR-Cas9 system is as used in the above documents such as WO 2014/093622 (PCT/US2013/074667) and refers collectively to transcripts and other elements involved in the expression or directing the activity of a CRISPR-associated ("Cas") of an enzyme, e.g., Cas9, including sequences encoding the Cas enzyme (targeting DNA and/or RNA) or involved in its delivery, the tracr (CRISPR transactivating) sequence (e.g., tracr RNA or active partial tracr RNA), a tracr partner sequence (comprising a "forward repeat" and a tracr-RNA-processed partial direct repeat in the context of the endogenous CRISPR system), a targeting sequence (also called a "spacer" in the context of the endogenous CRISPR system), or "RNA molecule(s)" in as used herein (e.g., Cas9 targeting RNA molecule(s), such as CRISPR RNA (cr-RNA) and transactivating cr-RNA (tracr RNA) or single targeting sgRNA (chimeric RNA) or other sequences and transcripts of the CRISPR locus. In general, the CRISPR system is characterized by the presence of elements that stimulate the formation of the CRISPR complex at the site of the target sequence (also called protospacers in the context of the endogenous CRISPR system). In the context of CRISPR complex formation, "target sequence" means a sequence for which a targeting sequence is designed, in particular having complementarity, wherein hybridization between the target sequence and the targeting sequence stimulates the formation of a CRISPR complex. A certain section of the guide sequence, mediating the significance of complementarity to the target sequence for cleavage activity, is referred to in the present description as the seed-sequence (seed-sequence). The target sequence may include any polynucleotide, such as DNA or RNA polynucleotides, and be within the target locus of interest. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell. The invention described herein encompasses novel class 2 effector proteins of CRISPR-Cas systems, among which Cas 9 is an exemplary effector protein and hence the terms used in this application to describe novel effector proteins may correlate with the terms used to describe CRISPR systems. -Cas9.

[00192] The loci of the CRISPR-Cas system comprise more than 50 gene families and do not have strictly universal genes. Consequently, for the loci of the CRISPR-Cas system, no evolutionary tree will be the only true one, and a multifaceted approach is needed to identify new gene families. To date, cas genes have been exhaustively identified for 395 profiles of 93 Cas proteins. To classify CRISPR-Cas systems, gene profiles and characteristic features of loci architecture are used. A new classification of CRISPR-Cas systems is proposed in Figures 1A and 1B. Class 1 includes multi-subunit cr-RNA effector complexes (Cascade), and class 2 includes single-subunit cr-RNA effector complexes (Cas9-like). Figure 2 shows the molecular organization of CRISPR-Cas systems. Figure 3 depicts the structure of type I and III effector complexes: a common architecture/common ancestry is traceable despite extensive sequence divergence. Figure 4 CRISPR-Cas is depicted as a system based on RNA recognition motifs (RRM). Figure 5 shows the phylogeny of the Cas1 protein, showing the main aspect of the evolution of the CRISPR-Cas system - the recombination of cr-RNA effector modules for adaptation. Figure 6 presents a list of CRISPR-Cas systems, namely the distribution of types/subtypes of CRISPR-Cas systems among archaea and bacteria.

[00193] The operation of the CRISPR-Cas system is usually divided into three stages: (1) adaptation or integration of the spacer, (2) processing of the primary transcript of the CRISPR locus (pre-cr-RNA) and maturation of cr-RNA, which includes the spacer and variable regions, corresponding to fragments of CRISPR repeats at the 5' and 3' ends, and (3) RNA or DNA interference. Two proteins, Cas1 and Cas2, which are present in the vast majority of known CRISPR-Cas systems, are sufficient to insert spacers into CRISPR cassettes. These two proteins form a complex that is necessary for the adaptation process; Cas1 endonuclease activity is required for spacer integration, while Cas2 performs a non-enzymatic function. The Cas1-Cas2 complex is a highly conservative "information processing" module of the CRISPR-Cas system, which is quasi-autonomous from the rest of the system. (See Annotation and Classification of CRISPR-Cas Systems. Makarova KS, Koonin EV. Methods Mol Biol. 2015;1311:47-75).

[00194] Previously described class 2 systems, namely type II and putative type V, consisted of only three or four genes in the Cas operon, namely the cas1 and cas2 genes, constituting the adaptation module (the cas1-cas2 gene pair is not involved in interference), as well as a single multidomain effector protein responsible for interference but also facilitating pre-crRNA processing and adaptation, and often a fourth gene with uncharacterized functions that is optional in at least some type II systems (and in in some cases, the fourth gene is cas4 (data from biochemical or in silico experiments show that cas4 belongs to the PD-(DE)xK superfamily of nucleases with a cluster of three cysteine residues at the C-terminus; it has 5'-exonuclease activity for ssDNA), or csn2 , which codes for an inactivated ATPase). In most cases, the CRISPR sequence and the gene for a specific type of RNA known as tracr-RNA, a trans-encoded small cr-RNA, are adjacent to class 2 Cas operons. -RNA catalyzed by RNase III, a widespread bacterial enzyme not associated with the loci of the CRISPR-Cas system.

[00195] Cas1 is the most conserved protein that is present in most CRISPR-Cas systems and evolves more slowly than other Cas proteins. Accordingly, Cas1 phylogeny has been used to classify CRISPR-Cas systems. The results of biochemical experiments or experiments in silico show that Cas1 is a metal-dependent deoxyribonuclease. Deletions in the Cas1 gene in the E. coli genome result in increased susceptibility to DNA damage and reduced chromosome segregation, as described in "A dual function of the CRISPR-Cassystem in bacterial antivirus immunity and DNA repair," Babu M et al. Mol Microbiol 79:484-502 (2011). The results of biochemical experiments or in silico experiments show that Cas2 is an RNase characteristic of regions rich in U and is a double-stranded DNase.

[00196] Aspects of the invention relate to the identification and engineering of novel effector proteins associated with CRISPR-Cas class 2 systems. In a preferred embodiment, the effector protein comprises a single subunit effector module. In a future embodiment, the effector protein is functional in prokaryotic or eukaryotic cells for in vitro , in vivo or ex vivo use. One aspect of the invention encompasses computational methods and algorithms for predicting new Class 2 CRISPR-Cas systems and determining their constituent components.

[00197] In one embodiment, a computational method for identifying novel loci of the CRISPR-Cas class 2 system includes the following steps: detecting all contigs encoding the Cas1 protein; identification of all predicted protein-coding genes within 20 kb. from the cas1 gene, more specifically within 20 kb. from the beginning of the cas1 gene and 20 kb. from the end of the cas1 gene; comparing identified genes with specific profiles of Cas proteins and predicting CRISPR sequences by selecting partial and/or unclassified candidate CRISPR-Cas loci containing proteins with sequences greater than 500 amino acids (>500 a.k.); analysis of selected candidates using PSI-BLAST and HHPred assays to isolate and identify new CRISPR-Cas class 2 loci. In addition to the above steps, possible candidates can be further analyzed by searching for homologues in metagenomic databases.

[00198] One aspect of finding all contigs encoding the Cas1 protein is to use the GenemarkS program to predict genes, as described in more detail in "GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions," John Besemer, Alexandre Lomsadze and Mark Borodovsky, Nucleic Acids Research (2001) 29, pp 2607-2618, incorporated herein by reference.

[00199] In one aspect, all predicted protein-coding genes are identified by comparing the identified genes to specific Cas protein profiles and annotating them according to the NCBI Conservative Domains Database (CDD), which consists of a collection of well-annotated multiple sequence alignment models for ancient domains and full-length proteins. They are available as positional weight matrices (PSSM) for the rapid identification of conserved domains in protein sequences through RPS-BLAST analysis. CDD includes domains curated by NCBI that use 3D structure information to accurately define domain boundaries and provide insight into sequence/structure/function relationships, as well as domain models imported from many external databases (Pfam, SMART, COG, PRK , TIGRFAM). In the following aspect, PILER-CR, which is a publicly available CRISPR repeat search software, was used to predict CRISPR sequences, as described in "PILER-CR: fast and accurate identification of CRISPR repeats", Edgar, R.C., BMC Bioinformatics, Jan 20:8:18 (2007), incorporated herein by reference.

[00200] In the following aspect, an individual analysis of each case is carried out using PSI-BLAST (Position Based Basic Local Alignment Finder). PSI-BLAST analysis uses a positional weight matrix (PSSM) or multiple alignment profile of sequences found above a predetermined weight threshold in protein-protein BLAST analysis. This PSSM matrix is used to further search for new matches in the database and is updated for subsequent iterations with these newly discovered sequences. Thus, PSI-BLAST is a way to detect distant relationships between proteins.

[00201] In another aspect, individual analysis of each case involves the use of HHpred, a sequence database search and structure prediction method that is as easy to use as BLAST or PSI-BLAST, and at the same time much more sensitive in finding distant homologues. In fact, the sensitivity of the HHpred method is competitive with the most powerful structure prediction servers currently available. HHpred is the first server based on pairwise comparison of Hidden Markov Model (HMM) profiles. While most conventional sequence search methods search sequence databases such as UniProt or NR, HHpred searches alignment databases such as Pfam or SMART. This makes it much easier to obtain a list of matches with many families of sequences instead of disparate single sequences. All major public profile and alignment databases are available through HHpred. HHpred uses a sequence or multiple alignment as a single search query. Within minutes, HHpred presents search results in an easy-to-read PSI-BLAST-like format. Search options include local or global alignment and secondary structure weight similarity. HHpred can produce pairwise search and template sequence alignments, combined multiple search and template alignments (eg for transitive searches), and 3D structure models computed by the MODELLER software based on HHpred alignments.

[00202] The term "nucleic acid targeting system", where the nucleic acid is DNA or RNA, and in some aspects may also refer to RNA-DNA hybrids or derivatives thereof, refers collectively to transcripts and other elements involved in expression or targeting DNA or RNA targeting activities of genes associated with the CRISPR system ("Cas"), which may contain sequences encoding Cas proteins, targeting DNA or RNA, and targeting RNA, targeting DNA or RNA, including the sequence cr-RNA (cr-RNA ) and (in some but not all systems) an RNA sequence that trans-activates the CRISPR/Cas system (tracr-RNA), or other sequences and transcripts of the CRISPR locus that target DNA or RNA. In general, an RNA targeting system is characterized by elements that facilitate the formation of a DNA or RNA targeting complex at a target DNA or RNA sequence site. In the context of forming a DNA or RNA targeting complex, the term "target sequence" refers to a DNA or RNA sequence that is complementary to a DNA or RNA targeting guide RNA, where hybridization between the target sequence and the RNA targeting guide RNA promotes formation targeting the RNA complex. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell.

[00203] In one aspect of the invention, the novel DNA targeting systems, also referred to as DNA- or DNA-targeted CRISPR/Cas or CRISPR-Cas DNA targeting system, described herein, are based on identified Cas type V proteins (e.g. , subtype V-A, subtype V-B and subtype V-C) that do not require the creation of individual proteins to target specific DNA sequences, but instead a single effector protein or enzyme can be programmed by an RNA molecule to recognize a specific target DNA, in other words, the enzyme can be recruited to a specific target DNA using the specified RNA molecule. Aspects of the invention particularly relate to DNA-targeted RNA-guided CRISPR C2c1 or C2c3 systems.

[00204] In one aspect of the invention, the novel RNA targeting systems, also referred to as RNA or RNA targeting CRISPR/Cas or the RNA targeting system with the CRISPR-Cas system, described herein, are based on identified type VI Cas proteins, not requiring the creation of individual proteins to target specific RNA sequences, in addition to a single enzyme that can be programmed by an RNA molecule to recognize a specific target DNA, in other words, the enzyme can be designed to work with a specific target DNA using said RNA molecule.

[00205] The nucleic acid targeting systems, vector systems, vectors and compositions described herein can be used in the practice of nucleic acid targeting, alteration or modification of the synthesis of gene products such as proteins, nucleic acid cleavage, nucleic acid editing, splicing nucleic acids, target nucleic acid transfer, target nucleic acid tracking, target nucleic acid isolation, target nucleic acid imaging, etc.

[00206] As used herein, a Cas protein or CRISPR enzyme refers to any of the proteins featured in the new classification of CRISPR-Cas systems. In a preferred embodiment, the present invention encompasses effector proteins identified at CRISPR-Cas type V loci, it should be noted that Cpf1 encodes loci referred to as the V-A subtype. Currently, V-A subtype loci include cas1, cas2, a single gene designated cpf1, and the CRISPR cassette. Cpf1 (CRISPR-associated Cpf1 protein, PREFRAN subtype) is a large protein (about 1300 amino acids) containing a RuvC-like nuclease domain homologous to the corresponding Cas9 domain, along with an analogue of the characteristic arginine-rich Cas9 cluster. At the same time, Cpf1 lacks the HNH nuclease domain found in all Cas9 proteins, and the RuvC-like domain is continuous, in contrast to Cas9, where it contains long inserts containing the HNH domain. C2c1 and C2c3 are similar to Cpf1 in that they also encode for class V CRIPSR type II loci. Accordingly, in some embodiments, the CRISPR-Cas enzyme contains only a RuvC-like nuclease domain. The Cpf1 gene is found in several different bacterial genomes, mostly in the same locus containing the cas1, cas2, cas4 genes and the CRISPR cassette (for example, FNFX1_1431-FNFX1_1428 from Francisella cf. novicida Fx1). Thus, the structural organization of Cpf1 appears to be similar to type II-B. Moreover, similar to Cas9, the Cpf1 protein contains an easily identifiable C-terminal region homologous to the ORF-B transposon and includes an active RuvC-like nuclease, an arginine-rich region, and a zinc finger (absent in Cas9). However, unlike Cas9, Cpf1 is also present in several non-CRISPR-Cas genomes, and its relatively high similarity to ORF-B suggests that it may be a transposon component. It has been proposed that if this CRISPR-Cas system is true and Cpf1 is a functional analogue of Cas9, then it can thus be assigned to a new type of CRISPR-Cas, namely type V (see Annotation and Classification of CRISPR-Cas Systems. Makarova KS , Koonin EV Methods Mol Biol 2015;1311:47-75). However, as described herein, Cpf1 is assigned to the V-A subtype in order to distinguish it from C2c1p and C2c3p, which do not have an identical domain structure and therefore belong to the V-B and V-C subtypes, respectively.

[00207] In a preferred embodiment, the present invention relates to compositions and systems comprising effector proteins identified at C2c1 loci allocated to the V-B subtype. In this case, C2c1 refers to 1 candidate from class 2. All C2c1 loci encode a Cas1-Cas4 fusion protein, Cas2, and a large protein designated by Applicants as C2c1p, usually located near the CRISPR cassette.

[00208] In a preferred embodiment, the present invention relates to compositions and systems comprising effector proteins identified at C2c3 loci allocated to the V-C subtype. In this case, C2c3 refers to the 3rd candidate from the 2nd class. The C2c3 loci encode Cas1 and a large protein designated C2c3p.

[00209] In a preferred embodiment, the present invention encompasses effector proteins belonging to the type VI loci of the CRISPR-Cas system, for example, the C2c2 loci. As used herein, C2c2 refers to class 2 candidate 2. C2c2 loci include the cas1 and cas2 genes along with a large protein, which we refer to as C2c2p, and CRISPR sequences; however, unlike C2c1p, C2c2p is often encoded near CRISPR sequences, but not by cas1-cas2 genes (for comparison, FIG. 9 and FIG. 15).

[00210] Aspects of the invention also cover the methods and use of the compositions and systems described herein in genomic engineering, for example, for altering or directing the expression of one or more genes or one or more gene products in prokaryotic or eukaryotic cells in vitro , in vivo or ex vivo .

[00211] In embodiments of the invention, the terms "guide sequence" and "guide RNA" are used interchangeably, as in the documents mentioned above, such as WO 2014/093622 (PCT/US2013/074667). Broadly, a guide sequence is any nucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize to the target sequence and provide sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the level of complementarity between a guide sequence and a corresponding target sequence, when optimally aligned using an appropriate alignment algorithm, is about or greater than 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97 .5%, 99% or more. The optimal alignment can be determined using any suitable sequence alignment algorithm, non-limiting examples of which are the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, Burrows-Wheeler transform based algorithms (e.g. Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, the guide sequence is about or greater than 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 in length. , 29, 30, 35, 40, 45, 50, 75 or more nucleotides. In some embodiments, the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides in length. Preferably, the guide sequence is 10-30 nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence can be quantified by any suitable assay method. For example, components of the CRISPR system sufficient to form the CRISPR complex, including the targeting sequence under test, can be delivered into a cell containing the appropriate target sequence, e.g., by transfection with vectors encoding components of the CRISPR sequence, followed by quantification of preferential cleavage sites of the target sequence. in particular by Surveyor analysis as described herein. Similarly, cleavage of a target polynucleotide sequence can be assessed in a tube containing the target sequence, components of the CRISPR complex, including test guide sequence and control guide sequence, by comparing the binding or cleavage rate of the target sequence for reactions with test and control guide sequences. Other methods of analysis are also possible and will be understood by a person skilled in the art. In some embodiments, the target sequence is a sequence in the cell's genome. Illustrative target sequences include those that are unique within the target genome.

[00212] In general and in the description of the present invention, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid to which it is associated. Vectors include, but are not limited to, nucleic acid molecules that are single stranded, double stranded, or partially double stranded; nucleic acids that include one or more free ends that do not have free ends (eg, circular); nucleic acids, which are DNA, RNA, or both types of polynucleotides known in the art. One type of vector is called a "plasmid", which means a circular double-stranded DNA loop into which additional DNA segments can be introduced, including by standard molecular cloning techniques. Another type of vector is a viral vector that contains sequences derived from the viral DNA or RNA in the vector for packaging into a virus (eg, retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection of a host cell. Some vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episome vectors). Other vectors (eg, non-episomal mammalian vectors) are inserted into the genome of a host cell when introduced into the host cell and thereby replicate along with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are directly associated. Such vectors are referred to herein as "expression vectors". Vectors for expression and for expression in a eukaryotic cell may be referred to herein as "eukaryotic expression vectors". Frequently used vectors in recombinant DNA methods are plasmids.

[00213] Recombinant expression vectors may include a nucleic acid of the invention in a form suitable for the expression of such nucleic acid in a host cell, which means that such recombinant expression vectors contain one or more regulatory elements that can be selected in accordance with those used for expression by host cells and which are operably linked to the nucleic acid sequence to be expressed. In a recombinant expression vector, "operably linked" should mean that the target nucleotide sequence is linked to the regulatory element(s) in such a way that expression of such nucleotide sequence is possible (e.g., in an in vitro transcription/translation system or in a host cell). when the vector is introduced into the host cell).

[00214] The term "regulatory element" is used to refer to promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (eg, transcription termination signals such as polyadenylation signals and poly(U) sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that allow constitutive expression of a nucleotide sequence in various cell types and those that allow expression of a nucleotide sequence only in certain host cells (eg, tissue-specific regulatory sequences). A tissue-specific promoter can provide expression preferentially in the desired target tissue, such as muscle, neuron, bone, skin, blood, specific organs (eg, liver, pancreas), or specific cell types (eg, lymphocytes). Regulatory elements can also drive expression in a time dependent manner, eg depending on cell cycle stage or developmental stage, and may also be tissue or cell type specific. In some embodiments, the vector comprises one or more RNA polymerase III promoters (e.g., 1, 2, 3, 4, 5 or more RNA polymerase III promoters), one or more RNA polymerase II promoters (e.g., 1, 2 , 3, 4, 5 or more RNA polymerase II promoters), one or more RNA polymerase I promoters (eg, 1, 2, 3, 4, 5 or more RNA polymerase I promoters), or a combination thereof. Examples of RNA polymerase III promoters include, but are not limited to, the U6 and H1 promoters. Examples of RNA polymerase II promoters include, but are not limited to, Rous sarcoma virus (RSV) promoters (optionally with an RSV enhancer), cytomegalovirus (CMV) promoter, (optionally with a CMV enhancer) [see, for example, Boshart et al., Cell, 41:521-530 (1985)], SV40 promoter, dihydrofolate reductase promoter, p-actin promoter, phosphoglycerol kinase (PGK) promoter and EF1a promoter. The term "regulatory element" refers to enhancer elements such as WPRE; CMV enhancers; R-U5' segment in HTLV-I LTR (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); an SV40 enhancer and an intron sequence between exons 2 and 3 of rabbit P-globin (Proc. Natl, Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). One of skill in the art will appreciate that the design of the expression vector may depend on such factors as the host cell chosen to be transfected, the level of expression desired, and so on. The vector can be introduced into cells such that transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein are obtained (e.g., regularly clustered short palindromic repeat system (CRISPR) transcripts, their proteins , enzymes, mutants, fusion proteins, etc.)

[00215] Advantageous vectors include lentiviruses and adeno-associated viruses, and types of such vectors can also be selected to target specific cell types.

[00216] As used herein, the term "cr-RNA" or "guide RNA" or "single guide RNA" or "sg-RNA" or "one or more nucleic acid components" of an effector protein of a type V locus or a type VI system CRISPR Cas includes any polynucleotide sequence having sufficient complementarity with the target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct, sequence-specific binding of the nucleic acid-targeted complex to the target nucleic acid sequence. In some embodiments, the degree of complementarity when optimally aligned using a suitable alignment algorithm is approximately 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more. Optimal alignment can be performed using any suitable sequence alignment algorithm, examples of which include, but are not limited to, the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, Burrows-Wheeler transform based algorithms (e.g. Burrows Wheeler Aligner), ClustalW, Clustal X, BLAST, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA, USA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq. sourceforge.net). The ability of a guide sequence (within guide RNA targeting nucleic acids) to direct sequence-specific binding of a complex targeted to nucleic acids can be assessed by any suitable assay method. For example, nucleic acid-targeting CRISPR system components sufficient to form a nucleic acid-targeting complex, including the targeting sequence to be tested, can be provided to a host cell having the appropriate target nucleic acid sequence, for example, by transfection with vectors encoding components of a nucleic acid-targeting complex, followed by evaluation of preferential targeting (eg, cleavage) at the target nucleic acid sequence, such as in the Survey assay described herein. Similarly, cleavage of a target nucleic acid sequence can be assessed in vitro by presenting to a cell the target nucleic acid sequence, the components of the nucleic acid targeting complex, including the targeting sequence to be tested and a control targeting sequence different from the targeting sequence being tested, and comparing binding or the level of target sequence cleavage between the test and control guide sequence reactions. Other methods of analysis are possible and understandable to a person skilled in the art. The targeting sequence, and hence the targeting nucleic acid targeting RNA, can be chosen to target any target nucleic acid sequence. The target sequence may be DNA. The target sequence can be any RNA sequence. In some embodiments, the target sequence may be a sequence in an RNA molecule selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA, small interfering RNA ( miRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double-stranded RNA (dsRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA) and small cytoplasmic RNA (ssRNA). In some preferred embodiments, the target sequence may be a sequence in an RNA molecule selected from the group consisting of mRNA, pre-mRNA, and rRNA. In some preferred embodiments, the target sequence may be a sequence in an RNA molecule selected from the group consisting of ncRNA and dncRNA. In some more preferred embodiments, the target sequence may be a sequence in an mRNA molecule or a pre-mRNA molecule.

[00217] In some embodiments, the nucleic acid-targeted guide RNA is chosen such that the secondary structure of the RNA-targeted guide RNA is more compact. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less of nucleic acid-targeted guide RNA nucleotides , participates in self-complementary base pairing with optimal folding. The optimal folding can be determined by any suitable polynucleotide folding detection algorithm. Some programs are based on the calculation of the minimum Gibbs free energy. An example of one such algorithm is mFold as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). An example of another folding algorithm is the RNAfold online web server developed at the Institute for Theoretical Chemistry at the University of Vienna, which uses a centroid structure prediction algorithm (see, for example, A.R. Gruber et al, 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).

[00218] In certain embodiments, a guide RNA or crRNA may include, essentially consist of, or consist of a direct repeat (DR) sequence and a guide molecule or spacer sequence. In some embodiments, a guide RNA or crRNA may comprise, consist essentially of, or consist of a direct repeat (DR) sequence fused to or linked to a guide molecule sequence or a spacer sequence. In some embodiments, a direct repeat (DR) sequence may be located upstream (ie, 5') from a guide molecule sequence or a spacer sequence. In other embodiments, the direct repeat sequence may be located downstream (ie, 3') from the guide molecule or spacer sequence.

[00219] In certain embodiments, the crRNA includes a hairpin structure, preferably a single hairpin structure. In some embodiments, a hairpin structure, preferably a single hairpin structure, forms a direct repeat sequence.

[00220] In certain embodiments, the length of the guide RNA spacer is 15 to 35 nucleotides. In certain embodiments, the guide RNA spacer is at least 15 nucleotides long. In some embodiments, the spacer is 15 to 17 nucleotides long, such as 15, 16, or 17 nucleotides, 17 to 20 nucleotides, such as 17, 18, 19, or 20 nucleotides, 20 to 24 nucleotides, such as 20, 21, 22, 23 or 24 nucleotides, 23 to 25 nucleotides, such as 23, 24 or 25 nucleotides, 24 to 27 nucleotides, such as 24, 25, 26 or 27 nucleotides, 27 to 30 nucleotides, such as 27 , 28, 29 or 30 nucleotides, 30-35 nucleotides, for example 30, 31, 32, 33, 34 or 35 nucleotides, or 35 nucleotides or more.

[00221] The term "tracr-RNA sequence" or a similar term includes any polynucleotide that has sufficient complementarity to cr-RNA for subsequent hybridization. In general, the degree of complementarity refers to the optimal alignment of the sca sequence and the tracr sequence along the length of the shortest of the two sequences. The optimal alignment can be determined by any suitable alignment algorithm and, in addition, it can take into account the secondary structure, for example, self-complementarity within the sca sequence or the trascr sequence. In some embodiments, the degree of complementarity between the tracr sequence and the sca sequence along the length of the shorter of the two, when optimally aligned, is about or about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95 %, 97.5%, 99% or higher. In some embodiments, tracr-RNA may not be required.

[00222] Applicants also conducted a pilot experiment to test the ability of type V proteins such as C2c1 or C2c3 to target and cleave DNA. This experiment largely replicates similar work for heterologous expression of StCas9 in E. coli (Sapranauskas, R. et al. Nucleic Acids Res 39, 9275-9282 (2011)). Applicants introduced a plasmid containing both PAM and an antibiotic resistance gene into a heterologous strain of E. coli and then placed on a medium containing the appropriate antibiotic. In the case of plasmid DNA digestion, Applicants did not observe viable colonies.

[00223] In more detail, the analysis of the target DNA can be carried out as follows. Two strains of E. coli are used in the assay. One of the strains has a plasmid that encodes the endogenous effector protein locus of the bacterial strain. Another strain has an empty plasmid (eg pACYC184, control strain). All possible PAMs are 7 or 8 bp long. presented on an antibiotic resistance plasmid (pUC19 with the ampicillin resistance gene). PAM is located adjacent to the protospacer 1 sequence (target DNA for the first spacer at the endogenous effector protein locus). Two PAM libraries have been cloned. One library contains sequences from the 5' end of the protospacer, consisting of 8 random bp. (e.g. total 65,536 different PAM=complexity). Another library contains sequences of the 3'-end of the protospacer, consisting of 7 random bp. (for example, full complexity - 16,384 different PAMs). Both libraries were cloned to have an average of 500 plasmids containing each possible PAM. The test and control strains were transformed with the 5'-PAM and 3'-PAM libraries in separate transformations, and the transformed cells were cultured on a medium containing ampicillin. Recognition and subsequent cutting/interference by the plasmid makes the cell resistant to ampicillin and stops its growth. Approximately 12 hours after transformation, all colonies formed by the test and control strains were collected and plasmid DNA was isolated from them. Plasmid DNA was used as a template for PCR amplification and subsequent deep sequencing. The representation of all PAMs in non-transformed libraries showed the expected abundance of PAMs in transformed cells. The representation of all PAMs found in the control strains showed the actual representation. The representation of all PAMs in the test strain showed which PAMs are not recognized by the enzyme. Comparison with a control strain allows extraction of PAM depleted sequences.

[00224] In some embodiments of the CRISPR-Cas9 invention, the level of complementarity between the tracr-RNA and crRNA sequences is present along the length of the shortest of them with the most optimal alignment. As described herein, in some embodiments of the present invention, tracr-RNA is not required. In some embodiments of previously described CRISPR-Cas systems (eg, CRISPR-Cas9 systems), engineered chimeric guide RNA (sgRNA) patterns may include at least 12 bp. duplex structure between cr-RNA and tracr-RNA, however, such chimeric RNAs (chi-RNAs) are not possible in the CRISPR Cpf1 systems described herein because the system does not use tracrRNA.

[00225] In certain embodiments, mature crRNAs include a sequence element derived from the a repeat sequence (5' tag) of the CRISP locus that is important to function. See Marraffini et al., 28-Jan-2010, Nature Letters 463 (568-572), which is incorporated by reference.

[00226] In some embodiments, the degree of complementarity between a tracr-RNA sequence and a cr-RNA sequence along the length of the shorter one, when optimally aligned, is about or greater than about 25%, 30%, 40%, 50%, 60%, 70% , 80%, 90%, 95%, 97.5%, 99% or higher. In some embodiments, the tracr sequence is about or greater than about 5, 6, 7, 8, 9, 10, II, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40 , 50 or more nucleotides in length. In some embodiments, the tracr sequence and the crRNA sequence are contained in the same transcript such that hybridization between them creates a transcript having a secondary structure such as a hairpin. In one embodiment, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In preferred embodiments, the transcript has two, three, four or five hairpins. In a further embodiment of the invention, the transcript has no more than five hairpins. In the hairpin structure, part of the sequence from the 5' side of the final "N" and above the loop corresponds to the tracr sequence, and part of the sequence from the 3' side of the loop corresponds to the tracr sequence. In some embodiments, tracr-RNA may not be required.

[00227] In some embodiments of previously described CRISPR-Cas systems (eg, CRISPR-Cas9), engineered chimeric guide RNA (sgRNA) constructs may include at least 12 bp. duplex structure of crRNA and tracrRNA, however, in CRISPR Cpf1 systems, such chimeric RNA (chi-RNA) is not possible, since the system does not use tracr-RNA.

[00228] To minimize toxicity and non-specific effects, it is important to control the concentration of delivered guide RNA. Optimal concentrations of nucleic acid-targeted guide RNA can be determined by testing different concentrations in non-human cellular or eukaryotic animal models, deep sequencing can be used to analyze the degree of modification at potential non-target genomic loci. The concentration for in vivo delivery should be chosen to provide the highest level of target modification with the lowest level of off-target modification. It is preferred to derive the nucleic acid targeting system from a type V/type VI CRISPR system. In some embodiments of the invention, one or more elements of the nucleic acid targeting system are derived from a particular organism, including an endogenous RNA targeting system. In preferred embodiments, the RNA targeting system is a type V CRISPR system. In specific embodiments, the type V RNA targeting Cas enzyme is C2c1 or C2c3. Examples of Cas proteins include, but are not limited to, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologues or modified versions. In embodiments, a type V protein, such as C2c1 or C2c3, mentioned herein also includes homologues or orthologues of type V proteins, such as C2c1 or C2c3. The terms "ortholog" (also referred to herein as "ortologue") and "homolog" (also referred to herein as "homologue") are well known in the art. By way of further guidance, a "homologue" of a protein, as used herein, is a protein of the same species that performs the same or similar function as the protein of which it is a homologue. Homologous proteins may have a similar or partially similar structure, but this is not required. An "ortholog" of a protein, as used herein, is a protein of a different species that performs the same or a similar function as the protein of which it is orthologue. Orthologous proteins may have a similar or partially similar structure, but this is not required. In specific embodiments, the homologues or orthologues of type V proteins such as C2c1 or C2c3 mentioned herein have a homology or sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90% eg at least 95% with type V proteins such as C2c1 or C2c3. In further embodiments, homologues or orthologues of type V proteins such as C2c1 or C2c3 mentioned herein have sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, e.g. , at least 95% with wild type V proteins such as C2c1 or C2c3.

[00229] In an embodiment, type V RNA-targeting Cas proteins may be C2c1 or C2c3 orthologues of organisms from genera that include, but are not limited to: Corynebacter , Sutterella , Legionella , Treponema , Filifactor , Eubacterium , Streptococcus , Lactobacillus , Mycoplasma , Bacteroides , Flaviivola , Flavobacterium , Sphaerochaeta , Azospirillum , Gluconacetobacter , Neisseria , Roseburia , Parvibaculum , Staphylococcus , Nitratifractor , Mycoplasma and Campylobacter . The type of organism belonging to these genera may be discussed in this application in a different way.

[00230] Some methods for identifying CRISPR-Cas enzyme orthologues may include identifying the tracr sequence in target genomes. Identification of tracr sequences may involve the following steps: searching for direct repeats or a tracr helper sequence in a database to identify a CRISPR region including the CRISPR enzyme; search for homologous sequences in the CRISPR region flanking the CRISPR enzyme in the sense and antisense directions; search for transcription terminators and secondary structures; identifying any sequence that is not a direct repeat or tracr helper sequence, but has more than 50% identity with a direct repeat or tracr helper sequence as a potential tracr sequence; search for transcription terminator sequences associated with a potential tracr sequence.

[00231] It should be appreciated that any of the functional activities described herein can be engineered into CRISPR enzymes from other orthologs, including chimeric enzymes that include fragments of multiple orthologs. Examples of such orthologs are described in the present description. Thus, chimeric enzymes may include orthologue fragments of the organism's CRISPR enzyme, which includes, but is not limited to: Corynebacter , Sutterella , Legionella , Treponema , Filifactor , Eubacterium , Streptococcus , Lactobacillus , Mycoplasma , Bacteroides , Flaviivola , Flavobacterium , Sphaerochaeta , Azospirillum , Gluconacetobacter , Neisseria , Roseburia , Parvibaculum , Staphylococcus , Nitratifractor , Mycoplasma and Campylobacter . The chimeric enzyme may include a first fragment and a second fragment, the fragments may be derived from orthologues of the CRISPR enzyme of organisms belonging to the genera mentioned herein or the species mentioned herein; preferably, the fragments are derived from various species of CRISPR enzyme orthologues.

[00232] In embodiments, an RNA-targeting type V/type VI effector protein, specifically the C2c2 C2c1/C2c3 protein referred to herein, also encompasses a C2c1/C2c3 functional variant or a homologue or orthologue thereof. A "functional variant" of a protein, as used herein, refers to a variant of such a protein that retains, at least in part, the activity of that protein. Functional variants may include mutants (which may have insertions, deletions or substitutions), including polymorphs, and so on. Also included in functional variants are fusion products of such a protein with another, usually unrelated, protein, nucleic acid, polypeptide, or peptide. Functional options can be natural or artificial. Preferred embodiments of the invention may include a non-naturally occurring or engineered type V/type VI effector protein targeting RNA, eg C2c1/C2c3, or an orthologue or homologue thereof.

[00233] In an embodiment, the codon of the nucleic acid molecule(s) encoding an RNA-targeted type V/type VI effector protein, e.g., C2c1/C2c3, or an orthologue or homologue thereof, can be optimized for expression in a eukaryotic cell. The eukaryotic organism may be as described herein. The nucleic acid molecule(s) may be engineered or may not occur in nature.

[00234] In an embodiment, a type V/type VI effector protein targeted to RNA, e.g., C2c1/C2c3 (or an orthologue or homologue thereof), may include one or more mutations (hence the nucleic acid molecule(s) encoding that protein has the same mutation(s). Mutations may be introduced artificially and may include, but are not limited to, one or more mutations in the catalytic domain. Examples of catalytic domains for the Cas9 enzyme may include, but are not limited to, RuvC I, RuvC II, RuvC III, and HNH domains.

[00235] In an embodiment, a type V/type VI protein, such as C2c1 or C2c3, or an orthologue or homologue thereof, may include one or more mutations. Mutations may be introduced artificially and may include, but are not limited to, one or more mutations in the catalytic domain. Examples of catalytic domains for the Cas enzyme may include, but are not limited to, RuvC I, RuvC II, RuvC III, HNH, and HEPN domains.

[00236] In one embodiment, a type V/type VI protein, e.g., C2c1 or C2c3, or an orthologue or homologue thereof, can be used as a universal nucleic acid binding protein, in fusion or operably linked to a functional domain. Exemplary functional domains may include, but are not limited to, a translation initiator, a translation activator, a translation repressor, nucleases such as ribonucleases, spliceosomes, magnetic beads, a light inducible/controlled domain, or a chemically induced/controlled domain.

[00237] In some embodiments, the enzymatic activity of the unchanged nucleic acid-targeting effector protein is cleavage. In some embodiments, an RNA targeting effector protein may direct cleavage of one or both nucleic acids (DNA or RNA) of the strand at a location near the target sequence, such as within the target sequence and/or within a sequence complementary to the target sequence, or in sequences associated with the target sequence. In some embodiments, a nucleic acid-targeted Cas protein can direct cleavage of one or both DNA or RNA strands within about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs from the first or last nucleotide of the target sequence. In some embodiments, the split may be blunt, i.e. produce blunt ends. In some embodiments, the splitting may be staggered, ie. produce sticky ends. In some embodiments, the cleavage may be staggered with 5' overhangs, such as 1-5 nucleotide 5' overhangs. In some embodiments, the cleavage may be staggered with 3' overhangs, such as 1-5 nucleotide 3' overhangs. In some embodiments, the vector encodes a nucleic acid-targeted Cas protein that may be mutated from the corresponding wild-type enzyme such that the mutant nucleic acid-targeted Cas protein has a reduced ability to cleave one or both strands of DNA, or Target polynucleotide RNA containing the target sequence. As another example, two or more catalytic domains of the Cas protein (RuvC I, RuvC II and RuvC III or HNH domain or HEPN domain) can be mutated so that the mutated Cas protein has substantially lost RNA cleavage activity. As described herein, mutations can be made in the respective catalytic domains of the C2c1 or C2c3 effector protein so that the DNA cleavage activity of the mutant C2c1 or C2c3 effector protein is deficient or substantially reduced. In some embodiments, a nucleic acid-targeted effector protein is considered to have a significant lack of RNA cleavage activity when the RNA cleavage activity of the mutant enzyme is no greater than 25%, 10%, 5%, 1%, 0.1%, 0, 01% or less of the nucleic acid cleavage activity of the wild-type form of the enzyme; for example, when the nucleic acid cleavage activity of the mutant form is zero or negligible compared to the wild form. An effector protein can be identified with respect to a general class of enzymes that are homologous to the largest nuclease with multiple nuclease domains of the type V/type VI CRISPR system. Most preferably, a type V/type VI effector protein such as C2c1/C2c3 is used. By "derived" Applicants mean that the derived enzyme is largely based, in the sense of having a high degree of sequence homology, on the wild-type enzyme, but that it is mutated (modified) in some way, as is known in the art or as described herein.

[00238] Again, it should be appreciated that the terms "Cas and CRISPR enzyme", "CRISPR protein" and "Cas protein" are generally used interchangeably and, in all references herein, refer by analogy to new CRISPR effector proteins, hereinafter described in the present description, unless otherwise obvious, for example, with specific reference to Cas9. As mentioned above, many of the residue numbers used herein refer to the type V/type VI CRISPR locus effector protein. However, it should be appreciated that this invention includes many more effector proteins from other types of microorganisms. In some embodiments of the invention, Cas may be constitutively present, or inducibly present, or conditionally present, or be administered or delivered. Cas optimization can be used to enhance functions or develop new functions, and chimeric Cas proteins can be generated. Cas can be used as a versatile nucleic acid binding protein.

[00239] Generally, in the context of an endogenous nucleic acid-targeted system, formation of a nucleic acid-targeted complex (comprising a guide RNA hybridized to the target sequence and complexed with one or more nucleic acid-targeted effector proteins) results in cleavage of one or both strands of DNA or RNA within or near (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence . As used herein, the term "sequence(s) associated with a target locus" refers to sequences in the vicinity of the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from the target sequence, where the target sequence is at the target locus).

[00240] An example of a codon-optimized sequence here is a sequence optimized for expression in a eukaryotic organism, e.g., a human (i.e., optimized for expression in a human) or other eukaryotic organism, animal, or mammal, as described herein. description; see, for example, the human codon-optimized SaCas9 sequence in WO 2014/093622 (PCT/US2G13/074667) as an example of a codon-optimized sequence (s) acid(s), especially in the case of an effector protein (eg C2c2), is within the purview of the skilled artisan). While the above description is preferred, it will also be understood that other examples are possible and codon optimization for non-human host species or codon optimization for certain organs is known. In some embodiments, the enzyme-coding sequence encoding the DNA/RNA-targeted Cas protein is codon-optimized for expression in certain cells, particularly eukaryotic cells. Such eukaryotic cells of, or may be derived from, a particular organism, which may be a mammal, including but not limited to a human, non-human eukaryotic organism, animal, or mammal as described herein, e.g., mouse, rat, rabbit, dog , livestock or non-human mammal or primate. In some embodiments, processes for modifying the genetic makeup of an embryonic cell line of human individuals and/or processes for modifying the genetic makeup of an embryonic cell line of animals that are likely to cause suffering to them without significant benefit to human or animal medical applications, and also obtained by these processes animals can be excluded. In general, codon optimization refers to the process of altering a nucleic acid sequence to enhance expression in target host cells by changing at least one codon (e.g., about or more than 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of the native sequence by the codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Different species show a specific preference for certain codons of specific amino acids. Codon preference (differences in codon usage between organisms) is often correlated with the efficiency of messenger RNA (mRNA) translation, which in turn is thought to depend on, among other things, the properties of the translated codons or the availability of certain transfer RNA (tRNA) molecules. The predominance of certain tRNAs usually reflects the most commonly used codons in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a particular organism based on codon optimization. Tables of codon usage frequencies are freely available, for example, in the "Codon Usage Database" available at www.kazusa.oijp/codon/, and such tables can be adapted in various ways. See Nakamura, Y. et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res, 28:292 (2000). Computer algorithms are also available to optimize codons for a particular sequence for expression in a particular host cell, such as Gene Forge (Aptagen; Jacobus, PA, USA). In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, more, or all of the codons) in the sequence encoding the DNA/RNA-targeted Cas protein most frequently match the codons used for a particular amino acid. Regarding the use of codons in yeast, a link is provided to the Yeast Genome online database available at http:.Avww.yaastyeoome.org/eommuniyvcodon_rsage.shtml, or Codon selection in yeast, Bennetzen and Hall, J. Biol Chem. 1982 Mar 25;257(6):3026-31. With regard to codon usage in plants, including algae, reference is made to Codon usage in higher plants, green algae, and cyanobacteria, Campbell and Gowri, Plant Physiol. 1990 Jan; 92(1): 1-11. and also on Codon usage in plant genes, Murray et al, Nucleic Acids Res. 1989 Jan 25;17(2):477-98 or Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages, Morton BR, J. Mol. Evol. 1998 Apr;46(4):449-59.

[00241] In some embodiments, the vector encodes a nucleic acid-targeted effector protein, such as an RNA-targeted type V effector protein, particularly C2c2, or an orthologue or homologue thereof, containing one or more nuclear localization signal (NLS) sequences, e.g. about or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs. In some embodiments, such an RNA-targeted effector protein contains about or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the N-terminus, about or more than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the C-terminus, or a combination thereof (e.g., zero or at least one or more NLSs at the N-terminus or more NLSs at the C-terminus). In case there is more than one NLS, each of them can be selected independently of the others, in particular, a single NLS can be present in more than one copy and/or in combination with one or more NLS represented by one or more copies. In some embodiments, an NLS is considered to be near the N- or C-terminus when the nearest NLS amino acid is less than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more amino acids away. polypeptide chain from the N-terminus or C-terminus. Non-limiting examples of NLSs include NLS sequences derived from: SV40 large T antigen NLS having the PKKKRKV amino acid sequence; Nucleoplasmin NLS (eg, two-component NLS having the sequence KRPAATKKAGQAKKKK); c-myc NLS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP; NLS hRNPA1 M9 having the amino acid sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV of the importin-alpha IBB domain; VSRKRPRP and PPKKARED myoma T-protein sequences; human p53 POPKKKPL sequence; mouse SALIKKKKKMAP c-ab1 IV sequence; the DRLRR and PKQKKRK sequences of influenza virus NS1; the sequence RKLKKKIKKL of the hepatitis virus delta antigen; mouse Mx1 protein sequence REKKKFLKRR; poly(ADP-ribose) polymerase sequence KRKGDEVDGVDEVAKKKSKK; and steroid hormone receptor glucocorticoid sequence RKCLQAGMNLEARKTKK. In general, one or more NLSs are sufficient to cause DNA/RNA-targeted Cas protein to accumulate in detectable amounts in the nucleus of a eukaryotic cell. In general, the expression of nuclear localization activity may result from the activity of a number of NLSs in the nucleic acid-targeting effector protein, the use of certain NLSs, or a combination of these factors. Tracking accumulation in the core can be done using any suitable method. For example, a traceable marker can be fused to a nucleic acid-targeting protein so that its location in a cell can be visualized, particularly in combination with nuclear location methods (eg, using a nuclear-specific dye such as DAPI). Cell nuclei can also be isolated from cells, the contents of which can then be analyzed using any suitable protein tracking method, such as immunohistochemistry, Western blot, or protein activity analysis. Accumulation in the nucleus can also be determined indirectly, for example, by analyzing the effect of nucleic acid targeting complex formation (e.g., changes in expression activity upon formation of a DNA or RNA target complex and/or RNA targeting activity of the Cas protein), when compared with a control not exposed to the nucleic acid-targeted Cas protein or nucleic acid-targeted complex, or exposed to the nucleic acid-targeted Cas protein lacking one or more NLSs. In a preferred embodiment, the C2c1 or C2c3 effector protein complexes and systems described herein and the C2c1 or C2c3 effector protein complexes and systems include an NLS attached to the C-terminus of the protein.

[00242] In some embodiments, one or more vectors directing the expression of one or more nucleic acid targeting systems is introduced into a cell such that expression of elements of the nucleic acid targeting system directs the formation of a nucleic acid targeting complex at one or more target sites. . For example, a nucleic acid-targeting effector protein and a nucleic acid-targeting guide RNA can be operably linked to separate regulatory elements in separate vectors. The nucleic acid targeting system molecule(s) can be delivered to a transgenic nucleic acid targeting effector protein that is an animal or mammalian protein that constitutively, inducibly or conditionally expresses the nucleic acid targeting effector protein, or an animal or mammal that expresses an effector protein otherwise targeted to the nucleic acid, or having cells containing the effector protein targeted to the nucleic acid present there due to prior administration of the vector or vectors encoding and expressing in vivo the effector protein targeted to the nucleic acid. Alternatively, two or more elements expressed with one or different regulatory elements can be introduced into a single vector, with one or more additional vectors providing any components of the nucleic acid targeting system not included in the first vector. The components of the nucleic acid-targeting system combined into a single vector can be assembled in any suitable orientation, such as one element upstream from the 5' end or downstream from the 3' end of the second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of the second element and have the same or opposite orientation. In some embodiments, the same promoter controls the expression of transcripts encoding a nucleic acid-targeting effector protein and a nucleic acid-targeting guide RNA embedded in one or more intron sequences (e.g., each in a separate intron, two or more in at least one intron, or all in the same intron). In some embodiments, the nucleic acid-targeting effector protein and the nucleic acid-targeting guide RNA may be operably linked to or expressed from the same promoter. Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expressing one or more elements of a nucleic acid targeting system are used in the aforementioned documents, in particular WO 2014/093622 (PCT/US2013/074667). In some embodiments, the vector contains one or more insertion sites, such as restriction endonuclease recognition sequences (also referred to as a "cloning site"). In some embodiments, one or more insertion regions (e.g., about or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more insertion regions) are arranged in an ascending and/or descending direction relative to one or more elements of sequences of one or more vectors. In some embodiments, a vector comprising an insertion region upstream of the tracr partner sequence, and optionally downstream from a regulatory element operably linked to the tracr partner sequence, such that, upon introduction of the guide sequence, the insertion region when expressed, the guide sequence directs the sequence-specific binding of the nucleic acid-targeted complex to the target sequence in the eukaryotic cell. In some embodiments, the implementation of the vector includes two or more sections of inserts, which allows the introduction as inserts of the targeting sequences in each section. In this arrangement, two or more guide sequences may include two or more copies of a single guide sequence, two or more different guide sequences, or a combination thereof. By using many different targeting sequences, a single expression construct can be used to target nucleic acid binding activity to many different corresponding target sequences in a cell. For example, a single vector may include about or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more guide sequences. In some embodiments, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of these guide sequence-containing vectors may be provided, and optionally they may also be delivered to the cell. In some embodiments, the vector contains a regulatory element operably linked to an enzyme-coding sequence. The nucleic acid-targeting effector protein or nucleic acid-targeting guide RNA or nucleic acid-targeting guide RNAs can be delivered independently; preferably, at least one(one) of them was(a) delivered(a) through a complex of particles or nanoparticles. The mRNA of the nucleic acid-targeting effector protein may be delivered prior to the nucleic acid-targeting guide RNA in order to allow time for expression of the nucleic acid-targeting effector protein. The mRNA of the effector protein targeting the nucleic acid can be introduced 1-12 hours (preferably about 2-6 hours) prior to the introduction of the guide RNA targeting the nucleic acid. Alternatively, the nucleic acid-targeting effector protein mRNA and the nucleic acid-targeting guide RNA can be administered simultaneously. Preferably, the second booster dose of guide RNA is administered within 1-12 hours (preferably about 2-6 hours) after the initial administration of the effector protein-targeted nucleic acid+guide RNA mRNA. Additional administrations of a nucleic acid-targeted effector protein may be useful in order to achieve the most efficient levels of genome modification.

[00243] In one aspect, the invention relates to methods for using one or more components of a nucleic acid targeting system. Such a nucleic acid-targeting complex of the invention provides an effective tool for modifying a target DNA or RNA that is single or double stranded, linear or supercoiled. Such a nucleic acid-targeted complex of the invention has a variety of advantages, including modification (eg, removal, insertion, movement, inactivation, activation) of the target DNA or RNA in a variety of cell types. As such, the nucleic acid-targeting complex of the invention has a wide range of applications, for example in gene therapy, pharmacological screening, disease diagnosis and prognosis. An exemplary nucleic acid-targeting complex includes a DNA or RNA-targeted effector protein complexed with a guide RNA hybridized to a target sequence at the target locus of interest.

[00244] In one embodiment, the present invention relates to a method for cleaving a target RNA. The method may include modifying a target RNA using a nucleic acid-targeting complex that binds the target RNA and cleaves the target RNA. In one embodiment, the nucleic acid-targeting complex of the invention, when introduced into a cell, can introduce a break (eg, single or double strand break) into the RNA sequence. For example, such a method can be used to cleave disease-associated RNA in a cell. For example, an exogenous RNA template comprising the sequence to be inserted, flanked by an upstream sequence and a downstream sequence, can be introduced into a cell. Such upstream and downstream sequences are characterized by sequence similarity to both ends of the RNA insertion site. If desired, the donor RNA may be mRNA. The exogenous RNA template contains the sequence to be inserted (eg mutated RNA). The insertion sequence may be a sequence endogenous or exogenous to the cell. Examples of sequences for insertion are protein-coding RNA or non-coding RNA (eg microRNA). Thus, the insertion sequence can be operably linked to the appropriate control sequence or sequences. Alternatively, the insertion sequence may perform a regulatory function. The upstream and downstream sequences in the exogenous RNA are chosen to promote recombination between the target RNA and the donor RNA. An upstream sequence is an RNA sequence having sequence similarity to an RNA sequence upstream from the target integration site. Similarly, the downstream sequence is an RNA sequence having sequence similarity to an RNA sequence downstream from the target integration site. The upstream and downstream sequences in the exogenous RNA template may have 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the target RNA sequence. Preferably, the upstream and downstream sequences in the exogenous RNA template have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the target RNA sequence. In some methods, the upstream and downstream sequences of the exogenous template RNA have 99% or 100% sequence identity with the target RNA sequence. Upstream or downstream sequences may be from about 20 bp. up to about 2500 b.p. , 2000, 2100, 2200, 2300, 2400 or 2500 b.p. In some methods, an exemplary upstream or downstream sequence is from about 200 bp. up to about 2000 bp, from about 600 bp up to about 1000 b.p. or more specifically from about 700 b.p. up to about 1000 b.p. In some methods, the exogenous RNA template may further comprise a marker. Such a marker may facilitate screening for targeted embeddings. Examples of suitable markers include restriction sites, fluorescent proteins, or labels for which selection is possible. The exogenous RNA template of the invention can be obtained using recombinant methods (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996). According to a method for modifying a target RNA by inserting an exogenous target RNA, a break (e.g., a double-strand or single-strand break in single- or double-stranded DNA or RNA) is introduced into the DNA or RNA sequence by a nucleic acid-targeting complex, the break being repaired by homologous recombination with the exogenous RNA templates such that the template is incorporated into the target RNA. The presence of a double-strand break facilitates template insertion. In other embodiments, the present invention relates to a method for modifying RNA expression in a eukaryotic cell. Such a method includes increasing or decreasing the expression of a target polynucleotide by using a nucleic acid-targeted DNA or RNA (eg, mRNA or pre-mRNA) binding complex. In some methods, the target RNA may be inactivated in order to affect the modification of expression in the cell. For example, upon binding of an RNA-targeting complex to a target sequence in a cell, the target RNA is inactivated such that the sequence is not translated, the protein it encodes is not formed, or the sequence does not function as the wild-type sequence. For example, protein, or microRNA or pre-miRNA transcripts are not formed. The target RNA of the RNA-targeted complex can be any RNA, endogenous or exogenous to a given eukaryotic cell. For example, the target RNA may be RNA found in the nucleus of a eukaryotic cell. The target RNA can be a sequence (eg mRNA or pre-mRNA) encoding a gene product (eg protein) or a non-coding sequence (eg ncRNA, lnc-RNA, tRNA or rRNA). Examples of a target RNA include a sequence associated with signaling pathways, eg, associated with signaling RNA pathways. Examples of target RNA include disease-associated RNA. "Disease-associated" RNA refers to any RNA used to synthesize translation products at an abnormal level or in an abnormal form in cells derived from disease-affected tissues compared to tissues or cells of unaffected controls. It may be RNA transcribed from a gene that is expressed at an abnormally high level; it may be RNA transcribed from a gene that is expressed at an abnormally low level, the altered expression being correlated with the onset and/or progression of the disease. Disease-associated RNAs are also referred to as RNAs transcribed from a gene carrying a mutation(s) or genetic variation that is directly responsible for or related to a linkage disequilibrium with the gene(s) that is(are) involved in the etiology of the disease. The translation product may or may not be known, and its amount may be normal or abnormal. The target RNA of the RNA-targeting complex can be any RNA, endogenous or exogenous to such a eukaryotic cell. For example, the target RNA of an RNA-targeting complex may be RNA found in the nucleus of a eukaryotic cell. Such a target RNA can be a sequence (eg mRNA or pre-mRNA) encoding a gene product (eg protein) or a non-coding sequence (eg ncRNA, lnc-RNA, tRNA or rRNA).

[00245] In some embodiments, the method may allow a nucleic acid-targeted complex to bind to a target DNA or RNA to cleave said target DNA or RNA, thereby altering the target DNA or RNA where the target the nucleic acid complex comprises a nucleic acid-targeted effector protein complexed with a guide RNA hybridized to a target sequence in said target DNA or RNA. In one aspect, the invention relates to a method for modifying the expression of DNA or RNA in a eukaryotic cell. In some embodiments, the method allows the nucleic acid-targeted complex to bind to DNA or RNA such that said binding results in an increase or decrease in the expression of said DNA or RNA; wherein the nucleic acid-targeted complex comprises a nucleic acid-targeted effector protein in complex with a guide RNA. Applicable considerations and conditions similar to those indicated above for methods of modifying a DNA or RNA target. In fact, sampling, culturing, and re-introduction options apply to many aspects of the present invention. In one aspect, the invention relates to methods for modifying a target DNA or RNA in a eukaryotic cell, which may occur in vivo , ex vivo , or in vitro . In some embodiments, the method includes taking a cell or population of cells from a human or non-human animal and modifying the cell or cells. Cultivation ex vivo can occur at any stage. The cell or cells may be reintroduced into a non-human animal or plant. For reintroduced cells, it is particularly preferred that these cells are stem cells.

[00246] Indeed, in any aspect of the invention, the nucleic acid-targeted complex may include a nucleic acid-targeted effector protein complexed with a guide RNA that is hybridized to the target sequence.

[00247] The invention relates to the engineering and optimization of systems, methods and compositions used to control gene expression, including targeting DNA and RNA sequences that are associated with a nucleic acid targeting system and its components. In preferred embodiments, the effector protein (enzyme) is a type V protein, such as C2c1 or C2c3. An advantage of the methods of the present invention is that the CRISPR system minimizes or avoids non-specific binding and its side effects. This system is achieved using systems organized such that they have a high degree of specificity for the target DNA or RNA.

[00248] With respect to the nucleic acid-targeted complex or system, it is preferred that the tracr sequence has one or more hairpins and is 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length; the length of the crRNA sequence varied between 10 and 30 nucleotides, and the nucleic acid-targeted effector protein belonged to type V effector proteins.

[00249] In some modifications of the invention, the effector protein may be C2c1p from Alicyclobacillus sp., preferably C2c1p from Alicyclobacillus acidoterrestris , more preferably C2c1p of Alicyclobacillus acidoterrestris ATCC 49025, and the crRNA sequence may be 34 nucleotides long and contain a 5'-terminal straight a 14 nucleotide repeat (DR) and a 20 nucleotide spacer.

[00250] In some modifications of the invention, the effector protein may be Bacillus sp. C2c1p, preferably Bacillus thermoamylovorans C2c1p , more preferably Bacillus thermoamylovorans strain B4166 C2c1p, and the crRNA sequence may be 33 nucleotides long and contain a 5'-terminal straight a 14 nucleotide repeat (DR) and a 29 nucleotide spacer.

[00251] In some modifications, the effector protein may be an effector protein of type V-B loci, more specifically C2c1p, and the crRNA sequence may be 27 to 40 nucleotides in length, preferably 29 to 38 nucleotides or 30 to 37 nucleotides, more preferably 31 to 36 nucleotides or 32 to 35 nucleotides, most preferably 33 or 34 nucleotides. for example, a crRNA may include, consist mostly of, or consist only of a direct repeat (DR), preferably a 5'-terminal DR of 12 to 16 nucleotides in length, preferably 13 to 15 nucleotides, even more preferably 14 nucleotides, and also the spacer is 15 to 24 nucleotides in length, preferably 16 to 23 nucleotides, more preferably 17 to 22 nucleotides, even more preferably 18 to 21 nucleotides, and most preferably 19 or 20 nucleotides.

[00252] In some embodiments, the effector protein may be Listeria sp. C2c2p, preferably Listeria seeligeria C2c2p , more preferably Listeria seeligeria serovar 1/2b strain SLCC3954 C2c2p, and the crRNA sequence may be 44-47 nucleotides in length with straight lines. repeats (DR) at the 5'-end 29 nucleotides long and a spacer 15 nucleotides to 18 nucleotides long.

[00253] In some embodiments, the effector protein may be Leptotrichia sp. C2c2p, preferably Leptotrichia shahii C2c2p , more preferably Leptotrichia shahii DSM 19757 C2c2p, and the crRNA sequence may be 42-58 nucleotides in length with a 5' direct repeat -end 28 nucleotides long and spacer 14 nucleotides to 28 nucleotides long.

[00254] In some embodiments, the effector protein may refer to effector proteins of type VI loci, more specifically C2c2p, the crRNA sequence may be 36-63 nucleotides in length, preferably 37 nucleotides to 62 nucleotides, or 38 nucleotides to 61 nucleotides, or 39 nucleotides to 60 nucleotides, more preferably 40 nucleotides to 59 nucleotides, or 41 nucleotides to 58 nucleotides, most preferably 42 nucleotides to 57 nucleotides. For example, a crRNA may include, essentially consist of, or consist of a direct repeat (DR), preferably at the 5' end, 26 nt to 31 nt long, preferably 27 nt to 30 nt, even more preferably 28 nt or 29 nt long. nucleotides in length, and a spacer 10 nucleotides to 32 nucleotides in length, preferably 11 nucleotides to 31 nucleotides, more preferably 12 nucleotides to 30 nucleotides, even more preferably 13 nucleotides to 29 nucleotides, and most preferably 14 nucleotides to 28 nucleotides .

[00255] In some embodiments, the effector protein may be a C2c1p protein from Alicyclobacillus sp ., preferably C2c1p from Alicyclobacillus acidoterrestris, more preferably C2c1p from Alicyclobacillus acidoterrestris ATCC 49025 and the crRNA sequence may be at least 78 in length, e.g. 79 nucleotides in length or more than 79 nucleotides in length, for example, may be at least 80 nucleotides in length, at least 90 nucleotides in length, at least 100 nucleotides in length, at least 110 nucleotides in length, at least 120 nucleotides in length, at least 130 nucleotides in length, at least 140 nucleotides, at least 1500 nucleotides or more.

[00256] In some embodiments, the effector protein may be Bacillus sp. C2c1p protein, preferably Bacillus thermoamylovorans C2c1p , more preferably Bacillus thermoamylovorans C2c1p , strain B4166, and the tracr RNA sequence may be about 91 nucleotides in length, e.g., 91 nucleotide in length.

[00257] In some embodiments, the effector protein may be an effector protein of type V-B loci, more specifically C2c1p, and the tracr RNA sequence may be at least 60 nucleotides in length, in particular at least 65 nucleotides in length, or at least 70 nucleotides in length, in particular 60 to 70 nucleotides in length, or 60 to 70 nucleotides in length, 70 to 80 nucleotides in length, 80 to 90 nucleotides in length, 90 to 100 nucleotides in length, 100 to 110 nucleotides in length, 110 to 120 nucleotides in length, 120 to 130 nucleotides in length, 130 to 140 nucleotides in length, 140 to 150 nucleotides in length, or more than 150 nucleotides in length. See illustrative examples in Fig.17-21.

[00258] In some embodiments, the effector protein may refer to effector proteins of type VI loci, more specifically C2c2p, and the tracr RNA sequence may be at least 60 nucleotides in length, such as at least 65 nucleotides or at least 70 nucleotides , such as 60 nucleotides to 70 nucleotides, or 60 nucleotides to 70 nucleotides, or 70 nucleotides to 80 nucleotides, or 80 nucleotides to 90 nucleotides, or 90 nucleotides to 100 nucleotides, or 100 nucleotides to 110 nucleotides, or 110 nucleotides to 120 nucleotides, or 120 nucleotides to 130 nucleotides, or 130 nucleotides to 140 nucleotides, or 140 nucleotides to 150 nucleotides, or more than 150 nucleotides. See illustrative examples in Fig.22-37.

[00259] In certain embodiments, the effector protein may refer to effector proteins of type VI loci, more specifically C2c2p, so tracr-RNA may not be required for cleavage.

[00260] The use of two different aptamers (each associated with a separate nucleic acid-targeting guide RNA) allows the use of activator-adapter protein fusions and repressor-adapter protein fusions with different nucleic acid-targeting guide RNAs to activate the expression of only one DNA or RNA, suppressing the other. These, along with their various guide RNAs, can be co-administered, or substantially co-administered, in a multiplex approach. A large number of such modified nucleic acid-targeted guide RNAs can be used simultaneously, e.g. 10 or 20 or 30 etc., while only one (or at least a minimum number) of the effector protein molecule needs to be delivered, as comparatively a small number of effector protein molecules can be used with a large number of modified target molecules. The adapter protein may be associated with (preferably linked or fused to) one or more activators or one or more repressors. For example, the adapter protein may be associated with the first and second activators. The first and second activators may be the same, but preferably they are different activators. Three or more or even four or more activators (or repressors) may be used, however, the allowable package size limits their number to 5 different functional domains. The use of linkers is preferred over direct fusion to an adapter protein, where two or more functional domains are associated with the adapter protein. Suitable linkers may include a GlySer linker.

[00261] It is also contemplated that the nucleic acid-targeting complex of effector protein and guide RNA as a whole can be associated with two or more functional domains. For example, there may be two or more functional domains associated with a nucleic acid-targeted effector protein, or there may be two or more functional domains associated with a guide RNA (via one or more adapter proteins), or there may be one or more functional domains associated with a nucleic acid-targeted effector protein, and one or more functional domains associated with the guide RNA (via one or more adapter proteins).

[00262] The fusion between the adapter protein and the activator or repressor may include a linker. For example, GlySer GGGS linkers can be used. They can be used in repetitions of 3 ((GGGGS) ₃ ) or 6, 9 or even 12 or more to provide the appropriate length as required. Linkers can be used between guide RNAs and a functional domain (activator or repressor), or between a nucleic acid-targeted effector protein and a functional domain (activator or repressor). Linkers are used to give the protein the necessary "mechanical flexibility". The invention encompasses a nucleic acid-targeted complex comprising a nucleic acid-targeted effector protein and a guide RNA, wherein the nucleic acid-targeted effector protein comprises at least one mutation such that the nucleic acid-targeted Cas protein has no more than 5% of the activity of the targeted Cas protein. a nucleic acid of a Cas protein lacking at least one mutation and optionally at least one or more nuclear localization sequences; the guide RNA comprises a guide molecule sequence capable of hybridizing to a target sequence in an RNA of interest in a cell; and wherein the nucleic acid-targeted effector protein is associated with two or more functional domains, or at least one guide RNA hairpin structure is altered by insertion of a distinct RNA sequence(s) that is associated with one or more adapter proteins, and wherein the adapter protein is associated with two or more functional domains; or the nucleic acid-targeted effector protein is associated with one or more functional domains, and at least one guide RNA hairpin structure is altered by insertion of a distinct RNA sequence(s) that is associated with one or more adapter proteins, and wherein the adapter protein is associated with one or more more functional domains.

Enzyme mutations that reduce off-target effects

[00263] In one aspect, the invention relates to a non-naturally occurring or engineered CRISPR enzyme, preferably a class 2 CRISPR enzyme, preferably a type V or VI CRISPR enzyme, as described herein, in particular, preferably, but not limited to, to C2c1 or C2c3, as described herein, having one or more mutations leading to a reduction in off-target effects, ie. to improved CRISPR enzymes for use in order to induce modifications of target loci, but reducing or eliminating off-target activity, in particular in combination with guide RNAs, to improved CRISPR enzymes for increasing the activity of CRISPR enzymes, in particular in combination with guide RNAs. It should be understood that the mutant enzymes described below can be used in any of the methods of the invention as described herein. Any of the methods, products, compositions, and uses described herein are equally applicable to mutant CRISPR enzymes, as described in more detail below. It should be understood that in the aspects and embodiments of the invention described herein, when referring to or describing C2c1 or C2c3 as a CRISPR enzyme, it is preferred that the restoration of a functional CRISPR-Cas system does not require or is not dependent on the tracr sequence and/or a direct repeat was in the 5'(upstream) direction from the guide sequence (target or spacer).

[00264] The following specific aspects and embodiments are provided as further guidance.

[00265] The inventors have surprisingly found that it is possible to modify CRISPR enzymes to give them reduced off-target activity compared to unmodified enzymes and/or increased target activity compared to unmodified CRISPR enzymes. Thus, in certain aspects of the invention, improved CRISPR enzymes are provided within the scope of the present invention, which can be used for a wide range of gene modification applications. Also contemplated within the scope of the present invention are CRISPR complexes, compositions and systems, as well as methods and uses, all of which include the modified CRISPR enzymes described herein.

[00266] As used herein, the term "Cas" can mean "C2c1", "C2c3", or a CRISPR enzyme. The terms "C2c1p" and "C2c1" are used interchangeably and the terms "C2c3p" and "C2c3" are used interchangeably. The p in C2c1p and C2c3p is the protein designation. In the context of this aspect of the invention, the C2c1, C2c3, or CRISPR enzyme is mutated or modified, "whereby the enzyme of the CRISPR complex has a reduced ability to modify one or more non-target loci compared to an unmodified enzyme" (or similar statements); and when reading this description, the terms "C2c1", "C2c3", "Cas" or "CRISPR enzyme" and the like. are intended to include mutated or modified C2c1, C2c3, Cas or the CRISPR enzyme of the invention, i.e. "therefore, the CRISPR complex enzyme has a reduced ability to modify one or more non-target loci compared to an unmodified enzyme" (or similar statements).

[00267] In one aspect, engineered C2c1 or C2c3 proteins as defined herein are provided, specifically C2c1 or C2c3, wherein the proteins are complexed with a nucleic acid molecule comprising an RNA to form a CRISPR complex, and when in As part of the CRISPR complex, the nucleic acid molecule targets one or more target polynucleotide loci and the protein contains at least one modification compared to the unmodified C2c1 protein or the unmodified C2c3 protein, and the CRISPR complex containing the modified protein has an altered activity compared to that of the complex containing unmodified C2c1 protein or unmodified C2c3 protein. It should be understood that when referring to a CRISPR "protein" herein, it is preferred that the C2c1 protein or C2c3 protein be a modified CRISPR enzyme (e.g., have increased or decreased (or no) enzymatic activity), in particular including, but not limited to, C2c1 or C2c3. The term "CRISPR protein" may be used interchangeably with the term "CRISPR enzyme" whether or not the protein has been altered, such as increased or decreased (or no) enzymatic activity compared to the wild-type CRISPR protein.

[00268] In one aspect, the altered activity of the engineered CRISPR protein includes altered binding properties for a nucleic acid molecule comprising RNA or target polynucleotide loci, altered binding kinetics for a nucleic acid molecule comprising RNA or target polynucleotide loci, or altered binding specificity at in relation to a nucleic acid molecule comprising RNA or target polynucleotide loci versus non-target polynucleotide loci.

[00269] In some embodiments, unmodified Cas has DNA cleavage activity, such as C2c1 or C2c3 activity. In some embodiments, Cas provides for cleavage of one or both strands in a region of the target sequence, in particular within the target sequence and/or in a region complementary to the target sequence. In some modifications of the invention, Cas provides cleavage of one or both chains at a distance of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more nucleotides from the first or last nucleotide of the target sequence. In some embodiments, the vector encodes a Cas that is mutated from the corresponding wild-type enzyme such that the mutated Cas lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence. In some embodiments, a Cas is considered to be substantially devoid of any DNA cleavage activity if the mutant enzyme's cleavage activity is no more than about 25%, 10%, 5%, 1%, 0.1%, 0.01 % or less relative to the DNA cleavage activity of the wild-type form of the enzyme; for example, the DNA cleavage activity of the mutant form is absent or negligible compared to the non-mutant form. Thus, Cas may include one or more mutations and may be used as a general DNA-binding protein with or without a fusion with a functional domain. Such mutations may be artificially introduced or may be gain or loss of function mutations. In one aspect of the invention, the Cas enzyme may be fused to another protein, such as TAG and/or an inducible/controllable domain, in particular a chemically inducible/controllable domain. The Cas of the present invention may be chimeric Cas proteins; for example, Cas, which has enhanced function by being chimeric. Chimeric Cas proteins may be novel Cas containing fragments from more than one naturally occurring Cas. These may include chimeric proteins containing the N-terminal fragment(s) of one of the Cas9 homologues with the C-terminal fragment(s) of another Cas homologue. Such Cas can be delivered to the cell in the form of mRNA. Expression of Cas may be under the control of an inducible promoter. The absolute aim of the present invention is to avoid the use of known mutations. Indeed, the statement "according to which a CRISPR complex enzyme has a reduced ability to modify one or more non-target loci compared to an unmodified enzyme and/or whereby a CRISPR complex enzyme has an increased ability to modify one or more target loci compared to with unmodified enzyme" (or similar statements) is not intended to describe mutations whose only result is nickase, "dead" Cas, or known Cas9 mutations. However, it is not intended that the modification(s) or mutation(s) of the present invention "whereby the CRISPR complex enzyme has a reduced ability to modify one or more non-target loci compared to the unmodified enzyme and/or whereby the enzyme the CRISPR complex has an increased ability to modify one or more target loci compared to an unmodified enzyme" (or similar claims) cannot be combined with mutations that cause the enzyme to be a nicase or a non-functional ("killed") enzyme. Such a "dead" enzyme may have an increased ability to bind nucleic acids. Such a nikazah may be an improved nikazah. For example, substitution of neutral amino acid(s) in and/or near the groove and/or other charged residues in other Cas regions located in close proximity to the nucleic acid (e.g., DNA, cDNA, RNA, gRNA) with positively charged amino acids can result in to the fact that "in this regard, the enzyme of the CRISPR complex will have a reduced ability to modify one or more non-target loci compared to the unmodified enzyme", for example, more active cleavage. Since both targeted and non-targeted cleavage (over-cleavage of C2c1 or C2c3) can be enhanced in this regard, it is possible to use this technique together with what is known in the art as tru-guide or tru-sg-RNA (see e.g. Fu et at., "Improving CRISPR - Cas nuclease specificity using truncated guide RNAs," Nature Biotechnology 32, (2014) doi: 10.1038/nbt.2808, Received 17 November 2013, Accepted 06 January 7 2014, Published online 26 January 2014 , Corrected online 29 January 2014) to enhance targeted activity without enhancing off-target cleavage to produce over-cleaving nicases, or in combination with a mutation that renders Cas "killed" for an over-binding molecule.

[00270] In certain embodiments, the altered activity of the engineered C2c1 or C2c3 proteins includes increased targeting efficiency or reduced off-target binding. In certain embodiments, the modified activity of the engineered C2c1 or C2c3 proteins includes a modified cleavage activity.

[00271] In certain embodiments, the altered activity includes altered binding properties for a nucleic acid molecule containing target polynucleotide RNA or loci, altered binding kinetics for a nucleic acid molecule containing target polynucleotide RNA or loci, or altered binding specificity for the target polynucleotide. a nucleic acid comprising RNA or target polynucleotide loci, or an altered binding specificity for a nucleic acid molecule containing RNA or target polynucleotide loci compared to non-target polynucleotide loci.

[00272] In certain embodiments, the altered activity comprises an increase in targeting efficiency or a decrease in off-target binding. In some embodiments, the altered activity comprises an increase in cleavage activity at the loci of the target polynucleotide. In some modifications, the change in activity includes a decrease in the activity of cleavage z against loci of the target polynucleotide. In some embodiments, the altered activity comprises an increase in cleavage activity at the non-target polynucleotide loci.

[00273] Accordingly, in certain embodiments, there is increased specificity for target polynucleotide loci over non-target polynucleotide loci. In other embodiments, the specificity for target polynucleotide loci, as compared to non-target polynucleotide loci, is reduced.

[00274] In one aspect of the invention, the altered activity of the engineered C2c1 or C2c3 protein comprises altered helicase activity kinetics.

[00275] In one aspect of the invention, the engineered C2c1 or C2c3 proteins comprise a modification that alters the binding of the protein to a nucleic acid comprising RNA, or a target polynucleotide loci strand or a non-target polynucleotide loci strand. In one aspect of the invention, the engineered C2c1 or C2c3 proteins include a modification that alters the formation of the CRISPR complex.

[00276] In some embodiments, the modified C2c1 or C2c3 proteins include a modification that alters the targeting of a nucleic acid molecule to polynucleotide loci. In certain embodiments of the invention, the modification includes a mutation in a region of the protein that is involved in binding to the nucleic acid molecule. In some embodiments, such a modification includes a mutation in a region of the protein that is involved in binding to the chain of loci of the target polynucleotide. In some embodiments, such a modification includes a mutation in a region of the protein that is involved in binding to the non-target polynucleotide loci chain. In some embodiments, the modification or mutation comprises reducing the negative charge in a region of a protein that is involved in binding an RNA nucleic acid molecule, a target polynucleotide loci chain, or a non-target polynucleotide chain of loci. In some embodiments, the modification or mutation comprises reducing the negative charge in a region of a protein that is involved in binding an RNA nucleic acid molecule, a target polynucleotide loci chain, or a non-target polynucleotide chain of loci. In some embodiments, the modification or mutation includes an increased positive charge in a region of the protein that is involved in binding to a nucleic acid molecule comprising RNA, target polynucleotide loci chains, or non-target polynucleotide loci chains. In some embodiments, the modification or mutation comprises an increased negative charge in a region of the protein that is involved in binding an RNA nucleic acid molecule, target polynucleotide loci chains, or non-target polynucleotide loci chains. In some embodiments, the modification or mutation increases the steric interaction between the protein and the RNA-containing nucleic acid molecule, the target polynucleotide loci strand, or the non-target polynucleotide loci strand. In some embodiments, the modification or mutation comprises a replacement for Lys, His, Arg, Glu, Asp, Ser, Gly, or Thr. In some embodiments, the modification or mutation comprises a change to Gly, Ala, He, Glu, or Asp. In some cases, the modification or mutation is to change the amino acid in the binding groove.

[00277] In one aspect, the present invention relates to a non-naturally occurring CRISPR enzyme as defined herein, such as C2c1 or C2c3, where the enzyme forms a complex with a guide RNA to form a CRISPR complex, the guide RNA when it is part of a CRISPR complex , targets one or more loci of the target polynucleotide, and the enzyme makes changes to the loci of that polynucleotide, and the enzyme has at least one modification, whereby the enzyme in the CRISPR complex has a reduced ability to modify one or more non-target loci, according to compared to the unmodified protein, and whereby the enzyme in the CRISPR complex has an increased ability to modify one or more target loci compared to the unmodified enzyme.

[00278] In any of these non-naturally occurring CRISPR enzymes, the modification may include modification of one or more amino acid residues of the enzyme.

[00279] In any of these non-naturally occurring CRISPR enzymes, the modification may include modifying one or more amino acid residues located in a region containing residues that are positively charged in the unmodified enzyme.

[00280] In any of these non-naturally occurring CRISPR enzymes, the modification may include modifying one or more amino acid residues that are positively charged in the unmodified enzyme.

[00281] In any of these non-naturally occurring CRISPR enzymes, the modification may include modification of one or more amino acid residues that are not positively charged in the unmodified enzyme.

[00282] The modification may include modifying one or more amino acids that do not have a charge in the unmodified protein.

[00283] The modification may include modification of one or more amino acids having a negative charge in the unmodified protein.

[00284] The modification may include the modification of one or more amino acids that are hydrophobic in the unmodified protein.

[00285] The modification may include the modification of one or more amino acids that are polar in the unmodified protein.

[00286] In some of the non-naturally occurring CRISPR enzymes described above, the modification may include modification of one or more residues located in the binding groove.

[00287] In some of the non-naturally occurring CRISPR enzymes described above, the modification may include modification of one or more residues located outside the binding groove.

[00288] In some of the non-naturally occurring CRISPR enzymes described above, the modification may include the modification of one or more residues, where such one or more residues may be arginine, histidine, or lysine.

[00289] In any of the non-naturally occurring CRISPR enzymes described above, such an enzyme can be modified by mutating said one or more residues.

[00290] In some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutating one or more of these residues, where the mutation is the replacement of an unmodified enzyme residue with an alanine residue.

[00291] In some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutating one or more of these residues, where the mutation is the replacement of an unmodified enzyme residue with a glutamic acid residue.

[00292] In some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutating one or more of these residues, where the mutation is the replacement of an unmodified enzyme residue with a serine, threonine, asparagine, or glutamine residue.

[00293] In some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutating one or more of these residues, where the mutation is the replacement of an unmodified enzyme residue with an alanine, glycine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, or valine residue.

[00294] In some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutating one or more of these residues, where the mutation is the replacement of an unmodified enzyme residue with a polar amino acid residue.

[00295] In some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutating one or more of these residues, where the mutation is a replacement of an unmodified enzyme residue with a residue that is not a polar amino acid residue.

[00296] In some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutating one or more of these residues, where the mutation is the replacement of an unmodified enzyme residue with a negatively charged amino acid residue.

[00297] In some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutating one or more of these residues, where the mutation is the replacement of an unmodified enzyme residue with a residue that is not a negatively charged amino acid residue.

[00298] In some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutating one or more of these residues, where the mutation is the replacement of an unmodified enzyme residue with an uncharged amino acid residue.

[00299] In some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutating one or more of these residues, where the mutation is the replacement of an unmodified enzyme residue with a residue that is not a chargeless amino acid residue.

[00300] In some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutating one or more of these residues, wherein the mutation involves replacing an unmodified enzyme residue with a hydrophobic amino acid residue.

[00301] In the case of some of the above non-naturally occurring CRISPR enzymes, the enzyme is modified by mutating one or more of the residues mentioned, where the mutation may be the replacement of an unmodified enzyme residue with an amino acid residue that is not a hydrophobic amino acid residue.

[00302] In certain embodiments, the effector protein may be Alicyclobacillus sp. C2c1p protein, or more preferably Alicyclobacillus acidoterrestris C2c1p , or even more preferably Alicyclobacillus acidoterrestris C2c1p , ATCC 49025. In certain embodiments, the effector protein may be Bacillus C2c1p protein sp., or more preferably C2e1p Bacillus thermoamylovorans or even more preferably C2c1p Bacillus thermoamylovorans strain B4I66. In some embodiments, the effector protein may be an effector protein of VB-type loci, in particular C2c1p.

[00303] In some embodiments, the C2c1 or C2c3 protein contains one or more nuclear localization signal (NLS) domains. In some embodiments, the C2c1 or C2c3 protein contains at least two NLSs.

[00304] In some embodiments, the C2c1 or C2c3 protein is a chimeric CRISPR protein consisting of a first fragment from a first CRISPR orthologue and a second fragment from a second CRISPR orthologue, wherein the first and second CRISPR orthologues are different.

[00305] In some embodiments of the invention, the enzyme is modified or contains modifications, for example, contains, essentially consists of, or consists of modifications obtained by mutations of any of the residues listed herein or the corresponding residue in the appropriate orthologue: or the enzyme contains or essentially consists of or consists of modifications of any (single modification), two (double), three (triple), four (quadruple) positions, or more positions, as described herein, or the corresponding residues or positions in a CRISPR enzyme orthologue; for example, the enzyme contains or essentially consists of or consists of a modification of any of the C2c1 or C2c3 residues listed herein, or the corresponding residue or position in the CRISPR enzyme orthologue. In such an enzyme, each residue can be modified by substitution for an alanine residue.

[00306] Applicants have recently described a method for producing Cas9 orthologues with improved specificity (Slaymaker et al. 2015 "Rationally engineered Cas9 nucleases with improved specificity"). This strategy can be used to increase the specificity of C2c1 or C2c3 orthologues. Priority residues for mutagenesis are preferably all positively charged residues within the RuvC domain. Additional residues are positively charged residues that are conserved across different orthologues.

[00307] In some embodiments, the specificity of C2c1 or C2c3 can be increased by mutating residues that stabilize the non-target DNA strand.

[00308] In any of the non-naturally occurring CRISPR enzymes:

(a) there may be one mismatch between the target sequence and the corresponding sequence at one or more non-target loci; and/or

(b) there may be two, three, four or more mismatches between the target sequence and the corresponding sequence at one or more non-target loci; and/or

(c) two, three, four or more unpaired bases in (b) are adjacent.

[00309] In the case of any of the naturally occurring CRISPR enzymes, the enzyme in the CRISPR complex may have a reduced ability to modify one or more non-target loci compared to the unmodified enzyme, while the enzyme in the CRISPR complex has an increased ability to modify these target loci compared to the unmodified enzyme.

[00310] For any of the naturally occurring CRISPR enzymes, the relative difference between the ability of an enzyme in a CRISPR complex to modify a target locus and the ability to modify at least one non-target locus can be increased compared to an unmodified enzyme.

[00311] For any of the non-naturally occurring CRISPR enzymes, the CRISPR enzyme may contain one or more additional mutations, wherein the one or more additional mutations are in one or more catalytically active domains.

[00312] In the case of such non-naturally occurring CRISPR enzymes, the CRISPR enzyme may have reduced nuclease activity, compared to an enzyme that lacks these one or more additional mutations, or may not have it.

[00313] In the case of some of these non-naturally occurring CRISPR enzymes, the CRISPR enzyme does not cleave one or the other strand of DNA in the region of the target sequence.

[00314] In the case where the CRISPR enzyme contains one or more additional mutations in one or more catalytically active domains, one or more additional mutations may be in the catalytically active domain of the CRISPR enzyme, including the RuvCI, RuvCII and RuvCIII domains.

[00315] Without being limited by theory, in one aspect of the invention, the described methods and mutations involve increasing conformational rearrangements of CRISPR enzyme domains (e.g., C2c1 and C2c3 domains) into conformational states that result in cleavage at target sites and reducing the likelihood of these conformational states at target sites. that are not targets. Cleavage of the target DNA by the CRISPR enzyme occurs as a series of successive coordinated steps. Initially, the PAM-binding domain recognizes the PAM sequence from the 5' side of the target DNA. After binding to PAM, the first 10-12 nucleotides of the target sequence (primer sequence) are checked for complementarity of the guide RNA (gRNA) complex with DNA, a process dependent on separation of the DNA strands. If the nucleotides of the seed sequence are complementary to the gRNA, the remaining DNA is unpaired and the gRNA hybridizes to the target DNA strand over its entire length. Non-target grooves can stabilize the target DNA strand and promote unwinding through non-specific interactions with the positively charged DNA phosphate backbone. Interactions of RNA with complementary DNA and of the CRISPR enzyme with non-complementary DNA promote DNA unwinding by competing with the reverse hybridization of complementary and non-complementary DNA strands. Other domains of CRISPR enzymes can also influence the conformation of nuclease domains, such as linkers connecting different domains. Accordingly, these methods and mutations affect, without limitation, RuvCI, RuvCIll, RuvCIII, as well as linkers. Conformational changes, for example, C2c1 or C2c3, due to target DNA binding, including binding to the primer sequence and interactions with target and non-target DNA strands, determine whether the domains will be positioned to initiate nuclease activity. Thus, the methods and mutations described herein demonstrate and enable modifications beyond PAM recognition and RNA and DNA base pairing.

[00316] In one aspect, the invention relates to CRISPR nucleases as defined herein, e.g., C2c1 or C2c3, having improved equilibrium, biased towards the formation of conformations that promote cleavage in targeted interactions, and/or equilibrium, biased downward probabilities of formation of conformations associated with splitting during off-target interactions. In one aspect, the invention provides a Cas (eg, C2c1 or C2c3) nuclease with improved editing activity, i. a Cas nuclease (eg, C2c1 and C2c3) that adopts a conformation that promotes nuclease activity when at the target site and reduces its likelihood when at the non-target site. The authors of the article (Sternberg et al., Nature 327(7576): 110-3, doi: 10.1038/naturel3344, published on the Internet October 28, 2013. Epub 2013 Oct 28) used the FRET (fluorescence resonance energy transfer) method to determine the relative orientation of Cas9 catalytic domains upon association with target and non-target DNA, and these experiments can be extrapolated to the CRISPR enzymes of the present invention (eg, C2c1 or C2c3).

[00317] The present invention relates to methods and mutations for modulating nuclease activity and/or specificity using modified guide RNAs. As described, non-targeted nuclease activity can be increased or decreased. In addition, you can increase or decrease the specificity - the ratio of the target activity to non-target. Modified guide RNAs include, but are not limited to, truncated guide RNAs, non-functional guide RNAs, chemically modified guide RNAs, domain-linked guide RNAs, modified guide RNAs containing functional domains, aptamer-containing guide RNAs, adapter proteins containing guide RNAs, guides RNA containing added or modified loops. In some embodiments, one or more functional domains are associated with dead guide RNAs (dRNAs). In some embodiments, the dRNA-CRISPR enzyme complex regulates genes via a functional domain within a gene locus, while a guide RNA directs the CRISPR enzyme's DNA cleavage at another locus. In some embodiments, the dRNA is chosen to maximize the selectivity of regulation of the gene locus of interest over non-target regulation. In some embodiments, dRNAs are selected to maximize gene regulation and minimize target DNA cleavage.

[00318] For the purpose of further discussion, reference to a functional domain refers herein to a functional domain associated with a CRISPR enzyme or a functional domain associated with an adapter protein.

[00319] When practicing the present invention, gRNA loops can be extended without interacting with a Cas9 protein (e.g., C2c1 or C2c3) by inserting a separate RNA loop(s) or a separate sequence(s) that can recruit adapter proteins capable of bind to a single RNA loop(s) or a single sequence(s). Adapter proteins may include, but are not limited to, combinations of orthogonal RNA-binding proteins/aptamers that exist in a variety of bacteriophage envelope proteins. The list of such bacteriophage envelope proteins includes, but is not limited to, the following: Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, F1, ID2, NL95, TW19, AP205, ϕСb5, ϕСb8r, ϕСb12r, ϕСb2Зr, 7s and PRR1. Adapter proteins or orthogonal RNA binding proteins may further recruit effector proteins or fusion proteins that consist of one or more functional domains. In some embodiments, the functional domain may be selected from the group consisting of: transposase domain, integrase domain, recombinase domain, resolvase domain, invertase domain, protease domain, DNA methyltransferase domain, DNA hydroxymethylase domain, DNA demethylase domain, histone acetylase domain, histone deacetylase domain, nuclease domain, repressor domain, activation domain, domain containing a nuclear localization signal, domain of proteins that carry out transcriptional regulation or recruitment of the transcription complex, domain associated with the uptake of substances by the cell, nucleic acid binding domain, antigen presenting domain, histone- modifying domain that attracts histone-modifying proteins: inhibitor of histone-modifying enzymes, histone methyltransferase, histone demethylase, histone kinase, histone phosphatase, histone ribosylase, histone deribosylase, histone ubiquitinase, histone deubiquitinase, histone b iotinase, histone tail protease. In some preferred embodiments, the functional domain may be a transactivation domain, such as, but not limited to, VP64, p65, MyoD1, HSF1, RTA, SET7/9, or a histone acetyl transferase domain. In some embodiments, the functional domain may be a transcriptional repressor domain, preferably KRAB. In some embodiments, the transcriptional repressor domain may be a SID or SID concatemers (eg, SID4X). In some embodiments, the functional domain may be an epigenetic modification domain, such that an epigenetic modification enzyme is provided. In some embodiments, the functional domain may be an activation domain, which may be a P65 activation domain. In some embodiments, the functional domain may be a deaminase, such as cytidine deaminase. Cytidine deaminase can be directed to a target nucleic acid where it converts cytidine to uridine, resulting in the replacement of C (cytosine) by T (thymine) (and G (guanine) by A (adenine) in the complementary strand). In such an embodiment, the nucleotide substitution can be performed without DNA cleavage.

[00320] In one aspect, the present invention also provides methods and mutations for modulating Cas binding activity (eg, C2c1 or C2c3) and/or Cas binding specificity. In some embodiments, Cas proteins (eg, C2c1 or C2c3) are used that lack nuclease activity and/or binding specificity. In some embodiments, modified guide RNAs are used that promote binding but do not stimulate the nuclease activity of the Cas nuclease (eg, C2c1 or C2c3). In some embodiments, the level of targeted binding may be increased or decreased. Also, in some embodiments, the level of off-target binding can be increased or decreased. Moreover, there may be increased or decreased specificity, as determined by the ratio of targeted to non-targeted binding.

[00321] In some embodiments, the level of off-target cleavage is reduced by destabilizing strand separation, in particular by introducing mutations in the C2c1 or C2c3 enzymes that reduce the positive charge in regions interacting with DNA (as described hereinafter for Cas9 in Slaymaker et al. 2016 (Science, 1;351(6268):84-8) In further embodiments, the reduction of off-target cleavage is provided by introducing mutations in the C2c1 or C2c3 enzymes that affect the interaction between the target strand and the guide RNA sequence, in particular by disrupting interaction between C2c1 or C2c3 with the phosphate backbone of the target DNA strand such that target specific activity remains but off-target activity is reduced (as described for Cas9 in Kleinstiver et al. 2016, Nature, 28;529(7587):490-5 In some embodiments of the invention, off-target activity is reduced by modifying C2c1 (or modifying C2c3), while changing i interaction of both target strand and non-target strand compared to wild-type C2c1 (or wild-type C2c3).

[00322] Methods and mutations that can be used in various combinations to increase or decrease the activity and/or specificity of a target activity relative to a non-target one, or to increase or decrease the binding and/or specificity of a target binding relative to a non-target one, can be used to compensate for or enhance the effect of mutations or modifications made to enhance other effects. Such mutations or modifications made to enhance other effects include mutations and modifications to Cas (eg, C2c1 or C2c3) and/or mutations or modifications made to guide RNAs. In certain embodiments, methods and mutations are used on a chemically modified guide RNA. Examples of guide RNA chemical modifications include, but are not limited to, incorporation of 2'-O-methyl (M), 2'-O-methyl-3'-phosphorothioate (MIS), or 2'-O-methyl-3'-thio- PACE (MSP) to one or more terminal nucleotides. Such chemically modified guide RNAs may be more stable and active than unmodified guide RNAs, although the ratio of target to non-target specificity cannot be predicted. (See Hendel, 2015, Nat Biotechnol . 33(9):985-9, doi: 10.1038/nfot.3290., published online June 29, 2015). Chemically modified guide RNAs include, but are not limited to, RNAs with phosphorothioate linkages and closed nucleic acid nucleotides (LNAs) containing a methylene bridge between the 2'-4' carbon atoms of the ribose ring. The methods and mutations of the invention are used to modulate the activity of the Cas (or C2c3) nuclease (eg, C2c1 or C2c3) and or bind to chemically modified guide RNAs.

[00323] In one aspect, the invention relates to methods and mutations for modulating the binding and/or binding specificity of Cas proteins (e.g., C2c1 or C2c3) of the invention, as defined herein, including functional domains such as nucleases, transcription activators, repressors transcriptions, etc. For example, a Cas protein (eg, C2c1 or C2c3) can be designed to lack nuclease activity, or to have nuclease activity altered or reduced by introducing mutations, such as C2c1 or C2c3 mutations. Cas proteins (eg, C2c1 or C2c3) without nuclease activity can be useful for delivering functional domains, targeted RNA, and sequence-specific DNA. The invention relates to methods and mutations for modulating the binding of Cas proteins (eg C2c1 or C2c3). In one embodiment, the functional domain contains VP64, providing an RNA-guided transcription factor. In another embodiment, the functional domain contains FokI, which provides RNA-mediated nuclease activity. This is mentioned in US Patent Publication 2014/0356959, US Patent Publication 2014/0342456, US Patent Publication 2015/0031132 and Mali, P. et al., 2013, Science 339(6121):823-6, doi: 10.1126/science. 1.232033, published via the Internet on January 3, 2013, and through the ideas set forth in the present description, the invention covers the methods and materials of these documents used in connection with the ideas set forth in the present description. In some embodiments, targeted binding is increased. In some embodiments, off-target binding is reduced. In some embodiments, the level of targeted binding is reduced. In some embodiments, the level of off-target binding is increased. Accordingly, the invention also provides for an increase or decrease in the specificity of targeted versus non-targeted binding of functionalized Cas-binding proteins (eg, C2c1 or C2c3).

[00324] The use of Cas (eg, C2c1 or C2c3) as a targeting RNA binding protein is not limited to using Cas (eg, C2c1 or C2c3) without nuclease activity. Cas enzymes (eg, C2c1 or C2c3) with nuclease activity present can also function as guided RNA binding proteins when used with specific guide RNAs. For example, short guide RNAs and guide RNAs containing mismatched nucleotides can stimulate RNA-guided Cas (eg C2c1 or C2c3) binding to the target sequence with little or no cleavage (see e.g. Dahlman, 2015 , Nat Biotechnol 33(11):1159-1161, doi: 10.1038/nbt.3390, published online October 05, 2015). In some embodiments, the invention relates to methods and mutations for modulating the binding of Cas proteins (eg, C2c1 or C2c3) having nuclease activity. In some embodiments, the level of targeted binding is increased. In some embodiments, the level of off-target binding is reduced. In some embodiments, the level of targeted binding is reduced. In some embodiments, the level of off-target binding is increased. In some embodiments, the specificity of targeted binding relative to non-targeted binding is increased or decreased. In some embodiments, the nuclease activity of an enzyme comprising a guide RNA and Cas (eg, C2c1 or C2c3) is also modulated.

[00325] The formation of an RNA-DNA heteroduplex is important for the presence of cleavage activity and specificity across the entire target region, and not just the primer sequence region closest to the PAM. Thus, the truncated guide RNAs have a reduced level of activity and level of cleavage specificity. In one aspect, the invention relates to a method and mutations to increase cleavage activity and specificity using altered guide RNAs.

[00326] The invention also demonstrates that modifications to the nuclease specificity of Cas (eg, C2c1 or C2c3) can be made simultaneously with changes in the target region. Cas mutants (e.g., C2c1 or C2c3) can be engineered to have both increased target specificity and adaptive changes in PAM recognition, for example, selecting mutations that alter PAM specificity and combining these mutations with non-groove mutations. target, which can increase (or, if desired, reduce) specificity for target sequences in relation to non-target sequences. In one such embodiment, the PI domain residue is mutated to improve recognition of the PAM sequence of interest, while one or more non-target groove amino acids are mutated to change target specificity. The Cas methods and modifications (e.g., C2c1 or C2c3) described herein can be used to prevent the loss of specificity that occurs with a change in PAM recognition, increase the increased level of specificity that occurs with a change in PAM recognition, prevent an increase in the level of specificity, arising from changes in PAM recognition, or contribute to the loss of specificity that occurs when PAM recognition changes.

[00327] These methods and mutations can be used for any Cas enzymes (eg, C2c1 or C2c3) with altered PAM recognition. Non-limiting examples of PAMs included are as described herein.

[00328] In the following embodiments, modified proteins are used in methods and mutations.

[00329] In the case of any of the non-naturally occurring CRISPR enzymes, the CRISPR enzyme may contain one or more heterologous functional domains.

[00330] One or more heterologous functional domains may include one or more nuclear localization signal (NLS) domains. One or more heterologous functional domains may include at least two or more NLSs.

[00331] One or more heterologous functional domains may include one or more transactivation domains. The transactivation domain may be VP64.

[00332] One or more heterologous functional domains may contain one or more transcriptional repression domains. The transcriptional repression domain may include a KRAB domain or a SID domain.

[00333] One or more heterologous functional domains may include one or more nuclease domains. One or more nuclease domains may be FokI.

[00334] One or more heterologous functional domains may have only one or more of the following functions: methylase activity, demethylase activity, transactivation activity, transcriptional repression activity, transcription factor release activity, histone modification activity, nuclease activity, single-stranded RNA cleavage activity , double-stranded RNA cleavage activity, single-stranded DNA cleavage activity, double-stranded DNA cleavage activity, and nucleic acid binding activity.

[00335] At least one or more heterologous functional domains may be adjacent to or at the N-terminus of the enzyme and/or adjacent to or at the C-terminus of the enzyme.

[00336] One or more heterologous functional domains can be fused to the CRISPR enzyme or linked to the CRISPR enzyme or connected to the CRISPR enzyme through a linker portion.

[00337] In any of the non-naturally occurring CRISPR enzymes, the CRISPR enzyme may include the CRISPR enzyme from organisms of the genus such as: Alicyclobacillus, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacillus, Desulfatirhabdium, Citrobacter, and Methylobacterium.

[00338] For any of the non-naturally occurring CRISPR enzymes, the CRISPR enzyme may include a chimeric Cas enzyme (e.g., C2c1 or C2c3) containing a first fragment of a first Cas orthologue (e.g., C2c1 or C2c3) and a second fragment of a second Cas orthologue (e.g., C2c1 or C2c3), with the first and second Cas orthologues (for example, C2c1 or C2c3) being different. At least one of the orthologues (first and/or second) of Cas (for example, C2c1 or C2c3) may be Cas (for example, C2c1 or C2c3) of species such as: Alicyclobacillus acidoterrestris (for example, ATCC 49025), Alicyclobacillus contaminans (for example, DSM 17975), Desulfovihrio inopinatus (e.g. DSM 10711), Desulfonatronum thiodismutans (e.g. MLF-1 strain), Opitutaceae TAV5 bacteria, Tuberibacillus calidus ( e.g. DSM 17572), Bacillus thermoamylovorans (e.g. B4166 strain), Brevibacillus sp. CF112, Bacillus sp. NSP2.1, Desulfatirhabdium butyrativorans (eg DSM 18734), Alicyclobacillus herbarius (eg DSM 13609), Citrobacter freundii (eg ATCC 8090), Brevibacillus agri (eg BAB-2500) and Methylobacterium nodulans (eg ORS 2060).

[00339] In any of the non-naturally occurring CRISPR enzymes, the nucleotide sequence encoding the CRISPR enzyme may be codon-optimized for expression in eukaryotes.

[00340] In any of the non-naturally occurring CRISPR enzymes, the cell may be a eukaryotic cell or a prokaryotic cell, wherein the CRISPR complex is functional in the cell, and the CRISPR complex enzyme has a reduced ability to modify one or more non-target loci of the cell, according to compared to the unmodified enzyme, and/or the enzyme in the CRISPR complex has an increased ability to modify one or more non-target cell loci compared to the unmodified enzyme.

[00341] Accordingly, in one aspect, the invention provides a eukaryotic cell containing an artificial protein or CRISPR system as defined herein.

[00342] In certain embodiments, methods as described herein may include delivery of a Cas (e.g., C2c1 or C2c3) to a transgenic cell in which one or more nucleic acids encoding one or more guide RNA molecules are delivered or directly introduced, are associated in the cell with a regulatory element containing the promoter of one or more target genes. The term "Cas transgenic cell", as used herein, refers to a cell, in particular a eukaryotic cell, into whose genome the Cas gene is inserted. The nature, type or origin of such a cell is not particularly limited in accordance with the present invention. Also, the method of introducing the Cas transgene into the cell may vary and be any of the methods available in the art. In some embodiments, a transgenic Cas cell is obtained by introducing a Cas transgene into an isolated cell. In some other embodiments, a transgenic Cas cell can be obtained by isolating cells from a transgenic Cas organism. As a non-limiting example, a transgenic Cas cell as used herein can be derived from a Cas transgenic eukaryotic organism, such as a eukaryotic organism with an inserted Cas. See WO 2014/093622 (PCT/US13/74667), incorporated herein by reference. The methods of publication of US Pat. The methods of US Pat. No. 20130236946 assigned to Cellectis to target the Rosa locus can also be modified to use the CRISPR-Cas system of the present invention. For the following example, a reference is made to an article by Platt et. al. (Cell; 159(2):440-455 (2014)) describing a mouse with a mouse Cas9 insert, which is incorporated herein by reference and which can be extrapolated to the CRISPR enzymes of the present invention characterized herein. The Cas transgene can also include a Lox-Stop-polyA-Lox(LSL) cassette, which makes Cas expression driven by the Cre recombinase. Alternatively, a Cas transgenic cell can be obtained by introducing a Cas transgene into an isolated cell. Transgene delivery systems are well known in the art. An example would be the delivery of a Cas transgene, for example, into a eukaryotic cell via a vector (eg AAV, adenovirus, lentivirus) and/or particle and/or nanoparticle, as also described elsewhere herein.

[00343] It will be understood by the skilled artisan that a cell, such as a Cas transgenic cell as used herein, may contain further genome changes in addition to having an inserted Cas gene or mutations resulting from the sequence-specific action of Cas in complex with RNA capable of directing Cas to the target locus, such as, for example, one or more oncogenic mutations, as described by way of non-limiting example in Platt et al. (2014), Chen et al., (2014), or Kumar et al. (2009).

[00344] The invention also provides a composition comprising the engineered CRISPR protein described herein as described in this section.

[00345] The invention also relates to a non-naturally engineered composition comprising a CRISPR-Cas complex containing any of the non-naturally occurring CRISPR enzymes described above.

[00346] In one aspect, the invention relates to a vector system comprising one or more vectors, where one or more vectors contain:

a) a first regulatory element operably linked to one or more nucleotide sequences encoding an engineered CRISPR protein as defined herein, and optionally

b) a second regulatory element operably linked to one or more nucleotide sequences, encoding one or more nucleic acids, including a guide RNA containing a guide sequence, a direct repeat sequence, and optionally where components (a) and (b) are located in the same vector or different vectors.

[00347] The invention also relates to a non-naturally occurring composition comprising:

a delivery system functionally optimized to deliver components of a CRISPR-Cas complex or one or more polynucleotide sequences containing or encoding said components into a cell, said CRISPR-Cas complex being functional inside the cell.

components of the CRISPR-Cas complex or one or more polynucleotide sequences encoding for transcription and/or translation in a cell components of the CRISPR-Cas complex, including:

non-naturally occurring CRISPR enzymes (eg, engineered by C2c1 or C2c3 engineering methods) described herein;

a CRISPR-Cas guide RNA containing a guide sequence, a direct repeat sequence, wherein the enzyme in the CRISPR complex has a reduced ability to modify one or more non-target loci compared to the unmodified enzyme, and/or the enzyme in the CRISPR complex has an increased ability to modify one or more target loci compared to the unmodified enzyme.

[00348] In one aspect, the invention also provides a system comprising said engineered CRISPR protein, such as described in this section.

[00349] In any of these compositions, the delivery system may include: a yeast system, a lipofection system, a microinjection system, a gene gun, virosomes, liposomes, immunoliposomes, polycations, nucleic acid lipid conjugates, or artificial virions as described herein and elsewhere. .

[00350] In any of these compositions, the delivery system may include a vector system containing one or more vectors, wherein component (II) contains a first regulatory element operably linked to a polynucleotide sequence that contains a guide sequence and a direct repeat sequence, and optionally component ( I) contains a second regulatory element operably linked to a polynucleotide sequence encoding the CRISPR enzyme.

[00351] In any of these compositions, the delivery system may include a vector system containing one or more vectors, wherein component (II) contains a first regulatory element operably linked to the direct repeat sequence and sequence, and component (I) contains a second regulatory element, operably linked to a polynucleotide sequence encoding the CRISPR enzyme.

[00352] In any of these compositions, the composition may include more than one guide RNA, and each guide RNA has a different target, providing a multiplex approach.

[00353] In any of these compositions, the polynucleotide sequence(s) may reside on a single vector.

[00354] The invention also relates to a non-naturally occurring vector system of short palindromic repeats regularly clustered in CRISPR-Cas ((CRISPR-CRISPR-associated protein (Cas) (CRISPR-Cas)) comprising one or more vectors that contain:

a) a first regulatory element operably linked to a polynucleotide sequence encoding a non-naturally occurring CRISPR enzyme of any of the constructs of the invention described herein;

b) a second regulatory element operably linked to a polynucleotide sequence encoding one or more guide RNAs, and this guide RNA contains a guide sequence, a direct repeat sequence, where: components (a) and (b) are located in the same or different vectors, a complex is formed CRISPR guide RNAs target polynucleotide target loci and the enzyme acts on target polynucleotide loci and the enzyme in the CRISPR complex has a reduced ability to modify one or more non-target loci compared to an unmodified enzyme, and/or the enzyme in the CRISPR complex has an increased ability to modify one or more target loci compared to an unmodified enzyme.

[00355] In such a system, component (II) may comprise a first regulatory element operably linked to a polynucleotide sequence that contains a guide sequence, a direct repeat sequence, and wherein component (II) may comprise a second regulatory element operably linked to a polynucleotide sequence encoding CRISPR enzyme. In such a system, when possible, the guide RNA may include a chimeric RNA.

[00356] In such a system, component (I) may contain a first regulatory element operably linked to a guide sequence and a direct repeat sequence, while component (II) may contain a second regulatory element operably linked to a polynucleotide sequence encoding a non-naturally occurring enzyme CRISPR. Such a system may include more than one guide RNA and each guide RNA may target different targets, thus providing a multiplex approach. Components (a) and (b) may be located on the same vector.

[00357] In any of these vector systems, one or more vectors may contain one or more viral vectors, such as retroviral, lentiviral, adenovirus, adeno-associated virus, or herpes simplex virus.

[00358] In any of these systems containing regulatory elements, at least one of these regulatory elements may contain a tissue-specific promoter. The tissue-specific promoter can direct expression in a mammalian blood cell, a mammalian liver cell, or a mammalian eye.

[00359] In any of the mixtures or systems described above, the direct repeat sequence may comprise one or more protein-binding RNA aptamers. One or more aptamers may be in a tetraloop. One or more aptamers may have the ability to bind bacteriophage MS2 coat protein.

[00360] In any of the formulations or systems described above, the cell may be a eukaryotic cell or a prokaryotic cell: wherein the CRISPR complex is functional in the cell and the CRISPR complex enzyme has a reduced ability to modify one or more non-target loci compared to the unmodified enzyme, and /or this enzyme in the CRISPR complex has an increased ability to modify one or more target loci compared to an unmodified enzyme.

[00361] The invention also relates to the CRISPR complex of any of the compositions described above or any of the systems described above.

[00362] The invention also relates to a method of modifying a locus of interest in a cell, comprising contacting the cell with any of the engineered CRISPR enzymes described herein (e.g., engineered C2c1 or C2c3), compositions, or any described herein systems or vector systems, or the cell contains any of the CRISPR complexes described herein. In the case of such methods, the cell may be a eukaryotic cell or a prokaryotic cell. In the case of such methods, the cell may be a prokaryotic cell or a eukaryotic cell, preferably a eukaryotic cell. In such methods, the organism may contain a cell. In such methods, the organism may not be a human or other animal.

[00363] Any of these methods may be performed ex vivo or in vitro .

[00364] In some embodiments, a nucleotide sequence encoding at least one of said Cas guide RNA or protein is operably linked in a cell to a regulatory element containing a promoter of the gene of interest, wherein expression of at least one component of the CRISPR-Cas system is induced the promoter of the gene of interest. "Operably linked" means that a nucleotide sequence encoding a guide RNA and/or a Cas protein is linked to the regulatory element(s) such that expression of the nucleotide sequence occurs, as also referred to herein. The term "regulatory element" is also described in the present description. According to the invention, the regulatory element comprises a gene of interest promoter, preferably such as an endogenous gene of interest promoter. In some embodiments, the promoter is at its endogenous genomic position. In such embodiments, the nucleic acid encoding CRISPR and/or Cas is under transcriptional regulation by the promoter of the gene of interest at its native genomic position. In other specific embodiments, the promoter is provided on a (single) nucleic acid molecule, such as a vector or plasmid, or other extrachromosomal nucleic acid, i.e. the promoter is not present in its natural genomic position. In some embodiments, the implementation of the promoter is integrated into the genome not in its natural genomic position.

[00365] In any of these methods, these modifications include the modulation of gene expression. Said modulation of gene expression may include activation of gene expression and/or repression of gene expression. Accordingly, in one aspect, the invention relates to a method for modulating gene expression, wherein the method comprises introducing into a cell an engineered protein or CRISPR system as described herein.

[00366] The invention also relates to a method of treating a disease, disorder, or infection in an individual in need thereof, comprising administering an effective amount of any of the engineered CRISPR enzymes (e.g., C2c1 or C2c3), CRISPR compositions, systems, or complexes described herein. description. Diseases, disorders or infections may include a viral infection. The viral infection may be hepatitis B virus (HBV).

[00367] The invention also relates to the use of one of the engineered CRISPR enzymes (eg C2c1 or C2c3), CRISPR compositions, systems or complexes described above for genetic or genomic engineering.

[00368] The invention also relates to a method of altering the expression of a genomic locus of interest in a mammalian cell, including interacting the cell with engineered CRISPR enzymes (e.g., C2c1 or C2c3), CRISPR mixtures, systems, or complexes described herein, and thereby delivering the CRISPR-Cas (vector) and allowing the CRISPR-Cas complex to form and bind to the target; and determining whether the expression of the genomic locus being changed is changed, for example, whether there is an increase or decrease in expression or a modification of the gene product.

[00369] The invention also relates to any engineered CRISPR enzyme (eg, C2c1 or C2c3), composition, system, or CRISPR complex described above for therapeutic use. The therapeutic application may be for gene or genome editing or for gene therapy.

[00370] In some embodiments, the activity of the artificial CRISPR enzymes (eg, C2c1 or C2c3) described herein includes cleavage of genomic DNA, optionally resulting in a reduction in gene transcription.

[00371] In one aspect, the invention relates to an isolated cell with altered expression of a gene locus by the method described herein, wherein the expression is altered compared to a cell not altered in expression of the gene locus by this method. In a related aspect, the invention relates to a cell line derived from such a cell.

[00372] In one aspect, the invention relates to a method for modifying an organism or non-human organism by manipulating a target sequence at a genomic locus of interest, e.g., a hematopoietic stem cell (HSC), in particular, in which the genomic locus of interest is associated with a mutation associated with aberrant protein expression or with a disease or condition, including:

delivery to the HSC, for example, by bringing the HSC into contact with a particle containing a non-naturally occurring composition, including:

polynucleotide sequence of the CRISPR-Cas guide RNA containing:

a) a guide sequence capable of hybridizing to a target sequence in the HSC;

b) direct repetition; and

a CRISPR enzyme, optionally containing at least one or more nuclear localization sequences;

where the guide sequence directs the sequence-specific binding of the CRISPR complex to the target sequence, and

wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) a guide sequence that hybridizes to the target sequence, and

said method may optionally include delivering an HDR template, for example, by contacting an HSC containing the HDR template, or by contacting the HSC with another particle containing the HDR template, the HDR template allowing expression of a normal or less aberrant form of the protein; where "normal form" is a wild-type protein and "aberrant form" is a protein that causes the development of a disease or condition, and

optionally, the method may include isolating or obtaining HSC from an organism or non-human organism, optionally increasing the population of HSCs, contacting the particle(s) with HSCs to obtain a population of modified HSCs, optionally increasing the population of modified HSCs, and optionally introducing the modified HSCs into the organism or not. a human organism.

[00373] One aspect of the invention is a method of modifying an organism or non-human organism by manipulating a target sequence at a genomic locus of interest, and in particular an HSC, for example, where the genomic locus of interest is associated with a mutation associated with aberrant protein expression or a disease or condition, including delivery to the HSC, in particular by binding to the HSC particles containing a non-naturally occurring or engineered composition that contains:

I. (a) a guide sequence capable of hybridizing to a target sequence in the HSC, and

(b) at least one or more direct repeats, and

II. a CRISPR enzyme, optionally comprising one or more NLSs, wherein the targeting sequence directs sequence-specific binding of a CRISPR complex comprising the CRISPR enzyme in complex with the targeting sequence that hybridizes to the target sequence, and

said method may optionally include delivering an HDR template, for example, by contacting an HSC containing the HDR template, or by contacting the HSC with another particle containing the HDR template, the HDR template allowing expression of a normal or less aberrant form of the protein; where "normal form" is a wild-type protein and "aberrant form" is a protein that causes the development of a disease or condition, and

optionally, the method may include isolating or obtaining HSC from an organism or non-human organism, optionally increasing the population of HSCs, contacting the particle(s) with HSCs to obtain a population of modified HSCs, optionally increasing the population of modified HSCs, and optionally introducing the modified HSCs into the organism or not. a human organism.

[00374] One or more polynucleotides encoding any one or more or all of the CRISPR complexes can be delivered, preferably linked to one or more regulatory elements for in vivo expression, e.g., via particles containing a vector containing the polynucleotide(s) operably linked with regulatory element(s). Any of these polynucleotide sequences encoding a CRISPR enzyme, guide sequence, direct repeat, may be RNA. It will be appreciated that if an RNA polynucleotide is mentioned that is said to include a feature such as a direct repeat, then the RNA also includes that feature. If the polynucleotide is DNA and is indicated to include a feature, such as a direct repeat sequence, then the DNA sequence is or can be transcribed into an RNA including the feature in question. When the trait is a protein, such as the CRISPR enzyme, the DNA or RNA sequence referred to is or may be translated (and in the case of DNA, first transcribed)

[00375] In some embodiments, the invention relates to a method for modifying an organism, in particular a mammal, including a mammal or an organism, whether or not a human, by manipulating a target sequence at a genomic locus of interest, and in particular an HSC, for example, in the case where the genomic locus of interest is associated with a mutation associated with aberrant protein expression or a disease or condition, including delivery to the HSC, in particular by bringing the HSC into contact with a non-naturally occurring or engineered composition that contains one or more particles containing a viral vector (viral vectors), a plasmid vector (plasmid vectors) or a vector (s) with a nucleic acid molecule (for example, RNA), functionally encoding the specified composition, for its expression, while the composition includes:

(A) I. the first regulatory element operably linked to an RNA polynucleotide sequence of the CRISPR-Cas system, where the polynucleotide sequence contains

(a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell,

(b) a direct repeat sequence, and

II. a second regulatory element operably linked to a coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization signals (or optionally at least one or more nuclear localization signals (NLS) as some embodiments may not include NLS)

and (a), (b) and (c) are located in the orientation from the 5' to the 3'-end, and components I and II are located on the same or different vector systems, while the transcribed guide sequence directs the sequence-specific binding of the CRISPR complex to a target sequence, wherein the CRISPR complex comprises the CRISPR enzyme complexed with a guide RNA hybridized to the target sequence, or

(B) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising:

I. the first regulatory element functionally related to

(a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell,

(b) at least one or more direct repeat sequences, and

II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and optionally, if applicable,

where components I and II are located on the same or different vectors of the system, while the transcribed guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence, and the CRISPR complex contains the CRISPR enzyme in complex with the guide sequence, which hybridizes with the target sequence; the method may optionally include delivering an HDR template, for example, by contacting an HSC containing the HDR template, or by contacting the HSC with another particle containing the HDR template, the HDR template allowing expression of a normal or less aberrant form of the protein; where "normal form" is a wild-type protein and "aberrant form" is a protein that causes the development of a disease or condition, and optionally the method may include isolating or obtaining HSC from an organism or a non-human organism, optionally increasing the HSC population, bringing particles ( s) contacting HSCs to obtain a population of modified HSCs, optionally increasing the population of modified HSCs, and optionally introducing the modified HSCs into an organism or non-human organism. In some embodiments, components I, II, and III are located on the same vector. In some embodiments, components I and II are located on the same vector while component III is located on a different vector. In some embodiments, components I and III are located on the same vector while component II is located on a different vector. In some embodiments, components II and III are located on the same vector while component I is located on a different vector. In some embodiments, components I, II, and III are located on different vectors. The invention also relates to a viral or plasmid vector system as described herein.

[00376] By manipulation of the target sequence, applicants also mean epigenetic manipulation of the target sequence. This can be manipulation of the chromatin state of the target sequence, such as modifying the methylation state of the target sequence (i.e., introducing or removing methylation or methylation pattern or CpG islands), modifying histones, increasing or decreasing the availability of the target sequence, or facilitating folding. into a three-dimensional structure. It is understood that when a method of modifying an organism or mammal, including a human or non-human mammal, by manipulation of a target sequence at a genomic locus of interest is mentioned, it is understood that this may refer to the organism (or mammal) as a whole, or to only one cell or population of cells from that organism (if the organism is multicellular). In the case of a human, for example, Applicants contemplate, inter alia , a single cell or population of cells, and these may preferably be modified ex vivo and then reintroduced. In this case, a biopsy or other tissue or body fluid may be needed. Stem cells are also preferred in this regard. But, of course, in vivo embodiments are also contemplated. This invention is particularly advantageous in the case of HSC.

[00377] In some embodiments, the invention relates to a method for modifying an organism or a non-human organism by manipulating first and second target sequences on opposite strands of a DNA duplex at an HSC genomic locus of interest, in particular, in particular, where the genomic the locus is associated with a mutation associated with aberrant protein expression or with a disease or syndrome involving delivery, for example, by bringing the HSC into contact with a particle(s) containing a non-naturally occurring or engineered composition containing:

I. polynucleotide sequence of the guide RNA of the first CRISPR-Cas system (for example, C2c1 or C2c3), and the first polynucleotide sequence contains:

(a) a first guide sequence capable of hybridizing to the first target sequence,

(b) a first direct repeat sequence, and

II. a guide RNA polynucleotide sequence of a second CRISPR-Cas system (e.g., C2c1 or C2c3), wherein the second polynucleotide sequence contains:

(a) a second guide sequence capable of hybridizing to a second target sequence,

(b) a second direct repeat sequence, and

III. a polynucleotide sequence encoding a CRISPR enzyme containing at least one or more nuclear localization signals and containing one or more mutations, wherein (a), (b) and (c) are in 5' to 3' order; or

IV. expression product(s) of one or more components I to III, eg first and second direct repeat sequence, CRISPR enzyme;

wherein, upon transcription, the first and second guide sequences direct the sequence-specific binding of the first and second CRISPR complex to the first and second target sequence, respectively, wherein the first CRISPR complex comprises a CRISPR enzyme in complex with (1) the first guide sequence that hybridizes to the first target sequence, and the second CRISPR complex comprises the CRISPR enzyme in complex with (1) a second target sequence that hybridizes to the second target sequence, wherein the polynucleotide sequence encoding the CRISPR enzyme may be DNA or RNA, wherein the first target sequence directs cleavage of one strand of the DNA duplex near the first target sequence, and the second guide sequence directs cleavage of the other strand of the DNA duplex near the second target sequence, causing the formation of a double strand break, thus m modifying an organism or an organism that does not belong to a person; the method may optionally include the delivery of a homologous recombination repair template, in particular by particles that bind to the HSC or bind to the HSC via another particle and contain a homologous recombination repair template, wherein the homologous recombination repair template provides expression of a normal or less aberrant protein forms; wherein "normal form" is a wild-type protein and "aberrant form" is a protein that causes the development of a disease or syndrome; also, optionally, the method may include isolating or obtaining HSCs from an organism or a non-human organism, optionally expanding a population of HSCs, binding particle(s) to HSCs to obtain a population of modified HSCs, optionally expanding a population of modified HSCs, optionally introducing modified HSCs into an organism or non-human organism. In some methods of the invention, any or all of the polynucleotide sequences encode a CRISPR enzyme, first and second guide sequences, first and second direct repeats. In further embodiments of the invention, the polynucleotides encoding the sequence encoding the CRISPR enzyme, the first and second guide sequences, the first and second direct repeats, are RNA and will be delivered via liposomes, nanoparticles, exosomes, microvesicles or gene gun, but particle delivery has the greatest advantages. . In specific implementations of the invention, the sequences of the first and second repeats have a 100% level of identity. In some embodiments of the invention, the polynucleotides may be contained in a vector system comprising one or more vectors. In preferred embodiments, the first CRISPR enzyme contains one or more mutations such that the enzyme is a complementary strand ncase and the second CRISPR enzyme contains one or more mutations such that the enzyme is a non-complementary strand ncase. Alternatively, the first CRISPR enzyme contains one or more mutations such that the enzyme is a non-complementary strand ncase, and the second CRISPR enzyme contains one or more mutations such that the enzyme is a complementary strand ncase. In preferred methods of the invention, a first guide sequence directing the cleavage of one strand of the DNA duplex in the vicinity of the first target sequence and a second guide sequence directing the cleavage of one strand of the DNA duplex in the vicinity of the second target sequence result in the formation of sticky ends with 5'-overhanging nucleotides (overhangs). In embodiments of the invention, the sticky end with 5'-overhanging nucleotides is nearly 200 base pairs, preferably no more than 100 base pairs, or even more preferably no more than 50 base pairs. In embodiments of the invention, the sticky end with 5'-overhanging nucleotides (overhang) is at least 26 base pairs, preferably at least 30 base pairs, and most preferably 34-50 base pairs.

[00378] The present invention, in some embodiments, provides a method for modifying an organism or a non-human organism by manipulating first and second target sequences on opposite strands of a DNA duplex at an HSC genomic locus of interest, in particular, in particular, in which the genomic locus is associated with a mutation associated with aberrant protein expression or with a disease or syndrome, including delivery, for example, by binding of HSC to a particle (s) containing a non-naturally occurring or artificial composition, including:

I. the first regulatory element functionally related to

(a) a first targeting sequence capable of hybridizing to a first target sequence in a eukaryotic cell,

(b) at least one or more direct repeat sequences, and

II. the second regulatory element functionally associated with

(a) a second targeting sequence capable of hybridizing to a second target sequence in a eukaryotic cell,

(b) at least one or more direct repeat sequences, and

III. a third regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme (eg, C2el or C2c3),

IV. an expression product of one or more components I to IV, in particular the first and second direct repeats, a CRISPR enzyme;

wherein the components I, II, III and IV are located on the same or different vectors of the system, where during transcription the first and second guide sequences direct the sequence-specific binding of the first and second CRISPR complexes to the first and second target sequences, respectively, and the first CRISPR- the complex includes a CRISPR enzyme in complex with a first guide sequence hybridized to a first target sequence, and the second CRISPR complex includes a CRISPR enzyme in complex with a second guide sequence hybridized to a second target sequence, wherein the polynucleotide sequence encoding the CRISPR enzyme can be DNA or RNA, wherein the first guide sequence directs the cleavage of one strand of the DNA duplex in the vicinity of the first target sequence, and the second guide sequence directs the cleavage of the other strand of the DNA duplex in the vicinity of the second target sequence , causing a double-strand break to form, thus modifying an organism or a non-human organism; the method may optionally include the delivery of a homologous recombination repair template, in particular by particles that bind to the HSC or bind to the HSC via another particle and contain a homologous recombination repair template, wherein the homologous recombination repair template provides expression of a normal or less aberrant protein forms; wherein "normal form" is a wild-type protein and "aberrant form" is a protein that causes the development of a disease or syndrome; also, optionally, the method may include isolating or obtaining HSCs from an organism or a non-human organism, optionally expanding a population of HSCs, binding particle(s) to HSCs to obtain a population of modified HSCs, optionally expanding a population of modified HSCs, optionally introducing modified HSCs into an organism or non-human organism.

[00379] The invention also provides the vector system described here. The system may include one, two, three or four different vectors, with all combinations of localization of the components provided, for example, components I, II, III and IV may be in one vector; components I, II, III and IV may be on different vectors; components I, II, III and IV can be on two or three different vectors, in any possible combination. In some methods of the invention, any or all of the polynucleotide sequences encoding the CRISPR enzyme, the first and second guide sequences, the first and second direct repeats are RNA. In further implementations of the invention, the sequences of the first and second repeats have a 100% level of identity. In preferred embodiments, the first CRISPR enzyme contains one or more mutations in which (which) the enzyme is a complementary strand nicase, and the second CRISPR enzyme contains one or more mutations in which (which) the enzyme is a non-complementary strand nicase. Alternatively, the first enzyme may be a non-complementary chain case and the second enzyme may be a non-complementary chain case. In further implementations of the invention, one or more viral vectors will be delivered via liposomes, nanoparticles, exosomes, microvesicles or gene gun; but delivery by particles has the greatest advantages.

[00380] In the preferred methods of the present invention, a first guide sequence directing the cleavage of one strand of the DNA duplex in the vicinity of the first target sequence and a second guide sequence directing the cleavage of one strand of the DNA duplex in the vicinity of the second target sequence result in the formation of a sticky end with 5' overhanging nucleotides (overhang). In embodiments of the invention, the sticky end with 5'-overhanging nucleotides (overhang) is almost 200 base pairs, preferably not more than 100 base pairs, or even more preferably not more than 50 base pairs. In embodiments of the invention, the sticky end with 5'-overhanging nucleotides (ovehang) is at least 26 base pairs, preferably at least 30 base pairs, and most preferably 34-50 base pairs.

[00381] The invention in some embodiments provides a method for modifying a genomic locus of interest, in particular an HSC, for example when the genomic locus of interest is associated with a mutation associated with aberrant protein expression or a disease or syndrome, including delivery to the HSC by particle(s) , containing a Cas protein containing one or more mutations and two gRNAs whose targets are the first and second strands of the DNA molecule in the HSC, respectively, with the gRNA targeting the DNA molecule and the Cas protein introducing nick breaks in each (first and second) strand DNA molecules, and the target in HSC changes; and wherein the Cas protein and the two guide RNAs do not naturally occur together, and the method may optionally include delivery of a template for repair by homologous recombination, e.g., via particles that bind to an HSC or bind to an HSC via another particle and contain a template for repair by homologous recombination. recombination, and the template for repair by homologous recombination provides the expression of a normal or less aberrant form of the protein; wherein "normal form" is a wild-type protein and "aberrant form" is a protein that causes the development of a disease or syndrome, and optionally: the method may include isolating or obtaining HSC from an organism or a non-human organism, optionally - population propagation HSC, coupling the particle(s) to the HSC to obtain a population of modified HSCs, optionally propagating the population of modified HSCs, optionally introducing the modified HSCs into a non-human or non-human organism. In preferred methods of the invention, the nicase activity of the Cas protein on each (first and second) strands of the DNA molecule results in the formation of a sticky end with 5' overhanging nucleotides (overhang). In embodiments of the invention, the sticky end with 5'-overhanging nucleotides (overhang) is at least 26 base pairs, preferably at least 30 base pairs, and most preferably 34-50 base pairs. In an aspect of the invention, the codon composition of the Cas protein is optimized for expression in a eukaryotic cell, preferably a mammalian or human cell. Aspects of the invention are associated with a decrease in the expression of the gene product or the further introduction of a template polynucleotide into the DNA molecule encoding the gene product, or the precise excision of the interrupt sequence, allowing two 5'-overhangs to anneal to each other, followed by ligation, or a change in the activity or function of the gene product, or an increase in expression of the gene product. When implementing the invention, the gene product is a protein.

[00382] In some embodiments, the invention provides a method for modifying a genomic locus of interest, e.g., in an HSC, in particular, in which the genomic locus of interest is associated with a mutation associated with aberrant protein expression or with a disease or syndrome, including delivery, for example, by binding HSC to particle(s), including:

a) a first regulatory element operably linked to each of two CRISPR-Cas guide RNA systems that target, respectively, the first and second strands of the double-stranded DNA molecule in the HSC, and

b) a second regulatory element operably linked to the Cas protein (eg C2c1 or C2c3), or

c) expression product(s) of a) or b),

moreover, components (a) and (b) are located on the same or different vectors of the system, the target of gRNA is the HSC DNA molecule, and the Cas protein introduces nick breaks in each (first and second) strand of the HSC DNA molecule; the Cas protein and the two gRNAs do not occur together in nature; the method may further include delivering a homologous recombination repair template, e.g., via particles that bind to the HSC or bind to the HSC via another particle and containing the homologous recombination repair template, wherein the homologous recombination repair template allows expression of a normal or less aberrant form squirrel; wherein "normal form" is a wild-type protein and "aberrant form" is a protein that causes the development of a disease or syndrome, and optionally: the method may include isolating or obtaining HSC from an organism or a non-human organism, optionally - population propagation HSC, coupling the particle(s) to the HSC to obtain a population of modified HSCs, optionally propagating the population of modified HSCs, optionally introducing the modified HSCs into a non-human or non-human organism. In an aspect of the invention, guide RNAs may contain guide sequences fused to direct repeat sequences. Aspects of the invention are associated with a decrease in the expression of the gene product or the further introduction of a template polynucleotide into the DNA molecule encoding the gene product, or the precise excision of the interrupt sequence, allowing two 5'-overhangs to anneal to each other, followed by ligation, or a change in the activity or function of the gene product, or an increase in expression of the gene product. In carrying out the invention, the gene product is a protein. In preferred embodiments, the vectors of the system are viral vectors. In further embodiments of the invention, the polynucleotides encoding the sequence encoding the CRISPR enzyme, the first and second guide sequences, the first and second direct repeats, are RNA and will be delivered via liposomes, nanoparticles, exosomes, microvesicles, or gene gun, but particle delivery has the advantage. In one aspect, it relates to a method for modifying a target polynucleotide in an HSC. In some embodiments, the method allows a CRISPR complex to bind to a target polynucleotide to cleave the target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR complex contains a CRISPR enzyme in complex with a guide sequence capable of hybridizing to the target sequence within said target polynucleotide. polynucleotide; wherein said guide sequence is connected to a forward repeat sequence. In some embodiments, said cleavage includes cleaving one or two strands at the location of the target sequence with said CRISPR enzyme. In some embodiments, said cleavage results in a decrease in the level of transcription of the target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein said repair results in a mutation involving the insertion, deletion, or substitution of one or more nucleotides in said target polynucleotide. In some embodiments, said mutation results in one or more amino acid substitutions in the resulting protein upon expression of a gene containing the target sequence. In some embodiments, the implementation of the method includes the introduction of one or more vectors or the subsequent expression of the product (products) in the HSC, in particular by particle (s), and one or more vectors induce the expression of one or more of the following components from the list: CRISPR enzyme, guide sequence connected to a direct repeat sequence. In some embodiments, said vectors are delivered specifically to a subject's HSC. In some embodiments, these HSC modifications are implemented in cell culture. In some embodiments, the method further comprises isolating said HSCs from the subject prior to said modifications. In some embodiments, the method further comprises administering said HSCs and/or cells derived from them back to said subject.

[00383] In one embodiment, refers to a method for obtaining, in particular, HSC containing a mutant gene associated with the disease. In some embodiments, such a disease-associated gene may be associated with a risk of occurrence or development of a disease. In some embodiments, the method comprises (a) introducing one or more vectors or subsequently expressing the product(s), particularly via particles, in the HSC, wherein the one or more vectors induce the expression of one or more of the following: a CRISPR enzyme, a directing a sequence connected to a direct repeat sequence; and (b) providing binding of the CRISPR complex to a target polynucleotide that promotes cleavage of the target polynucleotide within said disease-associated gene, wherein the CRISPR complex includes a CRISPR enzyme in complex with a targeting sequence capable of hybridizing to a target sequence within said target polynucleotide, and, optionally, if applicable, thus resulting in HSCs containing a mutant gene associated with the disease. In some embodiments, said cleavage includes cleaving one or two strands at the location of the target sequence with said CRISPR enzyme. In some embodiments, said cleavage results in a decrease in the level of transcription of the target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein said repair results in a mutation involving the insertion, deletion, or substitution of one or more nucleotides in said target polynucleotide. In some embodiments, said mutation results in one or more amino acid substitutions in the resulting protein upon expression of a gene containing the target sequence. In some embodiments, the modified HSCs are introduced into an animal, thereby resulting in a model organism.

[00384] In one aspect, relates to a method for modifying a target polynucleotide in, for example, HSC. In some embodiments, the method allows a CRISPR complex to bind to a target polynucleotide to cleave the target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR complex contains a CRISPR enzyme in complex with a guide sequence capable of hybridizing to the target sequence within said target polynucleotide. a polynucleotide, wherein said guide sequence is linked to a direct repeat sequence. In other embodiments, the invention provides a modification of the expression of a polynucleotide in a eukaryotic cell that is derived from, for example, an HSC. The method of the invention includes increasing or decreasing the expression of a target polynucleotide using a CRISPR complex that binds to a polynucleotide in an HSC; delivery of the CRISPR complex by particle(s) has the greatest advantage.

[00385] In some methods, the target polynucleotide can be inactivated to affect expression in, for example, hematopoietic stem cells. For example, upon binding of a CRISPR complex to a target sequence in a cell, the target polynucleotide is inactivated such that the sequence is not transcribed, the encoded protein is not produced, or the sequence does not function as the wild-type sequence does.

[00386] In some embodiments of the invention, the specified RNA CRISPR-Cas system, for example, gRNA can be modified; for example, include aptamers and a functional domain. An aptamer is a synthetic nucleotide that binds to a specific target molecule; for example, a nucleic acid molecule that has been created by repeated rounds of in vitro selection, or by SELEX (systematic evolution of ligands by exponential enrichment, systematic evolution of ligands by exponential enrichment) to bind various molecular targets such as small molecules, proteins, nucleic acids and even cells, tissues and organisms. Aptamers are convenient to use, since their recognition characteristics of molecules are comparable to those of antibodies. In addition to their fine recognition ability, the advantages of aptamers over antibodies include low or no immunogenicity in therapeutic applications. Accordingly, in the practice of the invention, the enzyme and/or RNA may include a functional domain.

[00387] In some embodiments, the functional domain is a transactivation domain, preferably VP64. In some embodiments, the functional domain is a transcriptional repression domain, preferably KRAB. In some embodiments, the transcriptional repression domain is a SID or SID concatemers (eg, SID4X). In some embodiments, the functional domain is an epigenetic modification domain that provides for the operation of an epigenetic modification enzyme. In some embodiments, the functional domain is an activation house, which may be a P65 activation domain. In some embodiments, the functional domain may have nuclease activity. In one embodiment, the functional domain includes Fok1.

[00388] The invention also relates to an in vitro or ex vivo cell containing any of the modified CRISPR enzymes, compositions or complexes described above, obtained by any of the above methods. A cell can be either eukaryotic or prokaryotic. The invention also relates to the progeny of such cells. The invention also relates to the product of any cell, or any such progeny, where the product is the product of said one or more target loci modified with a CRISPR enzyme or CRISPR complex. The product may be a peptide, polypeptide or protein. Some of these products can be modified with a modified CRISPR enzyme or CRISPR complex. In the case of some of these modified products, the product of the target locus is physically different from the product of said target locus that has not been modified with the modified CRISPR enzyme or CRISPR complex.

[00389] The invention also relates to a polynucleotide molecule containing a polynucleotide sequence encoding any of the non-naturally occurring CRISPR enzymes described above.

[00390] Any such polynucleotide may further comprise one or more regulatory elements that are operably linked to a polynucleotide sequence encoding non-naturally occurring CRISPR enzymes.

[00391] In any such polynucleotide that contains one or more regulatory elements, one or more regulatory elements can be operably arranged to express a non-naturally occurring CRISPR enzyme in a eukaryotic cell. The eukaryotic cell may be a human cell. A eukaryotic cell could be a rodent cell, possibly a mouse cell. The eukaryotic cell may be a yeast cell. The eukaryotic cell may be a Chinese hamster ovary (CHO) cell. The eukaryotic cell may be an insect cell.

[00392] In any such polynucleotide that contains one or more regulatory elements, one or more regulatory elements can be functionally optimized for expression of non-naturally occurring CRISPR enzymes in a prokaryotic cell.

[00393] In any such polynucleotide that contains one or more regulatory elements, the one or more regulatory elements can be functionally optimized for expression in an in vitro system.

[00394] The invention also relates to an expression vector containing one of the polynucleotide molecules described above. The invention also relates to such polynucleotide molecule(s), for example polynucleotide molecules, operably arranged to express white and/or nucleic acid component(s), as well as such vector(s).

[00395] In addition, the invention relates to a method for introducing mutations in Cas (for example, C2c1 or C2c3) or mutant or modified Cas (for example, C2c1 or C2c3), which is an orthologue of CRISPR enzymes, according to the invention described in the present description, including the definition amino acid(s) in this orthologue, which may be adjacent to or in contact with a nucleic acid molecule, e.g., DNA, RNA, gRNA, etc., and/or amino acid(s) similar to or corresponding to the amino acid(s) described herein ) in the CRISPR enzymes of the invention described herein for mutations and/or modifications, or the synthesis or production or expression of ortholog(s) consisting or essentially consisting of modification(s) and/or mutation(s), or introducing a mutation as described herein, for example, a modification, for example, substitution or mutation of a neutral amino acid with a charged one, in particular, a positively charged amino acid, such as alanine. The ortholog modified in this way can be used in the CRISPR-Cas system; and the nucleic acid molecule(s) expressing it can be used in a vector or other delivery systems that deliver molecules or encode components of the CRISPR-Cas systems described herein.

[00396] In one aspect, the invention provides effective target activity and minimized off-target activity. In one aspect, the invention provides efficient targeted cleavage by the CRISPR protein and minimized off-target cleavage by the CRISPR protein. In one aspect, the invention provides guide RNA-specific binding of a CRISPR protein at a gene locus without DNA cleavage. In one aspect, the invention provides efficient guide RNA targeting of the CRISPR protein at a gene locus and minimized off-target binding of the CRISPR protein. Accordingly, in this aspect, the invention provides specific regulation of the target gene. In one aspect, the invention relates to guide RNA-specific binding of the CRISPR enzyme at a gene locus in the absence of DNA cleavage. Accordingly, in one aspect, the invention provides for cleavage at one gene locus and gene regulation at another gene locus using a single CRISPR enzyme. In one aspect, the invention provides orthogonal activation and/or inhibition and/or cleavage of multiple targets using one or more CRISPR proteins and/or enzymes.

[00397] In another embodiment, the present invention relates to a method for functional screening of the genome in a population of cellsex vivo orin vivo, including the introduction or expression of a library containing multiple guide RNA (gRNA) of the CRISPR-Cas system, and the screening further includes the use of the CRISPR enzyme, and the CRISPR complex is modified to contain a heterologous functional domain. In one aspect, the invention relates to a method for screening a genome, comprising introduction into a host or expression in a host organism. librariesin vivo. In one aspect, the invention relates to the method described in the present description, in which the activator is introduced into the host organism or expressed in the host organism. In one aspect, the invention relates to the method described in the present description, in which the activator is associated with a CRISPR protein. In one aspect, the invention relates to the method described in the present description, in which the activator is associated with the N-terminus or C-terminus of the CRISPR protein. In one aspect, the invention relates to the method described in the present description, in which the activator is associated with a loop in the guide RNA. In one aspect, the invention relates to the method described in the present description, including a repressor that is introduced into the host organism or expressed in the host organism. In one aspect, the invention relates to the method described in the present description, in which the screening includes changing and detecting gene activation, gene inhibition or cleavage at the locus.

[00398] In one aspect, the invention relates to the method described in the present description, in which the host organism is a eukaryotic cell. In one aspect, the invention relates to the method described in the present description, in which the host organism is a mammalian cell. In one aspect, the invention relates to the method described in the present description, in which the host organism is a eukaryotic cell that is not a human cell. In one aspect, the invention relates to the method described herein, wherein the non-human eukaryotic cell is a non-human mammalian cell. In one aspect, the invention relates to the method described herein, wherein the non-human mammalian cell may include, but is not limited to, primate, bovine, bird, pig, dog, rodent, hare, such as monkey, cows, sheep, pigs, dogs, rabbits, rats or mice. In one aspect, the invention relates to the method described herein, wherein the cell may be a cell of a non-mammalian eukaryotic organism, such as a poultry (eg, chicken), vertebrate fish (eg, salmon), or a shellfish (eg, oyster, lobster, shrimp). In one aspect, the invention relates to the method described herein, wherein the non-human eukaryotic cell may be a plant cell. The plant cell may be a monocot or dicot, or a crop or cereal plant such as cassava, corn, sorghum, soybean, wheat, oats or rice. A plant cell may also belong to an algae, a tree or food plant, a fruit or vegetable (for example, trees such as citrus trees, in particular orange, grapefruit or lemon trees; peach or nectarine trees, apple or pear trees; nuts such as almonds, hazelnuts or pistachio; nightshade; plants of the genus Brassiccr, plants of the genus Lactuca, plants of the genus Spinada, plants of the genus Capsicum, cotton, tobacco, asparagus, carrots, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, raspberries, strawberries, blueberries, blueberries, grapes, coffee, cocoa, etc.).

[00399] In one aspect, the invention provides a method as described herein, comprising delivering CRISPR-Cas complexes or component(s) thereof or nucleic acid molecule(s) encoding them, wherein said nucleic acid is operably linked to regulatory sequences and expressed in vivo . In this aspect, the invention relates to the method described in the present description, in which the expression in vivo occurs through a lentivirus, adenovirus or adeno-associated virus. In this embodiment, the invention relates to a method, as described in the present description, in which the delivery occurs through a particle, nanoparticle, lipid or cell penetrating peptide (CPP).

[00400] In specific embodiments, it may be of interest to target the CRISPR-Cas complex to the chloroplast. In many cases, this targeting can be done in the presence of an N-terminal extension called a chloroplast transit peptide (CTP) or a plastid transit peptide. Chromosomal transgenes from bacterial sources must have the sequence encoding the CTP sequence fused to the sequence encoding the expressed polypeptide if the expressed polypeptide is to be directed to the plant plastid (ie chloroplast). Accordingly, localization of the exogenous polypeptide in the chloroplast is often achieved by operably linking the polynucleotide sequence encoding the CTP sequence to the 5' region of the polynucleotide encoding the exogenous peptide. However, the amino acid sequence of the CTP and the sequences adjacent to the N-terminus of the peptide can affect the efficiency of processing. Other chloroplast targeting options that have been described are the maize cab-m7 signal sequence (US Patent 7022896, WO 97/41228), the pea glutathione reductase signal sequence (WO 97/41228), and CTP described in US2009029861.

[00401] In one aspect, the invention relates to a pair of CRISPR-Cas complexes, each containing a guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell, where at least one loop of each a single guide sequence (sg-RNA) is modified by inserts of a single RNA sequence(s) that bind to one or more adapter proteins, the adapter protein being associated with one or more functional domains, with each gRNA of each CRISPR-Cas complex containing a functional domain, with DNA cleavage activity. In one aspect, the invention relates to paired CRISPR-Cas complexes, such as described herein, wherein the DNA cleavage activity is provided by the Fok1 nuclease.

[00402] In this aspect, the invention relates to a method for cleaving a target sequence at a genomic locus of interest, comprising delivering CRISPR-Cas complexes or their component(s) or nucleic acid molecule(s) encoding them, said nucleic acid being operably linked to regulatory sequence and is expressed in vivo . In one aspect, the invention relates to the method described in the present description, in which the delivery is due to lentivirus, adenovirus or adeno-associated virus. In this aspect, the invention relates to the method described herein, or to the paired CRISPR-Cas complexes described herein, in which the target sequence for the first complex of the pair is on the first strand of double-stranded DNA, and the target sequence for the second complex of the pair located on the second strand of double-stranded DNA. In this aspect, the invention relates to the method described herein, or the paired CRISPR-Cas complexes described herein, in which the target sequences for the first and second complex are adjacent to each other such that the DNA is cut so as to provide homologous reparation. In one aspect, this method or these CRISPR-Cas pair complexes may include such CRISPR-Cas complexes each containing a CRISPR enzyme mutated such that it has no more than 5% of the nuclease activity of the wild-type CRISPR enzyme.

[00403] In one aspect, the invention relates to a library, method, or complex as described herein, wherein the gRNA is modified to have at least one non-coding functional loop, for example, where at least one non-coding functional loop is repressive; for example, where at least one non-coding functional loop contains Alu.

[00404] In one aspect, the invention relates to a method for altering or modifying the expression of gene products. Said method may include introducing into a cell containing or expressing a DNA molecule encoding a gene product of a non-naturally occurring CRISPR-Cas system containing a Cas protein and a guide RNA targeting a DNA molecule, where the guide RNA is targeting a DNA molecule encoding the product gene, and the Cas protein cleaves the DNA molecule encoding the gene product, and the expression of the gene product is changed; nor does the Cas protein and guide RNA occur together in nature. The invention further includes a Cas protein that is codon-optimized for expression in a eukaryotic cell. In a preferred embodiment, the eukaryotic cell is a mammalian cell, and in an even more preferred embodiment, the mammalian cell is a human cell. In a further embodiment of the invention, the expression of the gene product is reduced.

[00405] In one aspect, the invention relates to altered cells and the progeny of these cells, as well as to the products of these cells. The proteins and CRISPR-Cas systems (eg, C2c1) and of the present invention can be used to obtain cells containing a modified target locus. In some embodiments, the method provides for the binding of a nucleic acid-targeted complex to a target DNA or target RNA to cleave said target DNA or target RNA, thereby modifying said target DNA or target RNA, wherein the complex , the target of which is a nucleic acid, includes an effector protein, the target of which is a nucleic acid, in complex with a guide RNA hybridized with a target sequence within said target DNA or target RNA. In one aspect, the invention relates to a method for repairing a gene locus in a cell. In another aspect, it relates to a method for altering the expression of DNA or RNA at a gene locus of a cell. In some embodiments, the method provides for binding of a nucleic acid-targeted complex to a target DNA or target RNA such that said binding results in an increase or decrease in the expression level of said DNA or RNA, wherein the nucleic acid-targeted complex comprises an effector protein, the target of which is a nucleic acid, in complex with a guide RNA. For modification of a target DNA or target RNA, considerations and conditions similar to those described above apply. In fact, these conditions of selection, cultivation and reintroduction are applicable in various aspects of the present invention. In one aspect, the invention relates to a method for modifying DNA or RNA in a eukaryotic cell, which can be carried out in vivo, ex vivo or in vitro . In some embodiments, the method includes selecting a cell or population of cells from a human or non-human body and modifying the cell or cells. Cultivation can take place at any stage ex vivo. Such cells can be, without any limitation, a plant cell, an animal cell, a specific cell type of any organism, including stem cells, immune cells, T cells, B cells, dendritic cells, cells of the cardiovascular system, epithelial cells, etc. P. Cells can be modified according to the invention to produce gene products, for example in controlled amounts, which can be increased or decreased depending on the purpose of use, or mutated. In certain embodiments, the genetic locus of the cell is repaired. The cell or cells can even be reintroduced into a non-human organism or plant. In the case of reintroduced cells, it may be preferable to use stem cells.

[00406] In one aspect, the invention relates to cells that temporarily contain CRISPR systems or components thereof. For example, CRISPR proteins or enzymes and nucleic acids are temporarily introduced into cells and the genetic locus is changed, followed by a decrease in the amount of one of the components of the CRISPR system. Then, cells, cell progeny, and organisms containing CRISPR-generated cells contain less of one or more components of the CRISPR system, or no more of any of the components of the CRISPR system. One non-limiting example is the CRISPR-Cas self-inactivating system as described herein. Thus, the invention relates to cells, cell progenitors and organisms containing cells that contain one or more genetic loci modified by the CRISPR system, but which are essentially missing one or more components of the CRISPR system. In some embodiments, there are no CRISPR system components. Such cells, tissues, and organisms predominantly contain the desired or selected genetic change, but have lost CRISPR-Cas components or residues that could potentially act non-specifically and lead to safety issues or prevent regulatory approval. The invention also relates to products of activity of cells, organisms, cell precursors and organisms.

Inducible CRISPR-Cas systems C2c1 or C2c3 ("split C2c1" or "split C2c3")

[00407] In one aspect, the invention relates to a non-naturally occurring or engineered inducible CRISPR-Cas C2c1 or C2c3 system comprising:

a first C2c1 or C2c3 fusion coupled to the first half of the inducible dimer and a second C2c1 or C2c3 fusion coupled to the second half of the inducible dimer;

wherein the first C2c1 or C2c3 fusion construct is operably linked to one or more nuclear localization signals;

wherein the second fusion construct C2c1 or C2c3 is operably linked to one or more nuclear localization signals;

while contact with the inducing energy source brings the first and second halves of the inducible dimer together;

wherein bringing the first and second halves of the inducible dimer together allows the first and second C2c1 fusion constructs to constitute a functional C2c1 CRISPR-Cas system, or the first and second C2c3 fusion constructs to constitute a C2c3 functional CRISPR-Cas system;

wherein the CRISPR-Cas C2c1 system or the CRISPR-Cas C2c3 system comprises a guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence at a cell genomic locus of interest; and

wherein the functional CRISPR-Cas C2c1 system or the CRISPR-Cas C2c3 system binds to the target sequence and optionally edits the genomic locus to alter gene expression.

[00408] In one possible aspect of the invention, in an inducible CRISPR-Cas C2c1 system, the inducible dimer is, or contains, or essentially consists of, or consists of, an inducible heterodimer. In one possible aspect, in an inducible CRISPR-Cas C2c1 system, the first half or first portion or first fragment of the inducible heterodimer is, contains, essentially consists of, or consists of a FKBP protein, optionally FKBP12. In one possible aspect of the invention, in an inducible CRISPR-Cas C2c1 system, the second half or second portion or second fragment of the inducible heterodimer is, contains, essentially consists of, or consists of a FRB protein. In one possible aspect of the invention, in an inducible CRISPR-Cas C2c1 system, the structure of the first C2c1 fusion construct is, contains, essentially consists of, or consists of a C2c1-FRB-NES NES-N-terminal region. In one possible aspect of the invention, in an inducible CRISPR-Cas C2c1 system, the structure of the second C2c1 fusion construct is, contains, essentially consists of, or consists of a C2c1-FRB-NLS NLS-C-terminal region. In one possible aspect, a linker may be present in the CRISPR-Cas C2c1 inducible system separating the C2c1 portion from a half, portion, or fragment of the inducible dimer. In one possible aspect, in an inducible CRISPR-Cas C2c1 system, the source of induction is, contains, essentially consists of, or consists of rapamycin. In one possible aspect, in an inducible CRISPR-Cas C2c1 system, the inducible dimer is an inducible homodimer. In one possible aspect, in the C2c1 inducible CRISPR-Cas system, C2c1 is AacC2c1. In one possible aspect, in an inducible C2c1 CRISPR-Cas system, one or more functional domains are associated with one or both parts of C2c1, in particular, the functional domains may optionally include a transcriptional activator, a transcriptional repressor, or a nuclease such as Fok1 nuclease. In one possible aspect, in an inducible CRISPR-Cas C2c1 system, a functional CRISPR-Cas C2c1 system binds to a target sequence and the enzyme is a dead C2c1, optionally having a nuclease activity reduced by at least 97% or 100% (respectively, having no more than 3% nuclease activity or predominantly 0% nuclease activity), compared with C2c1, not containing any mutation. In addition, the invention relates to and one of the possible aspects of the invention provides a polynucleotide encoding the inducible CRISPR-Cas C2c1 system described in the present description.

[00409] In one possible aspect of the invention, in an inducible CRISPR-Cas C2c3 system, the inducible dimer is, or contains, or essentially consists of, or consists of, an inducible heterodimer. In one possible aspect, in an inducible CRISPR-Cas C2c3 system, the first half or first portion or first fragment of the inducible heterodimer is, or contains, or consists essentially of, or consists of a FKBP protein, optionally FKBP12. In one possible aspect of the invention, in an inducible CRISPR-Cas C2c3 system, the second half or second portion or second fragment of the inducible heterodimer is, or contains, or essentially consists of, or consists of a FRB protein. In one possible aspect of the invention, in an inducible CRISPR-Cas C2c3 system, the structure of the first C2c3 fusion construct is, or contains, or essentially consists of, or consists of a C2c3-FRB-NES NES-N-terminal region. In one possible aspect of the invention, in an inducible CRISPR-Cas C2c3 system, the structure of the second C2c3 fusion construct is, or contains, or essentially consists of, or consists of a C2c3-FRB-NLS NLS-C-terminal region. In one possible aspect, a linker may be present in an inducible CRISPR-Cas C2c1 system separating a portion of C2c3 from a half, portion, or fragment of the inducible dimer. In one possible aspect, in an inducible CRISPR-Cas C2c1 system, the source of induction is, or contains, or essentially consists of, or consists of, rapamycin. In one possible aspect, in an inducible CRISPR-Cas C2c3 system, the inducible dimer is an inducible homodimer. In one possible aspect, in an inducible C2c3 CRISPR-Cas system, one or more functional domains are associated with one or both parts of C2c3, in particular, the functional domains may optionally include a transcriptional activator, a transcriptional repressor, or a nuclease such as Fok1 nuclease. In one possible aspect, in an inducible CRISPR-Cas C2c3 system, a functional CRISPR-Cas C2c3 system binds to a target sequence and the enzyme is a dead C2c3, optionally having a nuclease activity reduced by at least 97% or 100% (respectively, having no more than 3% nuclease activity or predominantly 0% nuclease activity), compared to C2c3 that does not contain any mutation. In addition, the invention relates to and one of the possible aspects of the invention provides a polynucleotide encoding the inducible CRISPR-Cas C2c3 system described in the present description.

[00410] In one possible aspect of the invention, the invention relates to a vector for delivering a first C2c1 fusion construct associated with the first half or portion or fragment of an inducible dimer and operably linked to one or more nuclear localization signals as described herein. In one possible aspect, the invention relates to a vector for delivery of a second C2c1 fusion associated with the second half or part or fragment of an inducible dimer and operably linked to one or more nuclear export signals.

[00411] In one possible aspect of the invention, the invention relates to a vector for delivering a first C2c3 fusion construct associated with the first half or portion or fragment of an inducible dimer and operably linked to one or more nuclear localization signals as described herein. In one possible aspect, the invention relates to a vector for delivering a second C2c3 fusion construct linked to the second half or portion or fragment of an inducible dimer and operably linked to one or more nuclear export signals

[00412] In one possible aspect of the invention, the invention relates to a vector for delivering both a first C2c1 fusion construct fused to the first half or portion or fragment of an inducible dimer and operably linked to one or more nuclear localization signals, and a second C2c1 fusion construct linked with a second half or portion or fragment of an inducible dimer and operably linked to one or more nuclear export signals as described herein.

[00413] In one possible aspect of the invention, the invention relates to a vector for delivering both a first C2c3 fusion construct fused to the first half or portion or fragment of an inducible dimer and operably linked to one or more nuclear localization signals, and a second C2c3 fusion construct linked with a second half or portion or fragment of an inducible dimer and operably linked to one or more nuclear export signals as described herein.

[00414] In one possible aspect, the vector may be a single plasmid or expression cassette.

[00415] In one possible aspect, the invention provides a eukaryotic host cell or cell line transformed with any of the vectors described herein or expressing the inducible CRISPR-Cas C2c1 or C2c3 system described herein.

[00416] In one possible aspect, the invention relates to a transgenic organism transformed with one of the vectors described herein or expressing an inducible CRISPR-Cas C2c1 or C2c3 system described herein, or descendants of this organism. In one possible aspect, the invention relates to a model organism that constitutively expresses the CRISPR-Cas C2c1 or C2c3 inducible system described herein.

[00417] In one possible aspect, the invention relates to a non-naturally occurring or engineered CRISPR-Cas C2c1 system comprising: a first C2c1 construct fusion associated with the first half of an inducible heterodimer and a second C2c1 fusion construct associated with the second half inducible heterodimer, wherein the first C2c1 fusion construct is operably linked to one or more nuclear localization signals, wherein the second C2c1 fusion construct is operably linked to one or more NES nuclear localization signals, wherein upon interaction with the induction source, the first and second halves of the inducible heterodimer combine, wherein the union of the first and second halves of the inducible heterodimer allows the first and second C2c1 fusion constructs to constitute a functional CRISPR-Cas C2c1 system, wherein the CRISPR-Cas C2c1 system includes a guide RNA (gRNA) containing a guide sequence capable of hybridizing interact with the target sequence at the genomic locus of interest in the cell, and the CRISPR-Cas C2c1 functional system edits the genomic locus by altering gene expression.

[00418] In one possible aspect, the invention relates to a non-naturally occurring or engineered CRISPR-Cas C2c3 system comprising: a first C2c3 construct fusion associated with the first half of an inducible heterodimer and a second C2c3 fusion construct associated with the second half inducible heterodimer, wherein the first C2c3 fusion construct is operably linked to one or more nuclear localization signals, wherein the second C2c3 fusion construct is operably linked to one or more NES nuclear localization signals, moreover, upon interaction with the induction source, the first and second halves of the inducible heterodimer combine, wherein the union of the first and second halves of the inducible heterodimer allows the first and second C2c3 fusion constructs to constitute a functional CRISPR-Cas C2c3 system, wherein the CRISPR-Cas C2c3 system includes a guide RNA (gRNA) containing a guide sequence capable of hybridizing interact with the target sequence at the genomic locus of interest in the cell, and moreover, the CRISPR-Cas C2c3 functional system edits the genomic locus by changing gene expression.

[00419] In one possible aspect, the invention provides a method of treating an individual in need thereof, comprising inducing gene editing by transforming the individual with a polynucleotide described herein or any of the vectors described herein, and administering to the individual a source of induction. The invention relates to the use of such a polynucleotide or vector in the manufacture of drugs, such as, for example, a drug for the treatment of an individual or for such a method of treating an individual. The invention relates to a polynucleotide described in the present description, or any of the vectors described in the present description, for use in a method of treating an individual in need thereof, including the induction of gene editing by transforming the individual with a polynucleotide described in the present description or any of the vectors described in the present description, and the introduction of an induction source to the individual. In one possible aspect of the method, a repair matrix is provided, for example delivered by a vector containing said repair matrix.

[00420] In one possible aspect, the invention relates to a method of treating an individual in need thereof, comprising the induction of activation or repression of gene transcription by transforming the individual with a polynucleotide described in the present description, or any of the vectors described in the present description, where the polynucleotide or vector encodes or contains a catalytically inactive C2c1 protein or an inactive C2c3 protein and one or more associated functional domains described herein; furthermore, the method includes administering to the individual a source of induction. The invention relates to the use of such a polynucleotide or vector in the manufacture of a medicament, such as, for example, a medicament for the treatment of an individual or for such a method of treating an individual. The invention relates to a polynucleotide described in the present description, or any of the vectors described in the present description, for use in a method of treating an individual in need thereof, including the induction of activation or repression of gene transcription by transforming the individual with a polynucleotide described in the present description, or any from the vectors described in the present description, and the introduction of an induction source to the individual.

[00421] Accordingly, the invention, among other things, covers homodimers, as well as heterodimers, dead C2c1 or C2c1, essentially without nuclease activity, in particular due to mutations, systems or complexes in which one or more NLSs are present and / or one or more NES; functional(s) domain(s) connected to the split C2c1; methods, including methods of treatment, and use.

[00422] Accordingly, the invention, inter alia, covers homodimers, as well as heterodimers, dead C2c3 or C2c3, essentially without nuclease activity, in particular due to mutations, systems or complexes in which one or more NLSs are present and / or one or more NES; functional(s) domain(s) connected to the split C2c3; methods, including methods of treatment, and use.

[00423] It is understood that when C2c1, C2c1 protein, or C2c1 enzyme is mentioned herein, this includes the separated C2c1 of the present invention. In one aspect, the invention relates to a method for altering or modifying the expression of a gene product. Said method may include introducing into a cell containing or expressing a DNA molecule encoding a gene product, an engineered non-naturally occurring C2c1 CRISPR-Cas system comprising a C2c1 protein and a guide RNA targeting the DNA molecule, where the guide RNA is targeted to the DNA molecule, encoding the gene product, and the C2c1 protein cleaves the DNA molecule encoding the gene product, thereby changing the expression of the gene product; moreover, the C2c1 protein and the guide RNA do not occur together in nature. The invention relates to a guide RNA containing a guide sequence linked to a DR sequence. In addition, the invention provides for the optimization of the codon composition of the C2c1 protein for expression in a eukaryotic cell. In a preferred embodiment, the eukaryotic cell is a mammalian cell, and in a more preferred embodiment, the mammalian cell is a human cell. In a further embodiment of the invention, expression of the gene product is reduced.

[00424] It is understood that when C2c3, C2c3 protein, or C2c3 enzyme is mentioned herein, this includes the separated C2c3 of the present invention. In one aspect, the invention relates to a method for altering or modifying the expression of a gene product. Said method may include introducing into a cell containing or expressing a DNA molecule encoding a gene product, an engineered non-naturally occurring C2c3 CRISPR-Cas system comprising a C2c3 protein and a guide RNA targeting the DNA molecule, where the guide RNA is targeted to the DNA molecule, encoding the gene product, and the C2c3 protein cleaves the DNA molecule encoding the gene product, thereby changing the expression of the gene product; moreover, the C2c3 protein and the guide RNA do not occur together in nature. The invention relates to a guide RNA containing a guide sequence linked to a DR sequence. In addition, the invention provides for the optimization of the codon composition of the C2c3 protein for expression in a eukaryotic cell. In a preferred embodiment, the eukaryotic cell is a mammalian cell, and in a more preferred embodiment, the mammalian cell is a human cell. In a further embodiment of the invention, expression of the gene product is reduced.

[00425] In one aspect, the invention relates to an engineered non-naturally occurring C2c1 CRISPR-Cas system comprising the C2c1 protein and a guide RNA targeting a DNA molecule encoding a gene product in a cell, the guide RNA targeting a DNA molecule encoding the gene product, and the C2c1 protein cleaves the DNA molecule encoding the gene product, thereby changing the expression of the gene product; moreover, the C2c1 protein and the guide RNA do not occur together in nature; and moreover, the system includes a separated C2c1 according to the present invention. The invention relates to a guide RNA containing a guide sequence linked to a DR sequence. In addition, the invention provides for the optimization of the codon composition of the C2c1 protein for expression in a eukaryotic cell. In a preferred embodiment, the eukaryotic cell is a mammalian cell, and in a more preferred embodiment, the mammalian cell is a human cell. In a further embodiment of the invention, expression of the gene product is reduced.

[00426] In one aspect, the invention relates to an engineered non-naturally occurring C2c3 CRISPR-Cas system comprising the C2c3 protein and a guide RNA targeting a DNA molecule encoding a gene product in a cell, the guide RNA targeting a DNA molecule encoding the gene product, and the C2c3 protein cleaves the DNA molecule encoding the gene product, thereby changing the expression of the gene product; moreover, the C2c3 protein and the guide RNA do not occur together in nature; and wherein the system comprises the separated C2c3 of the present invention. The invention relates to a guide RNA containing a guide sequence linked to a DR sequence. In addition, the invention provides for the optimization of the codon composition of the C2c3 protein for expression in a eukaryotic cell. In a preferred embodiment, the eukaryotic cell is a mammalian cell, and in a more preferred embodiment, the mammalian cell is a human cell. In a further embodiment of the invention, expression of the gene product is reduced.

[00427] In another aspect, the invention provides an engineered, non-naturally occurring system comprising one or more vectors comprising a first regulatory element operably linked to a CRISPR-Cas C2c1 or C2c3 guide RNA targeting a DNA molecule encoding a gene product and a second regulatory element operably linked to the C2c1 protein or the C2c3 protein; including the separated C2c1 or C2c3 of the present invention. Components (a) and (b) may be in the same or different system vectors. The guide RNA targets the DNA molecule encoding the gene product in the cell, and the C2c1 protein or C2c3 protein cleaves the DNA molecule encoding the gene product, whereby the expression of the gene product is altered; and wherein the C2c3 protein or the C2c1 protein and the guide RNA do not occur together in nature. The invention relates to a guide RNA containing a guide sequence linked to a DR sequence. In addition, the invention relates to a C2c1 protein or a C2c3 protein codon-optimized for expression in a eukaryotic cell. In a preferred embodiment, the eukaryotic cell is a mammalian cell, and in a more preferred embodiment, the mammalian cell is a human cell. In a further embodiment of the invention, expression of the gene product is reduced.

[00428] In one aspect, the invention relates to a vector system containing one or more vectors. In some embodiments, the system includes: a) a first regulatory element operably linked to a DR sequence and one or more insertion sites for inserting one or more targeting sequences downstream of the DR sequence, wherein the targeting sequence when expressed provides for sequence-specific binding of the CRISPR-Cas C2c1 complex or a CRISPR-Cas C2c3 complex with a target sequence in a eukaryotic cell, wherein the CRISPR-Cas C2c1 or CRISPR-Cas C2c3 complex contains C2c1 or C2c3 complexed with (1) a guide sequence that is hybridized to the target sequence, and (2) a sequence DR; and (b) a second regulatory element operably linked to an enzyme coding sequence encoding said C2c1 or C2c3 enzyme containing a nuclear localization sequence; moreover, the components (a) and (b) are located in one and more vectors of the system; including split C2c1 or C2c3. In some embodiments, component (a) further comprises two or more targeting sequences operably linked to the first regulatory element, wherein, when expressed, the two or more targeting sequences provide sequence-specific binding of C2c1 or C2c3 to different target sequences in a eukaryotic cell.

[00429] In some embodiments, the CRISPR-Cas C2c1 or CRISPR-Cas C2c3 complex comprises one or more nuclear localization sequences of sufficient strength to induce accumulation of said CRISPR-Cas C2c1 or CRISPR-Cas C2c3 complex in amounts to be found in the nucleus of a eukaryotic cell . Without wishing to be bound by theory, it is believed that a nuclear localization sequence is not necessary for the activity of the CRISPR-Cas C2c1 or CRISPR-Cas C2c3 complex in eukaryotic organisms, but that the inclusion of such sequences enhances the activity of the system, especially in targeting nucleic acid molecules to the nucleus.

[00430] In some embodiments, the C2c1 enzyme is a C2c1 bacterial species selected from the group consisting of: Alicyclobacillus acidoterrestris (e.g., ATCC 49025), Alicyclobacillus contaminans (e.g., DSM I7975), Desulfovibrio inopinatus (e.g., DSM I07II), Desulfonatronum thiodismutans (eg MLF-1 strain), Opitutaceae TAV5 bacterium, Tuberibacillus calidus (eg DSM I7572), Bacillus thermoamylovorans (eg B4I66 strain), Brevibacillus sp. CFII2, Bacillus sp. NSP2.I, Desulfatirhabdium butyrativorans (eg DSM I8734), Alicyclobacillus herbarius (eg DSM I3609), Citrobacter freundii (eg ATCC 8090), Brevibacillus agri (eg BAB-2500) and Methylobacterium nodulans (eg ORS 2060). The enzyme may be a homologue or orthologue of C2c1 or C2c3. In some embodiments, the C2c1 or C2c3 proteins are codon-optimized for expression in a eukaryotic cell. In some embodiments, C2c1 or C2c3 directs cleavage of one or two strands in the region of the target sequence. In a preferred embodiment, the chain break is a staggered cut with 5' overhanging nucleotides. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, the direct repeat has a minimum length of 16 nucleotides and contains a single hairpin. In further embodiments, the direct repeat is longer than 16 nucleotides, preferably greater than 17 nucleotides, and contains more than one hairpin or has an optimized secondary structure.

[00431] In one aspect, the invention relates to a eukaryotic host cell comprising a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more targeting sequences downstream of the DR sequence, wherein when expressed, the targeting sequence directs sequence-specific binding of a CRISPR-Cas C2c1 complex or a CRISPR-Cas C2c3 complex to a target sequence in a eukaryotic cell, wherein the CRISPR-Cas C2c1 or CRISPR-Cas C2c3 complex contains C2c1 or C2c3 in complex with (1) a targeting sequence that is hybridized to a target sequence, and (2) a DR sequence; and (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said C2c1 or C2c3 enzyme, comprising a nuclear localization sequence. In some embodiments, the host cell contains components (a) and (b); including split C2c1 or C2c3. In some embodiments, component (a) or (b), or components (a) and (b), are stably integrated into the genome of the eukaryotic host cell. In some embodiments, component (a) further comprises two or more targeting sequences operably linked to the first regulatory element, wherein, when expressed, the two or more targeting sequences direct sequence-specific binding of C2c1 or C2c3 to different target sequences in a eukaryotic cell. In some embodiments, the C2c1 or C2c3 proteins are codon-optimized for expression in a eukaryotic cell. In some embodiments, C2c1 or C2c3 directs cleavage of one or two strands in the region of the target sequence. In a preferred embodiment, the chain break is a staggered cut with 5' overhanging nucleotides. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, the direct repeat has a minimum length of 16 nucleotides and contains a single hairpin. In further embodiments, the direct repeat is longer than 16 nucleotides, preferably greater than 17 nucleotides, and contains more than one hairpin or has an optimized secondary structure. In one aspect, the invention relates to a non-human eukaryotic organism, preferably a multicellular eukaryotic organism, comprising a eukaryotic host cell according to the described embodiments. In other aspects, the invention relates to a eukaryotic organism, preferably a multicellular eukaryotic organism, including a eukaryotic host cell, according to the described embodiments. In some embodiments of these aspects, the organism may be an animal, such as a mammal. The organism may also be an arthropod, in particular an insect. An organism can also be a plant. In addition, the organism may be a fungus.

[00432] In one aspect, the invention relates to a kit containing one or more of the components described in the present description. In some embodiments, the implementation of the kit includes a system of vectors and instructions for use of the kit. In some embodiments, the system includes a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more targeting sequences downstream of the DR sequence, wherein the targeting sequence directs sequence-specific binding of the CRISPR-Cas C2c1 complex when expressed. or a CRISPR-Cas C2c3 complex with a target sequence in a eukaryotic cell, wherein the CRISPR-Cas C2c1 or CRISPR-Cas C2c3 complex contains C2c1 or C2c3 complexed with (1) a guide sequence that is hybridized to the target sequence, and (2) a sequence DR; and (b) a second regulatory element operably linked to an enzyme coding sequence encoding said C2c1 or C2c3 enzyme containing a nuclear localization sequence; predominantly including split C2c1 or C2c3. In some embodiments, the kit contains components (a) and (b) located on one or more vectors of the system. In some embodiments, component (a) further comprises two or more targeting sequences operably linked to the first regulatory element, wherein, when expressed, the two or more targeting sequences direct sequence-specific binding of C2c1 or C2c3 to different target sequences in a eukaryotic cell. In some embodiments, C2c1 or C2c3 contains one or more nuclear localization sequences of sufficient strength to induce accumulation of said C2c1 or C2c3 in detectable amounts in the nucleus of a eukaryotic cell. In some embodiments, the C2c1 enzyme is a C2c1 bacterial species selected from the group consisting of: Alicyclobacillus acidoterrestris (e.g. ATCC 49025), Alicyclobacillus contaminans (e.g. DSM I7975), Desulfovibrio inopinatus (e.g. DSM I07II), Desulfonatronum thiodismutans (e.g. , strain MLF-1), bacterium Opitutaceae TAV5, Tuberibacillus calidus (eg DSM I7572), Bacillus thermoamylovorans (eg strain B4I66), Brevibacillus sp. CFII2, Bacillus sp. NSP2.I, Desulfatirhabdium butyrativorans (eg DSM I8734), Alicyclobacillus herbarius (eg DSM I3609), Citrobacter freundii (eg ATCC 8090), Brevibacillus agri (eg BAB-2500) and Methylobacterium nodulans (eg ORS 2060). The enzyme may be a homologue or orthologue of C2c1 or C2c3. In some embodiments, the C2c1 or C2c3 proteins are codon-optimized for expression in a eukaryotic cell. In some embodiments, C2c1 or C2c3 directs cleavage of one or two strands in the region of the target sequence. In a preferred embodiment, the chain break is a staggered cut with 5' overhanging nucleotides. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, the direct repeat has a minimum length of 16 nucleotides and contains a single hairpin. In further embodiments, the direct repeat is longer than 16 nucleotides, preferably greater than 17 nucleotides, and contains more than one hairpin or has an optimized secondary structure.

[00433] In one possible aspect, the invention relates to modifications of a target polynucleotide in a eukaryotic cell. In some embodiments, the method includes allowing a C2c1 or C2c3 CRISPR-Cas complex to bind to a target polynucleotide to cleave said target polynucleotide, thereby modifying the target polynucleotide, wherein the C2c1 or C2c3 CRISPR-Cas complex contains C2c1 or C2c3 in the complex with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said target polynucleotide is linked to a direct repeat sequence. In some embodiments, said cleavage includes cleaving one or two strands in a region of said target sequence with said C2c1 or C2c3; including split C2c1 or split C2c3 according to the present invention. In some embodiments, said cleavage results in a decrease in the level of transcription of the target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, said repair resulting in a mutation involving the insertion, deletion, or substitution of one or more nucleotides in said target polynucleotide. In some embodiments, said mutation results in one or more amino acid substitutions in a protein resulting from expression of a gene containing the target sequence. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cell, wherein the one or more vectors induce the expression of one or more of C2c1 or C2c3 and a targeting sequence associated with a DR sequence. In some embodiments, said vectors are delivered to a eukaryotic cell within an individual. In some embodiments, said modification occurs in said eukaryotic cell in cell culture. In some embodiments, the method further comprises isolating said eukaryotic cell from an individual prior to said modification. In some embodiments, the method further comprises reintroducing said eukaryotic cell and/or cells subsequently derived from said eukaryotic cell to said subject.

[00434] In one aspect, the invention relates to a method for modifying the expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method includes allowing a C2c1 or C2c3 CRISPR-Cas complex to bind to a polynucleotide such that said binding results in an increase or decrease in the expression of said polynucleotide, wherein the C2c1 or C2c3 CRISPR-Cas complex contains C2c1 or C2c3 in complex with a targeting sequence capable of hybridize to a target sequence within said target polynucleotide, wherein said target polynucleotide is linked to a direct repeat sequence; this includes the split C2c1 or split C2c3 of the present invention. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cell, the one or more vectors inducing the expression of one or more of C2c1 or C2c3 and a guide sequence fused to a DR sequence.

[00435] In one aspect, the invention relates to a method for obtaining a model eukaryotic cell containing a mutant gene associated with a disease. In some embodiments, such a disease-associated gene may be associated with a risk of occurrence or development of a disease. In some embodiments, the method comprises (a) introducing one or more vectors into a eukaryotic cell, wherein the one or more vectors drive the expression of one or more of C2c1 or C2c3 and a targeting sequence fused to a direct repeat sequence; and (b) allowing the C2c1 or C2c3 CRISPR-Cas complex to bind to the target polynucleotide, causing cleavages of the target polynucleotide within said disease-associated gene, wherein the C2c1 or C2c3 CRISPR complex includes C2c1 or C2c3 in complex with (1) a guide sequence , hybridized to a target sequence within said target polynucleotide, and (2) a DR sequence, thus resulting in a model eukaryotic cell containing a disease-associated mutant gene, this includes a separated C2c1 or C2c3 of the present invention. In some embodiments, C2c1 or C2c3 provides one or two strand cleavage in the target sequence region. In a preferred embodiment, the chain break is a staggered cut with 5' overhanging nucleotides. In some embodiments, said cleavage results in decreased transcription of the target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, said repair resulting in a mutation involving the insertion, deletion, or substitution of one or more nucleotides in said target polynucleotide. In some embodiments, said mutation results in one or more amino acid substitutions in the resulting protein upon expression of the gene containing the target sequence.

[00436] In one possible aspect, the invention relates to a method for developing a biologically active agent that modulates signaling events in a cell associated with a gene associated with a disease. In some embodiments, such a disease-associated gene may be associated with a risk of occurrence or development of a disease. In some embodiments, the method includes a) bringing the test compound into contact with a model cell according to any of the described embodiments; and b) determining a data change indicative of a decrease or increase in cell signaling events associated with said mutation in said disease-associated gene, thereby developing a biologically active agent that modulates gene-associated cell signaling events, associated with the disease.

[00437] In one possible aspect, the invention provides a recombinant polynucleotide comprising a targeting sequence downstream of a direct repeat sequence, wherein, when expressed, the targeting sequence directs sequence-specific binding of a CRISPR-Cas C2c1 complex or a CRISPR-Cas C2c3 complex to an appropriate eukaryotic target sequence. cell. In some embodiments, the target sequence is a proto-oncogene or an oncogene.

[00438] In one possible aspect, the invention relates to a method for selecting one or more cell(s), the method comprising: introducing one or more vectors into the cell(s), wherein the one or more vectors induce the expression of one or more of C2c1 or C2c3 , the guide sequence associated with the direct repeat sequence, and the editing matrix; moreover, the editing matrix contains one or more mutations that prevent cleavage by the C2c1 or C2c3 protein, which allows homologous recombination of the editing matrix with the target polynucleotide in the cell to be selected; this allows the CRISPR-Cas C2c1 or C2c3 complex to bind to the target polynucleotide so as to cleave said target polynucleotide, wherein the CRISPR-Cas C2c1 or C2c3 complex contains C2c1 or C2c3 in complex with (1) a targeting sequence hybridized to a a target within said target polynucleotide, and (2) a direct repeat sequence, wherein binding of the CRISPR-Cas C2c1 or C2c3 complex to the target polynucleotide induces cell death, thereby allowing selection of one or more cells in which the mutation is introduced; this includes the split C2c1 or split C2c3 of the present invention. In another preferred embodiment, the cell being selected is a eukaryotic cell. Aspects of the invention allow the selection of specific cells without the use of a selectable marker or a two-step process that may include a counter-selection system.

[00439] The phrase "this includes a split C2c1 or split C2c3 according to the present invention" or similar text occurs in this application to indicate that C2c1 in embodiments of the present invention may be a split C2c1 and C2c3 may be a split C2c3 as described in the present description.

[00440] In one possible aspect, the invention relates to a non-naturally occurring or engineered CRISPR-Cas C2c1 system comprising a first C2c1 fusion coupled to the first half of the inducible heterodimer and a second C2c1 fusion coupled to the second half of the inducible heterodimer , wherein the first C2c1 fusion construct is operably linked to one or more nuclear localization signals, wherein the second C2c1 fusion construct is operably linked to one or more nuclear export signals, wherein upon interaction with the induction source, the first and second halves of the inducible heterodimer combine, wherein the union of the first and second half of the inducible heterodimer allows the first and second C2c1 fusion constructs to constitute a functional CRISPR-Cas C2c1 system, wherein the CRISPR-Cas C2c1 system includes a guide RNA (gRNA) containing a guide sequence capable of hybridizing with the sequence target in the genomic locus of interest in the cell, and moreover, the CRISPR-Cas C2c1 functional system edits the genomic locus by changing gene expression. In one possible aspect of the invention, in an inducible C2c1 CRISPR-Cas1 system, the first half of the inducible heterodimer is FKBP12 and the second half of the inducible heterodimer is FRB. In another embodiment, the induction energy source is rapamycin.

[00441] In one possible aspect, the invention relates to a non-naturally occurring or engineered CRISPR-Cas C2c3 system comprising a first C2c3 fusion construct associated with the first half of the inducible heterodimer and a second C2c3 fusion construct associated with the second half of the inducible heterodimer , wherein the first C2c3 fusion construct is operably linked to one or more nuclear localization signals, wherein the second C2c3 fusion construct is operably linked to one or more nuclear export signals, wherein upon interaction with the induction source, the first and second halves of the inducible heterodimer combine, wherein the union of the first and second half of the inducible heterodimer allows the first and second C2c3 fusion constructs to constitute a functional CRISPR-Cas C2c3 system, wherein the CRISPR-Cas C2c3 system includes a guide RNA (gRNA) containing a guide sequence capable of hybridizing with the sequence target in the genomic locus of interest in the cell, and moreover, the CRISPR-Cas C2c3 functional system edits the genomic locus by changing gene expression. In one possible aspect of the invention, in an inducible CRISPR-Cas C2c3 system, the first half of the inducible heterodimer is FKBP12 and the second half of the inducible heterodimer is FRB. In another embodiment, the induction energy source is rapamycin.

[00442] The source of induction energy can be considered a simple inductor or dimerizing agent. The term "induction energy source" is used throughout this specification for consistency. The induction energy source (or inductor) restores C2c1 or C2c3. In some embodiments, the induction energy source combines two parts of C2c1 or C2c3 through the action of two halves of an inducible dimer. Therefore, the two halves of the inducible dimer combine in the presence of an induction energy source. The two halves of a dimer do not dimerize (do not form a dimer) without an induction energy source.

[00443] Thus, the two halves of the inducible dimer cooperate with an induction energy source to dimerize the dimer. This in turn reconstructs C2c1 or C2c3 by concatenating the first and second parts of C2c1 or C2c3.

[00444] Each CRISPR enzyme fusion construct includes a portion of a split C2c1 or split C2c3. They are fused, preferably via a linker such as Gly-Ser described herein, to one of the two halves of the dimer. The two halves of a dimer may be substantially the same monomers which together form a homodimer, or may be different monomers which together form a heterodimer. In this case, two monomers can be considered half of a complete dimer.

[00445] C2c1 or C2c3 are separated in the sense that the two parts of the C2c1 or C2c3 enzyme essentially constitute a functional C2c1 or C2c3. This C2c1 or C2c3 may function as a genome-modifying enzyme (by forming a complex with the target DNA and guide nucleic acid), such as a nickase or nuclease (cutting both strands of DNA), or it may be a dead C2c1 or dead C2c3, which is essentially is a DNA-binding protein with very little or no catalytic activity, usually due to mutation(s) in the catalytic domain.

[00446] The two parts of a split C2c1 or split C2c3 can be considered an N-terminal part and a C-terminal part of a split C2c1 or split C2c3. The merger usually occurs at the split point C2c1 or C2c3. In other words, both the C-terminus of the N-terminal portion of the split C2c1 or split C2c3 is fused to one of the halves of the dimer, while the N-terminus of the C-terminal portion is fused to the other half of the dimer.

[00447] C2c1 or C2c3 should not be separated in the sense that the gap is created again. The split point is usually constructed in silico and cloned into constructs. Together, the two portions of the split C2c1 or split C2c3 construct, the N-terminal portion and the C-terminal portion, form a complete C2c1 or C2c3 comprising preferably at least 70% or more of the wild-type amino acids (or nucleotides encoding them), preferably at least 80% or more, preferably at least 90% or more, preferably at least 95% or more, preferably at least 99% or more of wild-type amino acids (or nucleotides encoding them). Shortenings are possible and mutants are contemplated. Non-functional domains can be completely removed. The important thing is that the two parts can be combined and that the desired function of C2c1 or C2c3 is restored.

[00448] A dimer may be a homodimer or a heterodimer.

[00449] One or more, preferably two, NLSs can be used as operably linked to the first C2c1 construct. One or more, preferably two, NES can be used as operably linked to the first C2c1 construct. The NLS and/or NES preferably flank the split C2c1 dimer (i.e., half of the dimer) fusion construct, i.e. The NLS may be located at the N-terminus of the first C2c1 construct, and the NLS may be located at the C-terminus of the first C2c1 construct. Similarly, NES may be located at the N-terminus of the second C2c1 construct, and NES may be located at the C-terminus of the second C2c1 construct. It will be appreciated that when an N' or C' terminus is mentioned, this corresponds to the 5' and 3' ends of the respective nucleotide sequence.

[00450] One or more, preferably two, NLSs can be used as operably linked to the first C2c3 construct. One or more, preferably two, NES can be used as operably linked to the first C2c3 construct. The NLS and/or NES preferably flank the split C2c3 dimer (i.e., half of the dimer) fusion construct, i.e. The NLS may be located at the N-terminus of the first C2c3 construct, and the NLS may be located at the C-terminus of the first C2c3 construct. Similarly, NES may be located at the N-terminus of the second C2c3 construct, and NES may be located at the C-terminus of the second C2c3 construct. It will be appreciated that when an N' or C'' terminus is mentioned, this corresponds to the 5' and 3' ends of the respective nucleotide sequence.

[00451] The preferred organization of the first C2c1 construct is: 5'-NLS-(N-terminus of C2c1)-linker-(first half of the dimer)-NLS-3' or the following organization of the C2c3 construct: 5'-NLS-(N- terminal part C2c3)-linker-(first half of dimer)-NLS-3'. The preferred organization of the second C2c1 construct is: 5'-NES-(C-terminus of C2c1)-linker-(second half of the dimer)-NES-3' or the organization of the second C2c3 construct is: 5'-NES-(C-terminus C2c3)-linker-(second half of dimer)-NES-3'.

[00452] In some embodiments, one or more NES operably associated with the second C2c1 construct may be replaced with NLS. However, this is generally not preferred, and in other embodiments, the localization signal operably associated with the second C2c1 is NES. In some embodiments, one or more NES operably associated with the second C2c3 construct may be replaced by NLS. However, this is generally not preferred, and in other embodiments, the localization signal operably associated with the second C2c3 is NES.

[00453] It would be desirable for NES to be operably linked to the N-terminal fragment of the split C2c1 or split C2c3 and for the NLS to be operably linked to the C-terminal fragment of the split C2c1 or split C2c3. However, there may be a preferred arrangement where the NLS is operably linked to the N-terminal fragment of the split C2c1 or split C2c3 and the NES is operably linked to the C-terminal fragment of the split C2c1 or split C2c3.

[00454] The NES allows the second C2c1 or C2c3 fusion to be localized outside the core at least as long as the induction energy source is present (eg, at least as long as the energy source is provided to the inductor to perform its function). The presence of the inducer stimulates the dimerization of the two C2c1 or C2c3 fusions in the cytoplasm and thermodynamically favors nuclear localization of the dimerized first and second C2c1 or C2c3 fusion proteins. Without wishing to be bound by theory, Applicants believe that NES restricts the second C2c1 or C2c3 fusion construct to the cytoplasm (ie, out of the nucleus). NLS on the first C2c1 or C2c3 fusion contributes to its nuclear localization. In both cases, applicants use NES or NLS to move the equilibrium (nuclear transport equilibrium) in the desired direction. Dimerization usually occurs outside the nucleus (a very small proportion can occur in the nucleus), and NLS on the dimerized complex shifts the nuclear transport equilibrium towards nuclear localization, so that the dimerized and thus reduced C2c1 or C2c3 protein enters the nucleus.

[00455] Applicants' ability to restore function of the split C2c1 or split C2c3 is advantageous. For proof of concept, transient transfection is used and in the presence of an induction energy source, dimerization occurs at a background level. Activity is not detected with separate fragments of C2c1 or C2c3. Then stable expression via lentiviral delivery is used to develop and demonstrate that a split C2c1 or C2c3 approach can be used.

[00456] This provided split C2c1 or split C2c3 approach is advantageous in that it provides an inducible pattern of C2c1 or C2c3 activity, thus providing control over time. Moreover, different localization sequences (ie preferred NES and NLS) can be used to reduce the level of background activity of self-assembling complexes. Tissue-specific promoters can also be used, eg one promoter for each of the C2c1 or C2c3 fusion constructs, for tissue-specific targeting, thus providing spatial control. Two different tissue-specific promoters can be used if finer regulation is required. The same approach can be used for developmental stage-specific promoters, or a combination of tissue-specific and developmental-specific promoters can be used. Moreover, one pair of C2c1 or C2c3 fusion constructs (first and second) is under the control (i.e., operably linked or includes) a tissue-specific promoter, while the other first and second C2c1 or C2c3 constructs are under control (i.e., functionally linked to or includes) a promoter specific to a particular developmental stage.

[00457] An inducible CRISPR-Cas C2c1 or C2c3 system includes one or more nuclear localization sequences (NLS) as described herein, for example, operably linked to a first C2c1 or C2c3 fusion construct. These nuclear localization sequences ideally have sufficient strength to induce the accumulation of said C2c1 or C2c3 fusion construct in the nucleus of a eukaryotic cell in detectable amounts. Without wishing to be bound by theory, it is believed that a nuclear localization sequence is not necessary for the activity of the CRISPR-Cas C2c1 or CRISPR-Cas C2c3 complex in eukaryotes, but also that the inclusion of such sequences enhances the activity of the system, especially by targeting nucleic acid molecules to the nucleus, and promotes functioning of the presented two-component system.

[00458] Similarly, the second C2c1 or C2c3 fusion construct is operably linked to the nuclear export sequence. Indeed, it may be associated with one or more NES. In other words, the number of NES used in the case of a second C2c1 or C2c3 fusion is preferably 1, 2, or 3. Generally, 2 NES are preferred, but in some embodiments, 1 is sufficient and thus preferred. Suitable examples of NLS and NES are known in this area. For example, the preferred nuclear export signal (NES) belongs to the human tyrosine kinase 2 protein. Preferred signals are species specific.

[00459] In a system that uses FRBs and FKBPs, it is preferred that the FKBPs be flanked by nuclear localization sequences (NLSs). In a system using FRB and FKBP, the preferred location is: N-terminal C2c1-FRB-NES:C-terminal C2c1-FKBP-NLS or N-terminal C2c3-FRB-NES:C-terminal C2c3-FKBP-NLS. Thus, the first C2c1 or C2c3 fusion construct contains a C2c1 or C2c3 C-terminal portion and the second C2c1 or C2c3 fusion construct contains a C2c1 or C2c3 N-terminal portion.

[00460] Another advantageous aspect of the present invention is that it can be started quickly, ie. has a fast response. Without wishing to be bound by theory, it is believed that C2c1 or C2c3 activity can be induced by dimerization of an existing (already present) fusion construct (by contact with an induction energy source) faster than by expression (especially translation) of a new fusion construct. As such, the first and second C2c1 or C2c3 fusion constructs can be expressed in the target cell in advance, i. before C2c1 or C2c3 activity is needed. C2c1 or C2c3 activity can be controlled over time and then triggered by the addition of an induction energy source that ideally acts faster (dimerizing the heterodimer and thus providing C2c1 or C2c3 activity) than by expression (including transcriptional induction) of C2c1 or C2c3 delivered , for example, using a vector.

[00461] The terms "C2c1" or "C2c1 enzyme" or "CRISPR enzyme" are used interchangeably herein, unless otherwise evident. The terms "C2c3" or "C2c3 enzyme" or "CRISPR enzyme" are used interchangeably herein unless otherwise evident.

[00462] Applicants demonstrate that C2c1 or C2c3 can be separated into two components that restore a functional nuclease when combined. Using rapamycin-responsive dimerization domains, Applicants create chemically inducible C2c1 or C2c3 to control the timing of C2c1-dependent genome editing or C2c3-dependent genome editing or transcriptional modulation. In other words, Applicants demonstrate that C2c1 or C2c3 can become chemically inducible when split into two fragments, and rapamycin-responsive dimerization domains can be used to control the assembly of C2c1 or C2c3. Applicants show that combined C2c1 or C2c3 can be used to mediate genome editing (via nuclease/nickase activity) as well as to modulate transcription (via the DNA binding domain - the so-called "dead C2c1" or "dead C2c3 ").

[00463] As such, the use of dimerization domains sensitive to rapamycin is preferred. Reassembly can be determined by restoring the binding activity. When C2c1 or C2c3 functions as a nickase or forms a double strand break, appropriate percentages are provided herein for comparison with wild type.

[00464] Treatment with rapamycin can last up to 12 days. The dose may be 200 nM. This temporal and/or molar dosage may exemplify a suitable dosage for the human embryonic kidney cell line 293FT (HEK293FT) and may be used for other cell lines. This number may be modified for in vivo therapeutic use, being expressed in, for example, mg/kg. However, it is also contemplated that a unit dose of rapamycin may also be used by an individual. By standard dosage is meant the normal therapeutic dose of rapamycin or the dose used in the main appointment (ie, the dose used when rapamycin is taken to prevent organ rejection).

[00465] It should be noted that the preferred organization of the C2c1-FRB/FKBP or C2c3-FRB/FKBP portions is such that the portions are separate and inactive until rapamycin-induced FRB and FKBP dimerization results in the assembly of a functional full-length C2c1 nuclease, or C2c3. Thus, it is preferred that the C2c1 or C2c3 fusion construct be linked to the first half of the inducible heterodimer.

[00466] In order to confine the C2c1(N)-FRB or C2c3(N)-FRB fragment to the cytoplasm where there is less chance of dimerization with a nuclear localized C2c1(C)-FKBP or C2c3(C)-FKBP fragment, it is preferable to use for C2c1(N)-FKBP )-FRB one NES from human tyrosine kinase 2 protein (C2c1(N)-FRB-NES) or for C2c3(N)-FRB one NES from human tyrosine kinase 2 protein (C2c3(N)-FRB-NES). In the presence of rapamycin, C2c1(N)-FRB-NES dimerizes with C2c1(C)-FKBP-2xNLS or C2c3(N)-FRB-NES dimerizes with C2c3(C)-FKBP-2xNLS, reducing the full-length C2c1 or C2c3 protein, which displaces balance of nuclear transport towards import into the nucleus and allows DNA targeting.

[00467] A high dosage of C2c1 or C2c3 can increase the insertion-deletion rate in non-target sequences that have slightly non-guide base bases. Such sequences are particularly sensitive if the mismatched bases are out of sequence and/or outside of the target nucleic acid seed sequence. Accordingly, temporal control of C2c1 or C2c3 activity may be used to reduce dosage in long-term expression experiments and therefore may provide a reduction in untargeted insertions/deletions compared to constitutively active C2c1 or C2c3.

[00468] Viral delivery is preferred. In particular, delivery is contemplated using a lentiviral vector or an adeno-associated virus vector. Applicants create a C2c1-cleaved or C2c3-cleaved lentiviral construct similar to the lentiCRISPR plasmid. These separated portions must be small enough to meet size limitations imposed by an adeno-associated vector of approximately 4.7 kb.

[00469] Applicants show that stable low-copy expression of split C2c1 or split C2c3 can be used to induce large insertions/deletions at the target locus without significant mutations at non-target sites. Applicants clone C2c1 fragments (2 parts based on split system 5 described herein) or C2c3 fragments.

[00470] A dead C2c1 or C2c3 including the VP64 transactivation domain, such as added to C2c1(C)-FKBP-2xNLS (killed-C2c1(C)-FKBP-2xNLS-VP64) or C2c3(C)-FKBP- 2xNLS (killed-C2c3(C)-FKBP-2xNLS-VP64). Transcriptional activation is induced by VP64 in the presence of rapamycin, inducing dimerization of the C2c1(C)-FKBP fusion and C2c1(N)-FRB fusion or the C2c3(C)-FKBP fusion and C2c3(N)-FRB fusion. In other words, Applicants test the feasibility of inducing cleaved dead C2c1-VP64 or cleaved dead C2c3-VP64 and demonstrate that transcriptional activation is induced by cleaved dead C2c1-VP64 or cleaved dead C2c3-VP64 in the presence of rapamycin. As such, the provided inducible C2c1 or C2c3 may be associated with one or more functional domains, such as a transcriptional activator or repressor, or a nuclease (such as FokI). The functional domain may be associated with or fused to one part of a split C2c1 or split C2c3.

[00471] The preferred organization is as follows: the first construct is organized as follows: 5'-1st localization signal-(N-terminal C2c1)-linker-(1st dimer part)-1st localization signal-3' or the first C2c3 construct is organized as follows: 5'-1st localization signal-(N-terminal part of C2c3)-linker-(1st part of dimer)-1st localization signal-3', and the second construct C2c1 is organized as follows : 5'-2nd localization signal-(2nd half of dimer)-linker-(C-terminus of C2c1)-2nd localization signal-functional domain-3' or second C2c3 construct organized as follows: 5'- 2nd localization signal-(2nd half of dimer)-linker-(C-terminal part of C2c3)-2nd localization signal-functional domain-3'. Here, the functional domain is located at the 3' end of the second C2c1 or C2c3 construct. Alternatively, a functional domain may be placed at the 5' end of the first C2c1 or C2c3 construct. One or more functional domains may be located at the 3' end or 5' end, or both ends. A suitable promoter is preferably located upstream of these sequences. Localization signals can be NLS or NES, but different signals cannot be on the same construct.

[00472] In one possible aspect, the invention provides an inducible CRISPR-Cas C2c1 or C2c3 system described herein, wherein C2c1 or C2c3 has at least 97% or 100% reduced nuclease activity compared to C2c1 or C2c3 that does not contain at least one mutation.

[00473] Accordingly, it is also preferred that C2c1 or C2c3 be dead C2c1 or dead C2c3. Ideally, the separation should be such that the catalytic domain(s) are not affected. In the case of dead C2c1 or dead C2c3, DNA binding is expected to occur, but no cleavage or nicase activity is present.

[00474] In one possible aspect, the invention relates to an inducible CRISPR-Cas C2c1 or C2c3 system described herein, in which one or more functional domains are associated with C2c1 or C2c3. This functional domain may be associated with (eg, associated or fused with) one or both parts of the split C2c1, or one part of the split C2c3 or both. One of them can be associated with each of the two parts of a split C2c1 or a split C2c3. Therefore, they can typically be provided as part of the first and/or second fusion construct C2c1 or C2c3, being fused to that construct. The functional domains are typically fused via a linker such as the GlySer linker described herein. One or more functional domains may be a transactivation domain or a transcriptional repressor domain. Although the functional domains may vary, it is preferred that all functional domains be either transactivation or repressor domains and not be used in the same mixture.

[00475] The transactivation domain may be: VP64, p65, MyoDl, HSF1, RTA, or SET7/9.

[00476] In one possible aspect, the invention provides an inducible C2c1 or C2c3 CRISPR-Cas system as described herein, wherein one or more functional domains associated with C2c1 or C2c3 are a transcriptional repressor domain.

[00477] In one possible aspect, the invention relates to the inducible CRISPR-Cas C2c1 or C2c3 system described herein, in which the repressor domain is the KRAB domain.

[00478] In one possible aspect, the invention provides an inducible CRISPR-Cas C2c1 or C2c3 system as described herein, wherein the transcriptional repressor domain is a NuE domain, an NcoR domain, a SID domain, or a SID4X domain.

[00479] In one possible aspect, the invention relates to an inducible CRISPR-Cas C2c1 or C2c3 system described herein, in which one or more functional domains are associated with an adapter protein having one or more activities, including methylase activity, demethylase activity, transactivation activity , transcriptional repressor activity, transcription factor release activity, histone modifying activity, RNA cleaving activity, DNA cleaving activity, DNA integrase activity, or nucleic acid binding activity.

[00480] Histone modifying domains are also preferred in some embodiments. Exemplary histone-modifying domains are discussed below. Transposase domains, homologous recombination machinery domains, recombinase domains and/or integrase domains are also preferred as functional domains within the scope of the present invention. In some embodiments, DNA integration activity comprises homologous recombination machinery domains, integrase domains, recombinase domains, and/or transposase domains.

[00481] In one possible aspect, the invention relates to the CRISPR-Cas C2c1 or C2c3 inducible system described herein, the DNA-cleaving activity of which is due to the presence of a nuclease.

[00482] In one possible aspect, the invention relates to the CRISPR-Cas C2c1 or C2c3 inducible system described herein, wherein the nuclease is a FokI nuclease.

[00483] The use of such functional domains that are preferred for the split C2c1 or split C2c3 system of the present invention is also discussed in detail in Konermaim et al. ("Genome-scale transcriptional activation with an engineered CRISPR-Cas9 complex" Nature, published December 11, 2014).

[00484] The system of the present invention can be used with any guide sequence.

[00485] In some embodiments, modified guide sequences may be used. Particularly preferred are guide sequences that are an implementation of the concept of the referenced article Konermann Nature 11 Dec 2014. These guide sequences are modified to include protein-binding portions of RNA (such as aptamers). Appropriate RNA-binding protein domains can be used to recognize RNA and recruit functional domains to the guide sequence, such as those described herein. This can be used primarily together in the case of dead C2c1 or dead C2c3, leading to transcriptional activation or repression or DNA cleavage by nucleases such as FokI. The use of such targeting sequences in combination with dead C2c1 or dead C2c3 is highly effective, especially when C2c1 or C2c3 is itself linked to its own functional domain, as described herein. When the reduction of dead C2c1 or dead C2c3 (with or without its own associated functional domain) is induced in accordance with the present invention, i.e. split C2c1 or split C2c3, this tool is especially useful.

[00486] A guide RNA (gRNA), which is preferably used in the context of the present invention, may contain a guide sequence capable of hybridizing to a target sequence at a genomic locus of interest in the cell, the gRNA being modified by inserting a separate RNA sequence(s) that binds to one or more adapter proteins, wherein the adapter protein is associated with one or more functional domains. C2c1 or C2c3 may contain at least one mutation such that the C2c1 or C2c3 enzyme has no more than 5% of the nuclease activity of the C2c1 or C2c3 enzyme without the mutation; and/or at least one or more NLSs. Also provided is a non-naturally occurring or engineered composition comprising: one or more guide RNAs (gRNAs) containing a guide sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell, a C2c1 or C2c3 enzyme containing at least one or more NLS, and the C2c1 or C2c3 enzyme may contain at least one mutation, such that the C2c1 or C2c3 enzyme has no more than 5% of the nuclease activity of the C2c1 or C2c3 enzyme that does not contain at least one mutation, while at least gRNA modified by inserting a separate RNA sequence(s) that binds to one or more adapter proteins, wherein the adapter protein is associated with one or more functional domains.

[00487] The gRNAs are preferably modified by inserting a separate RNA sequence(s) that binds to one or more adapter proteins. The insertion of a single RNA sequence(s) that binds to one or more adapter proteins is preferably aptamer sequences specific to one or more adapter proteins. The adapter protein can be: MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP50G, KU1, MU, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205 , ϕСb5, ϕСb8г, ϕСb12г, ϕСb2Зr, 7s and PRR1. Cell lines that stably express inter alia split dead C2c1 or split dead C2c3 may be useful.

[00488] Applicants show that a C2c1 or C2c3 protein can be split into two distinct fragments that constitute a functional full-length C2c1 or C2c3 nuclease when combined by chemical induction. A split C2c1 or split C2c3 architecture will be useful in a variety of applications. For example, split C2c1 may allow genetic strategies to restrict C2c1 activity to certain cell populations by placing each fragment under the control of a tissue-specific promoter, or split C2c3 may allow genetic strategies to restrict C2c1 activity to certain cell populations by placing each fragment under the control of a different tissue-specific promoter. In addition, various chemically inducible dimerization domains such as APA and gibberellin can be used.

[00489] The induction energy source is preferably chemical induction.

[00490] The position or site of separation is the point at which the first part of the C2c1 or C2c3 enzyme is separated from the second part. In some embodiments, the first part includes or encodes amino acids 1 to X, while the second part includes amino acids X+1 to the last. In this example, the numbering is continuous, but this may not always be necessary because the amino acids (or the nucleotides encoding them) can be cut off at each of the split ends so that sufficient DNA binding activity and optionally no DNA or cleavage activity is present. for example, at least 40%, 50%, 60%, 70%, 80%, 90%, or 95% activity compared to wild-type C2c1 or wild-type C2c3.

[00491] The exemplary numbering described herein may refer to a wild-type protein, preferably wild-type C2c1 or wild-type C2c3. However, it is contemplated that wild-type C2c1 mutants, such as the Bacillus C2c1 protein, or wild-type C2c3 mutants can be used. The numbering may not correspond exactly to C2c1 or C2c3 numbering, in particular some N- and C-terminal truncations or deletions may be used, but this can be accounted for using standard sequence alignment tools. Orthologues are also preferred sequence alignment tools.

[00492] Thus, the separation position can be selected using standard methods, for example, based on crystal structure data and/or structure prediction based on computer simulations.

[00493] Ideally, the separation position should be inside the loop. Preferably, the separation position is where interruption of the amino acid sequence does not lead to partial or complete destruction of the structural element (eg alpha helix or beta sheet). Often the best options are unstructured regions (areas that are not visible in crystal structures, because these regions are not structured enough to be "frozen" in the crystal). Applicants may, for example, make separations in unstructured areas that are exposed to the C2c1 or C2c3 surface.

[00494] Applicants may follow the following methodology, which is provided as a preferred example and guide. Since unstructured regions are not visible on crystal structures, applicants correlate the surrounding amino acid sequence of the crystal with the primary amino acid sequence of C2c1 or C2c3. Each unstructured region may consist of, for example, 3-10 amino acids that are not visible in the crystal structure. In such a case, applicants make a division in this region of the amino acid sequence. To include more potential split sites, Applicants include splits located on loops outside of C2c1 or C2c3 using the same criteria as for unstructured patches.

[00495] In some embodiments, the separation position is on the outer loop of C2c1 or C2c3. In other preferred embodiments, the separation position is in the C2c1 or C2c3 unstructured region. The unstructured region is typically a highly mobile region outside of the loop, the structure of which cannot be determined from the crystal lattice.

[00496] Once the separation position is identified, suitable compositions can be designed.

[00497] Typically, the NES is not located at the N-terminus of the first portion of the divided amino acid sequence (or at the 5'-terminal nucleotide encoding it). In such a case, the NLS is located at the C-terminus of the second part of the split amino acid sequence (or at the 3'-terminal nucleotide encoding it), thus the first C2c1 or C2c3 fusion construct can be operably linked to one or more NES, and the second fusion construct C2c1 or C2c3 may be operably linked to one or more NLSs.

[00498] Of course, the reverse order can be envisaged, in which the NLS is at the N-terminus of the first part of the separated amino acid sequence (or at the 5'-terminal nucleotide encoding it). In such a case, the NES is located at the C-terminus of the second part of the split amino acid sequence (or at the 3'-terminal nucleotide encoding it), thus the first C2c1 or C2c3 fusion construct can be operably linked to one or more NLSs, and the second fusion construct C2c1 or C2c3 may be operably linked to one or more NES.

[00499] Separated systems in which the two parts (each of the sides of the separated system) are approximately the same length may have packaging advantages. For example, it is believed that it is easier to maintain stoichiometry between two parts if the transcripts are approximately the same size.

[00500] In specific examples, the N- and C-terminal portions of a codon-optimized human C2c1, such as C2c1, are fused to the FRB and FKBP dimerization domains, respectively. Such an organization may be preferred. They can be interchanged (ie N-terminus fused to FKBP and C-terminus fused to FRB). In specific examples, the N- and C-terminal portions of a codon-optimized human C2c3, such as C2c3, are fused to the FRB and FKBP dimerization domains, respectively. Such an organization may be preferred. They can be interchanged (ie N-terminus fused to FKBP and C-terminus fused to FRB).

[00501] Use of linkers such as (GGGGS)₃, is preferred for fragment separation C2c1 or C2c3 fragment from the dimerization domain. Glycine residues are the most flexible, and serine residues increase the likelihood that the linker will be on the outer surface of the protein. Linkers (GGGGS) can be used as an alternative₆, (GGGGS)₉ or (GGGGS)₁₂. Other preferred alternatives are GGGGS)_one, (GGGGS)₂, (GGGGS)_four, (GGGGS)₅, (GGGGS)₇, (GGGGS)_eight, (GGGGS)_ten or (GGGGS)_eleven.

[00502] For example, (GGGGS) ₃ may be included between the N-terminus of a C2c1 fragment or a C2c3 fragment and the FRB. For example, (GGGGS) ₃ may be included between FKBP and the C-terminus of a C2c1 fragment or a C2c3 fragment.

[00503] Alternative linkers are also possible, but it is believed that highly mobile linkers function best, providing the maximum likelihood of association C2c1 or C2c3 and thus restore C2c1 or C2c3 activity. One alternative is to use nucleoplasmin as the NLS linker.

[00504] A linker can also be used to connect C2c1 or C2c3 and any functional domain. Again, a linker (GGGGS) can be used between C2c1 or C2c3 or a functional domain₃ (or 6-, 9- or 12-fold repeats) or NLS of nucleoplasmin.

[00505] Alternatives to the system are contemplated FRB/FKBP, e.g. ABA system and gibberellin.

[00506] Accordingly, preferred examples of the family FKBPs are any of the following inducible systems: FKBP which dimerizes with calcineurin A (CNA) in the presence of FK506 FKBP which dimerizes with CyP-Fas in the presence of FKCsA FKBP which dimerizes with FRB in the presence of rapamycin GyrB which dimerizes with GryB in the presence of cumermycin, GAI, which dimerizes with GID1 in the presence of gibberellin, Snap-tag, which dimerizes with HaloTag in the presence of HaXS.

[00507] Alternatives within the family FKBPs are also preferred. For example, FKBP that homodimerizes (ie one FKBP dimerizes with another FKBP) in the presence of FK1012. Thus, a non-naturally occurring or engineered C2c1 or CRISPR-Cas C2c3 system is also provided, comprising:

the first C2c1 fusion or the first C2c3 fusion associated with the first half of the inducible homodimer, and the second C2c1 fusion or the second C2c3 fusion associated with the second half of the inducible homodimer,

wherein the first C2c1 fusion construct or the first C2c3 fusion construct is operably linked to one or more NLSs, wherein the first C2c1 fusion construct or the first C2c3 fusion construct is operably linked to (optionally one or more) nuclear export signal(s),

moreover, the interaction with the induction energy source combines the first and second halves of the inducible homodimer,

wherein the combination of the first and second halves of the inducible homodimer allows the first and second C2c1 fusion constructs to restore the C2c1 CRISPR-Cas functional system or the first and second C2c3 fusion constructs to restore the C2c3 CRISPR-Cas functional system,

moreover, the C2c1 or CRISPR-Cas C2c3 system contains a guide RNA (gRNA) containing a guide sequence capable of hybridizing with a target sequence at the genomic target locus of the cell, and the C2c1 or CRISPR-Cas C2c3 functional system binds to the target sequence and optionally edits the genomic locus by changing gene expression.

[00508] In one embodiment, the homodimer is preferably FKBP, and the induction energy source is preferably FK1012. In another embodiment, the homodimer is preferably GryB and the induction energy source is preferably cumermycin. In another embodiment, the homodimer is preferably ABA and the induction energy source is preferably gibberellin.

[00509] In other embodiments, the dimer is a heterodimer. Preferred heterodimer variants are any of the following inducible systems: FKBP that dimerizes with calcineurin A (CNA) in the presence of FK506, FKBP that dimerizes with CyP-Fas in the presence of FKCsA, FKBP that dimerizes with FRB in the presence of rapamycin, GyrB that dimerizes with GryB in the presence of cumermycin, GAI that dimerizes with GID1 in the presence of gibberellin, Snap-tag, which dimerizes with HaloTag in the presence of HaXS.

[00510] Applicants used FKBP/FRB as this system is well characterized and both domains are small enough (less than 100 amino acids) to facilitate packaging. Moreover, rapamycin has been used for a long time and its side effects are well known. Large dimerization domains (greater than 300 a.a.) should also work, but in this case longer linkers may be required to allow recovery of C2c1 or C2c3.

[00511] Paulmurugan and Gambhir (Cancer Res, August 15, 2005 65; 7413) discuss the history of the FRB/FKBP/rapamycin system. Another helpful article is Crabtree et al. (Chemistry & Biology 13, 99-107, Jan 2006).

[00512] For example, one vector, one expression cassette (plasmid) is constructed. gRNA is under the control of the U6 promoter. Two different split C2c1 or split C2c3 are used. The split C2c1 or split C2c3 design is based on the first C2c1 or C2c3 fusion construct flanked by NLS in which FKBP is attached to the C-terminus of the split C2c1 or split C2c3 via a glycine-serine linker; and on a second NES-flanked C2c1 or C2c3 fusion construct in which the FRB is attached to the N-terminal portion of the split C2c1 or split C2c3 via a glycine-serine linker. Separation of the first and second C2c1 fusion constructs occurs during transcription by P2A. The rate of insertion-deletion formation in the case of split C2c1 or split C2c3 in the presence of rapamycin is approximately the same as in the case of wild type, but significantly lower than in the case of wild type in the absence of rapamycin.

[00513] Accordingly, a single vector is provided. The vector contains:

a first C2c1 fusion or first C2c3 fusion coupled to the first half of the inducible dimer and a second C2c1 fusion or second C2c3 fusion coupled to the second half of the inducible dimer,

wherein the first C2c1 fusion construct or the first C2c3 fusion construct is operably linked to one or more nuclear localization signals, wherein the first C2c1 fusion construct or the first C2c3 fusion construct is operably linked to one or more nuclear export signals,

moreover, the interaction with the induction energy source combines the first and second halves of the inducible homodimer,

wherein the combination of the first and second halves of the inducible homodimer allows the first and second C2c1 fusion constructs to restore the C2c1 CRISPR-Cas functional system or the first and second C2c3 fusion constructs to restore the C2c3 CRISPR-Cas functional system,

moreover, the C2c1 or CRISPR-Cas C2c3 system contains a guide RNA (gRNA) containing a guide sequence capable of hybridizing with a target sequence at the genomic target locus of the cell, and the C2c1 or CRISPR-Cas C2c3 functional system binds to the target sequence and optionally edits the genomic locus by changing gene expression.

These elements are preferably provided on a single construct, for example as part of an expression cassette.

[00514] The first C2c1 fusion or the first C2c3 fusion is preferably flanked by at least one nuclear localization signal at each end. The second C2c1 fusion or the second C2c3 fusion is preferably flanked by at least one nuclear export signal at each end.

[00515] Also provided is a method of treating an individual in need thereof, comprising inducing gene editing by transforming the individual with a polynucleotide encoding the system, or any of the vectors described herein, and administering to the individual an induction energy source. In one possible aspect of the application of said method, a repair matrix may also be provided, for example delivered by a vector containing said repair matrix.

[00516] Also provided is a method of treating an individual in need thereof, comprising inducing gene editing by transforming the individual with a polynucleotide encoding the system or any of the vectors described herein, said polynucleotide encoding or comprising a catalytically inactive C2c1 or C2c3 and one or more associated functional domains; the method further comprises administering to the individual an induction energy source.

[00517] Also provided are compositions containing the system of the present invention for use in said treatment method. Also provided is a method for using the system of the present invention in the manufacture of medicaments for such treatments.

[00518] Examples of syndromes that can be treated using the system of the present invention are described in the present description or in the documents cited in the present description.

[00519] A single vector may include a transcript separating agent, such as P2A. P2A bisects the transcript, separating the first and second C2c1 fusion constructs or the first and second C2c3 fusion constructs. Separation occurs due to the passage of the ribosome. Essentially, the ribosome skips an amino acid during translation, which causes the amino acid chain to terminate and results in two different polypeptides/proteins. The use of a single vector is also useful when low background activity is not necessary but high induction activity is desired.

[00520] One example is the generation of clonal embryonic stem cell lines. The normal procedure is transient transfection with plasmids encoding wild type C2c1 or C2c1 nickase or wild type C2c3 or C2c3 nickase. These plasmids produce C2c1 or C2c3 molecules that remain active for several days and have a higher chance of off-target activity. The use of a single C2c1-cleaved or C2c3-cleaved expression vector allows the "high" level of C2c1 or C2c3 activity to be limited to a shorter period of time (eg, a single dose of an inducer such as rapamycin). Without continuous (daily) administration of an inducer (eg, rapamycin), the activity of a single C2c1-cleaved or C2c3-cleaved expression vector is low, thus reducing the likelihood of unwanted off-target effects.

[00521] Peak induced C2c1 or C2c3 activity is advantageous in some embodiments and can be easily achieved using a single delivery vector, but this is also possible using a dual vector system (each vector delivers one half of split C2c1 or split C2c3). The peak may be high activity and a short period of time, usually coinciding with the half-life of the inductor.

[00522] Accordingly, a method is provided for obtaining clonal stem cell lines, comprising transfecting one or more embryonic stem cells with a polynucleotide encoding a system of the present invention, or one of the vectors of the present invention for expressing a split C2c1 or split C2c3, and introducing or interacting with one or more stem cells with an induction energy source of the present invention to induce C2c1 or C2c3 reduction. A reparation matrix may be provided.

[00523] As with all methods described herein, it is desirable that suitable gRNA or guide molecules be required.

[00524] In the case where the functional domains and the like "associated" with one and the other part of the enzyme, they are usually fused constructs. The term "associated with" is used herein to refer to how one molecule associates with another, such as between C2c1 moieties and a functional domain, or between C2c3 moieties and a functional domain. In the case of protein-protein interactions, this association can be considered in terms of recognition, similar to how an antibody recognizes an epitope. Alternatively, one protein can be associated with another protein by fusion of both proteins, for example where one subunit is fused to the other subunit. Fusion is usually accomplished by adding the amino acid sequence of one subunit to the amino acid sequence of the other, for example by splicing both nucleotide sequences encoding each protein or subunit. Alternatively, it can essentially be viewed as a binding of two molecules, or a direct connection, such as a fusion protein; in either case, the fusion protein may include a linker between the two subunits of interest (ie, between the enzyme and the functional domain, or between the adapter protein and the functional domain). Thus, in some embodiments, the C2c1 or C2c3 portions are linked to functional domains as they are fused to each other, optionally via an intermediate linker. Examples of linkers include the mentioned GlySer linkers.

[00525] Other examples of inductors include light and hormones. In the case of light, the inducible dimers may be heterodimers and comprise a first light-inducible half of the dimer and a second (or complementary) light-inducible half of the dimer. A preferred example of the first and second light-inducible halves of the dimer is the CIBI and CRY2 system. The CIBI domain is a heterodimeric protein that binds to photosensitive cryptochrome 2 (CRY2).

[00526] In another example, the blue light-responsive magnetic dimerization system (pMag and nMag) can be fused to two parts of a split C2c1 protein or a split C2c3 protein. In response to light stimulation, pMag and nMag dimerize and assemble C2c1 or C2c3. For example, such a system is described for Cas9 in Nihongaki et al. (Nat. Bjofechnol. 33, 755-790, 2015).

[00527] The invention contemplates that the source of induction energy may be thermal energy, ultrasound, electromagnetic radiation energy, or chemical energy. In a preferred embodiment, the induction energy source may be abscisic acid (ABA), doxycycline (DOX), cumate, rapamycin, 4-hydroxy tamoxifen (4-OHT), estrogen or ecdysone. The invention provides at least one switch, which may be selected from the group consisting of antibiotic-induced systems, electromagnetic radiation-induced systems, nuclear receptor-based inducible systems, and hormone-based inducible systems. In a more preferred embodiment, at least one switch may be selected from the group consisting of tetracycline/doxycycline (Tet/DOX) inducible system, light inducible system, ABA inducible system, cumate repressor/operator inducible system, 4-OHT/extrogen inducible system , an ecdysone-based inducible system, and an FKBP12/FRAP (FKBP12-rapamycin complex) inducible system. Such inductors are also discussed herein and in PCT/US20I3/051418, incorporated herein by reference.

[00528] In general, C2c1 or C2c3, either wild-type or nickase, or dead C2c1/C2c3 (bound or not bound to functional domains) can be used using the split C2c1 or split C2c3 approach of the present invention.

[00529] As a further example, split C2el or C2c3 fusion constructs with a fluorescent protein such as GFP can be created. This could allow imaging of genomic loci (see "Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System" Chen B et al. Cell 2013), but in an induced manner. As such, in some embodiments, one or more C2cl or C2c3 moieties may be linked (eg, fused) to a fluorescent protein, such as GFP.

[00530] The following experiments are designed to see if there is a difference in off-target cleavage between wild-type C2cl and separated C2c1 or wild-type C2c3 and separated C2c3. To do this, Applicants use transient transfection of plasmids with C2c1 wt and C2c1 split or with C2c3 wt or C2c3 split and collect at different time points. Applicants search for off-target activation after finding a set of samples in which the target cut is within 5%. Applicants create cell lines with stable expression of the wild-type or separated enzyme (C2c1 or C2c3) without guide nucleic acids (using a lentivirus). Following antibiotic selection, guide nucleic acids are delivered with a separate lentivirus, and cells are harvested at various time points to determine target and non-target cuts.

[00531] Applicants introduce a destabilizing sequence (PEST, see "Use of mRNA- and protein-destabilizing elements to develop a highly responsive reporter system" Voon DC et al. Nucleic Acids Research 2005) into the FRB(N)C2c1-NES fragment or FRB(N)C2c3-NES fragment, which contributes to faster degradation and subsequent decrease in the stability of the separated dead C2c1-VP64 complex or the separated dead C2c3-VP64 complex.

[00532] Such destabilizing sequences described elsewhere in this application (including PEST) may be advantageous for use in split C2c1 or split C2c3 systems.

[00533] Cell lines stably expressing split dead C2c1-VP64 and MS2-p65-HSFl+guide sequences have been developed. Screening for PLZ resistance can demonstrate that irreversible, timely transcriptional activation can be effective for screening small molecular weight substances. This approach may be advantageous when split dead C2c1-VP64 is irreversible.

[00534] Cell lines stably expressing split dead C2c3-VP64 and MS2-p65-HSFl+guide sequences have been developed. Screening for PLZ resistance can demonstrate that irreversible, timely transcriptional activation can be effective for screening small molecular weight substances. This approach may be advantageous when split dead C2c3-VP64 is irreversible.

[00535] In one aspect, the invention relates to a non-naturally occurring or engineered CRISPR-Cas C2c1 or C2c3 system that may include at least one switch, wherein the activity of said CRISPR-Cas C2c1 or C2c3 system is controlled by contacting at least with one induction energy source as a switch. In one embodiment of the invention, the regulation of at least one switch or the activity of said C2c1 or C2c3 CRISPR-Cas system can be up-regulated, up-regulated, stopped or repressed. The first effect may be one or more of the events of import into the nucleus, export from the nucleus, attraction of a secondary component (such as an effector molecule), conformational change (of a protein, DNA or RNA), cleavage, release of a cargo (such as an attached molecule or cofactor), association or dissociation. The second effect may be one or more events of activation, enhancement, termination or repression of regulation of at least one switch or activity of said C2c1 or C2c3 CRISPR-Cas system.

[00536] In another aspect of the invention, the CRISPR-Cas C2c1 or C2c3 system may further include at least one nuclear localization signal (NLS), nuclear export signal (NES), functional domain, movable linker, mutation, deletion, alteration, or truncation. One or more NLS, NES or functional domains can be activated or inactivated under certain conditions. In another embodiment, the mutation may be one or more mutations in the region of transcription factor homology, mutations in the DNA binding domain (such as a mutation in core residues of the basic helix-loop-helix motif), mutations in endogenous NLS, or mutations in endogenous NES. The invention provides that the induction energy source may be thermal energy, ultrasound, electromagnetic radiation energy or chemical energy. In a preferred embodiment, the induction energy source may be an antibiotic, small molecule, hormone, hormone derivative, steroid or steroid derivative, or in a more preferred embodiment, the induction energy source may be abscisic acid (ABA), doxycycline (DOX), cumate , rapamycin, 4-hydroxy tamoxifen (4-OHT), estrogen, or ecdysone. The invention provides that at least one switch may be selected from the group consisting of inducible systems based on antibiotics, inducible systems based on electromagnetic radiation, inducible systems based on small molecular weight compounds, inducible systems based on nuclear receptors, inducible systems based on hormones. In more preferred embodiments, at least one switch may be selected from the group consisting of tetracycline/doxycycline (Tet/DOX) inducible system, light induced system, ABA induced system, cumate repressor/operator inducible system, 4-OHT/extrogen inducible system, the ecdysone-based inducible system, and the FKBP12/FRAP (FKBP12-rapamycin complex) inducible system.

[00537] Aspects of regulation detailed in this application relate to at least one or more switch(s). As used herein, the term "switch" refers to a system or set of components that act in concert to cause a change that encompasses all aspects of a biological function, such as activation, repression, enhancement, or termination of that function. In one aspect, the term "switch" encompasses genetic switches that include the basic components of proteins involved in gene regulation and the specific DNA sequences recognized by those proteins. In one aspect, the switches are associated with inducible and repressor systems used in gene regulation. In general, an inducible system can be turned off until some molecule (called an inducer) is present that allows gene expression. This molecule is referred to as "expression inducing". The way in which this phenomenon is mediated depends on both regulatory mechanisms and differences in cell types. The repressor system is turned on except when some molecule (called a corepressor) is present that suppresses gene expression. This molecule is called "repressive expression". How this happens depends on regulatory mechanisms as well as differences in cell types. As used herein, the term "inducible" can encompass all aspects of a switch, regardless of the molecular mechanism involved. Accordingly, the switch provided by the invention may include, but is not limited to, antibiotic-based inducible systems, electromagnetic radiation-based inducible systems, small molecule-based inducible systems, nuclear receptor-based inducible systems, hormone-based inducible systems. In more preferred embodiments, the switch may be selected from the group consisting of tetracycline/doxycycline (Tet/DOX) inducible system, light inducible system, ABA inducible system, cumate inducible repressor/operator system, 4-OHT/extrogen inducible system, inducible system based on ecdysone and induced by FKBP12/FRAP (FKBP12-rapamycin complex) system.

[00538] The CRISPR-Cas C2c1 or C2c3 system of the present invention can be designed to modulate or change the expression of individual endogenous genes at precisely defined time and space intervals. The CRISPR-Cas C2c1 or C2c3 system can be designed to bind to promoter sequences of a target gene and alter gene expression. C2c1 or C2c3 can be split in two, with one half fused to one half of the cryptochrome-2 heterodimer (CIB1), while the remaining cryptochrome partner is fused to the other half of C2c1 or C2c3. In some aspects, a transcriptional repressor domain may also be included in the CRISPR-Cas C2c1 or C2c3 system. Effector domains can be either activators such as VP16, VP64 or p65 or repressors such as KRAB, EnR and SID. In the absence of stimulation, one half of the C2c1-cryptochrome-2 protein or C2c3-cryptochrome-2 protein is located in the promoter region of the target gene, but is not associated with the CIB1 effector protein. Upon stimulation with light in the blue region of the spectrum, cryptochrome-2 is activated and undergoes a conformational change, and its binding domain becomes available. CIB1, in turn, binds to cryptochrome-2, which leads to the localization of the second half of C2c1 or the second half of C2c3 in the promoter region of the target gene and initiation of genome editing, which can lead to gene overexpression or silencing. Aspects of LITE are further described in Liu, H et al., Science, 2008 and Kennedy M et al. Nature Methods 2010, the contents of which are incorporated herein by reference in their entirety.

[00539] Activator and repressor domains that can further modulate functions can be selected based on type, strength, mechanism, duration, size, or any number of other parameters. Preferred effector domains include, but are not limited to, transposase domain, integrase domain, recombinase domain, resolvase domain, invertase domain, protease domain, DNA methyltransferase domain, DNA demethylase domain, histone acetylase domain, histone deacetylase domain, nuclease domain, repressor domain , an activation domain, a domain containing a nuclear localization signal, a domain of proteins recruiting transcriptional proteins, a domain associated with the uptake of substances by a cell, a nucleic acid-binding domain, an antigen-presenting domain.

[00540] There are also several ways to obtain chemically inducible systems:

1. ABI-PYL based system induced by abscisic acid (ABA) (see e.g. website stke.sciencemag.org/cgi/content/abstract/sigtrans;4/1.64/rs2),

2. FKBP-FRB based system induced by rapamycin (or rapamycin analogs) (see e.g. nature.com/nmeth/joumal/v2/n6/full/nmeth763.html),

3. Gibberellin (GA) inducible GID1-GAI based system (see, for example, nature.com/nchembio/journal/v8/n5/full/nchembio.922.html).

[00541] Another system provided by the present invention is a chemically induced system based on changes in intracellular localization. Applicants also provide a CR1SPR-Cas C2c1 or C2c3 system designed to target a genomic target locus, wherein the CR1SPR-Cas C2c1 or C2c3 enzyme is split into two fusion constructs that are further linked to different portions of a chemically or energetically sensitive protein. A chemically or energetically sensitive protein induces changes in the intracellular localization of either half of the C2c1 or C2c3 enzyme (i.e. transport of either half of the C2c1 or C2c3 enzyme from the cytoplasm to the cell nucleus) upon binding a chemical compound or transferring energy to a chemically or energetically sensitive protein. This transport of fusion constructs from one intracellular compartment or organelle, in which activity is sequestered due to the lack of a substrate for the restored C2c1 or C2c3 CRISPR-Cas system, to another, in which the substrate is present, allows the components to combine and restore its functional activity and then interact with the substrate -target (i.e. genomic DNA in the mammalian nucleus) and lead to activation or repression of expression of the target gene.

[00542] Other inducible systems are contemplated, such as, but not limited to, heavy metal regulation [Mayo KE et al., Cell AC 29:99-108; Searle PF et al., Mol Cell Biol. 1985, 5:1480-1489 and Brinster RL et al., Nature (London) 1982, 296:39-42], steroid hormones [Hynes NE et al., Proc Natl Acad Sci USA 181, 78:2038-2042; Klock G et al., Nature. (London) 1987, 329:734-736 and Lee F et al.. Nature (London) 1981, 294:228-232.], heat shock [Nouer L: Heat Shock Response. Boca Raton, FL: CRC; 1991] and other developed reagents [Mullick A, Massie B: Transcription, translation and the control of gene expression. In Encyclopedia of Cell Technology Edited by: Speir RE. Wiley; 2000:1140-1164 and Fussenegger M,. Biotechnol Prog 2001, 17:1-51]. However, in the case of these inducible mammalian promoters, there is a limitation, consisting in the "leakage" of promoters in the "off" state and the pleiotropic effect of inducers (heat shock, heavy metals, glucocorticoids, etc.). The use of insect hormones (ecdysone) has been proposed in an attempt to reduce the effect on intracellular processes in mammalian cells [No D et al., Proc Natl Acad Sci USA 1996, 93:3346-3351]. Another preferred and well thought out system uses rapamycin as an inducer [Rivera VM et al., Nat Med 1996. 2:1028-1032], but the immunosuppressive effect of rapamycin is a major limitation to its use in vivo and therefore it was necessary to find a biologically inert a compound [Saez E et al., Proc Natl Acad Sci USA 2000, 97:14512-14517] to regulate gene expression.

[00543] In specific embodiments, the gene editing systems described herein are under the control of a coded kill switch, which is a mechanism that effectively causes host cell death when cell conditions change. This is provided by the introduction of hybrid transcription factors of the LacI-GalR family, the inclusion of which requires the presence of IPTG (Chan et al. 2015 Nature Nature Chemical Biology doi: 10.1038/nchembio.1979), which can be used to induce the gene encoding the enzyme necessary for survival cells. By combining different transcription factors that are sensitive to different chemicals, a "code" can be created. This system can be used to temporally and spatially control the spread of CRISPR-induced genetic modifications, which may be of interest when it is necessary to avoid GMO "exit" from the intended niche.

Delivery in general

Editing a gene or changing the target locus with C2c1 or C2c3

[00544] A double-strand break or a single-strand break in one of the strands must be sufficiently close to the target position such that repair occurs. In one of the possible embodiments, the distance should not exceed 50, 100, 200, 300, 350, or 400 nucleotides. Without wishing to be bound by theory, it is believed that the break must be close enough to the target position that the break is within the region that is intended to be removed by exonucleases when the ends are removed. If the distance between the target position and the break is too great, the mutation may not be included in the deletion and therefore cannot be repaired, since the template nucleic acid sequence can only be used to repair the sequence within the region of the deletion.

[00545] In one possible embodiment, in which the guide RNA and a type V/type VI molecule, in particular C2c1/C2c3, or an ortholog or homologue thereof, preferably a C2c1 or C2c3 nuclease, induces a double-strand break to induce homologous repair, the plot cleavage is in the range of 0-200 nucleotides (e.g., 0-175, 0-150, 0-125, 0-100, 0-75, 0-50, 0-25, 25-200, 25-175, 25-150 25-125, 25-100, 25-75, 25-50, 50-200, 50-175, 50-150, 50-125, 50-100, 50-75, 75-200, 75-175, 75 -150, 75-1 25, 75-100 nucleotides) from the target position. In one possible embodiment, the cleavage site is in the range of 0-100 nucleotides (e.g., 0-75, 0-50, 0-25, 25-100, 25-75, 25-50, 50-100, 50-75 or 75-100 nucleotides) from the target position. In a further embodiment, two or more guide RNAs in complex with C2c1 or C2c3, or an ortholog or homologue thereof, can be used to introduce multiple breaks to induce homologous repair.

[00546] The homology arm should span at least the region where end deletion may occur, for example, to allow the deleted single stranded overhangs to search for a complementary region within the donor template. The overall length may be limited by parameters such as plasmid size or viral packaging limits. In one possible embodiment, the homology arm may not reach repeating elements. A typical homology arm length is at least 50, 100, 250, 500, 750 or 1000 nucleotides.

[00547] The target position referred to herein refers to a site in a target nucleic acid or target gene (e.g., chromosome) that is modified by a type V/type VI molecule, in particular C2c1/C2c3 or their orthologue or homologue, preferably during the process , depending on the C2c1 or C2c3 molecule. For example, cleavage of the target nucleic acid by a modified C2c1 or C2c3 molecule may occur at the target position, and modification directed by the template nucleic acid. The modification may be, for example, a correction (repair) of the target position. In one possible embodiment, the target position may be a site between two nucleotides, such as adjacent nucleotides, on a nucleic acid to which one or more nucleotides are added. The target position may contain one or more nucleotides that are subject to change, such as repair with a template nucleic acid. In one possible embodiment, the target position may be within the target sequence (eg, the sequence to which the guide binds). In one possible embodiment, the target position is above or below the target sequence (eg, the sequence to which the guide RNA binds).

[00548] Template nucleic acid, as the term is used herein, refers to a nucleic acid sequence that can be used in combination with a type V/type VI molecule, preferably a C2c1/C2c3 molecule and a guide RNA molecule, to change the structure of the target position . In one possible embodiment, the target nucleic acid is modified to contain part or all of the template nucleic acid sequence, typically near or within the cleavage site(s). In one possible embodiment, the template nucleic acid is double-stranded. In one possible embodiment, the template nucleic acid is DNA. In another embodiment, the template nucleic acid is single stranded DNA.

[00549] In one possible embodiment, the implementation of the template nucleic acid changes the structure of the target position, participating in homologous recombination. In one of the possible embodiments, the implementation of the template nucleic acid changes the structure of the target position. In one possible embodiment, the template nucleic acid results in the incorporation of a modified or non-naturally occurring base into the target nucleic acid.

[00550] The template sequence may undergo breaks mediated or catalyzed by recombination with the target sequence. In one possible embodiment, the implementation of the template nucleic acid may contain a sequence corresponding to the site on the target sequence, which is mediated by C2c1 or C2c3 cleavage. In one possible embodiment, the template nucleic acid may comprise a sequence corresponding to both a first site in the target sequence cleaved in the first C2c1 or C2c3 mediated event and a second site in the target sequence cleaved in the second C2c1 or C2c3 mediated event. C2c3 events.

[00551] In one possible embodiment, the template nucleic acid may contain a sequence that results in a change in the coding sequence of the translated sequence, e.g., a sequence that results in the substitution of one amino acid for another in the protein product, e.g. type, transforming a wild-type allele into a mutant allele and/or introducing a stop codon, insertion of an amino acid residue or deletion of an amino acid residue, or nonsense mutation. In one possible embodiment, the template nucleic acid may contain a sequence that results in a change in a non-coding sequence, such as a change in an exon or a 5' or 3' untranslated region (UTR) or non-transcribed region. Such changes may include a change in a regulatory element, such as a promoter, enhancer, or a change in a cis or trans regulatory element.

[00552] A template nucleic acid having homology to a target position in a target gene can be used to change the structure of the target sequence. A template nucleic acid can be used to change an unwanted structure, such as an unwanted or mutated nucleotide. The template nucleic acid may contain a sequence which, when integrated, results in: decreased activity of the positive regulatory element; an increase in the activity of a negative regulatory element; decrease in gene expression; increase in gene expression; increased resistance to a syndrome or disease; increase resistance to viral infection; correcting a mutation or change in an undesirable amino acid residue that provides, increases, prevents, or reduces a biological property of a gene product, such as increasing the enzymatic activity of an enzyme or increasing the ability of the gene product to interact with another molecule.

[00553] The template nucleic acid may contain a sequence that results in changes in the sequence of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides of the target sequence. In one possible embodiment, the template nucleic acid may have a length of 20+/-10, 30+/-10, 40+/-10, 50+/-10, 60+/-10, 70+/-10, 80+ /-10, 90+/-10, 100-+/-10, 110+/-10, 120+/-10, 130+/-10, 140+/-10, 150+/-10, 160+/ -10, 170+/-10, 180+/-10, 190+/-10, 200+/-10, 210+/-10, or 220+/-10 nucleotides. In one possible embodiment, the template nucleic acid may have a length of 30+/-20, 40+/-20, 50+/-20, 60+/-20, 70+/-20, 80+/-20, 90+ /-20, 100+/-20, 1 10+/-20, 120+/-20, 130+R20, 140+/-20, I 50+/-20, 160+/-20, 170+/- 20, 180+/-20, 190+/-20, 200+/-20, 210+/-20, 220+/-20 nucleotides. In one possible embodiment, the template nucleic acid may be 10-1000, 20-900, 30-800, 40-700, 50-600, 50-500, 50-400, 50-300, 50-200, or 50- 100 nucleotides.

[00554] The template nucleic acid includes the following components: [5' homology arm]-[substitution sequence]-[3' homology arm]. Homology arms allow for recombination in a chromosome, thus replacing an undesirable element, such as a mutation or motif, with a substitution sequence. In one embodiment, the homologous arms flank the furthest cleavage sites. In one embodiment, the 3' end of the 5' arm of the homology is a position adjacent to the 5' end of the substitution sequence. In an embodiment, the 5' arm of the homology can be extended by at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides from the 5' -sides from the 5'end of the substitution sequence. In an embodiment, the 5' end of the 3' arm of the homology is a position adjacent to the 3' end of the substitution sequence. In an embodiment, the 3'-arm homology may include at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides with 3 '-sides from the 3'-end of the substitution sequence.

[00555] In certain embodiments, one or both of the homology arms may be truncated to avoid including certain repetitive sequences. For example, the 5' homology arm can be truncated to avoid including a repetitive sequence. In other embodiments, the 3' arm of the homology may be truncated to avoid including a repetitive sequence. In some embodiments, both the 5' and 3' homology arms can be truncated to avoid including certain repetitive sequences.

[00556] In certain embodiments, the implementation of the template nucleic acid for the correction of mutations can be designed for use as single-stranded oligonucleotides. When single stranded oligonucleotides are used, the 5' and 3' arm homology can be up to 200 nucleotides in length, eg at least 25, 50, 75, 100, 125, 150, 175 or 200 nucleotides.

C2c1 or C2c3 effector protein complex system-mediated nonhomologous end joining

[00557] In some embodiments, nuclease-induced non-homologous end joining (NHEJ) can be used to provide specific gene knockouts. Nuclease-induced NHEJ can also be used to remove a sequence in a target gene. In general, NHEJ repairs a double-strand break in DNA by joining the two ends; however, in general, the original sequence is restored only if two compatible ends, exactly as they were formed at the double-strand break, are accurately ligated. The ends of DNA double-strand breaks are often subject to enzymatic processes, resulting in the addition or removal of nucleotides in the case of one or both strands before rejoining the ends. This leads to the presence of mutations with insertions and/or deletions (insertions-deletions) in the DNA sequence in the region of NHEJ-dependent repair. Two-thirds of these mutations typically change the reading frame and therefore lead to the production of a non-functional protein. In addition, mutations that retain the reading frame, but which involve the insertion or deletion of a significant portion of the sequence, can render the protein non-functional. This depends on the locus, since it is likely that mutations in critical functional domains will lead to more significant consequences than in non-critical regions of proteins. Insertion-deletion mutations introduced by NHEJ are inherently unpredictable; however, in the case of a particular break, particular insertion-deletion sequences have greater advantages and are more represented in the population, most likely due to short microhomology regions. The length of deletions can vary considerably; most often it is in the range of 1-50 nucleotides, but it can also exceed 50 nucleotides, for example, reaching a length in excess of 100-200 nucleotides. Inserts are usually shorter and often include short sequence duplications immediately surrounding the break site. However, it is possible to provide long insertions, in which case the inserted sequence can often be tracked elsewhere in the genome or on plasmid DNA present in cells.

[00558] Since NHEJ-dependent repair is a mutagenic process, it can be used to remove sequence motifs if generation of a specific final sequence is not required. If the target of a double-strand break is to be adjacent to a short target sequence, deletion mutations caused by NHEJ-dependent repair often span and therefore remove unwanted nucleotides. For the deletion of longer DNA segments, the introduction of two double-strand breaks, one on each side of the sequence, can result in non-homologous end joining (NHEJ), followed by complete removal of the sequence lying between them. Both of these approaches can be used to remove specific DNA sequences; however, the nature of the non-homologous end joining, leading to a high error rate, can cause insertion-deletion formation at the repair site.

[00559] Both double-strand break-inducing type V/type VI molecules, in particular C2c1/C2c3, or their orthologues or homologues, preferably C2c1 or C2c3 molecules, or single-strand break-inducing (nickase) type V/type VI molecules, in particular, C2c1/C2c3, or their orthologues or homologues, preferably C2c1 or C2c3 molecules, can be used in the methods and compositions described herein to form NHEJ-repair mediated insertions/deletions. NHEJ-mediated insertions-deletions targeting a gene, eg a coding region, eg an early coding region of a target gene, can be used to knock out (ie eliminate expression) of the target gene. For example, the early coding region of a target gene includes the sequence immediately after the transcription start site within the first exon of the coding sequence, or within 500 base pairs of the transcription start point (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150 , 100 or 50 nucleotides).

[00560] In one possible embodiment, in which the guide RNA and a type V/type VI molecule, in particular C2c1/C2c3, or an orthologue or homologue thereof, preferably a C2c1 or C2c3 nuclease, induces a double-strand break to induce insertions-deletions, mediated by non-homologous end joining (NHEJ) repair, guide RNAs can be designed so that the double-stranded position is near the nucleotide of the target position. In one possible embodiment, the cleavage site may be within 0-500 nucleotides of the target position (e.g., less than 500, 400, 300, 200, 100, 50, 40, 30, 25, 20, 15, 10, 9, 8 , 7, 6, 5, 4, 3, 2 or 1 nucleotides from the target position).

[00561] In one possible embodiment, in which two guide RNAs in complex with type V/type VI molecules, in particular C2c1/C2c3, or their orthologues or homologues, preferably C2c1 or C2c3 nicases, induce two single strand breaks to induce In non-homologous end joining (NHEJ) repair-mediated insertion-deletion, the two guide RNAs can be designed so that the positions of the two nicks are such as to allow repair of the nucleotide at the target position by NHEJ.

C2c1 or C2c3 effector protein complexes can deliver functional effector proteins

[00562] In contrast to CRISPR-Cas mediated gene knockout, which permanently eliminates mutant gene expression at the DNA level, CRISPR-Cas knockdown allows for a temporary reduction in gene expression through the use of artificial transcription factors. Mutations of key residues in both DNA-cleaving domains of the C2c1 or C2c3 protein result in the formation of catalytically inactive C2c1 or C2c3. Catalytically inactive C2c1 or C2c3 forms a complex with the guide RNA and localizes to the DNA sequence defined by the target domain of the guide RNA, but does not cleave the target DNA. Fusion of an inactive C2c1 or C2c3 protein to an effector domain, such as a transcriptional repressor domain, allows the recruitment of the effector to any DNA region determined by the guide RNA. In certain embodiments, C2c1 or C2c3 can be fused to a transcriptional repressor domain and brought into the promoter region of a gene. In particular, with regard to gene repression, it is believed that blocking the binding site of an endogenous transcription factor may contribute to a decrease in the level of gene expression. In another embodiment, an inactive C2c1 or C2c3 may be fused to a chromatin modifying protein. A change in the state of chromatin can lead to a decrease in the expression of the target gene.

[00563] In one possible embodiment, the guide RNA molecule can be targeted to known transcriptional response elements (e.g., promoters, enhancers, etc.), known activating sequences upstream of the transcription start, and/or sequences known or unknown functions that are thought to be able to control the expression of the target DNA.

[00564] In some methods, the target polynucleotide can be inactivated to effect modification of expression in the cell. For example, upon binding of a CRISPR complex to a target sequence in a cell, the target polynucleotide is inactivated such that the sequence is not transcribed, the encoded protein is not produced, or the sequence does not function as the wild type sequence does. For example, the coding sequence for a protein or microRNA can be inactivated so that the protein is not produced.

[00565] In some embodiments, the implementation of the CRISPR enzyme contains one or more mutations selected from the list consisting of substitutions D917A, E1006A and D1225A, and/or one or more mutations in the RuvC domain of the CRISPR enzyme, or is another mutation described herein description. In some embodiments, the CRISPR enzyme contains one or more mutations in the catalytic domain in which, during transcription, the direct repeat sequence forms a single hairpin and the guide sequence directs sequence-specific binding to the target sequence of the CRISPR complex, the enzyme further comprising a functional domain. In some embodiments, the functional domain is a transcriptional repressor domain, preferably KRAB. In some embodiments, the transcriptional repressor domain is a SID and SID concatemers (SID4X). In some embodiments, the functional domain is an epigenetic modification domain, if an epigenetic modification protein is provided. In some embodiments, the functional domain is an activation domain, which may be a P65 activation domain.

Delivery of C2c1 or C2c3 effector protein complex or its components

[00566] As used herein, as well as in the prior art, it is contemplated that TALE nucleases, CRISPR-Cas systems, or components thereof, or nucleic acid molecules thereof (including, for example, an HDR template), or nucleic acid molecules encoding or components delivering them, can be delivered by the delivery system described herein both in general terms and in more detail.

[00567] Vector delivery, e.g., plasmid, viral delivery: A CRISPR enzyme, e.g., a type V protein, such as C2c1 or C2c3, and/or any of the RNAs of the present invention, e.g., a guide RNA, can be delivered using any suitable vector, for example, plasmid or viral vectors such as adeno-associated virus (AAV), lentivirus, adenovirus, or other types of viral vectors, or combinations thereof. The effector proteins and one or more guide RNAs can be packaged in one or more vectors, such as plasmid or viral vectors. In some embodiments, the vector, e.g., a plasmid or viral vector, is delivered to the target tissue, e.g., by intramuscular injection, while in other cases, delivery is by intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. Such delivery may be in the form of a single dose or in the form of multiple doses. One skilled in the art will recognize that the actual dosage to be delivered can vary greatly depending on a number of factors such as choice of vector, type of target cell, organism or tissue itself, general condition of the patient, degree of transformation/modification desired, method administration, mode of administration, type of transformation/modification desired, etc.

[00568] Such a preparation may further include, for example, a carrier (comprising water, aqueous sodium chloride solution, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc. .d.), a solvent, a pharmacological carrier (eg phosphate buffered saline), a pharmacological excipient and/or other compounds known in the art. The preparation may also contain one or more salts used in pharmacology, such as, for example, an inorganic acid salt such as hydrochloride, hydrobromide, phosphate, sulfate, etc., and organic acid salts such as acetates, propionates, malonates. , benzoates, etc. In addition, adjuvants such as wetting or emulsifying agents, buffer solutions, gels or gelling agents, flavoring agents, coloring agents, microspheres, polymers, suspension agents, etc. may also be used. In addition, one or more other commonly used pharmaceutical additives such as preservatives, humectants, suspending agents, surfactants, antioxidants, anti-caking agents, fillers, chelating agents, coating agents, chemical stabilizers, etc. may also be used, especially if the dosage is in dilute form. Suitable illustrative components include microcrystalline cellulose, sodium carboxymethyl cellulose, polysorbate 80, phenylethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, gelatin, albumin, and combinations thereof. A full discussion of excipients used in pharmacology is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub, Co., N.J. 1991), which is incorporated herein by reference.

[00569] In one embodiment described herein, delivery is by adenovirus, which can be delivered as a single booster dose containing at least 1 x 10 ⁵ particles (also referred to as particle units, ppm) of the adenoviral vector. In one embodiment of the invention of the present invention, the preferred dose is at least about 1x10 ⁶ particles (for example, about 1x10 ⁶ -1x10 ¹² particles), more preferably at least about 1x10 ⁷ particles, more preferably at least about 1x10 ⁸ particles (e.g., about 1x10 ⁸ -1x10 ¹¹ particles or about 1x10 ⁸ -1x10 ¹² particles), and most preferably at least about 1x10 ⁹ particles ( for example, about 1x10 ⁹ -1x10 ¹⁰ particles or about 1x10 ⁹ -1x10 ¹² particles), or even at least about 1x10 ¹⁰ particles (for example, about 1x10 ¹⁰ -1x10 ¹² particles) of an adenoviral vector. Alternatively, the dose comprises no more than about 1x10 ¹⁴ particles, preferably no more than about 1x10 ¹³ particles, even more preferably no more than about 1x10 ¹² particles, even more preferably no more than about 1x10 ¹¹ particles , and most preferably no more than about 1x10 ¹⁰ particles (eg, no more than about 1x10 ⁹ particles). Thus, a dose may contain a single dose of an adenoviral vector, for example, with about 1x10 ⁶ particle units (pch), about 2x10 ⁶ pch, about 4x10 ⁶ pch, 1x10 ⁷ pch, about 2x10 ⁷ pm, approximately 4×10 ⁷ pm, approximately 1×10 ⁸ pm, approximately 2×10 ⁸ pm, approximately 4×10 ⁸ pm, approximately ¹ ×10 ⁹ pm, approx ^. , approximately 1×10 ¹⁰ hr, approximately 2×10 ¹⁰ hr, approximately 4×10 ¹⁰ hr, approximately 1×10 ¹¹ hr, approximately 2×10 ¹¹ hr, approximately 4×10 ¹¹ hr, approximately 1×10 ¹² hr, approximately 2×10 ¹² u or approximately 4×10 ¹² u adenoviral vector. See, for example, adenoviral vectors in US patent application No. 8454972, B2 Nabel, et al., filed June 4, 2013, incorporated herein by reference, and in its column 29, lines 36-58. In one embodiment of the invention described herein, adenovirus is delivered via multiple doses.

[00570] In the embodiment described herein, delivery is via AAV. A therapeutically effective dosage for in vivo delivery of AAV to humans is believed to be in the range of about 20 to about 50 ml of saline containing about 1 x 10 ¹⁰ to about 1 x 10 ¹⁰ functional AAV solution/ml. The dosage may be adjusted to balance the therapeutic effect and any side effects. In one embodiment described herein, the dose of AAV is typically in the following concentration range: from about 1x10 ⁵ to 1x10 ⁵⁰ AAV genomes, from about 1x10 ⁸ to 1x10 ²⁰ AAV genomes, from about 1x10 ¹⁰ to about 1x10 ¹⁶ genomes, or about 1x10 ¹¹ to about 1x10 ¹⁶ AAV genomes. The human dosage may be approximately 1×10 ¹⁶ AAV genomes. At such concentrations, about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml of carrier solution can be delivered. Other effective dosages can be easily determined by one of skill in the art through standard tests, from which a dose-response curve is constructed. See, for example, US Patent Application No. 8,404,658 B2 to Hajjar, et al., filed March 26, 2013, column 27, lines 45-60.

[00571] In one embodiment of the invention described in the present description, the delivery is via a plasmid. In the case of such plasmid constructs, their dosage should be sufficient to elicit a response. For example, a suitable amount of plasmid DNA in plasmid constructs can be from about 0.1 to about 2 mg, or from about 1 pg to about 10 pg per 70 kg person. Plasmids of the invention typically include (i) a promoter; (ii) a sequence encoding a nucleic acid-targeted CRISPR enzyme operably linked to said promoter; (iii) a selectable marker; (iv) origin of replication; and (v) a transcription terminator downstream of and operably linked to (ii). The plasmid may also encode RNA components of the CRISPR complex, but one or more of them may be encoded by another vector.

[00572] The doses described herein are based on an average human weight of 70 kg. Determining the frequency of administration is within the purview of the medical or veterinary practitioner (eg, physician, veterinarian) or a scientist skilled in the art. It is also noted that the mice used in the experiments typically have a mass of approximately 20 g, and the results of experiments on mice can be extrapolated to an individual weighing 70 kg.

[00573] In some embodiments, the RNA molecules of the invention are delivered in a liposome formulation or in a formulation containing lipofectin and the like. and can be obtained using methods known to the person skilled in the art. Such methods are described, for example, in US Pat. Delivery systems for enhanced and improved siRNA delivery to mammalian cells have been developed (see e.g. Shen et al. FEBS Let. 2003, 539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006- 1010; Reich et al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol. Biol. 2003, 327: 761-766; Lewis et al., Nat. Gen. 2002, 32 : 107-108 and Simeoni et al., NAR 2003, 31, 11: 2717-2724) and can be used in the context of the present invention. Recently, siRNA has been successfully used to inhibit gene expression in primates (see, for example, Tolentino et al., Retina 24(4):660) and can also be used in the context of the present invention.

[00574] Indeed, RNA delivery is an efficient method for in vivo delivery. It is possible to deliver nucleic acid-targeted Cas, Cas9 protein and guide RNA-gRNA (and, for example, HR repair template) into cells using liposomes or particles. Thus, delivery of a nucleic acid-targeted Cas protein/CRISPR enzyme, such as the Cas protein Cas9 and/or delivery of guide RNAs of the invention can be done in the form of RNA using microvesicles, liposomes, or particles. For example, Cas mRNA and guide RNA can be packaged into liposomal particles for in vivo delivery. Liposomal transfection reagents such as Lipofectamine from Life Technologies and other commercially available reagents can efficiently deliver RNA molecules to the liver.

[00575] Preferably, RNA delivery vehicles also include nanoparticle delivery of RNA (Cho, S, Goldberg, M, Son, S, Xu, Q, Yang, F, Mei, Y, Bogatyrev, S, Langer, R, and Anderson , D, Lipid-like nanoparticles for small interfering RNA delivery to endothelial cells, Advanced Functional Materials, 19; 3112-3118, 2010) or exosomes (Schroeder, A, Levins, C, Cortez, C, Langer, R, and Anderson, D, Lipid-based nanotherapeutics for siRNA delivery, Journal of Internal Medicine, 267: 9-21, 2010, PMID: 20059641). Indeed, exosomes have been shown to be particularly useful in the delivery of siRNA, a system that bears some resemblance to an RNA targeting system. For example, El-Andaloussi S et al. ("Exosome-mediated delivery of siRNA in vitro and in vivo Nat Protoc. 2012 Dec;7(12):2112-26. doi: 10.1038/nprot.2012.131. Epub 2012 Nov 15.) described exosomes as a promising delivery tool drugs through various biological barriers and can be used to deliver siRNAs in vitro and in vivo.This approach consists in the production of target exosomes by transfection of an expression vector comprising an exosomal protein fused to a ligand-peptide.The exosomes are further purified and separated from the cellular supernatant, then RNA is loaded into exosomes.Delivery or administration according to the invention can be made using exosomes, for example, but not limited to the brain.Vitamin E (α-tocopherol) can be associated with nucleic acid-targeted Cas protein and delivered to the brain along with high-density lipoprotein (HDL), for example, in a manner similar to that done by Uno et al.(HUMAN GENE THERAPY 22:711-719 (June 2011)) to deliver and cute interfering RNA (siRNA) into the brain. Mice were infused using an osmotic micropump (model 1007D; Alzet, Cupertino, CA, USA) with phosphate buffered saline (PBS) or free TocsiBACE or Toc-siBACE/HDL and connected to a brain infusion kit (Brain Infusion Kit 3, Alzet) . The cannula used for cerebral infusion was placed approximately 0.5 mm posterior to the bregma in the midline for infusion into the third dorsal ventricle. Uno et al. found that no more than 3 nmol Toc-siRNA with HDL can cause a targeted reduction in a degree comparable to that when using the intracranial route of administration (ICV). A similar dosage of nucleic acid-targeted effector protein conjugated to α-tocopherol and co-administered with brain-targeted HDL is contemplated in humans within the scope of the present invention, for example, about 3 nmol to about 3 µmol of nucleic acid-targeted effector protein may be contemplated, aimed at the brain. Zou et al. (HUMAN GENE THERAPY 22:465-475 (April 2011)) describe a method for lentivirus-mediated delivery of shRNA targeting PKCγ to suppress gene expression in vivo in rat spinal cord. Zou et all. approximately 10 μl of recombinant lentivirus having a titer of 1×10 ⁹ transducing particles (PM)/ml was injected through an intrathecal catheter. A similar dosage of nucleic acid-targeted effector protein expressed from a brain-targeted lentiviral vector can also be contemplated within the scope of the present invention, e.g., about 10-50 ml of nucleic acid-targeted brain-targeted effector protein with a lentivirus titer of 1 is also contemplated. ×10 ⁹ transducing particles (PM)/ml.

[00576] With respect to local delivery to the brain, various pathways are possible. For example, the material can be delivered to the striatum, for example by injection. The injection can be made stereotactically during craniotomy.

[00577] Increasing the efficiency of NHEJ or HR is also useful for such delivery. Preferably, the efficacy of NHEJ is enhanced by co-expressed end processing enzymes such as Trex2 (Dumitrache et al. Genetics. 2011 August: 188(4): 787-797). Preferably, the effectiveness of HR is increased by transient inhibition by the NHEJ machinery, in particular Ku70 and Ku86. The efficiency of HR can also be increased by the simultaneous expression of prokaryotic or eukaryotic recombination enzymes such as RecBCD, RecA.

General Characteristics of Packaging and Promoters

[00578] Methods for packaging nucleic acid molecules encoding a nucleic acid-targeted effector protein (such as a type V protein, e.g. C2c1 or C2c3), e.g. DNA, into vectors, e.g. viral vectors, to mediate in vivo genome modification include:

To achieve NHEJ mediated gene knockout:

Vector based on a single virus:

A vector containing two or more expression cassettes:

Promoter guide RNA terminator

Promoter-guide RNA (N-terminus)-terminator (up to vector size limit)

Double viral vector:

Vector 1 containing one expression cassette to direct the expression of a nucleic acid-targeted effector protein (such as a type V protein, eg C2c1 or C2c3)

Nucleic acid promoter molecule encoding a nucleic acid-targeted effector terminator protein

Vector 2 containing one or more expression cassettes for driving the expression of one or more guide RNA(s)

Promoter-guide RNA 1-terminator

Promoter-guide RNA 1 (N-terminus)-terminator (up to vector length limit)

To mediate homologous repair.

In addition to the single and double vector approaches, an additional vector is used to deliver a homology-based repair matrix.

[00579] A promoter used to direct the expression of a nucleic acid targeted to the nucleic acid by an effector protein (such as a type V protein such as C2c1 or C2c3) may include:

The AAV ITR can act as a promoter: this is preferable in order to get rid of the extra promoter element (which takes up space in the vector). The resulting extra space can be used to direct the expression of additional elements (guide RNA, etc.). In addition, the activity of ITR is relatively weaker so that it can be used to reduce potential toxicity due to overexpression of a nucleic acid-targeted effector protein (such as a type V protein such as C2c1 or C2c3).

For ubiquitous expression, promoters can be used: CMV, CAG, CBh, PGK, SV40, ferritin heavy or light chains, etc.

For brain or other CNS expression, promoters can be used: synapsin I promoter for all neurons, CaMKIIalpha for excitatory neurons, GAD67, GAD65 or VGAT for GABAergic neurons, etc.

For expression in the liver, an albumin promoter can be used.

For expression in the lungs, SP-B can be used.

For endothelial cells, ICAM can be used.

For hematopoietic cells, IFN-beta or CD45 can be used.

For osteoblasts, OG-2 can be used.

[00580] The promoter used to target the guide RNA may include:

an RNA polymerase III promoter such as U6 or H1,

use of the RNA polymerase II promoter and intron guide RNA expression cassettes.

Adeno-associated virus (AAV)

[00581] A nucleic acid-targeted effector protein (such as a type V protein such as C2c1 or C2c3) and one or more guide RNAs can be delivered using adeno-associated virus (AAV), lentivirus, adenovirus, or other plasmid or viral types of vectors, in particular, using the dosage forms and dosages given, for example, in US patent applications Nos. plasmids) and clinical trials and publications on clinical trials including lentivirus, AAV and adenovirus. By way of example, for AAV, the route of administration, dosage form, and dose are given in US Patent Application No. 8,454,972 and in clinical trials involving AAV. For adenovirus, the route of administration, dosage form, and dose are given in US Patent Application No. 8,404,658 and in clinical trials involving adenovirus. For plasmid delivery, the route of administration, dosage form, and dose are given in US Patent Application No. 5,846,946 and in clinical trials involving plasmids. Doses can be developed based on an average human weighing 70 kg (eg, adult male), and can be adapted for patients, individuals, mammals of different weights and species. The frequency of administration is within the purview of the medical or veterinary practitioner (eg, physician, veterinarian), depending on common factors including age, sex, general health, other conditions of the patient or individual, and the particular condition and symptoms of interest. Viral vectors can be introduced into the target tissue. For cell type-specific genome modification, expression of a nucleic acid-targeted effector protein (such as a type V protein such as C2c1 or C2c3) may be under the control of a cell type-specific promoter. For example, liver-specific expression may be under the control of an albumin promoter, and neuronal-specific expression (e.g., to target disorders of the central nervous system) may be under the control of the synapsin I promoter.

[00582] In terms of in vivo delivery, AAV is preferred over other viral vectors for two reasons:

-low toxicity (possibly due to a purification method that does not require ultracentrifugation of cell parts that can trigger an immune response), and

- low probability of occurrence of insertional mutagenesis, because AAV does not integrate into the host genome.

[00583] The packaging limit of AAV is 4.5 kb or 4.75 kb. This means that the nucleic acid-targeted effector protein (such as a type V protein such as C2c1 or C2c3) as well as the promoter and transcription terminator must be placed in the same viral vector. Therefore, embodiments of the invention include the use of homologs of a nucleic acid-targeted effector protein (such as a type V protein such as C2c1 or C2c3) that are shorter.

[00584] With regard to AAV, AAV may be AAV1, AAV2, AAV5, or any combination. AAV can be selected based on the cells to be targeted; for example, serotypes AAV 1, 2, 5 or AAV1, AAV2, AAV5 with a hybrid capsid, or any combination thereof, can be selected for targeting brain cells or neurons; AAV4 can be chosen to target cardiac tissue, AAV8 is suitable for liver delivery. Within the scope of the present invention, individual promoters and vectors are preferred. The correspondence between specific AAV serotypes and cell types (see Grimm, D. et al, J. Virol. 82: 5887-5911 (2008)) is as follows:

cell line AAV-1 AAV-2 AAV-3 AAV-4 AAV-5 AAV-6 AAV-8 AAV-9 Huh-7 13 100 2.5 0.0 0.1 ten 0.7 0.0 HEK293 25 100 2.5 0.1 0.1 5 0.7 0.1 HeLa 3 100 2.0 0.1 6.7 one 0.2 0.1 HepG2 3 100 16.7 0.3 1.7 5 0.3 ND HepLA twenty 100 0.2 1.0 0.1 one 0.2 0.0 911 17 100 eleven 0.2 0.1 17 0.1 ND CHO 100 100 fourteen 1.4 333 fifty ten 1.0 COS 33 100 33 3.3 5.0 fourteen 2.0 0.5 mewo ten 100 twenty 0.3 6.7 ten 1.0 0.2 NIH3T3 ten 100 2.9 2.9 0.3 ten 0.3 ND A549 fourteen 100 twenty ND 0.5 ten 0.5 0.1 HT1180 twenty 100 ten 0.1 0.3 33 0.5 0.1 Monocytes 1111 100 ND ND 125 1429 ND ND Immature DC 2500 100 ND ND 222 2857 ND ND Mature DC 2222
jLd jLu jh Au 100 ND ND 333 3333 ND ND

Lentivirus

[00585] Lentiviruses are complex retroviruses capable of infecting and expressing their genes in both mitotic and post-mitotic cells. The best known lentivirus is the human immunodeficiency virus (HIV), which uses the viral envelope glycoproteins of other viruses to target a wide range of cell types.

[00586] Lentiviruses can be obtained as follows. After cloning pCasES10 (which contains the lentivirus transfer plasmid backbone), HEK293FT at a low number of passages (p=5) were plated in a T-75 flask to 50% confluence the day before transfection in DMEM with 10% fetal bovine serum and no antibiotics. After 20 hours, culture media were replaced with OptiMEM medium (without serum) and transfection was carried out after 4 hours. Cells were transfected with 10 μg of the lentiviral transfer plasmid (pCasES10) and the following packaging plasmids: 5 μg of pMD2.G (VSV-g pseudotype) and 7.5 μg of psPAX2 (gag/pol/rev/tat). Transfection was performed in 4 ml OptiMEM with cationic lipid delivery vehicle (50 µl Lipofectamine 2000 and 100 µl Reagent Plus). After 6 hours, the culture medium was changed to DMEM without antibiotic with 10% fetal bovine serum. These methods use serum during culture, however, methods without serum are preferred.

[00587] The lentivirus can be purified as follows. Viral supernatants were collected after 48 hours. The supernatants were first cleared of debris and passed through a 0.45 μm low protein binding filter (PVDF). They were then ultracentrifuged for 2 hours at 24,000 rpm. The virus pellets were then resuspended in 50 μl DMEM overnight at 4°C. Then they were divided into aliquots and immediately frozen at -80°C.

[00588] In another embodiment, the invention also provides a minimal non-primate lentivirus vector based on equine infectious anemia virus (EIAV), especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med 2006; 8; 275-285). In another embodiment, the invention also provides RetinoStat®, a lentiviral equine infectious anemia virus gene therapy vector expressing the angiostatic proteins endostatin and angiostatin delivered via subretinal injection for the treatment of age-related macular degeneration (see, for example, Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)) and this vector can be modified for use in the nucleic acid targeting system of the present invention.

[00589] In another embodiment, self-inactivating lentiviral vectors with an siRNA targeting an exon common to the HIV tat/rev genes, a nucleolar TAR trap, and an anti-CCR5 specific "hammerhead" ribozyme (see, e.g., DiGiusto et al (2010) Sci Transl Med 2:36ra43) can be used and/or adapted for use in the nucleic acid targeting system of the present invention. A minimum of 2.5×10 ⁶ CD34+ cells per kilogram of patient weight can be obtained by pre-stimulation for 16-20 hours in X-VIVO 15 culture medium (Lonza) containing 2 μM L-glutamine, stem cell factor (100 ng/ml ), Flt-3 ligand (Flt-3L) (100 ng/ml) and thrombopoietin (10 ng/ml) (CellGenix) at a density of 2×10 ⁶ cells/ml. Pre-stimulated cells can be transduced with a lentiviral vector at a MOI of 5 for 16-24 hours in 75 cm ² tissue culture flasks coated with fibronectin (25 mg/cm ² ) (RetroNectin, Takara Bio Inc.).

[00590] Lentiviral vectors are described for the treatment of Parkinson's disease, see, for example, US Patent Application Publication No. 20120295960, No. 7303910, and No. 7351585. Lentiviral vectors are described as a method of treating diseases of the eye, see, for example, US Patent Application Publication No. 20060281180 20090007284, US20110117189; US20090017543; US20070054961, US20100317109. Lentiviral vectors have also been described as a means of delivery to the brain, see, for example, US Patent Application Publications No. US20110293571; US20110293571, US20040013648, US20070025970, US20090111106 and US Patent No. US7259015.

Delivery of RNA

[00591] RNA Delivery: A nucleic acid-targeted Cas protein, for example a type V protein such as C2c1 or C2c3, and/or a guide RNA can also be delivered in the form of RNA. mRNA for a nucleic acid-targeted Cas protein (eg, a type V protein such as C2c1 or C2c3) can be obtained using in vitro transcription. mRNA of a nucleic acid-targeted Cas protein (e.g., a type V protein such as C2c1 or C2c3) can be synthesized using a PCR cassette containing the following elements: T7 promoter-Kozak sequence (GCCACC)-effector protein-3'-untranslated region (UTR) beta-globin polyA sequences (sequence of 120 or more adenine residues). Such a cassette can be used for transcription with the participation of T7 polymerase. Guide RNAs can also be transcribed by in vitro transcription from a cassette containing the T7 promoter-GG guide RNA sequence.

[00592] To enhance expression and reduce potential toxicity, the sequence encoding the nucleic acid-targeted effector protein and/or guide RNA can be modified to contain one or more modified nucleosides, for example, using pseudo-U or 5-methyl- C.

[00593] mRNA delivery methods currently appear to be particularly promising for delivery to the liver.

[00594] Significant clinical efforts in RNA delivery have been focused on RNAi or antisense molecules, however, such systems can be adapted for RNA delivery in order to carry out the present invention. Accordingly, the references to RNA-I etc. below should be read.

Delivery systems for particles and/or formulations:

[00595] Several types of particle and/or formulation delivery systems have found use in a wide range of biomedical applications. In general, a particle is defined as a small object that behaves like a whole unit in terms of its movement and properties. The particles are further subdivided according to their diameter. Large particles correspond to the range from 2500 to 10000 nanometers. Fine particles range in size from 100 to 2500 nanometers. Ultrafine particles, or nanoparticles in general, are 1 to 100 nanometers in size. The basis for choosing a limit of 100 nm is the manifestation of new properties that distinguish particles from bulk material, which usually develop at a transitional length scale of less than 100 nm.

[00596] As used herein, a particle delivery system/dosage form is defined as any biological delivery system/dosage form that includes a particle in accordance with the present invention. A particle in accordance with the present invention is any object having a largest dimension (eg diameter) less than 100 microns (μm). In some embodiments, the particles of the invention have a largest dimension of less than 10 microns. In some embodiments, the particles of the invention have a largest dimension of less than 2000 nanometers (nm). In some embodiments, the particles of the invention have a largest dimension of less than 1000 nanometers (nm). In some embodiments, the particles of the invention have a largest dimension of less than 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, or 100 nm. Typically, the particles of the invention have a largest dimension (eg diameter) of 500 nm or less. In some embodiments, the particles of the invention have a largest dimension (eg, diameter) of 250 nm or less. In some embodiments, the particles of the invention have a largest dimension (eg, diameter) of 200 nm or less. In some embodiments, the particles of the invention have a largest dimension (eg, diameter) of 150 nm or less. In some embodiments, the designed particles have a largest dimension (eg, diameter) of 100 nm or less. Smaller particles, for example, having a largest dimension of 50 nm or less are used in some embodiments of the invention. In some embodiments of the invention, the particles according to the invention have the largest dimension in the range from 25 nm to 200 nm.

[00597] Characterization of particles (including, for example, morphology, measurement, etc.) is carried out using a number of different methods. Common methods are electron microscopy (TEM, SEM), atomic force microscopy (AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), X-ray powder diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), time-of-flight matrix-assisted laser desorption/ionization (MALDI-TOF), optical spectroscopy, dual polarization interferometry and nuclear magnetic resonance (NMR). Characterization (size measurement) can be performed on native particles (i.e., before loading) or after loading a cargo (in the present description, the cargo refers, for example, to one or more components of the CRISPR-Cas system, for example, a CRISPR enzyme, or mRNA, or guide RNA, or any combination thereof, and may include additional carriers and/or excipients) to provide particles of optimal size for delivery in any in vitro , ex vivo and/or in vivo application of the present invention. In certain preferred embodiments of the invention, the characterization of particle sizes (eg, diameter) is based on a measurement using dynamic light scattering (DLS). Reference is made to US Patent Application No. 8,709,843; US patent application No. 6007845, US patent No. 5855913; US patent No. 5985309; US patent application No. 5543158; and the publication of James E. Dahlman and Carmen Barnes et al. Nature Nanotechnology (2014) published on the Internet on May 11, 2014, doi:10.1038/nnano.2014.84, concerning particles, methods for their production and application and measurement.

[00598] The particle delivery systems of the present invention may be in any form, including, but not limited to, solid, semi-solid, emulsion, or colloidal particles. Substantially any such delivery systems described herein, including but not limited to, for example, systems based on lipids, liposomes, micelles, microvesicles, exosomes, or gene guns, can be provided for delivery of particles within the scope of the present invention.

Particles

[00599] CRISPR enzyme mRNA and guide RNA can be delivered simultaneously using particles or lipid envelopes; for example, the CRISPR enzyme and the RNA of the invention, for example, in the form of a complex, can be delivered using particles according to Dahlman et al., WO2015089419 A2 and documents cited therein, such as 7C1 (see, for example, James E. Dahlman and Carmen Barnes et al Nature Nanotechnology (2014) published online May 11, 2014, doi:10.1038/nnano.2014.84), e.g., a delivery particle comprising a lipid or lipidoid and a hydrophilic polymer, e.g., a cationic lipid, and a hydrophilic polymer, for example, where the cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-ditradecanoyl- sn -glycero-3-phosphocholine (DMPC) and/or where the hydrophilic polymer comprises ethylene glycol or polyethylene glycol ( PEG); and/or where the particle further comprises cholesterol (e.g., composition particle 1=DOTAP 100, DMPC 0, PEG 0, cholesterol 0; composition number 2=DOTAP 90, DMPC 0, PEG 10, cholesterol 0; composition number 3=DOTAP 90, DMPC 0, PEG 5, cholesterol 5), where the particles are formed using an efficient, multi-step process, where first, the effector protein and RNA are mixed together, for example, in a 1:1 molar ratio, for example, at room temperature, for example, for 30 minutes, eg in sterile, nuclease-free 1X PBS; and separately, DOTAP, DMPC, PEG and cholesterol, as applicable to the formulation, are dissolved in alcohol, eg 100% ethanol; and, the two solutions are mixed together to form complexed particles).

[00600] Nucleic acid-targeting effector proteins (such as type V proteins such as C2c1 or C2c3) mRNA and guide RNA can be delivered simultaneously using viral envelope particles or lipids.

[00601] For example, Su X, Fricke J, Kavanagh DG, Irvine DJ (" In vitro and in vivo mRNA delivery using lipid-enveloped pH-responsive polymer nanoparticles" Mol Pharm. 2011 Jun 6;8(3):774-87 doi: 10.1021/mpl00390w Epub 2011 Apr 1) describe biodegradable structured particles with a poly(β-aminoether) (PBAE) core surrounded by a phospholipid bilayer. They have been designed for in vivo mRNA delivery. The pH-dependent PBAE component was chosen to promote endosomal degradation, while the surface lipid layer was chosen to minimize polycation core toxicity. Therefore, such a method is preferred for delivering the RNA of the present invention.

[00602] In one embodiment, the invention contemplates particles based on self-assembly of bioadhesive polymers that can be used to deliver peptides to the brain by various delivery methods: oral, intravenous, nasal. Other embodiments are also contemplated, such as oral and ocular delivery of hydrophobic drugs. Molecular envelope technology involves an engineered polymeric envelope that is protected and delivered to the disease site (see e.g. Mazza, M. et al. ACSNano, 2013. 7(2): 1016-1026; Siew, A., et al. Mol Pharm, 2012. 9(1): 14-28, Lalatsa, A., et al., J Contr Rel, 2012. 161(2):523-36, Lalatsa, A., et al., Mol Pharm, 2012. 9(6): 1665-80 Lalatsa, A., et al Mol Pharm, 2012. 9(6): 1764-74, Garrett, N.L., et al J Biophotonics, 2012. 5(5-6): 458-68 Garrett, N.L., et al J Raman Spect, 2012. 43(5):681-688, Ahmad, S., et al J Royal Soc Interface 2010. 7:S423-33; Uchegbu, I.F. Expert Opin Drug Deliv 2006 3(5):629-40 Qu, X. et al Biomacromolecules 2006 7(12):3452-9 and Uchegbu IF. et al Int J Pharm 2001 224 :185-199). Doses of approximately 5 mg/kg are contemplated in single or multiple doses, depending on the target tissue.

[00603] In one embodiment, particles that can deliver RNA to a cancer cell to stop tumor growth, developed by Dan Anderson's laboratory at MIT, can be used/and adapted for the nucleic acid targeting system of the present invention. In particular, the Anderson laboratory has developed fully automated, combinatorial systems for the synthesis, purification, characterization and formulation of new biomaterials and nanodrugs. See, for example, Alabi et al., Proc Natl Acad Sci U S A. 2013 Aug 6; 110(32): 12881-6; Zhang et al., Adv Mater. 2013 Sep 6;25(33):4641-5; Jiang et al., Nano Lett. 2013 Mar 13;13(3): 1059-64; Karagiannis et al., ACS Nano. 2012 Oct 23;6(10):8484-7; Whitehead et al., ACS Nano. 2012 Aug 28;6(8):6922-9 and Lee et al., Nat Nanotechnol. 2012 Jun 3.7(6):389-93.

[00604] US Patent Application 20110293703 relates to lipidoid complexes, particularly useful in the administration of polynucleotides, which can be used to deliver the nucleic acid-targeted system of the present invention. In one aspect, the amino alcohol lipidoid compounds are combined with a cell or individual delivery agent to form microparticles, nanoparticles, liposomes, or micelles. The agent delivered by particles, liposomes, or micelles may be in the form of a gas, liquid, or solid, and the agent may be a polynucleotide, protein, peptide, or small molecule. Amino alcohol lipidoid compounds can be combined with other amino alcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, and the like. to form particles. These particles can then optionally be combined with a pharmaceutical excipient to form a pharmaceutical composition.

[00605] US Patent Application No. 20110293703 also describes methods for preparing an amino alcohol lipidoid compound. One or more equivalents of an amine is allowed to react with one or more equivalents of epoxy-terminated compounds, which, under suitable conditions, allows the formation of an amino alcohol lipidoid compound of the present invention. In some embodiments, all amine groups of the amine react completely with the epoxy end groups to form tertiary amines. In other embodiments of the invention, all of the amino groups of the amine do not fully react with the epoxy end groups, thus leading to the formation of primary or secondary amines in the amino alcohol lipidoid compound. These primary or secondary amines retain the ability to react with other electrophiles such as various epoxy terminated compounds. As one skilled in the art can appreciate, the reaction of an amine deficient compound with epoxy groups results in a variety of different amino alcohol lipidoid compounds with varying numbers of tails. Certain amines can be fully functionalized with two epoxy-terminated compounds to form two tails, while other molecules are not fully functionalized with epoxy-terminated compounds to form tails. For example, a diamine or polyamine may include one, two, three, or four epoxy-terminated tails attached to different amino groups of the molecule, resulting in primary, secondary, and tertiary amines. In some embodiments of the invention, all amino groups are not fully functionalized. In some embodiments of the invention, two compounds with the same type of epoxy end group are used. In other embodiments, two or more different epoxy terminated compounds are used. Synthesis of aminoalcohol lipidoid compounds can occur with or without a solvent, and the synthesis can be carried out at higher temperatures in the range of 30-100°C, preferably at about 50-90°C. The prepared amino alcohol lipidoid compounds may optionally be purified. For example, a mixture of amino-alcohol lipidoid compounds can be purified to provide an amino-alcohol lipidoid compound with a specific number of epoxide-derived moieties. Or the mixture may be purified to give a particular stereo or regio isomer. Amino alcohol lipidoid compounds can also be alkylated using an alkyl halide (eg, methyl iodide) or other alkylating agent, and/or they can be acylated.

[00606] US Patent Application No. 20110293703 also describes libraries of amino alcohol lipidoid compounds prepared by the methods of the invention. These amino-alcohol lipidoid compounds can be prepared and/or tested using high-throughput methods, including fluid manipulators, robots, microtiter plates, computers, and the like. In some embodiments, the amino alcohol lipidoid compounds are screened for their ability to transfect polynucleotides or other agents (eg, proteins, peptides, small molecules) into a cell.

[00607] U.S. Patent Application No. 20130302401 relates to a class of poly(beta-aminoalcohols) (PBAA), which are obtained using combinatorial polymerization. Developed PBAAs can be used in biotechnology and biomedical applications as coatings (such as film or multilayer film coatings for medical devices or implants), additives, materials, fillers, non-biofouling agents, microstructuring agents, and cell encapsulation agents. The use of PBAAs as surface coatings has made it possible to induce different levels of inflammation, both in vitro and in vivo , depending on the chemical structure. The great chemical diversity of this class of materials has allowed the identification of polymer coatings that inhibit macrophage activation in vitro . In addition, these coatings reduce the recruitment of inflammatory cells and reduce fibrosis after subcutaneous implantation of carboxylated polystyrene microparticles. These polymers can be used to form a capsule of a polyelectrolyte complex to encapsulate a cell. The invention may also have many other biological applications, such as antibacterial coatings, DNA or siRNA delivery, and stem cell-based tissue engineering. The recommendations of US Patent Application No. 20130302401 can be applied to the nucleic acid targeting system of the present invention.

[00608] In another embodiment, lipid nanoparticles (LNPs) are provided. Anti-transthyretin small interfering RNA has been encapsulated in lipid nanoparticles and delivered to human cells (e.g., see Coelho et al., N Engl J Med 2013;369:819-29), and such a system can be adapted and applied to nucleic acid-targeted system of the present invention. Doses contemplated are from about 0.01 to about 1 mg per kg of body weight when delivered intravenously. Drugs such as dexamethasone, acetaminophen, diphenhydramine, cetirizine, and ranitidine are considered to reduce the risk of infusion reactions. Considered the use of multiple doses of approximately 0.3 mg per kilogram of body weight every 4 weeks up to five doses.

[00609] LNPs have been shown to be highly effective in delivering siRNA to the liver (see, e.g., Tabemero et al, Cancer Discovery, April 2013, Vol. 3, No. 4, pages 363-470), therefore, LNPs can be used to deliver RNA, encoding a nucleic acid-targeted effector protein to the liver. The dosage may be approximately four doses of 6 mg/kg LNP every two weeks. Tabemero et al. demonstrated that tumor regression was observed after the first 2 cycles of LNP at a dosage level of 0.7 mg/kg, by the end of 6 cycles the patient achieved partial remission with complete regression of lymph node metastases and a significant decrease in liver tumors, complete remission was achieved after 40 doses, then the remission was maintained, the treatment was completed after 26 months. Two patients with RCC and extrahepatic sites of disease including kidney, lung, and lymph nodes who progressed after prior therapy with VEGF cascade inhibitors had stable disease in all areas for approximately 8-12 months, and the patient with PNET and liver metastases continued treatment in an additional study for 18 months (36 doses) with stable disease.

[00610] However, the charge of the LNP must also be taken into account. Cationic lipids are combined with negatively charged lipids to induce non-bilayer structures to facilitate intracellular delivery. Because charged LNPs leave the blood stream rapidly after intravenous injection, ionizable cationic lipids with pKa values below 7 have been developed (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011) . Negatively charged polymers such as RNA can be loaded into LNPs at low pH (eg pH 4) when the ionizable lipids are positively charged. However, at physiological pH values, LNPs have a low surface charge compatible with longer circulation times. The study focused on four kinds of ionizable cationic lipids, namely 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy -keto-N,N-dimethyl-3-aminopropane (DLinKDMA); and 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA). LNP siRNA systems containing these lipids have been shown to exhibit significantly different gene silencing properties in hepatocytes in vivo , with efficiencies varying according to the series DLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP using Factor VII gene silencing models (see below). eg Rosin et al, Molecular Therapy, vol, 19, no. 12, pages 1286-2200, Dec, 2011). A dosage of 1 µg/mL LNP or CRISP-Cas RNA within or in association with LNP may be considered, especially for a drug product containing DLinKC2-DMA.

[00611] LNP preparation and CRISPR-Cas encapsulation can be used or adapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec, 2011. Cationic lipids 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy -keto-N,N-dimethyl-3-aminopropane (DLinKDMA), 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA), (3-o-[2 "-(methoxypolyethylene glycol 2000) succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG) and R-3-[(co-methoxy-poly(ethylene glycol)2000) carbamoyl]-1,2-dimyristyloxypropyl -3-amine (PEG-C-DOMG) can be purchased from Tekmira Pharmaceuticals (Vancouver, Canada) or synthesized Cholesterol can be purchased from Sigma (St. Louis, MO) Specific Nucleic Acid Targeting Complex RNA (CRISPR-Cas ) can be encapsulated in LNP containing DLinDAP, DLinDMA, DLinK-DMA and DLinKC2-DMA (cationic lipid:DSPC:CHOL:PEGS-DMG or PEG-C-DOMG in a molar ratio of 40:10:40:10). Optionally, 0.2% SP-DiOC18 (Invitrogen, Burlington, Canada) may be included in the formulation to assess cellular uptake, intracellular delivery and biodistribution. Encapsulation can be performed by dissolving a lipid mixture consisting of cationic lipid:DSPC:cholesterol:PEG-c-DOMG (molar ratio 40:10:40:10) in ethanol to a final lipid concentration of 10 mmol/L. This lipid solution in ethanol can be added dropwise to 50 mmol/L citrate, pH 4.0, to form multilamellar vesicles to achieve a final ethanol concentration of 30% by volume. Large unilamellar vesicles can be formed by passing multilamellar vesicles through two 80 nm stacked Nuclepore polycarbonate filters using an extruder (Northern Lipids, Vancouver, Canada). Encapsulation can be achieved by adding RNA, dissolved to a concentration of 2 mg/ml, in 50 mmol/l citrate, pH 4.0, containing 30% ethanol by volume, dropwise to pre-formed large single-layer vials and incubation at 31°C in for 30 minutes with constant stirring until the final ratio of RNA/lipid by weight equal to 0.06/1. Removal of ethanol and neutralization of the resulting formulation with buffer can be performed by dialysis against sodium phosphate buffer (PBS), pH 7.4, for 16 hours using Spectra/Por 2 reconstituted cellulose dialysis membranes. Particle size distribution can be determined by dynamic scatter light using a NICOMP 370 granulometer, vesicle/intensity mode, and Gaussian fit (Nicomp Particle Sizing, Santa Barbara, CA). The particle size for all three LNP systems can be ~70 nm in diameter. The efficiency of RNA encapsulation can be determined by removing free RNA using a VivaPureD Mini column (Sartorius Stedim Biotech) from samples collected before and after dialysis. The encapsulated RNA can be recovered from the eluted particles and quantified at 260 nm. The ratio of RNA to lipids can be determined by measuring the cholesterol content of the vesicles using the cholesterol E enzymatic assay from Wako Chemicals USA (Richmond, Virginia). Together with the LNPs and PEG lipids discussed herein, pegylated liposomes or LNPs are also suitable for delivery of a nucleic acid-targeted system or components thereof.

[00612] The production of lipid nanoparticles (LNP) can be done according to and/or adapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011. A lipid blend pre-solution (with a total lipid concentration of 20.4 mg/mL) can be prepared in ethanol containing DLinKC2-DMA, DSPC and cholesterol in molar ratios of 50:10:38.5. Sodium acetate can be added to the pre-prepared lipid mixture solution in a molar ratio of 0.75:1 (sodium acetate: DLinKC2-DMA). Lipids can be further hydrated using a mixture of 1.85 volumes of citrate buffer (10 mmol/l, pH 3.0) with vigorous agitation leading to spontaneous formation of liposomes in an aqueous buffer containing 35% ethanol. The liposome solution can be incubated at 37° C. to allow a time dependent increase in particle size. Aliquots can be removed at different time points during incubation to study changes in liposome size using a dynamic light scattering method (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK). Upon reaching the desired particle size, an aqueous solution of PEG and lipids (stock solution=10 mg/ml PEG-DMG in 35% (v/v) ethanol) can be added to the liposome mixture to achieve a final PEG concentration of 3.5% of total lipid. When PEG lipids are added, the liposomes should be about their size and effectively inhibit further growth. Next, RNA can be added to empty liposomes at a ratio of RNA to total lipids of about 1:10 (w:w) followed by incubation for about 30 min at 37°C to form loaded lipid nanoparticles (LNP). This mixture can then be dialyzed overnight in PBS) and filtered through a 0.45 pm syringe filter.

[00613] Spherical Nucleic Acid (SNA™) constructs and other particles (particularly gold particles) are also contemplated as a means of delivering nucleic acid-targeted systems to intended targets. A significant body of data demonstrates the usefulness of AuraSense's (SNA™) therapeutic spherical nucleic acid constructs based on nucleic acid-functionalized gold particles.

[00614] Literature that may be used in conjunction with the teachings set forth herein includes: Cutler et al., J. Am. (Chem. Soc. 2011 133:9254-9257, Hao et al., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962-6970, Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al., Nano Lett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA 2012 109:11975-80, Mirkin, Nanomedicine 2012 7:635-638 Zhang et al., J. Am. Chem. Soc. 2012 134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choi et al., Proc. Natl. Acad. Sci. USA 2013 110(19):7625-7630, Jensen et al., Sci. Transl. Med. 5, 209ral52 (2013) and Mirkin, et al., Small, 10:186-192.

[00615] Self-assembling RNA particles can be made from polyethyleneimine (PEI) coupled to PEG through an Arg-Gly-Asp (RGB) peptide ligand attached to the far end of the PEG. Such a system has been used, for example, as a means of targeting tumor vessels expressing integrins and delivering siRNA that inhibits the expression of vascular endothelial growth factor receptor 2 (VEGFR2) and thereby achieving angiogenesis in the tumor (see, e.g., Sehiffelers et al, Nucleic Acids Research, 2004, Vol 32, No 19). Nanoplexes can be prepared by mixing equal volumes of aqueous solutions of cationic polymer and nucleic acid to produce a net molar excess of ionizable nitrogen (polymer) over phosphate (nucleic acid) ranging from 2 to 6. Electrostatic interactions between cationic polymers and nucleic acid lead to the formation of polyplexes with with an average size distribution of approximately 100 nm, which is why they are referred to herein as nanoplexes. A dosage of about 100 to 200 mg of nucleic acid targeting RNA complex is contemplated for delivery in self-assembling particles according to Sehiffelers et al.

[616] The nanoplexes described by Bartlett et al. (PNAS, September 25, 2007, vol. 104, no. 39) can also be used in the context of the present invention. The nanoplexes described by Bartlett et al. are prepared by mixing equal volumes of aqueous solutions of cationic polymer and nucleic acid to produce a net molar excess of ionizable nitrogen (polymer) over phosphate (nucleic acid) ranging from 2 to 6. Electrostatic interactions between cationic polymers and nucleic acid lead to the formation of polyplexes with an average size distribution of about 100 nm, and therefore here they are called nanoplexes. The DOTA siRNA described by Bartlett et al. was synthesized as follows: 1,4,7,10-tetrazacyclododecane-1,4,7,10-tetraacetic acid mono(N-hydroxysuccinimide ester) (NHS DOTA ester) was ordered in Macrocyclics (Dallas, Texas, USA). The amino-modified RNA coding strand with 100-fold molar excess of DOTA-NHS ester in carbonate buffer (pH 9) was placed in a microcentrifuge tube. The reaction of the contents was carried out with stirring for 4 hours at room temperature. Bound DOTA-coding RNAs were ethanol precipitated, resuspended in water, and annealed to the unmodified non-coding strand to obtain DOTA siRNAs. All fluids were pre-treated with Chelex-100 (Bio-Rad, Hercules, CA, USA) to remove trace metal contamination. Tf-targeted and non-targeted siRNA particles can be formed using cyclodextrin-containing polycations. Typically, particles are formed in water at a charge ratio (+/-) of 3 and an siRNA concentration of 0.5 g/l. One percent of the adamantane-PEG molecules on the surface of the targeted particles were modified (adamantane-PEG-Tf). The particles were suspended in 5% (w/v) of the resulting glucose transporter solution for injection.

[00617] Davis et al. (Nature, Vol 464, 15 April 2010) conducted RNA clinical trials using a targeted particle delivery system (clinical trial registration number NCT00689065). Patients with a solid type of malignant tumor refractory to standard therapies received doses of targeted particles on days 1, 3, 8, and 10 of a 21-day course by 30-minute intravenous infusion. Such particles include, essentially consist of, or consist of a synthetic delivery system containing: (1) a linear cyclodextrin-based polymer (CDP), (2) a ligand targeting the human transferrin (TF) protein outside the nanoparticle to activate TF receptors ( TFR) on the surface of malignant cells, (3) a hydrophilic polymer (polyethylene glycol, PEG) is used to increase the stability of nanoparticles in biological fluids), and (4) an siRNA designed to reduce the expression of RRM2 (the sequence used in clinical practice was previously designated as siR2B+5 ). It has been known for a long time that TFR is more actively expressed in cancer cells, so RRM2 is a proven target for anti-cancer therapy. Such particles (the clinically used version is designated CALAA-01) have been shown to be well tolerated in various dosage studies in non-human primates. Although previously one patient with chronic myeloid leukemia received siRNA by liposome delivery, clinical trials by Davis et al. are primary for the systematic delivery of siRNA with a targeted delivery system and for the treatment of patients with solid malignant tumors. To test whether a targeted delivery system can efficiently deliver functional siRNA to human tumors, Davis et al. studied biopsies of three patients from three dosage groups: patients A, B, and C, each of whom had metastatic melanoma and received CALAA-01 at doses of 18, 24, and 30 mg/m ² siRNA, respectively. Similar doses can also be expected for the nucleic acid targeting system of the present invention. Delivery according to the invention can be achieved using particles containing a linear cyclodextrin-based polymer (CDP), a ligand targeting human transferrin (TF) protein outside the nanoparticle to activate TF receptors (TFR) on the surface of malignant cells, and/or a hydrophilic polymer (e.g. polyethylene glycol (PEG) used to improve stability in biological fluids).

[00618] With respect to the present invention, it is preferred to have one or more components of a nucleic acid-targeted complex, for example, a nucleic acid-targeted effector protein or mRNA, or a guide RNA delivered by particles or lipid packaging. Other delivery systems or vectors may be used in conjunction with particles in appropriate embodiments of the invention.

[00619] In general, "nanoparticle" refers to any particle with a diameter less than 1000 nm. In certain preferred embodiments, the nanoparticles of the present invention have a long axis dimension (eg, diameter) of 500 nm or less. In other preferred embodiments described in this invention, the nanoparticles have the largest size in the range between 25 nm and 200 nm. In other preferred embodiments, the nanoparticles described herein have a long axis size of 100 nm or less. In other preferred embodiments, described in this invention, the nanoparticles have a size of from 35 nm to 60 nm.

[00620] Particles related to the present invention can be delivered in various forms, for example, solid particles (for example, metals such as silver, gold, iron, titanium), non-metals, lipid-based solid particles, polymers, suspensions of particles, or combinations thereof. . Particles can be made from metals, dielectrics, or semiconductors, as can hybrid structures (for example, with a core and a shell). Particles of semiconductor materials can also be labeled with quantum dots if they are small enough (usually less than 10 nm) to allow quantization of the electronic levels. Such nanoparticles are used in biomedical applications as drug carriers or imaging agents and can be adapted for similar purposes within the scope of the present invention.

[00621] Were produced semi-solid and soft particles included in the scope of the present invention. The prototype particle of a semi-solid nature is the liposome. Various types of liposome particles are currently being used in clinical practice as delivery systems for anti-cancer drugs and vaccines. Half hydrophilic and half hydrophobic particles are called Janus particles and are particularly effective in stabilizing emulsions. They are capable of self-assembly at water/oil interfaces and act as solid surfactants.

[00622] US Pat. No. 8,709,843, incorporated herein by reference, proposes a drug delivery system for targeted delivery of particles containing a therapeutic agent to tissues, cells, and intracellular compartments. The present invention relates to targeted particles containing a polymer associated with a surfactant, a hydrophilic polymer or a lipid.

[00623] US Pat. No. 6,007,845, incorporated herein by reference, discloses particles having a multiblock copolymer core formed by covalently linking a multifunctional compound to one or more hydrophilic polymers and containing a biologically active material.

[00624] US Pat. No. 5,855,913, incorporated herein by reference, proposes a particle composition comprising aerodynamically light particles with a density of less than 0.4 g/cm ³ and an average diameter of 5 pm to 30 pm, having surface active substance for delivering drugs to the lung system.

[00625] U.S. Pat. No. 5,985,309, incorporated herein by reference, provides particles containing a surfactant and/or a hydrophilic or hydrophobic complex with a positively or negatively charged drug or diagnostic agent and an oppositely charged charged particle for delivery to the lungs.

[00626] US Pat. No. 5,543,158, incorporated herein by reference, discloses biodegradable injectable particles with a biodegradable solid core containing biologically active material and poly(alkylene glycol) residues on the surface.

[00627] WO2012135025 (also published as US20120251560), incorporated herein by reference, describes polymers of conjugated polyethyleneimine (PEI) and conjugated azamacrocycles (collectively referred to as "conjugated lipomer" or "lipomers"). In some embodiments, it may be contemplated that such methods and materials from documents cited herein, for example, conjugated lipomers, may be used in the context of a nucleic acid-targeted system to achieve in vitro , ex vivo , and in vivo genome changes to modify expression. genes and protein expression.

[00628] In one embodiment, the particle may be an epoxy-modified lipid polymer, preferably 7C1 (see, e.g., James E. Dahlman and Carmen Barnes et al. Nature Nanotechnology (2014) published online May 11, 2014, doi:10.1038/ nnano.2014.84). C71 was synthesized by reacting Cl5 epoxy-terminated lipids with PEI600 at a molecular ratio of 14:1 and formulated with C14PEG2000 to give particles (diameter between 35 and 60 nm) stable in PBS solution for not less than 40 days.

[00629] An epoxy-modified lipid polymer can be used to deliver the nucleic acid-targeted system of the present invention to lung, cardiovascular, or kidney cells, however, the system can be adapted by one skilled in the art for delivery to other target organs. . Dosages in the range of about 0.05 to about 0.6 mg/kg are contemplated. Dosages administered over several days or weeks are also contemplated, with a total dose of approximately 2 mg/kg.

Exosomes

[00630] Exosomes are endogenous nanovesicles that transport RNA or proteins that can deliver RNA to the brain and other target organs. To reduce immunogenicity, Alvarez-Erviti et al. (2011, Nat Biotechnol 29:341) used their own mouse dendritic cells to produce exosomes. Targeting to the brain has been achieved by dendritic cell engineering resulting in the expression of Lamp2b, an exosomal membrane protein fused to a specific peptide in RVG neurons. Purified exosomes were loaded with exogenous RKN by electroporation. GAPDH siRNA delivered by intravenous RVG-targeted exosomes to neurons, microglia, and oligodendrocytes in the brain causes specific gene knockdown. Pretreatment with RVG exosomes did not attenuate knockdown, and no nonspecific uptake was observed in other tissues. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated by strong knockdown of mRNA (60%) and protein (62%) of BACE1, a therapeutic target of Alzheimer's disease.

[00631] To obtain a population of immunologically inert exosomes, Alvarez-Erviti et al. Bone marrow samples were taken from inbred C57BL/6 mice with a homogeneous major histocompatibility complex (MHC) haplotype. Because immature dendritic cells produce large volumes of exosomes lacking T cell activators such as MHC-II and CD86, Alvarez-Erviti et al. dendritic cells were harvested using granulocyte-macrophage colony-stimulating factor (GM-CSF) for 7 days. Exosomes were purified from the culture supernatant the next day using standard ultracentrifugation protocols. The resulting exosomes were physically uniform with a size distribution peaking at 80 nm diameter as determined by particle trajectory analysis (NTA) and electron microscopy. Alvarez-Erviti et al. received 6-12 µg of exosomes (measured based on protein concentration) per 10 ⁶ cells.

[00632] Further, Alvarez-Erviti et al. explored the possibility of loading modified exosomes with exogenous cargo using electroporation protocols adapted for nanoscale applications. Since the electroporation of membrane particles at the nanoscale is not well characterized, non-specific Cy5-tagged RNA has been used empirically to optimize the electroporation protocol. The amount of packaged RNA was estimated after ultracentrifugation and exosome lysis. Electroporation at 400 V and 125 pF resulted in maximum RNA retention and was used in all subsequent experiments.

[00633] Alvarez-Erviti et al. administered 150 μg of each BACE1 siRNA packaged in 150 pg of RVG exosomes to normal C57BL/6 mice and compared the knockdown efficiency with four controls: intact mice, mice injected with RVG exosomes alone, mice injected with BACE1 siRNA in combination with the in vivo reagent cationic liposomes and mice injected with BACE1 siRNA in complex with RVG-9R, an RVG peptide bound to 9 D-arginine residues electrostatically bound to the siRNA. Cortical tissue samples were analyzed after 3 days of administration and found to have significant protein knockdown (45%, P<0.05 vs. 62%, P<0.01) in both siRNA-RVG-9R and siRNA treated exosomes. -RVG, which was due to a significant decrease in BACE1 mRNA levels (66% [+ or -] 15%, P<0.001 and 61% [+ or -] 13%, respectively, P<0.01). Moreover, Applicants demonstrated a significant reduction (55%, P<0.05) in total levels of beta-amyloid 1-42, a major component of amyloid plaques in Alzheimer's disease pathology, in animals injected with RVG exosomes. The observed decrease was superior to that for amyloid beta 1-40 in normal mice following intraventricular administration of BACE1 inhibitors. Alvarez-Erviti et al. performed rapid amplification of the 5' ends of the cDNA (RACE) for the BACE1 cleavage product, providing evidence for RNAi-mediated siRNA knockdown.

[00634] Finally, Alvarez-Erviti et al. examined whether RVG RNA exosomes elicit an immune response in vivo by measuring serum concentrations of L-6, IP-10, TNFα, and IFN-α. After injection of exosomes, minor changes were registered for all cytokines, similar to those occurring upon administration of the miRNA transfection reagent and opposite to those occurring upon treatment with miRNA-RVG-9R, which stimulates high secretion of IL-6, which confirms the immunological inertness upon administration of exosomes. Considering that exosomes contain only 20% siRNA, delivery by RVG exosomes appears to be more efficient than delivery by RVG-9R in achieving a comparable level of mRNA knockdown using 5-fold less siRNA without an appropriate level of immune stimulation. This experiment demonstrated the therapeutic potential of RVG exosome technology, which may be suitable for long-term silencing of genes associated with neurodegenerative diseases. Delivery system using exosomes according to Alvarez-Erviti et al. can be used to deliver the nucleic acid-targeted system of the present invention to therapeutic targets, especially in the case of neurodegenerative diseases. A dose in the range of about 100 to 1000 mg of a nucleic acid targeting system packaged in ~100 to ~1000 mg of RVG exosomes can be contemplated within the scope of the present invention.

[00635] El-Andaloussi et al. (Nature Protocols 7,2112-2126(2012)) have established how exosomes derived from cultured cells can be used to deliver RNA in vitro and in vivo . This protocol first describes the production of targeted exosomes by transfection of an expression vector containing an exosomal protein fused to a peptide ligand. Further El-Andaloussi et al. describe the purification and characterization of exosomes from the supernatant of transfected cells. Further El-Andaloussi et al. detail the key steps for loading RNA into exosomes. At the end of El-Andaloussi et al. briefly describe the use of exosomes for efficient in vitro and in vivo delivery of RNA to the mouse brain. Examples of expected outcomes for which exosome-mediated delivery has been quantified by functional analysis and imaging are also provided. The full protocol takes ~3 weeks. Delivery or administration according to the invention can be performed using exosomes obtained from the patient's dendritic cells. In accordance with the ideas described in the present description, this can be used in the practice of using the invention.

[00636] In another embodiment, plasma exosomes from Wahlgren et al. (Nucleic Acids Research, 2012, Vol. 40, No. 17 el30). Exosomes are nanosized vesicles (30-90 nm in size) produced by many cell types, including dendritic cells (DC), B cells, T cells, mast cells, epithelial cells, and tumor cells. Such vesicles are formed by invagination and separation of late endosomes and are then released into the extracellular environment upon fusion with the plasma membrane. Since exosomes naturally carry RNA between cells, this property may be useful for gene therapy, and based on this description, exosomes can be used in the practice of the present invention.

[00637] Exosomes from plasma can be obtained by centrifuging a fraction of platelets and white blood cells at 900g for 20 minutes to isolate plasma, followed by collection of supernatant, centrifugation at 300g for 10 minutes to remove cells, and at 16500g for 30 minutes, followed by filtered through a 0.22 mm filter. Exosomes are precipitated by ultracentrifugation at 120,000g for 70 min. Chemical transfection of miRNAs into exosomes was performed according to the manufacturer's recommendations for the human/mouse RNAi starter kit (RNAi Human/Mouse Starter Kit, Quiagen, Hilden, Germany). siRNA is added to 100 ml PBS at a final concentration of 2 mmol/ml. After adding the HiPerFect transfection reagent, the mixture is incubated for 10 min under normal conditions. To remove excess micelles, exosomes are re-isolated using aldehyde/sulfate latex beads. Chemical transfection of the nucleic acid-targeted system into exosomes can be carried out in a manner similar to that for miRNAs. Such exosomes can be co-cultured with monocytes and lymphocytes isolated from the peripheral blood of healthy donors. Therefore, it can be hypothesized that exosomes containing a nucleic acid-targeting system can be introduced into monocytes and lymphocytes and autologously reintroduced into humans. Accordingly, delivery or administration according to the invention can be carried out using plasma exosomes.

Liposomes

[00638] Delivery or administration according to the invention can be performed using liposomes. Liposomes are spherical vesicular structures composed of a single or multilayer lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes have attracted significant attention as drug delivery vehicles because they are biocompatible, non-toxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and carry their cargo across biological membranes and the blood-brain barrier (BBB). (See, for example, Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi: 10.1155/2011/469679 for a review).

[00639] Liposomes can be composed of several different types of lipids; however, phospholipids are commonly used as drug carriers for the production of liposomes. Liposomes form spontaneously when a lipid film is mixed with an aqueous solution, but this process can be accelerated by mechanical shaking with a homogenizer, sonicator or extruder (see e.g. Spuch and Navarro, Journal of Drug Delivery, vol. 2011 , Article ID 469679, 12 pages, 2011. doi: 10.1155/2011/469679 for review).

[00640] Several other additives can be added to liposomes to alter their structure and properties. For example, cholesterol or sphingomyelin can be added to the liposomal mixture to stabilize the structure of the liposomes and prevent loss of internal cargo. Further, liposomes can be prepared from hydrogenated egg phosphatidylcholine or egg phosphatidylcholine, cholesterol and diacetyl phosphate, and the average vesicle sizes can be adjusted to about 50 and 100 nm. (See, for example, Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi: 10.1155/2011/469679 for a detailed review).

[00641] The liposome dosage form may consist primarily of naturally occurring phospholipids and lipids such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg phosphatidylcholines, and monosialoganglioside. Since this dosage form is composed only of phospholipids, many problems have arisen in the creation of liposomes, including their instability in plasma. Several attempts have been made to overcome these problems, mainly by changing the composition of the lipid membrane. One of these attempts was to add cholesterol. The addition of cholesterol to conventional dosage forms slows down the release of the encapsulated biologically active substance into plasma, or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increases stability (see, for example, Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi: 10.1155/2011/469679 for review).

[00642] In a particularly preferred embodiment, the use of Trojan horse liposomes (also known as molecular Trojan horses) is desirable, and protocols can be found at http://cshprotocols.cshlp.org/content/2010 /4/pdb.prot5407.long. These particles provide delivery of the transgene to the entire brain after intravascular injection. Without limitation, neutral lipid particles with specific antibodies bound to their surface are considered to be able to cross the blood-brain barrier by endocytosis. Applicant claims that Trojan horse liposomes can be used to deliver the CRISPR family of nucleases to the brain by intravascular injection, which would allow the creation of transgenic brain animals without the need for manipulation of embryos. Approximately 1-5 g of DNA or RNA can be used for in vivo administration in liposomes.

[00643] In another embodiment, the nucleic acid-targeted system or components thereof can be delivered by liposomes, e.g., a stable nucleolipid particle (SNALP) (see e.g., Morissey et al. No. 8, August 2005). Daily intravenous injections of approximately 1, 3, or 5 mg/kg/day of a specific nucleic acid-targeted system targeted at SNALP are contemplated. Daily treatment can be given for about three days and then weekly for about five weeks. In another embodiment, the invention also provides a specific nucleic acid-targeting system embodied in SNALP administered by intravenous injection at doses of about 1 or 2.5 mg/kg (see, for example, Zimmerman et al., Nature Letters, Vol. 441, May 4, 2006). SNALP formulation may contain lipids 3-N-[(vmethoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine (PEG-C-DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a ratio of 2:40:10:48 mole percent (see, e.g., Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006).

[00644] In another embodiment, stable nucleolipid particles (SNALPs) have been shown to be effective molecules for delivery to highly vascularized liver tumors derived from HepG2, but not to poorly vascularized liver tumors derived from HCT-116 (see eg Li, Gene Therapy (2012) 19, 775-780). SNALP liposomes can be prepared by mixing D-Lin-DMA and PEG-C-DMA with disteroylphosphatidylcholine (DSPC), cholesterol and siRNA used in a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio for cholesterol/D -Lin-DMA/DSPC/PEG-C-DMA. The resulting SNALP liposomes are about 80-100 nm in size.

[00645] In yet another embodiment, the stable nucleic acid-lipid (SNALP) particles may include synthetic cholesterol (Sigma-Aldrich, St. Louis, MO, USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, Alabama, USA), 3- N-[(w-methoxy-poly(ethylene glycol)2000)carbamoyl]-1,2-dimyristoyloxypropylamine and cationic 1,2-dilyenoyloxy-3-N,N-dimethylaminopropanan (see e.g. Geisbert et al., Lancet 2010 ; 375: 1896-905). A dosage of about 2 mg/kg of the nucleic acid-targeted system may be contemplated, administered, for example, by intravenous bolus infusion.

[00646] In yet another embodiment, SNALP may include synthetic cholesterol (Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids Inc.), PEG-cDMA, and 1, 2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA) (see, for example, Judge, J. Clin. Invest. 119:661-673 (2009)). Formulations used for in vivo studies may include a final lipid/RNA weight ratio of approximately 9:1.

[00647] The safety of RNAi nanodrugs has been reviewed by Barros and Gollob of Alnylam Pharmaceuticals (see, for example, Advanced Drug Delivery Reviews 64 (2012) 1730-1737). A stable nucleolipid acid particle (SNALP) is composed of four different lipids: an ionizable lipid (DLinDMA) that is cationic at low pH, a neutral helper lipid, cholesterol, and a diffusible polyethylene glycol (PEG) lipid. The particle is approximately 80 nm in diameter and is neutrally charged at physiological pH. The ionizable lipid serves to bind the lipid to the anionic RNA during particle formation. As acidity increases within the endosome, the ionizable lipid becomes positively charged and mediates SNALP fusion with the endosome membrane, releasing RNA into the cytoplasm. The PEG-containing lipid stabilizes the particle and reduces aggregation during assembly, subsequently providing a neutral hydrophilic surface that improves pharmacokinetic properties.

[00648] To date, two clinical programs have been initiated using mixtures of SNALP with RNA. Tekmira Pharmaceuticals recently completed a phase I clinical trial using a single dose of SNALP-ApoB in adult volunteers with elevated LDL cholesterol levels. ApoB is predominantly expressed in the liver and jejunum and is required for the assembly and secretion of VLDL and LDL. Seventeen study participants received a single dose of SNALP-ApoB (gradual dose escalation over 7 levels). There was no evidence of liver toxicity (expected as potential dose-limiting toxicity based on preclinical studies). One (of two) subjects at the highest dose experienced flu-like symptoms consistent with immune system stimulation and the decision was made to terminate the trial.

[00649] Alnylam Pharmaceutcals has also been promoting ALN-TTR01, which uses the SNALP technology described above and targets hepatocyte production of mutant and wild-type TTR for the treatment of TTR amyloidosis (ATTR). Three ATTR syndromes have been described: familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC) - both caused by autosomal dominant mutations in the TTR; and senile systemic amyloidosis (SSA) caused by wild-type TTR. A phase I placebo-controlled trial in patients with ATTR has recently been completed with a single dose of ALN-TTR01 and its gradual increase. ALN-TTR01 was administered as a 15-minute intravenous infusion to 31 patients (23 study drug and 8 placebo) over a dose range of 0.01 to 1.0 mg/kg (based on siRNA). The subjects tolerated the treatment well without significant increases in liver function on tests. Infusion reactions were noted in 3 of 23 patients at doses greater than 0.4 mg/kg; all responded to a slow infusion rate and all continued to participate in the study. Minimal and transient increases in serum cytokines IL-6, IP-10, and IL-1ra were noted in two patients at the highest dose of 1 mg/kg (as expected from preclinical and primate studies). By lowering serum TTR levels, the expected pharmacodynamic effect of ALN-TTR01 administration was observed at a dose of 1 mg/kg.

[00650] In yet another embodiment of the invention, SNALP can be assembled by dissolving the cationic lipid, DSPC, cholesterol and PEG lipid, for example, in ethanol, for example, in a molar ratio of 40:10:40:10, respectively (see, for example, Semple et al., Nature Biotechnology, Volume 28 Number 2 February 2010, pp. 172-177). The lipid mixture was added to an aqueous buffer (50 mM citrate, pH 4) to reach a final concentration of ethanol and lipids of 30% (v/v) and 6.1 mg/mL, respectively, and was equilibrated at 22°C for 2 minutes before extrusion. Hydrated lipids were extruded through two stacked 80 nm pore filters (Nuclepore) at 22° C. using a Lipex Extruder (Northern Lipids) until a vesicle diameter of 70-90 nm was reached, as determined by dynamic light scattering analysis. This usually required 1-3 passes. siRNA (dissolved in 50 mM citrate, pH 4 aqueous solution containing 30% ethanol) was added to the pre-equilibrated (35° C.) vesicles at a rate of 5 ml/minute with mixing. After reaching a final target siRNA/lipid ratio of 0.06 (w/w), the mixture was incubated for 30 minutes at 35° C. to allow for vesicle reorganization and siRNA encapsulation. The ethanol was then removed and the external buffer changed to PBS (155 mM NaCl, 3 mM Na2HPO4, 1 mM KH2PO4, pH 7.5) either by dialysis or tangential flow filtration. siRNA was encapsulated from SNALP using a controlled stepwise dissolution process. The lipid components of KC2-SNALP were DLin-KC2-DMA (cationic lipid), dipalmitoylphosphatidylcholine (DPPC; Avanti Polar Lipids), synthetic cholesterol (Sigma), and PEG-C-DMA in a molar ratio of 57.1:7.1:34.3 :1,4. After the formation of loaded particles SNALP were dialyzed against PBS, the filtrate was sterilized through a 0.2 μm filter before use. The average particle sizes were 75-85 nm and 90-95% of the siRNAs were encapsulated in lipid particles. The final ratio of siRNA/lipid in the formulations used for in vivo testing was ~0.15 (w/w). LNP-siRNA systems containing anti-factor VII siRNA were immediately dissolved to appropriate concentrations in sterile PBS prior to use, and formulations were administered intravenously via the lateral tail vein in a total volume of 10 ml/kg. This method and these delivery systems can be extrapolated to the nucleic acid targeting system of the present invention.

[00651] Other cationic lipids such as the aminolipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA) can be used to encapsulate the nucleic acid-targeting system, components of that system, or nucleic acid molecule(s) encoding components of this system, such as miRNA-like (see, for example, Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533) and, therefore, can be used in the practice of the invention. A preformed vesicle with the following lipid composition may be considered: aminolipid, distearoylphosphatidylcholine (DSPC), cholesterol, and (R)-2,3-bis(octadecyloxy)propyl-1-(methoxypoly(ethylene glycol)2000)propylcarbamate (PEG lipid) in a molar ratio of 40/10/40/10, respectively, and a coagulation factor VII (FVII) mRNA/total lipid ratio of approximately 0.05 (w/w). To ensure a narrow particle size distribution in the 70-90 nm range and a low polydispersity index of 0.11+0.04 (n=56), particles can be extruded up to three times through 80 nm membranes prior to the addition of guide RNA. Can be used particles containing a potent amino lipid 16 in which the molar ratio of the four lipid components is 16, DSPC, cholesterol and PEG lipid (50/10/38.5/1.5), which can be further optimized to increase activity in vivo .

[00652] Michael S D Kormann et al. ("Expression of therapeutic proteins after delivery of chemically modified mRNA in mice: Nature Biotechnology, Volume:29, Pages: 154-157 (2011)) describe the use of lipid envelopes for RNA delivery. The use of lipid envelopes is also preferred in the present invention.

[00653] In another embodiment, lipids can be mixed with the nucleic acid-targeted system of the present invention, its component(s), or nucleic acid(s) molecule(s) encoding such(s) to form lipid nanoparticles (LNP). Lipids include, but are not limited to, DLin-KC2-DMA4, C12-200, and disteroylphosphatidylcholine colipids, cholesterol, and PEG-DMG can be mixed with an RNA-targeted system in place of siRNA (see, e.g., Novobrantseva, Molecular Therapy-Nucleic Acids (2012 ) I, e4; doi:10.1038/mtna.2011.3) using the spontaneous vesicle formation procedure. The molar ratio of the components may be about 50/10/38.5/1.5 (DLin-KC2-DMA or C12-200/disteroylphosphatidylcholine/cholesterol/PEG-DMG). The final lipid:RNA weight ratio can be 12:1 and 9:1 for lipid particles (LNP) with DLin-KC2-DMA and C12-200, respectively. Such compositions can contain particles with an average diameter of ~80 nm with a capture efficiency of >90%. A dose of 3 mg/kg may be considered.

[00654] Tekmira has a portfolio of approximately 95 patent families in the US and other countries relating to various aspects of lipid nanoparticles (LNPs) and mixtures thereof (see, for example, US Pat. Nos. 7,982,027; 7,799,565; 8,058,069; 7745651; 7803397; 8101741; 8188263; 7915399; 8236943 and 7838658 and European patents No. 1766035; 1519714; 1781593 and 1664316), all can be used and/or adapted for use in the present invention.

[00655] The nucleic acid-targeting system or components thereof, or the molecule(s) encoding(s) thereof, can be delivered packaged in PLGA microspheres in the manner described in U.S. Patent Publications 20130252281, 20130245107 and 20130245107 and 20130244279 (attributed to Modema Therapeutics) relating to aspects of formulating compositions comprising modified nucleic acid molecules that may encode a protein, a protein precursor, or a partially or fully truncated form of a protein or protein precursor. The mixture may have a molar ratio of 50:10:38.5:1.5:3.0 (cationic lipid: fusogenic lipid: cholesterol: PEG lipid). The PEG lipid may be selected from, but not limited to, PEG-c-DOMG, PEG-DMG. The fusogenic lipid may be DSPC. See also Schrum et aJ., Delivery and Formulation of Engineered Nucleic Acids, published Supplement US 20120251618.

[00656] Nanomerics technologies are designed to overcome the challenges associated with bioavailability for a wide range of therapeutic applications, including small molecule hydrophobic drugs, peptides and nucleic acid-based drugs (plasmids, miRNAs, microRNAs). Specific routes of administration for which significant advantages of this technology have been shown include oral administration, transport across the blood-brain barrier, delivery to solid tumors, and the eye. See, for example, Mazza et al., 2013, ACS Nano. 2013 Feb 26;7(2): 1016-26; Uchegbu and Slew, 2013, J Pharm Sci. 102(2):305-10 and Lalatsa et al., 2012, J Control Release, 2012 Jul 20; 161(2):523-36.

[00657] US Patent Application Publication No. 20050019923 describes cationic dendrimers for delivering bioactive molecules, such as polynucleotide, peptide or polypeptide molecules, and/or pharmacological agents, into the body of a mammal. Such dendrimers are suitable for directing the delivery of bioactive molecules to, for example, the liver, spleen, lungs, kidney or heart (or even the brain). Dendrimers are three-dimensional macromolecules obtained in several stages from simple branched monomer units, the nature and functionality of which can be easily controlled and changed. Dendrimers are synthesized by repeated addition of structural units to the multifunctional core (divergent synthesis method) or their extension towards the multifunctional core (convergent synthesis method), and each addition of structural units to the three-dimensional shell leads to the formation of the next generation of dendrimers. Polypropyleneimine dendimers are formed starting from a diaminobutane core, to which twice as many amino groups are attached in a Michael reaction with the addition of acrylonitrile to primary amines and subsequent hydrogenation of nitriles. This leads to a doubling of the number of amino groups. Polypropyleneimine dendrimers contain 100% protonated nitrogens and up to 64 terminal amino groups (generation 5, DAB 64). The protonable groups are usually amino groups which are capable of accepting protons at neutral pH. The use of dendrimers as gene delivery agents has largely focused on the use of polyamidoamine, and phosphorus-containing compounds with a mixture of amines/amides or N-P(02)S as conjugating units, respectively, with no reports of previous generations of polypropyleneimine dendrimers being used for gene delivery. . Polypropyleneimine dendrimers have also been investigated as pH sensitive controlled release systems for drug delivery and packaging as guest molecules when chemically modified with peripheral amino acid groups. The cytotoxicity and interaction of polypropyleneimine dendrimers with DNA, as well as the transfection efficiency of DAB 64, were also studied.

[00658] U.S. Patent Publication No. 20050019923 is based on the observation that, contrary to earlier reports, cationic dendrimers such as polypropyleneimine dendrimers exhibit suitable properties, such as specific targeting and low toxicity, for use in targeted delivery of bioactive molecules. such as molecules of genetic material. In addition to this, derivatives of cationic dendrimers also show suitable properties for targeted delivery of bioactive molecules. See also Bioactive Polymers, U.S. Published Application 20080267903, which describes: "Various polymers, including cationic polyamine polymers and dendrimer polymers, have been shown to have anti-proliferative activity, and therefore may be useful in the treatment of diseases characterized by unwanted cell proliferation, such as such as neoplasms and tumors, inflammatory diseases (including autoimmune diseases), psoriasis and atherosclerosis Such polymers can be used either alone as active agents or as a "container" for the delivery of other therapeutic agents such as drug molecules or nucleic acids for gene therapy In such cases, the polymer's inherent antitumor activity may complement that of the delivered agent.The statements of this patent publication may be used in conjunction with the teachings herein to deliver a nucleic acid-targeted system. theme(s) or component(s) thereof, or a nucleic acid molecule(s) encoding such.

Supercharged Proteins

[00659] Supercharged proteins are a class of engineered or naturally occurring proteins with exceptionally high positive or negative net theoretical charge that can be used to deliver a nucleic acid-targeted system(s), or component(s), or molecule(s) thereof. nucleic acid that encodes them. Proteins carrying both superpositive and supernegative charges show outstanding resistance to thermally or chemically induced aggregation. Superpositively charged proteins are also able to enter mammalian cells. Binding to these proteins of a cargo, such as plasmid DNA, RNA, or other proteins, may allow functional delivery of these macromolecules to mammalian cells in vitro and in vivo . The laboratory led by David Liu reported the creation and characterization of supercharged proteins in 2007 (Lawrence et. al., 2007, Journal of the American Chemical Society 129, 10110-10112).

[00660] Non-viral delivery of plasmid RNA and DNA into mammalian cells is of value for both research and therapeutic applications (Akinc et al., 2010, Nat. Biotech. 26, 561-569). The purified +36 GFP protein (or other super positively charged protein) was mixed with RNA on appropriate nutrient media without serum and left to form complexes before being added to cells. The addition of serum at this stage inhibits the formation of supercharged protein-RNA complexes and reduces the effectiveness of the treatment. The protocol below has demonstrated efficacy in a number of cell lines (McNaughton et al., 2009, Proc. Natl. Acad. Sci, USA 106, 6111-6116). However, experimental experiments altering the dose of protein and RNA must be performed in order to optimize the procedure for specific cell lines. However, trial experiments with protein and RNA dose changes should be performed to optimize the use of the procedure for specific cell lines.

(1) One day before treatment, 1×10 ⁵ cells per well were seeded in a 48-well plate.

(2) On the day of treatment, dissolve the purified +36 GFP protein in nutrient media without serum to a final concentration of 200 nM. RNA is added to a final concentration of 50 nM. Mix with a shaker and incubate at room temperature for 10 minutes.

(3) During incubation, remove the cell culture medium and wash with PBS once.

(4) After +36 GFP incubation with RNA, protein-RNA complexes are added to the cells.

(5) Incubate cells with complexes at 37°C for 4 hours.

(6) After incubation, culture media are removed and washed three times with a solution of heparin in PBS (20 U/ml). Incubate cells with serum-containing nutrient media for 48 hours or more, depending on the activity assay.

(7) Cells are analyzed by immunoblotting, q-PCR, phenotype analysis, or other appropriate method.

[00661] The laboratory led by David Liu has also found that the +36 GFP protein is an effective agent for delivering plasmids to various cells. Because plasmid DNA is a larger load than siRNA, a proportionately larger amount of +36 GFP proteins is required for effective complexation with plasmids. For efficient plasmid delivery, delivery applicants have developed a +36 GFP variant carrying the C-terminal tag of HA2, a peptide known to disrupt endosomes derived from influenza virus hemagglutinin protein. The following protocol has been shown to be effective in a range of cells, however, as recommended above, doses of plasmid DNA and supercharged protein need to be optimized for specific cell lines and different delivery method applications.

(1) The day before treatment, seed 1×10 ⁵ cells per well in a 48-well plate.

(2) On the day of treatment, dissolve the purified protein+36 GFP in culture media without serum to a final concentration of 2 nM. Add 1 mg of plasmid DNA. Mix with a shaker and incubate at room temperature for 10 minutes.

(3) During the incubation, cell culture media are removed and washed with PBS once.

(4) After incubation with +36 GFP and plasmid DNA, the protein-DNA complexes are carefully added to the cells.

(5) Incubate cells with complexes at 37°C for 4 hours.

(6) After incubation, remove the culture media and wash the cells with PBS. Cells are incubated in serum-containing nutrient media and incubated for a further 24-48 hours.

(7) Analyze plasmid delivery (eg, by plasmid-driven gene expression) as appropriate.

[00662] See also, for example, McNaughton et al., Proc. Natl. Acad. sci. USA 106, 6111-6116 (2009); Cronican et al., ACS Chemical Biology 5, 747-752 (2010); Cronican et al., Chemistry & Biology 18, 833-838 (2011); Thompson et al., Methods in Enzymology 503, 293-319 (2012); Thompson, D.B., et al., Chemistry & Biology 19 (7), 831-843 (2012). Supercharged protein methods can be used and/or adapted to deliver the nucleic acid-targeted system of the present invention. Such systems Dr. Lui and the documents cited herein, in conjunction with the teachings set forth herein, can be used in the delivery of a nucleic acid-targeted system(s) or component(s) or encoding nucleic acid molecule(s).

Cell Penetrating Peptides (CPP)

[00663] In yet another embodiment, the use of cell penetrating peptides (CPPs) to deliver a CRISPR-Cas system is contemplated. CPPs are short peptides that facilitate the capture of various molecular cargoes (from nanosized particles to small chemical molecules and large DNA fragments) by cells. The term "cargo" as used herein includes, but is not limited to, consisting of therapeutic agents, diagnostic probes, peptides, nucleic acids, antisense oligonucleotides, plasmids, proteins, particles including nanoparticles, liposomes, chromophores, small molecule compounds, and radioactive materials. Aspects of the invention also contemplate that the payload may also include any component of a CRISPR-Cas system or an entire functional CRISPR-Cas system. In one aspect, the invention relates to the following methods of delivering a desired cargo to an individual, including: (a) obtaining a complex comprising a peptide that is able to penetrate cells and the desired cargo, and (b) oral, intra-articular, intraperitoneal, intrathecal, intra-arterial, intranasal, intraparenchymal, subcutaneous, intramuscular, intravenous, dermal, rectal or topical administration of the complex to an individual. The cargo is linked to the peptides through chemical bonding (covalent) or through non-covalent interactions.

[00664] The function of cell penetrating peptides (CPPs) is to deliver cargo to cells, a process that is typically accomplished by endocytosis of cargo delivered to the endosomes of living mammalian cells. Cell-permeating peptides (CPPs) vary in size, amino acid sequence, and charge, but all CPPs share one distinctive feature, which is the ability to move across the plasma membrane and facilitate the delivery of various molecular cargoes to the cytoplasm or organelles. Such movement of CPPs can be divided into three main groups according to entry mechanisms: direct membrane entry, endocytosis-mediated entry, and transitional movement. CPPs have found numerous uses in medicine as drug delivery agents in the treatment of various diseases, including anti-cancer agents and agents for the treatment of viral diseases, as well as contrast agents for cell labeling. Examples of the latter include the use of GFP as a carrier, MRI contrast agents or quantum dots. CPPs have great potential as both in vitro and in vivo delivery vectors for research and use in medicine. CPPs typically either have an amino acid composition with a high relative content of positively charged amino acids such as lysine or arginine, or contain sequences having an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphiphilic, respectively. The third class of CPPs are hydrophobic peptides containing only non-polar residues with a low net charge or having hydrophobic amino acid groups that are essential for cellular uptake. One of the first CPPs discovered is the human immunodeficiency virus 1 (HIV-1) transactivating transcriptional activator (Tat), which is efficiently captured from culture media by numerous cultured cell types. More recently, the number of known CPPs has increased significantly, and small molecular synthetic analogs have been obtained that are more efficient at protein transduction. CPPs include, but are not limited to: penetratin, Tat (48-60), transportan, and (R-AhX-R4) (Ahx=aminohexanoyl).

[00665] US Pat. No. 8,372,951 describes a CPP derived from an eosinophilic cationic protein (ECP) that exhibits high cell entry efficiency and low toxicity. The features of CPP delivery with a load in representatives of vertebrates are also described. Future aspects of CPPs and their delivery are described in US Pat. Nos. 8,575,305; 8614194 and 8044019. CPP can be used to deliver the CRISPR-Cas system or its components. The use of CPP to deliver the CRISPR-Cas system or its components is also described in the manuscript "Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA", by Suresh Ramakrishna, Abu-Bonsrah Kwaku Dad, Jagadish Beloor, et al. Genome Res. 2014 Apr 2, [Epub in press], incorporated herein by reference in its entirety, demonstrating that treatment with recombinant CPP-conjugated Cas9 protein and guide RNAs in complex with CPP results in endogenous gene disorders in cell lines person. In the article, the Cas9 protein was linked to CPP via a thioether bond, while the guide RNA was complexed with CPP to form dense, positively charged particles. Simultaneous and sequential treatment of human cells, including embryonic stem cells, skin fibroblasts, HEK293T cells, HeLa cells, and embryonic carcinoma cells, with modified Cas9 and guide RNA has been shown to lead to efficient gene changes with fewer off-target mutations compared to transfection with plasmids. .

Implantable Devices

[00666] In another embodiment of the invention, implantable devices are also provided for delivery of a nucleic acid-targeted system, component(s) thereof, or nucleic acid molecule(s) encoding them. For example, US Patent Application Publication 20110195123 describes an implantable medical device that locally elutes a drug and resides in a person for a long time, including several types of such device, methods of treatment, and methods of implantation. A device including a polymeric substrate such as a matrix, for example, which is used as the body of the device, and a drug and, in some cases, additional scaffold materials such as metals or additional polymers and materials to increase visibility and imaging. An implantable device may be preferred for local drug delivery over an extended period, where the drug is delivered directly to the extracellular matrix (ECM) of a diseased area such as a tumor, an area of inflammation, degeneration, or for symptomatic treatment, or to injured smooth muscle cells, or for prevention. One type of drug is RNA, as described above, and this system can be used/and or adapted for the nucleic acid-targeted system of the present invention. Implantation methods in some embodiments of the invention are existing implantation methods that have been developed and are currently used for other treatments, including brachytherapy and needle biopsy. In such cases, the dimensions of the new implant described in this invention are similar to the original implant. Typically, multiple devices can be implanted during a single treatment session.

[00667] U.S. Patent Publication 20110195123 describes a drug that provides an implantable or insertion system, including systems applicable to cavities such as the abdomen and/or any other type of administration, in which the drug delivery system is loose or attached, containing biostable and/or degradable and/or bioabsorbable polymeric substrate, which in some cases may be, for example, a matrix. It should be noted that the term "installation" also includes implantation. The drug delivery system is preferably implemented as a "Loder" as described in US Patent Publication 20110195123.

[00668] The polymer or a plurality of polymers are biocompatible, comprising an agent and/or a plurality of agents, which allows the release of the agent at a controlled rate, and the total volume of the polymeric substrate, such as a matrix, for example, in some embodiments, optionally and preferably does not exceed the maximum volume, which allows reaching the therapeutic level of the agent. As a non-limiting example, such a volume is preferably in the range from 0.1 m ³ to 1000 mm ³ as required by the volume for loading the agent. The "Loder" may be larger in some cases, such as when included in a device whose size is determined by functionality, such as, but not limited to, a knee joint, a cervical ring, and the like.

[00669] The drug delivery system (for formulation delivery) is designed in some embodiments to preferably use degradable polymers, where the primary release mechanism is volumetric erosion; or in some embodiments, insoluble or slowly degrading polymers are used in which the primary release mechanism is diffusion rather than bulk erosion, such that the outer portion functions as a membrane and its inner portion functions as a drug reservoir that is substantially unaffected by the environment over a long period of time (for example, from a week to several months). Combinations of different polymers with different release mechanisms can also be used. The surface concentration gradient is preferably effectively kept constant for a significant portion of the total drug release period, and therefore the diffusion rate is effectively constant (referred to as "zero mode" diffusion). By "constant" is meant a diffusion rate that is preferably maintained above the lower threshold of therapeutic efficacy, but which may still in some cases have an initial spike and/or may fluctuate, eg increase and decrease to a certain extent. The diffusion rate is preferably maintained over a long period of time and may be considered constant to a certain level in order to optimize the therapeutically effective period, eg the effective period of gene silencing.

[00670] The drug delivery system is optionally and preferably designed to protect the nucleotide-based therapeutic agent from degradation, whether chemical in nature or due to the action of enzymes and other factors in the body of the individual.

[00671] The drug delivery system described in US Patent Publication 20110195123 is in some cases associated with sensing and/or activating devices that operate during and/or after device implantation via non-invasive and/or minimally invasive activation and/or acceleration/deceleration, for example, optionally including, but not limited to, heating and cooling, laser beams, and ultrasound, including focused ultrasonic and/or radio frequency (RF) methods or devices.

[00672] According to some embodiments of U.S. Patent Publication 010195123, the area for local delivery may optionally include target areas characterized by high abnormal cell proliferation and suppressed apoptosis, including tumors, active and chronic inflammations and infections, including autoimmune diseases, tissue degeneration, including muscle and nervous tissue, chronic pain, areas of degeneration and sites of bone fractures and other wounds to improve tissue regeneration and damaged cardiac, smooth and striated muscles.

[00673] The area for implantation of the composition, or target area, is preferably a radius, area and/or volume that is small enough for targeted local delivery. For example, the target area arbitrarily has a diameter in the range of about 0.1 mm to about 5 cm.

[00674] The location of the target area should be chosen preferably for maximum therapeutic efficacy. For example, the drug delivery system composition (optionally with an implantation device as described above) is optionally and preferably implanted directly into the tumor itself or its immediate environment or associated blood vessels.

[00675] For example, the composition (optionally together with the device) is optionally implanted directly into or near the pancreas, prostate, mammary glands, liver, through the nipple, into the vascular system, etc.

[00676] The position of the target region may be selected from the group including, essentially consisting of, or consisting of the following examples (non-limiting, as any body site may be suitable for Loder implantation): 1. degenerative areas of the brain in the basal ganglia, white and gray substance in Parkinson's or Alzheimer's disease, 2. spine, for example in the case of amyotrophic lateral sclerosis (ALS); 3. cervix to prevent human papillomavirus (HPV) infection; 4. joints with acute and chronic inflammation; 5. skin, for example in the case of psoriasis; 6. sympathetic and sensory nerves to obtain an analgesic effect; 7. intraosseous implantation; 8. areas of acute and chronic infection; 9. intravaginal implantation; 10. inner ear - auditory system, inner ear labyrinth, vestibular system; 11. intratracheal implantation; 12. intracardiac, coronary, epicardial implantation; 13. implantation in the bladder; 14. implantation in the biliary system; 15. parenchymal tissue, including, but not limited to, kidney, liver, spleen; 16. lymph nodes; 17. salivary glands; 18. gums; 19. intra-articular implantation (in the joints); 20. intraocular implantation; 21. brain tissue; 22. ventricles of the brain; 23. cavities, including the abdominal cavity (including, but not limited to, ovarian cancer); 24. inside the esophagus and 25. rectally.

[00677] In some cases, the introduction of a system (for example, a device containing a composition) involves the injection of material into the extracellular matrix at and near the target area to change the local pH value, and / or temperature, and / or other biological factors affecting on the diffusion of the drug and / or pharmacokinetics in the extracellular matrix of the target area and near it.

[00678] In some cases, according to some embodiments of the invention, the release of the specified agent may be associated with sensors and/or activating devices that are controlled before and/or during and/or after implantation, non-invasive and/or minimally invasive and/ or other activation methods and/or acceleration/deceleration methods, including the use of laser beams, radiation, heating and cooling, and ultrasound, including focused ultrasound and/or radio frequency methods or devices, as well as chemical activators.

[00679] According to other embodiments of the invention described in US patent publication 20110195123, the preparation used preferably includes RNA, for example for localized cases of breast, pancreas, brain, kidney, bladder, lung and prostate cancers, as described below. Although RNA interference is cited as an example, many drugs are useful when encapsulated in "Loder", and can be used in conjunction with this invention, provided that such drugs themselves can be encapsulated in the "Loder" substrate, for example, such as a template, and this system can be used and/or adapted to deliver the nucleic acid-targeted system of the present invention.

[00680] As another example of a specific application, nerve and muscle degenerative diseases that develop due to abnormal gene expression can be cited. Local delivery of RNA may have a therapeutic property to alter abnormal gene expression. Local delivery of anti-apoptotic, anti-inflammatory and anti-degenerative drugs, including small molecule drugs and macromolecules, may also be therapeutic. In such cases, "Loder" is applied for sustained release at a constant rate and/or through a specialized device that is implanted separately. All of these can be used and/or adapted for the nucleic acid targeting system of the present invention.

[00681] As another example of a specific application, psychiatric and cognitive diseases that can be treated with gene activity modifiers can be given. Gene knockdown is a treatment option for these diseases. Loders, which topically deliver therapeutic agents to central nervous system sites, are therapeutic options for the treatment of psychiatric and cognitive illnesses including, but not limited to, psychosis, bipolar disorders, neurotic illnesses, and behavioral disorders. Loders can also deliver drugs topically, including small molecule drugs and macromolecules after implantation in specific areas of the brain. All of these can be used and/or adapted for the nucleic acid targeting system of the present invention.

[00682] Another example of a specific application is the suppression of the expression of innate and/or adaptive immune mediators in local areas, which helps prevent transplant rejection. Local delivery of RNA and immunomodulatory reagents using Loder implanted in the transplanted organ and/or site of implantation promotes local immune suppression by reflecting immunocytes, such as CD, activated against the transplanted organ. All of these can be used/and adapted for the nucleic acid targeting system of the present invention.

[00683] As another example of a specific application, vascular growth factors, including VEGF, angiogenin, and others required for neovascularization, can be given. Local delivery of factors, peptides, peptidomimetics or suppression of their repressors is an important therapeutic approach; suppression of repressors and local delivery of factors, peptides, macromolecules and small molecules of drugs that stimulate angiogenesis using "Loder" is therapeutic for peripheral, systemic and cardiovascular diseases.

[00684] The mode of administration, such as implantation, may already be used for other types of tissue implantation and/or for the administration and/or sampling of tissues, in some cases without modification, or alternatively in some cases with only minor modifications in such ways. Such methods include, but are not limited to, brachytherapy, biopsy, endoscopy with or without the use of ultrasound, such as retrograde cholangiopancreatography (ERCP,) stereotaxic methods for brain tissues, laparoscopy, including laparoscope implantation in joints, abdominal organs, bladder wall and body cavity.

[00685] The implantable device technology discussed herein may be used within the scope of the present invention and, therefore, with this information and knowledge in the art, the CRISPR-Cas system, its components, their nucleic acid molecules, or the components encoding or providing them may be delivered via an implantable device.

Patient-specific screening methods

[00686] A nucleic acid targeting system that targets RNA, eg, trinucleotide repeats, can be used to screen patients or patient cell/tissue samples for the presence of such repeats. Repeats can be targeted by an RNA targeting system for nucleic acid, and if there is binding to it by the targeting system for nucleic acid, this binding can be detected, thereby indicating that such a repeat is present. Thus, the nucleic acid targeting system can be used to screen patients or patient cell/tissue samples for repeats. The appropriate compound(s) may then be administered to the patient to treat the condition; or a nucleic acid targeting system can be introduced for binding that results in an insertion, deletion, or mutation and alleviation of the condition.

[00687] The invention relates to nucleic acids for binding target RNA sequences.

CRISPR effector protein mRNA and guide RNA

[00688] CRISPR effector protein mRNA and guide RNA can also be delivered separately. CRISPR effector protein mRNA can be delivered upstream of the guide RNA to allow time for CRISPR protein expression. CRISPR effector protein mRNA can be administered 1-12 hours (preferably about 2-6 hours) prior to administration of the guide RNA.

[00689] Alternatively, the CRISPR effector protein mRNA and guide RNA can be administered together. Preferably, a second booster dose of guide RNA can be administered 1-12 hours (preferably about 2-6 hours) after initial administration of the CRISPR effector protein+guide RNA mRNA.

[00690] The CRISPR effector protein of the present invention, namely the C2c1 or C2c3 effector protein, is sometimes referred to herein as the CRISPR enzyme. It is understood that an effector protein is based on or derived from an enzyme, so the term "effector protein" certainly includes "enzyme" in some embodiments of the invention. However, it should also be understood that the effector protein may, as required in some embodiments, bind DNA or RNA, but not necessarily cut or introduce a single strand break, which is a function of the "dead" Cas effector protein.

[00691] Additional introduction of CRISPR effector protein mRNA and/or guide RNA may be beneficial to achieve the most efficient levels of genome modification. In some embodiments, the phenotypic change preferably results from genome modification when the target is a genetic disease, especially in therapies, and preferably when a repair matrix is provided to correct or change the phenotype.

[00692] In some embodiments, diseases that may be targeted include diseases that are associated with splicing defects.

[00693] In some embodiments, cellular targets include hematopoietic stem/progenitor cells (CD34+); human T cells; and retinal cells - for example, photoreceptor progenitor cells.

[00694] In some embodiments, target genes include: human beta globin gene - HBB (for the treatment of sickle cell anemia, including stimulation of gene conversion (using a closely related HBD gene as an endogenous template)); CD3 (T cells) and CEP920 - retina (eyes).

[00695] In some embodiments, the target diseases also include: malignant tumor; sickle cell anemia (based on point mutation); HIV; beta thalassemia; and ophthalmic or ocular diseases such as Leber's amaurosis (LCA) caused by a splicing defect.

[00696] In some embodiments, delivery methods include: cationic lipid-mediated "direct" delivery of an enzyme-guide complex (ribonucleoprotein) and electroporation of plasmid DNA.

[00697] The methods of the invention may further include the delivery of matrices, such as repair matrices, which may be dsODN or ssODN, see below. Delivery of the templates can be made simultaneously or separately from the delivery of any or all CRISPR effector proteins or targeting molecules, and through the same delivery mechanism or any other. In some embodiments, it is preferred that the template be delivered together with a targeting molecule and also preferably a CRISPR effector protein. An example is the AAV vector.

[00698] The methods of the invention may further comprise: (a) delivering into the cell a double-stranded oligodeoxynucleotide (dsODN) comprising overhangs complementary to the overhangs created by said double strand break, wherein said dsODN is inserted into the target locus, or (b) delivering a single-stranded oligodeoxynucleotide (ssODN) into the cell, wherein said ssODN acts as a template for homologous directed double strand break repair. The methods of the invention may be for the prevention or treatment of a disease in an individual when said disease is caused by a defect in said target locus. The methods of the invention can be carried out in vivo in an individual or ex vivo on a cell taken from an individual, where in some cases the specified cell is returned to the individual.

[00699] To minimize toxicity and non-specific effects, it is important to control the concentration of delivered CRISPR effector protein mRNA and guide RNA. Optimal concentrations of CRISPR effector protein mRNA and guide RNA can be determined by testing different concentrations in a cellular or animal model and using deep sequencing to analyze the degree of modification at potentially non-target genomic loci. For example, for a targeting sequence targeting 5'-GAGTCCGAGCAGAAGAAGAA-3' in the EMX1 gene of the human genome, deep sequencing can be used to assess the level of modification at the following two non-targeted loci: 1: 5'-GAGTCCTAGC AGGAGAAG AA-3' and 2: 5'-GAGTCTAAGCAGAAGAAAGAA-3'. The concentration that gives the highest level of target modification while minimizing the level of off-target modification should be chosen for in vivo delivery.

Inducible systems

[00700] In some embodiments, the CRISPR effector protein may be a component of the inducible system. The inducible nature of the system allows spatiotemporal control of gene editing or gene expression using energy. The form of energy may include, but is not limited to, electromagnetic radiation, sound energy, chemical energy, and thermal energy. Examples of an inducible system include stimulating tetracycline promoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptional activation systems (FKBP, ABA, etc.) or light-inducible systems (phytochrome, LOV domains, or cryptochrome). In one embodiment, the CRISPR effector protein may be part of a light-induced transcription effector (LITE) to directly alter transcriptional activity in a sequence-specific manner. The light-inducible components may include a CRISPR effector protein, a light-sensitive cytochrome heterodimer (eg, Arabidopsis thaliana ), and a transcriptional activation/repression domain. Other examples of inducible DNA binding proteins and methods of their use are given in US 61/736465 and US 61/721283 and WO 2014018423 A2, which are incorporated herein by reference in their entirety.

Self-inactivating systems

[00701] Once all copies of a gene in a cell's genome have been edited, continued expression of the CRISPR/C2c1 or C2c3 effector protein in that cell is no longer required. Indeed, long-term expression is undesirable in terms of off-target effects on non-targeted regions of the genome, etc. Thus, time-limited expression is useful. One approach is inducible expression, but in addition, Applicants have constructed a self-inactivating CRISPR system that relies on the use of a non-coding targeting sequence in the CRISPR vector itself. Thus, after the start of expression, the CRISPR system will perform its own destruction, but before the destruction is complete, the system will have time to edit the genomic copies of the target gene (which, in the case of a normal point mutation in a diploid cell, requires no more than two edits). A simply self-inactivating CRISPR-Cas system includes an additional RNA (i.e. guide RNA) that targets the coding sequence of the CRISPR effector protein itself or targets one or more non-coding target guide sequences that are complementary to unique sequences found in one or more of the following terms:

(a) in a promoter driving the expression of non-coding RNA elements,

(b) in a promoter driving the expression of a C2c1 or C2c3 effector protein gene,

(c) in 100 b.p. from the ATG translation start codon in the sequence encoding the C2c1 or C2c3 effector protein,

(d) in an inverted terminal repeat (iTR) of a viral delivery vector, eg in the AAV genome.

[00702] In addition, such RNA can be delivered via a vector, for example, a separate vector or the same vector that encodes the CRISPR complex. When delivered via a single vector, the CRISPR RNA targeted for Cas expression can be administered sequentially or simultaneously. When administered sequentially, the CRISPR RNA targeted for Cas expression must be delivered after the CRISPR RNA that is destined for, for example, gene editing or genetic engineering. This period may last minutes (eg, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes). This period may last for hours (eg, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours). This period can last days (eg, 2 days, 3 days, 4 days, 7 days). This period may last weeks (eg, 2 weeks, 3 weeks, 4 weeks). This period can last for months (eg 2 months, 4 months, 8 months, 12 months). This period can last years (2 years, 3 years, 4 years). Where the guide RNA targets sequences encoding Cas protein expression, the effector protein becomes an obstacle and the system becomes self-inactivating. In the same way, CRISPR RNA targeting Cas expression applied through, for example, liposomes, lipofection, particles, microvesicles, as described herein, can be administered sequentially or simultaneously. Similarly, self-inactivation can be used to inactivate one or more guide RNAs used to target one or more targets.

[00703] In some aspects, a single gRNA is provided that is capable of hybridizing downstream of the start codon of the CRISPR effector protein, such that loss of expression of the CRISPR effector protein is observed after a period of time. In some embodiments, one or more gRNAs are provided that are capable of hybridizing to one or more coding or non-coding regions of a polynucleotide encoding a CRISPR-Cas system, resulting in inactivation of one or more, or in some cases all, CRISPR-Cas systems after a period of time. . In some aspects of the system, and without being limited by theory, a cell may include a plurality of CRISPR-Cas complexes, where the first subset of CRISPR complexes includes a first guide RNA capable of targeting a genomic locus or loci for editing, and the second subset of CRISPR complexes includes at least one a second guide RNA capable of targeting a polynucleotide encoding the CRISPR-Cas system, wherein the first subset of CRISPR-Cas complexes mediate editing of the target genomic locus or loci and the second subset of CRISPR complexes ultimately inactivate the CRISPR-Cas system, thereby inactivating further expression of CRISPR-Cas in the cell.

[00704] The invention thus relates to a CRISPR-Cas system comprising one or more vectors for delivery to a eukaryotic cell, wherein the vector(s) encodes: (i) a CRISPR effector protein; (ii) a first guide RNA capable of hybridizing to a target sequence in a cell; (iii) a second guide RNA capable of hybridizing to one or more target sequences in a vector encoding a CRISPR effector protein, thus differing only in the guide sequence, wherein, when expressed in a cell, the first guide RNA directs the sequence-specific binding of the first CRISPR complex to the sequence a target in the cell, the second guide RNA directs the sequence-specific binding of the second CRISPR complex to the target sequence in the vector that encodes the CRISPR effector protein; the CRISPR complexes include a CRISPR effector protein associated with the guide RNA so that the guide RNA can hybridize to its target sequence; and the second CRISPR complex inactivates the CRISPR-Cas system to prevent continued expression of the CRISPR effector protein by the cell.

[00705] The following characteristics of the vector(s), encoded enzyme, targeting sequences, etc. disclosed in other sections here. The system may encode (i) a CRISPR enzyme, more specifically C2c1 or C2c3; (ii) a first guide RNA comprising a sequence capable of hybridizing to a first target sequence in a cell, (ii) a second guide RNA capable of hybridizing to a vector that encodes a CRISPR enzyme. Similarly, an enzyme may include one or more NLS sequences, and so on.

[00706] Various coding sequences (CRISPR effector protein and guide RNAs) can be included in a single vector or in multiple vectors. For example, it is possible to encode an effector protein in one vector and different RNA sequences in another vector, or to encode an effector protein and one guide RNA in one vector and the remaining guide RNA in another vector, or any other permutation is possible. In general, a system using only one or two different vectors is preferred.

[00707] When using multiple vectors, it is possible to deliver them in unequal amounts, and ideally with an excess of the vector encoding the first guide RNA relative to the second guide RNA, thus helping to delay the final inactivation of the CRISPR system so that genome editing occurs.

[00708] The first guide RNA can be targeted to any target sequence of interest in the genome, as described elsewhere herein. The second guide RNA targets a sequence in the vector that encodes the C2c1 or C2c3 effector protein of the CRISPR system and thus inactivates expression of the effector protein c of that vector. Thus, the target sequence in the vector must be able to inactivate expression. Suitable target sequences can be located, for example, near or at the translation start codon of the C2c1 or C2c3 effector protein coding sequence, in a non-coding sequence in a promoter driving the expression of non-coding RNA elements, in a promoter driving the expression of a C2c1 or C2c3 effector protein gene, in 100 on. from the ATG translation start codon in the Cas coding sequence, and/or in the inverted terminal repeat (iTR) of a viral delivery vector, for example, in the AAB genome. A double-strand break near this site can cause a structural change in the Cas coding sequence, causing loss of protein expression. An alternative target sequence for the "self-inactivating" guide RNA is to edit/inactivate the regulatory regions/sequences necessary for the expression of the CRISPR system with the C2c1 or C2c3 effector protein or for vector stability. For example, if the promoter for the Cas coding sequence is disrupted, then transcription may be inhibited or stopped. Similarly, if the vector includes sequences for replication, maintenance, or stability, then they can be targeted. For example, in the AAB vector, the useful target sequence is in inverted terminal repeats (iTR). Other useful targeting sequences may be promoter sequences, polyadenylation sites, and the like.

[00709] In addition, if guide RNAs are expressed in a template format, "self-inactivating" guide RNAs that target both promoters simultaneously will result in excision of the nucleotides in between from the CRISPR-Cas expressing construct, effectively leading to its complete inactivation. . Similarly, excision of intermediate nucleotides will result in cases where guide RNAs target both inverted terminal repeats (ITRs), or target two or more other CRISPR-Cas components at the same time. Self-inactivation, as explained herein, is generally applicable to the regulation of CRISPR-Cas systems. For example, self-inactivation, as explained herein, can be used to repair mutations by the CRISPR system, eg, repeat expansion disorders. As a result of self-inactivation, repair using the CRISPR system has a temporary activity.

[00710] The addition of non-targeting nucleotides to the 5' end (e.g., 1-10 nucleotides, preferably 1-5 nucleotides) of a "self-inactivating" guide RNA can be used to delay its processing and/or alter its efficiency as a means of enabling editing at the genomic locus -targets until the termination of the CRISPR-Cas system.

[00711] In one aspect of the self-inactivating AAV-CRISPR-Cas system, plasmids coexpressing one or more guide RNAs targeting genomic sequences of interest (e.g., 1-12, 1-5, 1-10, 1-15, 1- 20, 1-30) can be constructed with "self-inactivating" guide RNAs that target the SpCas9 sequence directly at or near the designed ATG start codon (e.g., within 5 nucleotides, within 15 nucleotides, within 30 nucleotides, within within 50 nucleotides, within 100 nucleotides). The guide RNA can also target a regulatory sequence in the region of the U6 promoter. U6-gated guide RNAs can be designed in a template format such that multiple guide RNA sequences can be released at the same time. After initial delivery to the target tissue/cells (leaving the cell), guide RNAs begin to accumulate while Cas levels increase in the nucleus. Cas complexes with all guide RNAs mediate genomic editing and self-inactivation of CRISPR-Cas plasmids.

[00712] One aspect of the self-inactivating CRISPR-Cas system is the expression alone or in tandem matrix format of 1 to 4 or more different guide sequences; for example, up to about 20 or about 30 guide sequences. Each individual self-inactivating targeting sequence can target a different target. This can be processed from, for example, a single chimeric pol3 transcript. Pol3 promoters such as U6 or Hl promoters can be used. Pol2 promoters such as those described herein may also be used. Inverted terminal repeat (iTR) sequences can flank the Pol3 promoter guide(s) Pol2-Cas promoter RNA.

[00713] One aspect of the tandem template transcript is that one or more guide RNAs edit one or more target(s), while one or more self-inactivating guide RNAs inactivate the CRISPR-Cas system. Thus, for example, the described CRISPR-Cas system for repeat expansion repair can be directly combined with the self-inactivating CRISPR-Cas system described herein. Such a system may have, for example, two guide RNAs targeting the target site for editing, as well as at least a third guide RNA targeting CRISPR-Cas self-inactivation. See Application Serial No. PCT/US2014/069897 titled "Compositions And Methods Of Use Of Crispr-Cas Systems In Nucleotide Repeat Disorders", published December 12, 2014 as WO/2015/089351.

[00714] The guide RNA may be a control guide RNA. For example, it can be designed to target a nucleic acid sequence encoding the CRISPR enzyme itself, as described in US2015232881Al, the description of which is incorporated herein by reference. In some embodiments, the system or composition may contain only a guide RNA designed to target a nucleic acid sequence encoding a CRISPR enzyme. In addition, the system or composition may contain a guide RNA designed to target a nucleic acid sequence encoding a CRISPR enzyme, as well as a nucleic acid sequence encoding a CRISPR enzyme and optionally a second guide RNA and optionally a repair template. The second guide RNA may be the primary target of a CRISPR system or composition (such as therapeutic, diagnostic, knockout, etc., as defined herein). Thus, the system or composition is self-inactivating. This is illustrated with respect to Cas9 in US2015232881A1 (also published as WO2015070083(Al) referred to herein and can be extrapolated to C2c1 or C2c3.

Enzymes according to the invention used in the multiplex (tandem) targeting approach

[00715] The inventors have shown that CRISPR enzymes as defined herein can use more than one guide RNA without losing activity. This allows CRISPR enzymes, systems or complexes as defined herein to be used to target multiple target DNAs, genes or loci through a single enzyme, system or complex as defined herein. Guide RNAs can be arranged in tandem, and optionally separated by a nucleotide sequence, such as a direct repeat, as defined here. The position of different guide RNAs in tandem does not affect activity. It is noted that the terms "CRISPR-Cas system", "CRISPR Cas complex", "CRISPR complex" and "CRISPR system" are used interchangeably. Also, the terms "CRISPR enzyme", "Cas enzyme", or "CRISPR-Cas enzyme" may be used interchangeably. In preferred embodiments, said CRISPR enzyme, CRISPR-Cas enzyme, or Cas enzyme is C2c1 or C2c3, or any of the modified or mutated variants thereof described elsewhere herein.

[00716] In one aspect, the invention relates to a non-naturally occurring or engineered CRISPR enzyme, preferably a class 2 CRISPR enzyme, preferably a type V or VI CRISPR enzyme as described herein, such as, but not limited to, C2c1 or C2c3 described herein for use in tandem or multiplex targeting. It should be understood that any of the CRISPR enzymes (or CRISPR-Cas or Cas), complexes or systems of the invention, as described elsewhere herein, may be used in this approach. Any of the methods, products, compositions, and uses as described herein are equally applicable in a multiplex or tandem targeting approach, as detailed below. The following specific aspects and embodiments of the invention are provided as further guidance.

[00717] In one aspect, the invention relates to the use of a C2c1 or C2c3 enzyme, complex or system as defined herein, to target multiple gene loci. In one embodiment of the invention, this can be done with multiple (tandem or multiplex) guide RNA (gRNA) sequences.

[00718] In one aspect, the invention provides methods for using one or more C2c1 or C2c3 enzyme elements, complex or system as defined herein for tandem or multiplex targeting, wherein said CRISPR system includes multiple guide RNA sequences. Preferably, said gRNA sequences are separated by nucleotide sequences, such as a direct repeat, as defined herein.

[00719] A C2c1 or C2c3 enzyme, system or complex as defined herein provides an effective means for modifying multiple target polynucleotides. A C2c1 or C2c3 enzyme, system or complex as defined herein has a wide variety of uses, including modification (eg, deletion, insertion, translocation, inactivation, activation) of one or more target polynucleotides in a variety of cell types. As such, the C2c1 or C2c3 enzyme, system or complex of the invention, as defined herein, has a wide range of applications, for example, in gene therapy, drug screening, disease diagnosis, and prognosis, including targeting multiple gene loci through a single CRISPR system.

[00720] In one aspect, the invention provides a C2c1 or C2c3 enzyme, system or complex as defined herein, i. a CRISPR-Cas C2c1 or C2c3 complex having a C2c1 or C2c3 protein, furthermore having at least one destabilization domain associated therewith, and several guide RNAs that target multiple nucleic acid molecules, such as DNA molecules, whereby each of these multiple guide RNAs, it specifically targets the corresponding nucleic acid molecule, for example, a DNA molecule. Each target nucleic acid molecule, such as a DNA molecule, may encode a gene product or contain a locus. Therefore, the use of multiple guide RNAs allows targeting of multiple loci or more genes. In some embodiments, the C2c1 or C2c3 enzyme can cleave the DNA molecule encoding the gene product. In some embodiments, the expression of the gene product is altered. The C2c1 or C2c3 protein and guide RNAs do not naturally occur together. The invention encompasses guide RNAs comprising guide sequences arranged in tandem. The invention further encompasses coding sequences for a C2c1 or C2c3 protein that are codon-optimized for expression in a eukaryotic cell. In a preferred embodiment, the eukaryotic cell is a mammalian cell, a plant cell, or a yeast cell, and in a more preferred embodiment, the mammalian cell is a human cell. Expression of the gene product may be reduced. The C2c1 or C2c3 enzyme may be part of the CRISPR system. or a complex that further comprises tandem guide RNAs (gRNAs) comprising a series of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 25, 30 or more than 30 guide sequences, each of which is capable of specific hybridization to a target sequence at a genomic target locus in a cell. In some embodiments, the CRISPR C2c1 or C2c3 functional system or complex binds to multiple target sequences. In some embodiments, a functional CRISPR system or complex may edit multiple target sequences, for example, target sequences may include a genomic locus, and in some embodiments, gene expression may be altered. In some embodiments, a functional CRISPR system or complex may include additional functional domains. In some embodiments, the invention relates to a method for altering or modifying the expression of multiple gene products. The method may include introducing into a cell containing said target nucleic acids, eg DNA molecules, or containing and expressing target nucleic acids, eg DNA molecules; for example, target nucleic acids may encode gene products or provide expression of gene products (eg, regulatory sequences).

[00721] In preferred embodiments, the CRISPR enzyme used for multiplex targeting is C2c1 or C2c3, or a CRISPR system or complex that includes C2c1 or C2c3. In some embodiments, the CRISPR enzyme used for multiplex targeting is AacC2c1, or the CRISPR system or complex used for multiplex targeting includes AacC2c1. In some embodiments, the CRISPR enzyme is AacC2c1, or the CRISPR system or complex includes AacC2c1. In some embodiments, the C2c1 enzyme used for multiplex targeting cleaves both strands of DNA, introducing a double-strand break (DSB). In some embodiments, the CRISPR enzyme used for multiplex targeting is nicase. In some embodiments, the C2c1 or C2c3 enzyme used for multiplex targeting is a double nickase. In some embodiments, the C2c1 or C2c3 enzyme used for multiplex targeting is a C2c1 or C2c3 enzyme, such as a C2c1 or C2c3 enzyme as defined herein.

[00722] In some basic embodiments of the invention, the C2c1 or C2c3 enzyme used for multiplex targeting is associated with one or more functional domains. In some more specific embodiments, the CRISPR enzyme used for multiplex targeting is dead C2c1 or dead C2c3 as defined herein.

[00723] In one aspect, the present invention provides a means for delivering a C2c1 or C2c3 enzyme, a system or complex for use in multiple targeting as defined herein, or polynucleotides as defined herein. Non-limiting examples of such delivery vehicles include, for example, particle(s) delivering component(s) of the complex, vector(s) comprising the polynucleotide(s) discussed herein (e.g., encoding CRISPR enzymes providing nucleotides encoding the CRISPR complex) . In some embodiments, the vector may be a plasmid or a viral vector such as AAV or a lentivirus. Transient transfection with plasmids into, for example, HEK cells may be advantageous, especially given the size limitations of AAV and that while C2c1 or C2c3 is placed in AAV, an upper limit can be reached with additional guide RNAs.

[00724] Also provided is a model that constitutively expresses the C2c1 or C2c3 enzyme, complex or system used within the scope of the present invention for multiplex targeting. The organism may be transgenic and may be transfected with the vectors of the present invention, or may be a progeny of the organism so transfected. In a further aspect, the present invention relates to compositions comprising the CRISPR enzyme, the system and complex as defined herein, or the polynucleotides or vectors described herein. Also contemplated are CRISPR C2c1 or C2c3 systems or complexes comprising multiple guide RNAs, preferably organized in tandem format. These different guide RNAs can be separated by nucleotide sequences such as direct repeats.

[00725] Also provided is a method of treating an individual, e.g., an individual in need thereof, comprising inducing gene editing by transforming the individual with polynucleotides encoding a C2c1 or C2c3 CRISPR system or complex, or any of the polynucleotides or vectors described herein and administered to a subject. A suitable repair matrix is also provided, eg delivered by a vector comprising said repair matrix. Also provided is a method of treating an individual, e.g., an individual in need thereof, comprising inducing transcriptional activation or repression of multiple target loci by transforming the individual with the polynucleotides or vectors described herein, wherein said polynucleotides or vectors encode or include a C2c1 or C2c3 enzyme, a complex or a system comprising multiple guide RNAs, preferably arranged in tandem. If any treatment takes place ex vivo , for example in cell culture, then it is understood that the term "individual" can be replaced by the phrase "cell or cell culture".

[00726] Also provided are compositions comprising a C2c1 or C2c3 enzyme, a complex or system comprising multiple guide RNAs, preferably arranged in tandem, or a polynucleotide or vector encoding or comprising said C2c1 or C2c3 enzyme, a complex or system comprising multiple guide RNAs, preferably arranged in tandem, for use in methods of treatment as defined herein. A kit including such compositions may be provided. Also contemplated is the use of said composition for the manufacture of medicaments for such treatments. The invention also contemplates the use of the C2c1 or C2c3 CRISPR system in screening, eg, acquisition screening. Cells that are artificially forced to overexpress a gene are able to reduce gene expression over time (reverse rebalancing), for example, through negative feedback. By the time screening is started, expression of the unregulated gene may again be down. The use of an inducible C2c1 or C2c3 activator allows transcription to be induced right before screening and therefore minimizes the chance of false negatives. Thus, by using the present invention in a screening, such as a gain-of-function screening, the likelihood of false negative results can be minimized.

[00727] In one aspect, the invention relates to an engineered, non-naturally occurring CRISPR system comprising a C2c1 or C2c3 protein and multiple guide RNAs, each of which specifically targets a DNA molecule encoding a gene product in a cell, whereby each of several guide RNAs target a specific DNA molecule encoding the gene product, and the C2c1 or C2c3 protein cleaves the target DNA molecule encoding the gene product, whereby the expression of the gene product is altered, and where the CRISPR protein and guide RNAs do not naturally occur together. The invention encompasses multiple guide RNAs, including multiple guide sequences, preferably separated by a nucleotide sequence, such as a direct repeat. In one embodiment, the CRISPR protein is a type V or VI CRISPR-Cas protein, and in a more preferred embodiment, the CRIPSR protein is a C2c1 or C2c3 protein. In addition, the invention includes a C2c1 or C2c3 protein that is codon-optimized for expression in a eukaryotic cell. In a preferred embodiment, the eukaryotic cell is a mammalian cell, and in a more preferred embodiment, the mammalian cell is a human cell. In a further embodiment of the invention, the expression of the gene product is reduced.

[00728] In another aspect, the invention relates to an engineered, non-naturally occurring vector system comprising one or more vectors comprising a first regulatory element operably linked to multiple guide RNAs of the C2c1 or C2c3 CRISPR system, each of which specifically targets a DNA molecule. , encoding the gene product, and a second regulatory element operably linked to the sequence encoding the CRISPR protein. Both regulatory elements can be located on the same vector or on different vectors of the system. Multiple guide RNAs target multiple DNA molecules encoding multiple gene products in a cell, and the CRISPR protein can cleave multiple DNA molecules encoding gene products (it may cleave one or both strands, or essentially have no nuclease activity), whereby the expression of multiple gene products can be changed; and where the CRISPR protein and multiple guide RNAs do not naturally occur together. In a preferred embodiment, the CRISPR protein is a C2c1 or C2c3 protein, optionally codon-optimized for expression in a eukaryotic cell. In a preferred embodiment, the eukaryotic cell is a mammalian cell, a plant cell, or a yeast cell, and in a more preferred embodiment, the mammalian cell is a human cell. In a further embodiment of the invention, the expression of each of several gene products is altered, preferably reduced.

[00729] In one aspect, the invention relates to a vector system comprising one or more vectors. In some embodiments, the system includes: (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more targeting sequences upstream or downstream of the direct repeat sequences, where, when expressed, the one or more targeting sequences sequences direct the sequence-specific binding of a CRISPR complex to one or more targeting sequences in a eukaryotic cell, wherein the CRISPR complex comprises a C2c1 or C2c3 enzyme in complex with one or more targeting sequences hybridized to one or more target sequences, and (b) a second regulatory an element operably linked to an enzyme-coding sequence encoding said C2c1 or C2c3 enzyme, preferably including at least one nuclear localization sequence and/or at least one NE sequence S; where components (a) and (b) are located on the same or on different vectors of the system. In some embodiments, component (a) further comprises two or more targeting sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more targeting sequences directs sequence-specific binding of the C2c1 or C2c3 CRISPR complex to different target sequences in eukaryotic cell. In some embodiments, the CRISPR complex includes one or more nuclear localization sequences and/or one or more NES of sufficient strength to stimulate the accumulation of said C2c1 or C2c3 CRISPR complex in a detectable amount within or outside the eukaryotic cell nucleus. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments of the invention, each of the guide sequences is at least 16, 17, 18, 19, 20, 25 nucleotides in length, or 16-30, or 16-25, or 16-20 nucleotides.

[00730] Recombinant expression vectors may include polynucleotides encoding a C2c1 or C2c3 enzyme, system or complex for use in multiple targeting, as defined herein, in a form suitable for the expression of such a nucleic acid in a host cell, which means that such recombinant expression vectors contain one or more regulatory elements which may be selected according to the host cells used for expression and which are operably linked to the nucleic acid sequence to be expressed. In a recombinant expression vector, "operably linked" shall mean that the target nucleotide sequence is linked to the regulatory element(s) in such a manner that expression of such nucleotide sequence is possible (e.g., in an in vitro transcription/translation system or in a host cell). when the vector is introduced into the host cell).

[00731] In some embodiments, the host cell is transiently or permanently transfected with one or more vectors comprising polynucleotides encoding a C2c1 or C2c3 enzyme, system or complex for use in multiple targeting as defined herein. In some embodiments of the invention, a cell naturally occurring in an individual is transfected. In some embodiments, the cell that is transfected is from an individual. In some embodiments, the cell is derived from cells derived from an individual, such as a cell line. A wide variety of tissue culture cell lines are known in the art and are illustrated herein. Cell lines are available from a variety of sources known to those skilled in the art (see, for example, American Type Culture Collection (ATCC) (Manassus, Virginia)). In some embodiments, a cell transfected with one or more vectors comprising polynucleotides encoding a C2c1 or C2c3 enzyme, a system or complex for use in multiple targeting as defined herein, is used to generate a new cell line comprising one or more derived from sequence vectors. In some embodiments, a cell transiently transfected with components of a C2c1 or C2c3 CRISPR system or complex for use in multiple targeting as described herein (e.g., via transient transfection of one or more vectors or RNA transfection) and modified by CRISPR C2c1 system activity or C2c3 or complex, is used to obtain a new cell line, including cells containing the modification, but devoid of any other exogenous sequence. In some embodiments, cells transiently or permanently transfected with one or more vectors comprising polynucleotides encoding a C2c1 or C2c3 enzyme, a system or complex for use in multiple targeting as defined herein, or cell lines derived from such cells are used in the evaluation of one or more test compounds.

[00732] the Term "regulatory element" is defined in the present description in other sections.

[00733] Advantageous vectors include lentiviruses and adeno-associated viruses, and such types of vectors can also be selected to target specific cell types.

[00734] In one aspect, the invention relates to a eukaryotic host cell comprising (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more guide RNA sequences upstream or downstream (depending on as applicable) relative to a direct repeat sequence, where, when expressed, the targeting sequence(s) direct sequence-specific binding of the CRISPR C2c1 or C2c3 complex to the corresponding target sequence(s) in a eukaryotic cell, where the CRISPR C2c1 or C2c3 complex includes an enzyme C2c1 or C2c3 in complex with one or more guide sequences hybridized to the corresponding target sequence(s); and/or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said C2c1 or C2c3 enzyme, preferably comprising at least one nuclear localization sequence and/or NES. In some embodiments, the host cell includes components (a) and (b). In some embodiments, component (a), component (b), or components (a) and (b) are stably integrated into the genome of the eukaryotic host cell. In some embodiments, component (a) further comprises two or more targeting sequences operably linked to the first regulatory element, and optionally separated by a direct repeat, wherein when expressed, each of the two or more targeting sequences directs sequence-specific binding of the C2c1 or C2c3 CRISPR complex with different target sequences in the eukaryotic cell. In some embodiments, the C2c1 or C2c3 enzyme comprises one or more nuclear localization sequences and/or nuclear export sequences (NES) of sufficient strength to stimulate the accumulation of said CRISPR enzyme in a detectable amount within and/or outside the nucleus of a eukaryotic cell.

[00735] In some embodiments, the C2c1 or C2c3 enzyme is a type V or VI CRISPR system enzyme. In some embodiments, the C2c1 enzyme is an AacC2c1 enzyme. In some embodiments, the C2c1 enzyme is derived from Alicyclobacillus acidoterrestris (e.g., ATCC 49025), Alicyclobacillus contaminans (e.g., DSM 17975), Desulfovibrio inopinatus (e.g., DSM 10711), Desulfonatronum thiodismutans (e.g., strain MLF-1), bacteria Opitutaceae TAV5 , Tuberibacillus calidus (e.g. DSM 17572) , Bacillus thermoamylovorans (e.g. strain B4166), Brevibacillus sp. CF112, Bacillus sp. NSP2.1, Desulfatirhabdium butyrativorans (eg DSM 18734), Alicyclobacillus herbarius (eg DSM 13609), Citrobacter freundii (eg ATCC 8090), Brevibacillus agri (eg BAB-2500), Methylobacterium nodulans (eg ORS 2060), and may include further changes or mutations of C2c1 as defined herein, and may be a chimeric C2c1. In some embodiments, the C2c1 or C2c3 enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme directs the cleavage of one or two strands at a specific position in the target sequence. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, one or more guide sequences (each) are at least 16, 17, 18, 19, 20, 25 nucleotides, or 16-30, or 16-25, or 16-20 nucleotides in length. When multiple guide RNAs are used, they are preferably separated by a direct repeat sequence. In one aspect, the invention relates to a non-human eukaryotic organism; preferably a multicellular eukaryotic organism, including a eukaryotic host cell according to any of the described embodiments of the invention. In other aspects, the invention relates to a eukaryotic organism, preferably a multicellular eukaryotic organism, including a eukaryotic host cell according to any of the described embodiments of the invention. In some embodiments, the implementation of the organism according to these aspects of the invention may be an animal, such as a mammal. In addition, the organism may be an arthropod, such as an insect. An organism can also be a plant. In addition, the organism may be a fungus.

[00736] In one aspect, the invention relates to a kit that includes one or more of the components described in the present description. In some embodiments, the kit includes a vector system and instructions for using the kit. In some embodiments, the vector system includes (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more guide sequences up or down (as applicable) relative to the direct repeat sequence. wherein, when expressed, the guide sequence controls the sequence-specific binding of a CRISPR C2c1 or C2c3 complex to a target sequence in a eukaryotic cell, wherein the CRISPR C2c1 complex includes a C2c1 or C2c3 enzyme in complex with the guide sequence hybridized to the target sequence; and/or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said C2c1 or C2c3 enzyme, comprising a nuclear localization sequence. In some embodiments of the invention, the kit includes components (a) and (b) located on the same or on different vectors of the system. In some embodiments, set (a) further comprises two or more targeting sequences operably linked to a first regulatory element, wherein when expressed, each of the two or more targeting sequences directs sequence-specific binding of the CRISPR complex to different target sequences in a eukaryotic cell. . In some embodiments, the C2c1 or C2c3 enzyme comprises one or more nuclear localization sequences of sufficient strength to stimulate the accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. In some embodiments, the CRISPR enzyme is a type V or VI CRISPR enzyme. In some embodiments, the CRISPR enzyme is a C2c1 or C2c3 enzyme. In some embodiments, the C2c1 enzyme is derived from Alicyclobacillus acidoterrestris (e.g., ATCC 49025), Alicyclobacillus contaminans (e.g., DSM 17975), Desulfovibrio inopinatus (e.g., DSM 10711), Desulfonatronum thiodismutans (e.g., strain MLF-1), bacteria Opitutaceae TAV5 , Tuberibacillus calidus (eg DSM 17572), Bacillus thermoamylovorans (eg strain B4166), Brevibacillus sp. CF112, Bacillus sp. NSP2.1, Desulfatirhabdium. butyrativorans (eg DSM 18734), Alicyclobacillus herbarius (eg DSM 13609), Citrobacter freundti (eg ATCC 8090), Brevibacilhis agri (eg BAB-2500), Methylobacterium nodulans (eg ORS 2060) (eg modified to have or be associated with at least one destabilization domain ("DD"), and may include a further change or mutation of C2c1, and may be a chimeric C2c1 In some embodiments, the DD-CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the DD-CRISPR enzyme directs the cleavage of one or two strands at a specific position in the target sequence.In some embodiments, the DD-CRISPR enzyme lacks DNA strand cleavage enzymatic activity (e.g., has no more than 5% nuclease activity at compared to a wild-type enzyme or an enzyme that does not have a mutation or change that reduces nuclease a activity). In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the second regulatory element is a polymerase II promoter. In some embodiments, the guide sequence is at least 16, 17, 18, 19, 20, 25 nucleotides, or 16-30 or 16-25 or 16-20 nucleotides in length.

[00737] In one aspect, the invention relates to a method for modifying multiple target polynucleotides in a host cell, such as a eukaryotic cell. In some embodiments, the method allows a C2c1 or C2c3 CRISPR complex to bind to multiple target polynucleotides, for example, cleaving said multiple target polynucleotides, thereby modifying the multiple target polynucleotides, wherein the C2c1 or C2c3 CRISPR complex includes the C2c1 or C2c3 enzyme in the complex with multiple guide sequences, each of which is hybridized with a specific target sequence in the specified target polynucleotide, where these multiple guide sequences are associated with a direct repeat sequence. In some embodiments of the invention, said cleavage includes cleaving one or two strands at a particular position in each target sequence with said C2c1 or C2c3 enzyme. In some embodiments of the invention, the specified cleavage leads to a decrease in the transcription of several target genes. In some embodiments, the method further comprises repairing one or more of said cleaved target polynucleotides by homologous recombination with an exogenous template polynucleotide, wherein said repair results in a mutation involving an insertion, deletion, or substitution of one or more nucleotides in one or more of said target polynucleotides. . In some embodiments, said mutations result in one or more changes in the amino acid sequence of a protein expressed from a gene comprising one or more target sequences. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cell, wherein the one or more vectors express one or more of a C2c1 or C2c3 enzyme and multiple guide RNA sequences associated with a direct repeat sequence. In some embodiments of the invention, these vectors are delivered to a eukaryotic cell in an individual. In some embodiments of the invention, said modification occurs in said eukaryotic cell in cell culture. In some embodiments, the method further comprises removing said eukaryotic cell from an individual prior to making said modification. In some embodiments, the method further comprises reintroducing said eukaryotic cell and/or cells derived therefrom into said individual.

[00738] In one aspect, the invention relates to a method for modifying the expression of several polynucleotides in a eukaryotic cell. In some embodiments, the method allows a C2c1 or C2c3 CRISPR complex to bind to multiple polynucleotides such that said binding results in an increase or decrease in the expression of said polynucleotides; wherein the CRISPR C2c1 or C2c3 complex comprises a C2c1 or C2c3 enzyme complexed with multiple guide sequences each specifically hybridized to its own target sequence in said polynucleotide, wherein said guide sequences are linked to a direct repeat sequence. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cells, wherein the one or more vectors express one or more of a C2c1 or C2c3 enzyme and multiple targeting sequences linked to direct repeat sequences.

[00739] In one aspect, the invention provides a recombinant polynucleotide comprising multiple guide RNA sequences upstream or downstream (as applicable) of a direct repeat sequence, where each of the guide sequences, when expressed, directs sequence-specific binding of the complex CRISPR C2c1 or C2c3 with its corresponding target sequence present in a eukaryotic cell. In some embodiments, the target sequence is a viral sequence present in a eukaryotic cell. In some embodiments, the target sequence is a proto-oncogene or an oncogene.

[00740] Aspects of the invention encompass a non-naturally occurring or engineered composition that may include a guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell, and a C2c1 or C2c3 enzyme, as defined in the present description, which may include at least one or more sequences of nuclear localization.

[00741] One aspect of the invention encompasses methods for modifying a genomic locus of interest to alter the expression of a gene in a cell by introducing any of the compositions described herein into the cell.

[00742] One aspect of the invention is that the aforementioned elements are contained in a single composition or are contained in individual compositions. These compositions can be successfully applied to the host to reveal a functional effect at the genomic level.

[00743] As used herein, the term "guide RNA" or "gRNA" has the same meaning as elsewhere and includes any polynucleotide sequence having sufficient complementarity with a target nucleic acid sequence to hybridize to a nucleic acid sequence. target acid and direct sequence-specific binding of the nucleic acid-targeting complex to the target nucleic acid sequence. Each gRNA can be designed to include multiple binding recognition sites (eg, aptamers) specific to one or different adapter proteins. Each gRNA can be designed to bind to a -1000-+1 nucleotide promoter region upstream of the transcription start site (ie TSS), preferably in the -200 nucleotide region. This arrangement serves to improve functional domains that affect gene activation (eg, transcriptional activators) or gene inhibition (eg, transcriptional repressors). The modified gRNA may comprise one or more modified gRNAs targeting one or more target loci (e.g., at least 1 gRNA, at least 2 gRNA, at least 5 gRNA, at least 10 gRNA, at least 20 gRNA , at least 30 gRNA, at least 50 gRNA) in the composition. These multiple gRNA sequences can be arranged in tandem format and are preferably separated by a direct repeat.

[00744] Thus, each of the gRNA, the CRISPR enzyme, as defined herein, can be individually formulated and administered to the host individually or concurrently. Alternatively, these components may be contained in a single composition for administration to the host. Administration to the host may be by means of viral vectors known to the skilled artisan or described herein for delivery to the host (eg, lentiviral vector, adenovirus vector, AAV vector). As explained herein, the use of different selectable markers (eg, to select for lentiviral gRNA) and concentration of gRNA (eg, depending on whether multiple gRNAs are used) may be advantageous to detect an improved effect. Based on this concept, there are several options for detecting events at the genomic locus, including DNA cleavage, gene activation and inactivation. Using the proposed compositions, one skilled in the art can advantageously and specifically target one or more loci using the same or different functional domains to detect one or more events at a genomic locus. The compositions can be used in a wide variety of cell library screening and in vivo functional modeling (e.g., gene activation by long intergenic non-coding RNAs (lincRNAs) and function identification; modeling gain of function; modeling loss of function; use of compositions of the invention to generate cell lines and transgenic animals for optimization and screening purposes).

[00745] The present invention encompasses the use of the compositions of the present invention to create and use transgenic, conditionally or inducibly CRISPR cells/animals; see, for example, Platt et al., Cell (2014), 159(2): 440-455 or the PCT patent publication cited herein, such as WO 2014/093622 (PCT/US2013/074667). For example, cells or animals such as non-human animals such as vertebrates or mammals such as rodents such as mice, rats or other laboratory or non-laboratory animals such as cats, dogs, sheep, etc. ., may be "knock-in", resulting in the animal depending on the conditions or induced to express C2c1 or C2c3, as in the article by Platt et al. Thus, the target cell or target animal turns on the CRISRP enzyme (e.g., C2c1 or C2c3) depending on conditions or inducibly (e.g., in the form of a Cre-dependent composition), after expression of the vector introduced into the target cell, the expression of the vector induces or creates conditions for the expression of the CRISRP enzyme (for example, C2c1 or C2c3) in the target cell. Through the use of a guide and a composition as defined herein with a known method for creating a CRISPR complex, induced events in the genome are also an aspect of the present invention. Examples of such induced events are described in the present description in other sections.

[00746] In some embodiments, the phenotypic change preferably results from genome modification when the target is a genetic disease, especially in therapies, and preferably when a repair matrix is provided to correct or change the phenotype.

[00747] In some embodiments, diseases that may be targeted include diseases that are associated with splicing defects.

[00748] In some embodiments, cellular targets include hematopoietic stem/progenitor cells (CD34+); human T cells; and retinal cells - for example, photoreceptor progenitor cells.

[00749] In some embodiments, the target genes include: human beta globin gene - HBB (for the treatment of sickle cell anemia, including stimulation of gene conversion (using a closely related HBD gene as an endogenous template)); CD3 (T cells) and CEP920 - retina (eyes).

[00750] In some embodiments, the target diseases also include: malignant tumor; sickle cell anemia (based on point mutation); HIV; beta thalassemia; and ophthalmic or ocular diseases such as Leber's amaurosis (LCA) caused by a splicing defect.

[00751] In some embodiments, delivery methods include: cationic lipid-mediated "direct" delivery of an enzyme-guide complex (ribonucleoprotein) and electroporation of plasmid DNA.

[00752] The methods, products, and uses described herein may be used for non-therapeutic purposes. In addition, any of the methods described herein can be applied in vitro and ex vivo .

[00753] In one aspect, a non-naturally occurring or engineered composition is provided, comprising:

I. two or more polynucleotide sequences of the CRISPR-Cas system containing

(a) a first guide sequence capable of hybridizing to a first target sequence at the polynucleotide locus,

(b) a second guide sequence capable of hybridizing to a second target sequence at the polynucleotide locus,

(c) a direct repeat sequence, and

II. a C2c1 enzyme or a second polynucleotide sequence encoding it, or a C2c3 enzyme or a second polynucleotide sequence encoding it,

where, during transcription, the first and second guide sequences direct the sequence-specific binding of the first and second CRISPR complex C2c1 or C2c3 to the first and second target sequences, respectively,

where the first CRISPR complex comprises a C2c1 enzyme or a C2c3 enzyme in complex with a first guide sequence that is capable of hybridizing to the first target sequence,

wherein the second CRISPR complex comprises a C2c1 enzyme or a C2c3 enzyme in complex with a second guide sequence that is capable of hybridizing to the second target sequence, and

where the first guide sequence directs the cleavage of one strand of the DNA duplex in the vicinity of the first target sequence, and the second guide sequence directs the cleavage of the other strand in the vicinity of the second target sequence, causing a double strand break, thereby modifying the organism or non-animal or non-human organism. Similarly, compositions comprising more than two guide RNAs may be envisaged, for example, each RNA is specific for one target and all RNAs are arranged in tandem in a composition, or CRISPR system, or complex, as described herein.

[00754] In another embodiment, C2c1 or C2c3 is delivered to the cell as a protein. In another and particularly preferred embodiment of the invention, C2c1 or C2c3 is delivered to the cell as a protein or as a nucleotide sequence encoding it. Delivery to a cell as a protein may include delivery of a ribonucleoprotein (RNP) complex, where the protein forms a complex with multiple guide sequences.

[00755] In one aspect, host cells and cell lines modified or comprising compositions, systems, or modified enzymes of the present invention are provided, including stem cells and their progeny.

[00756] In one aspect, cell therapy methods are provided where, for example, a single cell or population of cells is harvested or cultured, the cell or cells are modified ex vivo as described herein, and then reintroduced (extracted cells) or injected ( cultured cells) into the body. Stem cells, embryonic or induced pluripotent or totipotent stem cells, are also particularly preferred in this regard. But, of course, in vivo embodiments are also contemplated.

[00654] The methods of the invention may further include the delivery of matrices, such as repair matrices, which may be dsODN or ssODN, see below. Delivery of the templates can be made simultaneously or separately from the delivery of any or all CRISPR effector proteins or targeting molecules, and through the same delivery mechanism or any other. In some embodiments, it is preferred that the template be delivered together with a targeting molecule and also preferably a CRISPR effector protein. An example is the AAV vector.

[00655] The methods of the invention may further comprise: (a) delivering into the cell a double-stranded oligodeoxynucleotide (dsODN) comprising overhangs complementary to the overhangs created by said double strand break, wherein said dsODN is inserted into the target locus, or (b) delivering a single-stranded oligodeoxynucleotide (ssODN) into the cell, wherein said ssODN acts as a template for homologous directed double strand break repair. The methods of the invention may be for the prevention or treatment of a disease in an individual when said disease is caused by a defect in said target locus. The methods of the invention can be carried out in vivo in an individual or ex vivo on a cell taken from an individual, where in some cases the specified cell is returned to the individual.

[00759] The invention also covers products made using a CRISPR enzyme, or a Cas enzyme, or a C2c1 enzyme, or a C2c3 enzyme, or a CRISPR-CRISPR enzyme, or a CRISPR-Cas system, or a CRISPR-C2c1 system, or a CRISPR-C2c3 system for use in tandem or multiple targeting, as defined in the present description.

Sets

[00760] In one aspect, the invention provides kits containing any one or more of the elements described for the above methods and compositions. In some embodiments, the kit includes a vector system as described herein and in the instructions for use of the kits. The elements may be provided individually or in combinations and may be provided in any suitable container such as a vial, bottle or tube, in some embodiments the kit includes instructions in one or more languages, such as in more than one language. The instructions may be specific to the applications and methods described herein.

[00761] In some embodiments of the invention, the kit includes one or more reagents for use in a method that uses one or more of the elements described in the present description. The reagents may be provided in any suitable container. For example, one or more buffers may be provided in a kit to store or carry out reactions. Reagents may be provided in a form that is applicable to a particular assay, or in a form that requires the addition of one or more other components prior to use (eg, in concentrate or lyophilized form). The buffer can be any buffer, including but not limited to sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. In some embodiments of the invention, the buffer is alkaline. In some embodiments, the buffer has a pH of about 7 to about 10. In some embodiments, the kit includes one or more oligonucleotides corresponding to the guide sequence for insertion into a vector to operably link the guide sequence and the regulatory element. In some embodiments, the kit includes a homologous recombination template polynucleotide. In some embodiments, the kit includes one or more vectors and/or one or more polynucleotides described herein. The kit advantageously makes it possible to provide all elements of the systems of the invention according to the invention.

[00762] In one aspect, the invention relates to methods for using one or more elements of a CRISPR system. The CRISPR complex of the invention provides an efficient means of modifying a target polynucleotide. The CRISPR complex of the invention has a wide variety of useful applications, including modification (eg, deletion, insertion, movement, inactivation, activation) of a target polynucleotide in a variety of cell types. As such, the CRISPR complex of the invention has a wide range of applications, for example, in gene therapy, drug screening, disease diagnosis, and prognosis. A typical CRISPR complex includes a CRISPR effector protein in complex with a target sequence hybridized to a target sequence in a target polynucleotide. In certain embodiments of the invention, the forward repeat sequence is associated with a guide sequence.

[00763] In one embodiment, the present invention relates to a method for cleaving a target polynucleotide. The method includes modifying a target polynucleotide using a CRISPR complex that binds to the target polynucleotide and cleaves said target polynucleotide. Typically, the CRISPR complex of the invention, when introduced into a cell, creates a break (eg, single or double strand break) in the genome sequence. For example, the method can be used to cleave a disease gene in a cell.

[00764] The break introduced by the CRISPR complex can be repaired by repair processes such as error prone non-homologous end joining (NHEJ) or high fidelity homologous repair (HDR). During these repair processes, an exogenous polynucleotide template can be inserted into the genome sequence. In some methods, the HDR process is used to modify the genome sequence. For example, an exogenous polynucleotide template containing the sequence to be inserted, flanked by an underlying sequence and an upstream sequence, is introduced into a cell. The upstream and downstream sequences are similar to each side of the integration site in the chromosome.

[00765] Where desired, the donor polynucleotide may be DNA, e.g., plasmid DNA, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), viral vector, linear DNA fragment, PCR fragment, naked nucleic acid, or nucleic acid in complex with a delivery vehicle such as a liposome or poloxamer.

[00766] The exogenous polynucleotide template includes a sequence to be inserted (eg, a mutated gene). The sequence for integration may be a sequence endogenous or exogenous with respect to the cell. Examples of sequences for integration include polynucleotides encoding a protein or non-coding RNA (eg miRNA). Thus, the sequence to be integrated can be operably linked to the corresponding control sequence or sequences. Alternatively, the integration sequence may have a regulatory function.

[00767] The upstream and downstream sequences in the exogenous polynucleotide template are selected to allow for recombination between the chromosomal sequence of interest and the donor polynucleotide. An upstream sequence is a nucleic acid sequence that resembles a genome sequence upstream of the target integration site. Similarly, a downstream sequence is a nucleic acid sequence that bears similarity to a chromosomal sequence downstream of the target integration site. The upstream and downstream sequences in the exogenous polynucleotide template may have 75%, 80%, 85%, 90%, 95%, or 100% sequence similarity to the target genome sequence. Preferably, upstream and downstream sequences in the exogenous polynucleotide template have approximately 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to the target genome sequence. In some methods, the upstream and downstream sequences in the exogenous polynucleotide template have approximately 99% or 100% sequence similarity to the target genome sequence.

[00768] The upstream or downstream sequence may include from about 20 bp. up to about 2500 bp, for example, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 2100, 2200, 2300, 2400, or 2500 bp In some methods, a typical upstream or downstream sequence is from about 200 bp. up to about 2000 bp, from about 600 bp up to about 1000 bp, or more specifically from about 700 bp. up to about 1000 p.

[00769] In some methods, the exogenous polynucleotide template may further include a marker. Such a marker may facilitate screening for a given integration. Examples of suitable markers include restriction sites, fluorescent proteins, or selectable markers. The exogenous polynucleotide template of the invention can be constructed using recombinant techniques (see, for example, Sambrook et al, 2001 and Ausubel et al., 1996).

[00770] In a typical method for modifying a target polynucleotide by integrating an exogenous polynucleotide template, a double strand break is introduced into the genome sequence by the CRISPR complex, the break is repaired by homologous recombination with the exogenous polynucleotide template, such that the template integrates into the genome. The presence of a double-strand break facilitates template integration.

[00771] In other embodiments, the present invention relates to a method for modifying the expression of a polynucleotide in a eukaryotic cell. The method includes increasing or decreasing the expression of a target polynucleotide using a CRISPR complex that binds to the polynucleotide.

[00772] In some methods, the target polynucleotide can be inactivated to modify the expression in the cell. For example, upon binding of a CRISPR complex to a target sequence in a cell, the target polynucleotide is inactivated such that the sequence is not transcribed, the encoded protein is not synthesized, or the sequence does not function as a wild-type sequence. For example, the coding sequence for a protein or microRNA can be inactivated such that protein synthesis is stopped.

[00773] In some methods, the control sequence may be inactivated such that it no longer functions as a control sequence. As used herein, a "control sequence" refers to any nucleic acid sequence that produces transcription, translation, or accessibility of the nucleic acid sequence. Examples of a control sequence include a promoter, a transcription terminator, and an enhancer. An inactivated target sequence may include a deletion mutation (i.e., the deletion of one or more nucleotides), an insertion mutation (i.e., the insertion of one or more nucleotides), or a nonsense mutation (i.e., the replacement of a single nucleotide with another nucleotide, such in such a way that a stop codon is obtained). In some methods, inactivation of the target sequence results in "knockout" of the target sequence.

Exemplary Applications of the CRISPR Cas System

[00774] The invention relates to a non-naturally occurring or engineered composition, or one or more polynucleotides encoding components of said composition, or vectors or delivery systems containing one or more polynucleotides encoding components of said composition, for use in cell modification - targets in vivo , ex vivo , or in vitro , and this can be done in a manner that alters the cell such that, after modification, the progeny or cell line of the modified CRISPR cell retains the altered phenotype. The modified cells and their progeny can be part of a multicellular organism, such as a plant or animal, by applying an ex vivo CRISPR system to the desired cell types. The CRISPR invention may be a therapeutic method of treatment. Therapeutic treatment may include gene or genome editing, or gene therapy.

Use of an inactivated CRISPR C2c1 or C2c3 enzyme for detection methods such as FISH

[00775] In one aspect, the invention relates to an engineered non-naturally occurring CRISPR-Cas system comprising the catalytically inactivated Cas protein described herein, preferably C2c1 (dC2c1) inactivated or C2c3 (dC2c3) inactivated, and to the use of this systems in detection methods such as fluorescence in situ hybridization (FISH). dC2c1 or dC2c3 lacking the ability to make DNA double strand breaks can be associated with a marker such as a fluorescent protein such as enhanced green fluorescent protein (eEGFP) and co-expressed with small guide RNAs to target pericentric, centric and telomeric repeats in vivo . Systems with dC2c1 or dC2c3 can be used to visualize both repetitive sequences and individual genes in the human genome. Such new applications of labeled CRISPR-Cas systems may be important for cell imaging and the study of functional nuclear architecture, especially in the case of small nuclear volume or complex 3D structures. (Chen B, Gilbert LA, Cimini BA, Schnitzbauer J, Zhang W, Li GW, Park J, Blackburn EH, Weissman JS, Qi LS, Huang B. 2013. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/ Cas Cell 155(7): 1479-91, doi: 10.1016/j.cel1.2013.12.001)

Modification of the Target by a CRISPR Cas system or complex (e.g. C2c1/C2c3-RNA complex)

[00776] In one aspect, the invention relates to methods for modifying a target polynucleotide in a eukaryotic cell, which may be in vivo , ex vivo , or in vitro . In some embodiments, the method includes taking a sample of a cell or population of cells from a human or non-human animal and modifying the cell or cells. Cultivation can take place at any stage ex vivo . The cell or cells may be reintroduced into a non-human animal or plant. For newly introduced cells, it is particularly preferred that the cells are stem cells.

[00777] In some embodiments, the method includes allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of said target polynucleotide, resulting in a modification of the target polynucleotide, wherein the CRISPR complex comprises a CRISPR effector protein and a targeting sequence hybridized or capable of to hybridization with the target sequence in the specified target polynucleotide.

[00778] In one embodiment, the invention relates to a method for modifying the expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method includes allowing the CRISPR complex to bind to a polynucleotide such that said binding results in an increase or decrease in the expression of said polynucleotide; wherein the CRISPR complex comprises a CRISPR effector protein combined with a guide sequence hybridized or capable of hybridizing to a target sequence in said polynucleotide. Similar considerations and conditions apply as noted above for methods for modifying a target polynucleotide. In fact, these sampling, culturing and re-introduction options apply to all aspects of the present invention.

[00779] Indeed, in any aspect of the invention, the CRISPR complex may comprise a CRISPR effector protein in complex with a target sequence hybridized or capable of hybridizing to a target sequence. Similar considerations and conditions apply as noted above for methods for modifying a target polynucleotide.

[00780] Thus, any of the non-naturally occurring CRISPR effector proteins described herein has at least one modification, and thus the effector protein has certain enhanced capabilities. In particular, any of the effector proteins is able to form a CRISPR complex with a guide RNA. When such a complex is formed, the guide RNA is able to bind to the target polynucleotide sequence and the effector protein is able to modify the target locus. In addition, the effector protein in the CRISPR complex reduces the ability to modify one or more target loci compared to an unmodified enzyme/effector protein.

[00781] In addition, the modified CRISPR enzymes described herein encompass enzymes that provide an increase in the ability of the CRISPR complex effector protein to modify one or more target loci compared to an unmodified enzyme/effector protein. Such functionality may be provided alone or provided in combination with the reduced ability to modify one or more non-target loci described above. Any such effector proteins may be provided with any of the additional modifications to the CRISPR effector protein as described herein, for example, in combination with any activity conferred by one or more associated heterologous functional domains, any other mutations to reduce nuclease activity, and the like. .

[00782] In preferred embodiments, the modified CRISPR effector protein has a reduced ability to modify one or more non-target loci compared to the unmodified enzyme/effector protein and increases the ability to modify one or more target loci compared to the unmodified enzyme/effector protein. In combination with additional modifications to the effector protein, significantly increased specificity can be achieved. For example, the combination of such preferred embodiments with one or more additional mutations is provided if one or more additional mutations are in one or more active catalytically active domains. Such additional mutations in the catalytic domain can confer nicase functionality, as detailed herein. In such effector proteins, increased specificity can be achieved due to improved specificity for the activity of the effector protein.

[00783] Modifications to reduce off-target effects and/or enhance target effects, as described above, can be made to amino acid residues located in the positively charged region/groove located between the RuvC-III and HNH domains. It is understood that any of the functional effects described above can be achieved by modifying the amino acids in the aforementioned groove, as well as by modifying the amino acids adjacent to this groove or outside it.

[00784] Additional functionality that can be engineered into modified CRISPR effector proteins as described herein includes the following. 1. Modified CRISPR effector proteins that disrupt DNA:protein interactions without affecting the protein's tertiary or secondary structure. This includes residues that come into contact with any part of the RNA:DNA duplex. 2. modified CRISPR effector proteins that attenuate intraprotein interactions by maintaining C2c1 or C2c3 in the conformation required for nuclease cleavage in response to DNA binding (target or non-target). For example, a modification that mildly inhibits but still allows the HNH domain nuclease conformation (located on phosphate-cleaving), 3. modified CRISPR effector proteins that enhance intraprotein interactions by maintaining C2c1 or C2c3 in a conformation that inhibits nuclease activity in the DNA binding reaction ( targeted or non-targeted). For example: a modification that stabilizes the HNH domain into a cleaving phosphate conformation. Any such additional functional enhancement can be provided in combination with any other modification of the CRISPR effector protein, as detailed elsewhere in this application.

[00785] Any of the improved functionality described herein can be engineered into any CRISPR effector protein, such as the C2c1 or C2c3 effector protein. However, it will be understood that any of the functionality described herein can be engineered into C2c1 or C2c3 effector proteins from other orthologs, including chimeric effector proteins containing fragments from multiple orthologs.

Nucleic acids, amino acids and proteins, regulatory sequences, vectors, etc.

[00 786] The invention uses nucleic acids to bind target DNA sequences. This is promising because nucleic acids are much easier and cheaper to produce than proteins, and specificity can vary depending on the length of the region whose homology is required. For example, complex 3D positioning of multiple fingers is not required. The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid", and "oligonucleotide" are used interchangeably. They refer to the polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or their analogues. Polynucleotides can have any three-dimensional structure and can perform any of the possible known or unknown functions. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) determined from linkage analysis, exons, introns, messenger RNAs (mRNAs), transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), small interfering RNAs (siRNA), short hairpin RNA (shRNA), miRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. The term also covers nucleic acid-like structures with synthetic backbones, see, for example, Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; W097/03211; W096/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. A polynucleotide may contain one or more modified nucleotides such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may occur before or after polymer assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example by conjugation with a labeled moiety. As used herein, the term "wild type" is a term understood by those skilled in the art and means the typical form of an organism, strain, gene, or characteristic that occurs in nature, as opposed to mutated or variant forms. The "wild type" may be the baseline. As used in the present description, the term "variant" should be understood as the manifestation of qualities that are different from those that exist in nature. The terms "non-naturally occurring" or "engineered" are used interchangeably and indicate the involvement of a human hand. The terms referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or polypeptide is at least substantially free of at least one other component with which it is naturally associated in nature and exists in nature. "Complementarity" refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence, either by conventional Watson-Crick base pairing or other non-traditional types. Percent complementarity refers to the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 50%, 60 %, 70%, 80%, 90% and 100% complementary). "Totally complementary" means that all contiguous residues of a nucleic acid sequence are hydrogen bonded to the same number of contiguous residues in a second nucleic acid sequence. "Substantially complementary" as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98 %, 99% or 100% around 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 , 45, 50 or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions. As used herein, the term "stringent conditions" for hybridization refers to conditions under which a nucleic acid having complementarity with a target sequence preferentially hybridizes with the target sequence and does not substantially hybridize with non-target sequences. Stringent conditions tend to be sequence dependent and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are detailed in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, NY When a polynucleotide sequence is given, then complementary or partially complementary sequences are also contemplated. They are preferably capable of hybridizing to said sequence under very stringent conditions. Typically, relatively low hybridization conditions are chosen to maximize the hybridization rate: about 20-25°C lower than the thermal melting point (Tm). Tm is the temperature at which 50% of a particular target sequence hybridizes to a perfectly complementary probe in solution at a defined ionic strength and pH. Typically, in order to require at least ~85% nucleotide complementarity of the hybridized sequences, very stringent washing conditions are chosen, about 5-15° C. lower than the Tm. To require at least about 70% nucleotide complementarity of the hybridized sequences, moderately stringent wash conditions are chosen, about 15-30° C. lower than the Tm. High resolution conditions (very low stringency) can be as low as 50° C. below Tm, resulting in a high level of mismatch between hybridized sequences. Those of skill in the art will appreciate that other physical and chemical parameters during the hybridization and washing steps can also be changed to influence the result of a detected hybridization signal from a certain level of homology between target sequences and probes. Preferred very stringent conditions include incubation in 50% formamide, 5x SSC and 1% SDS at 42°C or incubation in 5×SSC and 1% SDS at 65°C with a wash of 0.2×SSC and 0.1% SDS at 65°C. "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding can occur by Watson-Crick base pairing, Hoogsteen bonding, or any other sequence-specific manner. The complex may contain two strands forming a duplex structure, three or more strands forming a multistrand complex, one strand that hybridizes to itself, or any combination thereof. The hybridization reaction may be a step in a broader process such as initiating a PCR or cleaving a polynucleotide with an enzyme. A sequence capable of hybridizing to a given sequence is said to be "complementary" to that sequence. As used herein, the term "genomic locus" or "locus" (plural: loci) is the specific location of a gene or DNA sequence on a chromosome. "Gene" refers to regions of DNA or RNA that code for a polypeptide or RNA chain that plays a functional role in an organism and is therefore the molecular unit of heredity in living organisms. For the purposes of the present invention, genes may be considered to include regions that regulate gene production, whether or not such regulatory sequences are "contiguous" with coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translation regulator sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, isolators, border elements, origins of replication, matrix attachment sites, and control regions. locus. As used herein, the term "expression of a genomic locus" or "expression of a gene" is the process by which the information contained in a gene is used to synthesize a functional gene product. Gene expression products are often proteins, but in non-protein genes such as rRNA genes or tRNA genes, the product is functional RNA. The process of gene expression is used by all known living organisms - eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea) and viruses - to produce functional products for survival. As used herein, the term "expression" of a gene or nucleic acid encompasses not only the expression of a cellular gene, but also the transcription and translation of the nucleic acid(s) in cloning systems and in any other context. As used herein, the term "expression" also refers to the method by which a polynucleotide is transcribed from a DNA template (e.g. into mRNA or other RNA transcript) and/or the process by which the transcribed mRNA is then translated into peptides, polypeptides or squirrels. Transcripts and encoded polypeptides may be collectively referred to as a "gene product". If the polynucleotide is derived from genomic DNA, expression may involve mRNA splicing in a eukaryotic cell. The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The terms also include a modified amino acid polymer; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation such as conjugation to a labeled moiety. As used herein, the term "amino acid" includes natural and/or non-natural or synthetic amino acids, including glycine and both D or L optical isomers, as well as amino acid analogs and peptidomimetics. As used herein, the term "domain" or "protein domain" refers to a portion of a protein sequence that can exist and function independently of the rest of the protein chain. As described for aspects of this invention, sequence identity is related to sequence homology. Homology comparisons can be made by eye or, most commonly, with readily available sequence comparison programs. These commercially available computer programs can calculate percent (%) homology between two or more sequences, and can also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences.

[00787] In certain embodiments, the term "guide RNA" refers to a polynucleotide sequence containing one or more putative or identified tracr sequences and putative or identified crRNA sequences or guide sequence. In specific embodiments, "guide RNA" includes a putative or identified crRNA sequence or guide sequence. In other embodiments, the guide RNA does not include a putative or identified tracr sequence.

[00788] As used herein, the term "wild type" is a term understood by those skilled in the art and means the typical form of an organism, strain, gene, or characteristic as it occurs in nature, as opposed to mutated or variant forms. The "wild type" may be the baseline.

[00789] As used in the present description, the term "variant" should be understood as the manifestation of properties that have characteristics in which these properties differ from natural properties.

[00790] The terms "non-natural" or "engineered" are used interchangeably and indicate human intervention. These terms referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or polypeptide is at least somewhat devoid of one other component with which it is naturally associated in its natural form. In all aspects and embodiments, whether or not they include these terms, it is to be understood that the use of these terms is optional and therefore there is no preference for their inclusion. In addition, the terms "non-natural" or "engineered" may be used interchangeably and may therefore be used singly or together, and one or the other may replace the mention of both together. In particular, "engineered" is preferred over "non-natural" or "non-natural and/or engineered".

[00791] Sequence homology can be determined by any of a number of computer programs known in the art, such as BLAST or FASTA et al. A suitable computer program for performing this alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software capable of performing sequence comparisons include, but are not limited to, BLAST (see Ausubel et al., 1999 ibid - Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403 -410) and the GENEWORKS Comparison Toolkit. Both BLAST and FASTA are searchable offline and online (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). In this case, it is preferable to use the GCG Bestfit program. Percent (%) sequence homology can be calculated for overlapping sequences, ie. one sequence is aligned against another and the amino acid or nucleotide of one sequence is directly compared to the corresponding other amino acid or nucleotide of the other sequence one(mu) at a time. In this case, it is called "gapless alignment". Typically, this gap-free alignment will only be performed for a relatively small number of residues. Although this is a very simple and logical way, it is not capable of considering, for example, cases of otherwise identical pairs of sequences where one insertion or deletion could cause the subsequent amino acid residue to be out of alignment consideration, thereby leading to a possible large reduction % homology when performing a global alignment. As a result, most sequence comparison methods are designed to perform optimal alignments for possible insertions and deletions without unduly penalizing overall homology or identity score. This is achieved by inserting "gaps" into the sequence alignment in order to maximize local homology or identity. However, these more sophisticated methods impose a "gap penalty" for each gap that occurs in the alignment, so that for the same number of identical amino acids, multiple gaps can be added - reflecting a closer relationship between the two compared sequences - and achieve a higher score. than in the case of one with many gaps. "Gap Affinity Value" is typically used to impose a relatively large penalty for the presence of a gap and a smaller penalty for each subsequent gap residue. This is the most commonly used pass scoring system. High gap penalties can, of course, result in optimized alignments with fewer gaps. Most alignment programs allow for changes to gap penalties. However, it is preferable to use raw values when using such sequence comparison software. For example, when using the GCG Wisconsin Bestfit package, the initial gap penalty in amino acid sequences is -12 for each gap and -4 for each insertion. The calculation of the maximum % homology thus first requires performing an optimal alignment with consideration of gap penalties. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit package (Devereux et al., 1984 Niic. Acids Research 12 p387). Examples of other software capable of performing sequence comparisons are, but are not limited to, BLAST packages (see Ausubel et al., 1999 Short Protocols in Molecular Biology, 4to Ed. - Chapter 18), FASTA (Altschul et al., 1990 J Mol Biol. 403-410) and the GENEWORKS Comparison Toolkit. Both BLAST and FASTA are searchable offline and online (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However, for some tasks, it is preferable to use the GCG Bestfit program. A new tool called BLAST 2 Sequences is also being used to compare protein and nucleotide sequences (see FEMS Microbiol Lett. 1999 174(2): 247-50; FEMS Microbiol Lett. 1999 177(1): 187-8 and website National Center for Biotechnology on the US National Institutes of Health website). Although the final % homology can be measured in terms of identity, the alignment process itself is not based on an all-or-nothing comparison of pairs. Instead, a scaled similarity score matrix is commonly used to calculate scores for each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is the BLOSUM62 matrix, the default matrix for the BLAST suite of programs. GCG Wisconsin programs typically use either the public defaults or a custom character comparison table if available (see user manual for further details). For some purposes, it is preferable to use the public defaults of the GCG package or, in the case of other software, a default matrix such as BLOSUM62. Alternatively, percent homology can be calculated using the multiple alignment property from DNASIS™ (Hitachi Software) based on an algorithm similar to CLUSTAL (Higgins DG & Sharp PIVI (1988), Gene 73(1), 237-244). Once the software has found the optimal alignment, it becomes possible to calculate % homology, preferably % sequence identity. This software usually does this during sequence comparison and gives a numerical result. Sequences may also have deletions, insertions, or substitutions of amino acid residues that result in change without notice and result in functionally equivalent states. Intentional amino acid substitutions can be made based on similarities in amino acid properties (such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues) and are therefore useful for grouping amino acids into functional groups. Amino acids can be grouped based on the properties of their side chains only. However, it is more appropriate to also include data on mutations. The sets of amino acids thus obtained are likely to be conservative due to their structure. Such sets can be described in the form of Venn diagrams (Livingstone C.D. and Barton G.J. (1993) "Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation" Comput. Appl. Biosci. 9: 745-756) (Taylor W.R. (1986 ) "The classification of amino acid conservation" J. Theor. Biol 119, 205-218). Can be made conservative substitutions, for example in accordance with the table below, which describes the conventional grouping of amino acids in the form of a Venn diagram.

Group Subgroup Hydrophobic F W Y H K M I L V A G C aromatic F W Y H Aliphatic I L V Polar W Y H K R E D C S T N Q Charged H K R E D positively charged H K R negatively charged E D small V C A G S P T N D Very small A G S

[792] The terms "subject", "individual" and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, mice, monkeys, humans, farm animals, sport animals, and pets. Also covered are tissues, cells and their progeny derived from a biological entity in vivo or cultured in vitro .

[00793] The terms "therapeutic agent", "therapeutically capable agent" or "therapeutic agent" are used interchangeably and refer to a molecule or compound that confers some beneficial effect when administered to an individual. Benefits include providing diagnostic definitions; alleviating the course of a disease, symptom, disorder or condition; reducing or preventing the occurrence of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder, or pathological condition.

[00794] As used herein, the terms "treatment" or "relief" or "improvement" are used interchangeably. These terms refer to an approach to obtain beneficial or desired results, including, but not limited to, therapeutic effect and/or prophylactic benefit. By therapeutic effect is meant any therapeutically significant improvement in or effect on one or more of the diseases, conditions or symptoms being treated. For prophylactic benefit, the constructs may be administered to an individual at risk for developing a particular disease, condition, or symptom, or to an individual exhibiting one or more physiological symptoms of a disease, although the disease, condition, or symptom may not yet have manifested.

[00795] The term "effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to produce beneficial or desired results. The therapeutically effective amount may vary depending on one or more of the individual and the condition of the disease being treated, the weight and age of the individual, the severity of the disease, the route of administration, and the like, which can be readily determined by one of ordinary skill in the art. The term also refers to a dose that will provide imaging for detection by any of the imaging modalities described herein. The specific dosage may vary depending on one or more of the particular agent selected, the dosage regimen to be followed, the administration of the combination with other compounds, the time of administration, the tissue to be imaged, and the physical delivery system.

[00796] In the practice of the present invention, unless otherwise indicated, conventional methods of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the competence of experts. See Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F, M. Ausubel, et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Flames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)).

[00797] Several aspects of the invention relate to vector systems containing one or more vectors, or vectors as such. Vectors can be designed to express CRISPR transcripts (eg, nucleic acid, protein, or enzyme transcripts) in prokaryotic or eukaryotic cells. For example, CRISPR transcripts can be expressed in bacterial cells such as Escherichia coli , insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro , for example, using the T7 promoter and T7 polymerase regulatory sequences.

[00798] Embodiments of the invention include sequences (both polynucleotide and polypeptide) that may contain a homologous substitution ("substitution" is used herein to refer to the exchange of an existing amino acid residue or nucleotide with an alternative residue or nucleotide), which can occur, for example , for a similar substitution in the case of amino acids, such as basic for basic, acidic for acidic, polar for polar, etc. Non-homologous substitution can also occur, i.e. from one class of residues to another or, alternatively, the incorporation of unnatural amino acids, such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyroarylanine, thienylalanine, naphthylalanine and phenylglycine. Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence, including alkyl groups such as methyl, ethyl or propyl groups, in addition to amino acid spacers such as glycine or β-alanine residues. Those of skill in the art may well understand a further form of variation which includes the presence of one or more amino acid residues in peptoid form. For the avoidance of doubt, "peptoid form" is used to refer to variants of amino acid residues in which the α-carbon substituent group is on the nitrogen atom of the residue and not on the α-carbon. Methods for preparing peptides in peptoid form are known in the art, for example, Simon RJ et al., PNAS (1992) 89 (20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134.

[00799] Homology Modeling: Corresponding residues in other C2c2 orthologues can be identified using the methods of Zhang et al., 2012 (Nature, 490 (7421): 556-60) and Chen et al., 2015 (PLoS Comput Biol; 11 ( 5): e1004248) is a protein-protein interaction (PP1) calculation method for predicting interactions mediated by domain-motif contact surfaces. PrePPI (Predicting PPI), a structure-based way of predicting PPI, combines structural evidence with non-structural data using a Bayesian statistical model. The method includes taking a pair of query proteins and using a structural alignment to identify structural representatives corresponding to either their experimentally determined structures or homologous patterns. Structural alignment is further used to identify both near and far structural neighbors by considering global and local geometric relationships. Whenever two neighbors of structural representatives form a complex, specified in the Protein Data Bank, this defines a template for modeling the interaction between two query proteins. Complex models are created by superimposing representative structures on the corresponding structural neighbor in the template. This approach is further described in Dey et al, 2013 (Prot Sci, 22: 359-66).

[00800] For the purposes of the present invention, amplification means any method using a primer and a polymerase capable of replicating a target sequence with reasonable fidelity. Amplification can be performed with natural or recombinant DNA polymerases such as TaqGold™ DNA polymerase, T7, the Klenow fragment of E. coli DNA polymerase, and reverse transcriptase. The preferred amplification method is PCR.

[00801] In some embodiments, the invention includes vectors. As used herein, the term "vector" is a tool that allows or facilitates the transfer of an object from one medium to another. It is a replicon such as a plasmid, phage or cosmid into which another segment of DNA can be inserted to cause the inserted segment to replicate. Typically, a vector is capable of replication when it is associated with the appropriate controls. In general, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single stranded, double stranded, or partially double stranded; nucleic acid molecules that contain one or more free ends, or do not contain free ends (for example, round); nucleic acid molecules that contain DNA, RNA, or both; and other types of polynucleotides known in the art. One type of vector is a "plasmid", which refers to a round, double-stranded loop of DNA into which additional DNA segments can be added by standard molecular cloning techniques. Another type of vector is a viral vector, in which viral DNA or RNA (eg, retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses (AAV)) are present in the vector to package the virus. Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Some vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) are integrated into the genome of the host cell upon introduction into the host cell and thus replicate along with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors". Conventional expression vectors used in recombinant DNA methods are often in the form of plasmids.

[00802] Recombinant expression vectors may contain the nucleic acids of the invention in a form suitable for expression of the nucleic acids in a host cell, which means that the recombinant expression vectors include one or more regulatory elements that may be selected based on the host cells used. for expression, and which are operably linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, "operably linked" means that the target nucleotide sequence is linked to the regulatory element(s) in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the cell). - host). With respect to recombination and cloning methods, reference may be made to US Patent Application 10/815730, published September 2, 2004 as US 2004-0171156 A1, the contents of which are incorporated herein by reference in its entirety.

[00803] Aspects of the invention relate to bicistronic vectors for guide RNA and wild-type RNA, modified or mutated CRISPR effector proteins/enzymes (eg, C2c2). Bicistronic expression vectors of guide RNA and wild-type RNA, modified or mutated CRISPR effector proteins/enzymes (eg C2c1 or C2c3). In general, and in particular in this embodiment and wild type, modified or mutated CRISPR effector proteins/enzymes (eg C2c1 or C2c3) are preferably under the control of the CBh promoter. The RNA may preferably be under the control of an RNA polymerase III promoter such as the U6 promoter. Ideally, these two promoters are combined.

[00804] In some embodiments, a loop is provided in the guide RNA. It can be a hairpin or a tetra-loop. The loop is preferably GAAA, but is not limited to this sequence or even 4 bp in length. Indeed, the preferred loop forming sequences for use in hairpin structures are four nucleotides in length and most preferably have the GAAA sequence. However, longer or shorter loop sequences, as well as alternative sequences, may be used. The sequences preferably include a nucleotide triplet (eg AAA) and an additional nucleotide (eg C or G). Examples of loop forming sequences include CAAA and AAAG.

[00805] In any of the methods described herein, a suitable vector may be introduced into a cell or embryo by one or more methods known in the art, including, but not limited to, microinjection, electroporation, sonoporation, biolistics, mediated calcium phosphate transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection, magnetotransfection, lipofection, impalefection, optical transfection, proprietary agent-enhanced uptake of nucleic acids, delivery via liposomes, immunoliposomes, virosomes, or artificial virions. In some methods, the vector is introduced into the embryo by microinjection. The vector or vectors can be introduced by microinjection into the nucleus or cytoplasm of the embryo. In some methods, the vector or vectors may be introduced into a cell by nucleofection.

[00806] The term "regulatory element" is used to refer to promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (eg, transcription termination signals such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulators include both those that directly control the constitutive expression of a nucleotide sequence in many types of host cells and those that control the expression of a nucleotide sequence only in certain host cells (eg, tissue-specific regulatory sequences). A tissue-specific promoter can drive expression primarily in the desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (eg, liver, pancreas), or specific cell types (eg, lymphocytes). Regulatory elements can also drive expression in a time dependent manner, eg depending on cell cycle stage or developmental stage, and may or may not be tissue or cell specific. In some embodiments, the vector contains one or more DNA polymerase III promoters (e.g., 1, 2, 3, 4, 5 or more DNA polymerase III promoters), one or more DNA polymerase II promoters (e.g., 1, 2, 3, 4, 5 or more DNA polymerase II promoters), one or more DNA polymerase I promoters (eg 1, 2, 3, 4, 5 or more DNA polymerase I promoters), or combinations thereof. Examples of DNA polymerase III promoters include, but are not limited to, the U6 and HI promoters. Examples of DNA polymerase II promoters include, but are not limited to, Rous sarcoma (RSV) RAR retroviral LTR promoter (optionally with an RSV enhancer), Cytomegalovirus (CMV) promoter (optionally with a CMV enhancer) [see, e.g., Boshart et al. ., Ceil, 41: 521-530 (1985)], SV40 promoter, dihydrofolate reductase promoter, 3-actin promoter, phosphoglycerol kinase (PGK) promoter and EF1a promoter. In addition, the term "regulatory element" encompasses enhancer elements such as WPRE; CMV enhancers; segment R-U5' in LTR HTLV-I (Mol, Cell. Biol, vol. 8(1), pp. 466-472, 1988); enhancer SV40; and an intron sequence between exons 2 and 3 of the rabbit P-globin protein (Proc. Natl. Acad. Sci. USA., vol. 78 (3), p, 1527-31, 1981). Those skilled in the art will appreciate that the design of the expression vector may depend on factors such as the choice of the host cell to be transfected, the level of expression desired, etc. The vector can be introduced into cells such that they produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., regularly clustered short palindromic repeat system (CRISPR) transcripts, their proteins, enzymes, their mutant forms, their fusion proteins, etc.). With regard to regulatory sequences, US Patent Application 10/491026 may be mentioned, the contents of which are incorporated herein by reference in their entirety. With respect to promoters, reference is made to PCT Publication WO 2011/028929 and US Appendix 12/511940, the contents of which are incorporated herein by reference in their entirety.

[00807] Vectors can be designed to express CRISPR transcripts (eg, nucleic acid, protein, or enzyme transcripts) in prokaryotic or eukaryotic cells. For example, CRISPR transcripts can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro , for example, using the T7 promoter and T7 polymerase regulatory sequences.

[00808] Vectors can be introduced and expanded in prokaryotic organisms or prokaryotic cells. In some embodiments, a prokaryotic organism is used to amplify copies of a vector to be introduced into a eukaryotic cell or as an intermediate vector in the formation of a vector to be introduced into a eukaryotic cell (eg, amplification of a plasmid as part of a viral vector packaging system). In some embodiments, a prokaryotic organism is used to amplify copies of a vector and express one or more nucleic acids, for example, to provide a source of one or more proteins for delivery to a host cell or host organism. Protein expression in prokaryotes most commonly occurs in Escherichia coli with vectors containing constitutive or inducible promoters that direct the expression of either fusion or non-fusion proteins. Fusion vectors insert a number of amino acids into the protein they encode, for example, at the N-terminus of the recombinant protein. Such fusion vectors may serve one or more purposes, such as: (i) increasing expression of the recombinant protein; (ii) increasing the solubility of the recombinant protein; and (iii) assisting in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced in fusion expression vectors at the junction of the fusion region and the recombinant protein to allow separation of the recombinant protein from the fusion fragment after purification of the fusion protein. Such enzymes and their related recognition sequences include factor Xa, thrombin, and enterokinase. Example fusion expression vectors include pGEX (Pharmacia Biotech Inc, Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, NJ), which produce glutathione fusion S-transferase (GST), maltose-binding protein E or protein A, respectively, with the target recombinant protein.

[00809] Examples of suitable inducible non- fused E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69: 301-315) and pET lid (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press , San Diego, CA (1990) 60-89).

[00810] In some embodiments, the vector is a yeast expression vector. Examples of expression vectors in the yeast Saccharomyces cerivisae include pYepSeel (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al. , 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, CA.) and picZ (InVitrogen Corp, San Diego, CA).

[00811] In some embodiments, the vector directs protein expression in insect cells using baculovirus expression vectors. Baculovirus vectors available for protein expression in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol., 3:2156-2165) and the pVL series (Lucklow and Summers , 1989. Virology 170: 31-39).

[00812] In some embodiments, the vector is capable of causing expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the control functions of an expression vector are typically performed by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, SV40 virus, and others described herein and known in the art. Other suitable expression systems for both prokaryotic and eukaryotic cells can be studied at: see, for example, chapters 16 and 17 of Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed., Laboratory Cold Spring Harbor, Laboratory Cold Spring Harbor Press, Cold Spring Harbor, NY, 1989.

[00813] In some embodiments, a recombinant mammalian expression vector is capable of directing the expression of nucleic acids preferentially in a particular cell type (eg, tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific: Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calarne and Eaton, 1988. Adv. Immunol 43: 235-275) , in particular T cell receptor promoters (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neurospecific promoters (e.g., neurofilament promoter, Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA, 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916) and mammary gland-specific promoters (eg, whey promoter, US Pat. No. 4,873,316 and EP Publication No. 264,166). Developmentally regulated promoters also include, for example, mouse hox promoters (Kessel and Grass, 1990. Science 249: 374-379) and the α-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546). With respect to these prokaryotic and eukaryotic vectors, US Pat. No. 6,750,509 may be mentioned, the contents of which are incorporated herein by reference in their entirety. Other embodiments of the invention may relate to the use of viral vectors, in respect of which US patent application 13/1002085 is mentioned, the contents of which are incorporated herein by reference in its entirety. Tissue-specific regulatory elements are known in the art and reference is made to US Pat. No. 7,776,321, the contents of which are incorporated herein by reference in their entirety.

[00814] In some embodiments, a regulatory element is operably linked to one or more elements of the CRISPR system to direct the expression of one or more elements of the CRISPR system. In general, CRISPR (Regularly Clustered Short Palindromic Repeats), also known as SPIDR (Scattered Between Spacer Direct Repeats), is a family of DNA loci that are usually specific to specific bacterial species. The CRISPR locus contains a distinct class of alternating short sequence repeats (SSRs) that have been described in E. coli (Ishino et al., J. Bacteriol. 169: 5429-5433 [1987] and Nakata et al., J. Bacteriol., 171 : 3553-3556 [1989]) and associated genes. Similar SSR regions have been identified in Haloferax mediterranei , Streptococcus pyogenes , Anabaena , and Mycobacterium tuberculosis (see Groenen et al., Mol. Microbiol., 10:1057-1065 [1993], Hoe et al., Emerg. Infect. Dis., 5: 254-263 [1999], Masepohl et al., Biochim Biophys Acta 1307: 26-30 [1996], Mojica et al., Mol. Microbiol., 17: 85-93 [1995]). CRISPR loci generally differ from other SSRs in the structure of repeats, which are called short regularly interrupted repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol., 6: 23-33 [2002], and Mojica et al. , Mol. Microbiol., 36: 244-246 [2000]). In general, repeats are short units that form clusters that are regularly distributed over unique, interspersed sequences of approximately constant length (Mojica et al., [2000], supra). Although the repeat sequences are very conserved across strains, the number of discontinuous repeats and spacer region sequences generally differ between strains (van Embden et al., J. Bacteriol, 182: 2393-2401 [2000]). CRISPR loci have been identified in more than 40 prokaryotes (see, for example, Jansen et al., Mol. Microbiol., 43: 1565-1575 [2002] and Mojica et al., [2005]), including, but not limited to, Aeropyrum, Pyrobacidum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staridium, Thermobacterco, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desidfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema and Thermotoga.

[00815] In general, the term "nucleic acid targeting system" used in the present invention refers collectively to transcripts and other elements involved in the expression or directing nucleic acid targeting activity of CRISPR-associated ("Cas") genes (also referred to as effector proteins herein), including sequences encoding a protein (effector) targeting a nucleic acid and a guide RNA (containing a crRNA sequence and a transactivating RNA sequence (tracr-RNA) of the CRISPR/Cas system) or other sequences and transcripts from CRISPR locus targeting nucleic acids. In some embodiments, one or more elements of a nucleic acid targeting system are derived from a CRISPR system targeting type V/VI nucleic acids. In some embodiments, one or more elements of the nucleic acid targeting system are derived from a particular organism, comprising an endogenous CRISPR system targeting nucleic acids. In general, a nucleic acid targeting system is characterized by elements that promote the formation of a nucleic acid targeting complex at the site of the target sequence. In the context of forming a complex targeting a nucleic acid, "target sequence" refers to a sequence for which the target sequence is intended and which has the complementarity necessary for hybridization between the target sequence and the target RNA, which promotes the formation of a complex with DNA or RNA. Complete complementarity is not required as long as there is sufficient complementarity to induce hybridization and promote formation of a nucleic acid-targeted complex. The target sequence may contain RNA polynucleotides. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell. In some embodiments, the target sequence may be within eukaryotic cell organelles, such as mitochondria or chloroplasts. A sequence or template that can be used for recombination at a target locus containing the target sequence is referred to as an "editing template or "editing RNA" or "editing sequence". In aspects of the invention, an exogenous RNA template may be referred to as an editing template. In any aspect of the invention, the recombination is homologous recombination.

[00816] Generally, in the context of an endogenous nucleic acid targeting system, formation of a nucleic acid targeting complex (comprising a guide RNA hybridized to a target sequence in complex with one or more nucleic acid targeting effector proteins) results in cleavage of one or both strands of RNA (about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs) of the target sequence. In some embodiments, one or more vectors directing the expression of one or more nucleic acid targeting system elements are introduced into a cell such that expression of the nucleic acid targeting system elements directs the formation of a nucleic acid targeting complex at one or more target sites. . For example, a nucleic acid-targeting effector protein and guide RNA can be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed with the same or different regulatory elements can be combined in one vector with one or more additional vectors providing other components of the nucleic acid targeting system not included in the first vector. The components of a nucleic acid targeting system combined in a single vector may be arranged in any suitable orientation, such as one element located 5' to (upstream) or 3' to (downstream) the other. element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of the second element and be oriented in the same or opposite direction. In some embodiments, a single promoter directs the expression of a transcript encoding an effector protein targeting a nucleic acid and a guide RNA inserted into one or more intron sequences (e.g., each in a separate intron, two or more in at least one intron, or all in one intron). In some embodiments, the nucleic acid targeting effector protein and the guide RNA are operably linked and expressed from the same promoter.

[00817] In general, a targeting sequence is any polynucleotide sequence having sufficient complementarity with the target polynucleotide sequence for hybridization to the target sequence and direct sequence-specific binding of the nucleic acid-targeted complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence when optimally aligned using a suitable alignment algorithm is about or greater than 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97 .5%, 99% or more. The optimal alignment can be determined using any suitable sequence alignment algorithm, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunch algorithm, Burroughs-Wheeler transform-based algorithms (e.g., Burrows Wheeler Aligner), ClustalW, Clustal X, BLAST, Novoalign (Novocraft Technologies, ELAND (iIllumina, San Diego, CA), SOAP (available from soap.genornics.org.cn), and Maq (available from maq.sourceforge.net). In some embodiments, the guide sequence is about or greater than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides in length In some embodiments, the targeting sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides in length. The ability of the guide sequence to drive with sequence-specific binding of a nucleic acid-targeted complex to a target sequence can be assessed by any suitable method. For example, components of a nucleic acid-targeting system sufficient to form the complex, including the targeting sequence to be tested, can be delivered into a cell having the appropriate target sequence, e.g., by transfection with vectors encoding CRISPR nucleic acid-targeting sequence components with subsequent assessment of preferred cleavage within or near the target sequence, such as the Surveyor assay described herein. Similarly, cleavage of a target polynucleotide sequence (or a sequence near it) can be assessed in vitro by providing the target sequence, the components of the nucleic acid targeting complex, including the targeting sequence to be tested, and a control targeting sequence other than the targeting sequence being tested, and comparing the binding or rate of cleavage occurring directly at or near the target sequence between the reactions of the test and control guide sequences. Other methods of analysis are also possible and can be developed by experts in this field.

[00818] The targeting sequence can be chosen to target any target sequence. In some embodiments, the target sequence is a sequence within a gene or mRNA transcript.

[00819] In some embodiments, the target sequence is a sequence in the cell's genome.

[00820] In some embodiments, a guide sequence is selected to compact the secondary structure within the guide sequence. The secondary structure may be determined by any suitable polynucleotide folding algorithm. Some programs are based on the calculation of the minimum Gibbs free energy. An example of such an algorithm is mFold as described by Zuker and Stiegler (Nucleic Acids Res., 9 (1981), 133-148). Another example of a folding detection algorithm is the RNAfold web server developed at the Institute for Theoretical Chemistry, University of Vienna, using a centroid prediction algorithm (see, e.g., AR Gruberera, 2008, Cell 106 (1): 23-24; PA Carr and GM Church, 2009, Nature Biotechnology 27 (12): 1151-62). Other algorithms can be found in US application serial number TBA (patent registry number 44790.11.2022, wide reference B1-2013/004A); included in the present description by reference.

[00821] In some embodiments, a recombination matrix is provided. The recombination template may be a component of another vector described herein, may be contained in a separate vector, or provided as a separate polynucleotide. In some embodiments, the recombination template is designed to serve as a homologous recombination template near or within a target sequence that is cleaved or nick-nipped by a nucleic acid-targeted effector protein as part of the nucleic acid-targeted complex. The template polynucleotide may be any suitable length of about or greater than 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000 or more nucleotides. In some embodiments, the template polynucleotide is complementary to a portion of the polynucleotide containing the target sequence. When optimally aligned, the template polynucleotide may overlap with one or more nucleotides of the target sequence (e.g., approximately or more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 , 100 or more nucleotides). In some embodiments, when the template polynucleotide is optimally aligned with the polynucleotide containing the target sequence, the nearest nucleotide of the template sequence is within 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000 or more nucleotides from the target sequence.

[00822] In some embodiments, the nucleic acid targeting effector protein is part of a fusion protein containing one or more heterologous protein domains (e.g., about or more than 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10 or more domains in addition to an effector protein targeting the nucleic acid). In some embodiments, the CRISPR effector protein/enzyme is part of a fusion protein containing one or more heterologous protein domains (e.g., about or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme). The CRISPR effector protein/enzyme may contain any additional protein sequence and optionally a linker sequence between any two domains. Examples of protein domains that can be fused to an effector protein include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase, demethylase, transcription activation, transcription suppression, transcription termination factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to: glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetitransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed , blue fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins, including blue fluorescent protein (BFP). An effector protein targeting a nucleic acid may be fused to a gene sequence encoding a protein or protein fragment that binds DNA molecules or binds other cellular molecules, including but not limited to the following: maltose binding protein (MBP), S-tag , Lex A DNA binding domain (DBD) fusion constructs, GAL4 DNA binding domain fusion constructs, and herpes simplex virus (HSV) BP16 protein fusion constructs. Additional domains that may be part of a fusion protein including an effector protein targeting a nucleic acid are described in US Patent Publication 20110059502, incorporated herein by reference. In some embodiments, a labeled effector protein that targets a nucleic acid is used to identify the location of a target sequence.

[00823] In some embodiments, the CRISPR enzyme may be a component of an inducible system. The inducible nature of the system allows spatiotemporal control of gene editing or gene expression using energy. The form of energy may include, but is not limited to, electromagnetic radiation, sound energy, chemical energy, and thermal energy. Examples of an inducible system include tetracycline inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptional activation systems (FKBP, ABA, etc.), light-inducible systems (phytochrome, LOV domains, or cryptochrome). In one embodiment of the invention, the CRISPR enzyme may be part of a light-induced transcription effector (LITE) to directly alter sequence-specific transcriptional activity. The light-inducible components may include the CRISPR enzyme, a photosensitive cytochrome heterodimer (eg, Arabidopsis thaliana ), and a transcriptional activation/inhibition domain. Other examples of inducible DNA-binding proteins and methods of using them are given in US 61/736465 and US 61/721283 and WO 2014/018423 and US8889418, US8895308, US20140186919, US20140242700, US20140273234, US20140332014, which are incorporated herein as references,9363 in full.

Delivery

[00 824] In some embodiments, the present invention relates to methods comprising delivering one or more polynucleotides, such as or one or more vectors as described herein, one or more transcripts thereof, and/or one or more proteins transcribed from them, in a cage. In some aspects, the invention further relates to cells obtained by such methods, and organisms (such as animals, plants or fungi) containing or derived from such cells. In some embodiments, a nucleic acid-targeting effector protein in combination with (and possibly complexed with) a guide RNA is delivered to a cell. Conventional viral and non-viral gene transfer methods can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to introduce nucleic acids encoding components of a nucleic acid targeting system into cells of a culture or organism. Delivery systems for non-viral vectors include DNA plasmids, RNA (eg, the transcript of the vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome. Viral vector delivery systems include DNA and RNA viruses that have either episomal or integrated genomes upon delivery into a cell. For a review of gene therapy methods, see Anderson, Science 256: 808-813 (1992); Nab el & Feigner, TIBTECH 11: 211-217 (1993), Mitani & Caskey, TIBTECH 11: 162-166 (1993); Dillon, TIBTESCH 11: 167-175 (1993); Miller, Nature 357: 455-460 (1992); Van Brunt, Biotechnology 6 (10): 1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8: 35-36 (1995), Kremer & Perricaudet, British Medical Bulletin 51 (1): 31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology, Doerfler and Bomm (eds) (1995) and Yu et al., Gene Therapy 1: 13-26 (1994).

[00825] Methods for non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycations or lipid-nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced DNA uptake. Lipofection is described, for example, in US patent No. 5049386, 4946787; and 4897355), and commercial lipofection reagents (eg Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient recognition of polynucleotide lipofection receptors include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (eg, in vitro or ex vivo ) or target tissues (eg, in vivo ).

[00826] The preparation of complexes of lipids with nucleic acids, including target liposomes, such as immunolipid complexes, is well known to the person skilled in the art (see, for example, Crystal, Science 270: 404-410 (1995), Blaese et al., Cancer Gene Ther 2: 291-297 (1995), Behr et at., Bioconjugate Chem., 5: 382-389 (1994), Remy et al., Bioconjugate Chem., 5: 647-654 (1994), Gao et al. ., Gene Therapy 2: 710-722 (1995), Ahmad et al., Cancer Res. 52: 4817-4820 (1992), US Pat. 4946787).

[00827] The use of RNA or DNA viral systems to deliver nucleic acids utilizes current techniques to target the virus to specific cells in the body and transfer the viral genome to the nucleus. Viral vectors can be administered directly to patients ( in vivo ) or they can be used to treat cells in vitro , and modified cells can be administered to patients ( ex vivo ). Conventional viral systems may include retroviral, lentiviral, adenovirus, adeno-associated, and herpes simplex viruses for gene transfer. Integration into the host genome is possible using gene transfer techniques for retroviruses, lentiviruses, and adeno-associated viruses, often resulting in long-term expression of the introduced transgene. In addition, high transduction efficiency has been observed in many other target cell and tissue types.

[00828] The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, thereby expanding the potential population of cells that can be targeted by the gene. Lentiviral vectors are retroviral vectors that are capable of transforming or infecting non-dividing cells and typically have high viral titers. Therefore, the choice of retroviral gene transfer system will depend on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats with a packaging capacity of up to 6-10 kb. foreign sequence. Minimal cis-acting LTRs are sufficient to replicate and package the vectors, which are then used to integrate the therapeutic gene into the target cell to ensure consistent transgene expression. Commonly used retroviral vectors include vectors based on murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol 66: 2731-2739 (1992), Johann et al., J. Virol 66: 1635-1640 (1992), Sommnerfelt et al., Virol 176: 58-59 (1990), Wilson et al. ., J. Virol, 63: 2374-2378 (1989), Miller et al., J. Virol 65: 2220-2224 (1991), PCT/US94/05700). In those embodiments where transient expression is preferred, adenoviral systems may be used. Adenovirus vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and expression levels have been obtained. This vector can be obtained in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors can also be used to transduce cells with target nucleic acids, for example, in vitro production of nucleic acids and peptides, as well as for in vivo and ex vivo gene therapy procedures (see, for example, West et al., Virology 160: 38-47 (1987), US 4797368, WO 93/24641, Kotin, Human Gene Therapy 5: 793-801 (1994), Muzyczka, J. Clin Invest.: 1351 (1994). The construction of recombinant AAV vectors has been described in a number of publications, including US Patent No. 5,173,414, Tratschin et al., Mol. Cell, Biol., 5: 3251-3260 (1985), Tratschin, et al., Mol. Cell, Biol., 4: 2072-2081 (1984), Hermonat & Muzyczka, PNAS 81: 6466-66470 (1984) and Samulski et al., J. Virol 63: 03822-3828 (1989).

Options for DNA/RNA or DNA/DNA or RNA/RNA or Protein/RNA

[00829] In some embodiments, components of CRISPR systems can be delivered in various forms such as DNA/RNA or RNA/RNA or protein/RNA combinations. For example, C2c1 or C2c3 can be delivered as a DNA encoding polynucleotide, or as an RNA encoding polynucleotide, or as a protein. The targeting molecule can be delivered as a DNA encoding polynucleotide or as RNA. All possible combinations are provided, including mixed forms of delivery.

[00830] In some embodiments, all such combinations (DNA/RNA, or DNA/DNA, or RNA/RNA, or protein/RNA) are contemplated.

[00831] In some embodiments, when C2c1 or C2c3 is delivered in the form of a protein, pre-assembly with one or more targeting molecules is possible.

Nanocoils

[00832] In addition, the CRISPR system can be delivered using nanocoils, such as those described in Sun W et al, Cocoon-like self-degradable DNA nanoclew for anticancer drag delivery., J Am Chem Soc. 2014 Oct 22; 136(42): 14722-5. doi: 10.1021/ja5088024. Epub 2014 Oct 13; or in Sun W et al, Self-Assembled DNA Nanoclews for the Efficient Delivery of CRISPR-Cas9 for Genome Editing., Angew Chem Int Ed En), 2015 Oct 5;54(41): 12029-33. doi: 10.1002/anie.201506030. Epub 2015 Aug 27.

[00833] The practice of the present invention uses, unless otherwise indicated, conventional methods of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA technology that are standard for those skilled in the art. See Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel, et al. eds., (1987)); METHODS IN ENZYMOLOGY series (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hanies and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, AND ANIMAL CELL CULTURE (RI Freshney, ed. (1987)).

Models of genetic and epigenetic conditions

[00834] The method of the invention can be used to create a plant, animal, or cell that can be used to model and/or study genetic or epigenetic conditions of interest, for example, through a mutation or disease model of interest. As used herein, the term "disease" refers to a disease, disorder, or symptom in an individual. For example, the method of the invention can be used to create an animal or cell that includes a modification in one or more disease-related nucleic acid sequences, or a plant, animal, or cell in which the expression of one or more disease-related nucleic acid sequences is changed. Such a nucleic acid sequence may encode a disease-associated protein sequence or may be a disease-associated control sequence. Accordingly, it is understood that in embodiments of the invention, the plant, individual, patient, organism, or cell may be a non-human patient, organism, or non-human cell. Thus, the invention relates to a plant, animal or cell obtained by the methods of the present invention, or their progeny. The offspring may be a clone of the produced plant or animal, or may be the result of sexual reproduction by crossing with other individuals of the same species to introduce other desirable traits into their offspring. The cell may be in vivo or ex vivo in the case of multicellular organisms, especially animals or plants. In the case where the cell is cultured, a cell line can be established if the appropriate culture conditions are met, and preferably if the cell is suitably adapted for the purpose (eg, a stem cell). Also contemplated are bacterial cell lines produced in accordance with the invention. Thus, cell lines are also envisaged.

[00835] In some ways, a disease model can be used to study the effects of mutations on an animal or cell, the development and/or progression of a disease, using measures commonly used in studying the disease. Alternatively, such a disease model is useful for studying the effect of pharmaceutically active compounds on disease.

[00836] In some ways, a disease model can be used to evaluate the effectiveness of a potential gene therapy strategy. That is, a disease-associated gene or polynucleotide can be modified such that the development of the disease and/or its progression is inhibited or reduced. In particular, the method includes modifying a disease-associated gene or polynucleotide such that an altered protein is produced and, as a result, the animal or cell has an altered response. Accordingly, in some ways, a genetically modified animal can be compared with an animal predisposed to the development of a disease, which will make it possible to evaluate the effect of gene therapy.

[00837] In another embodiment, the present invention relates to methods for developing a biologically active agent that alters the signal of cells associated with a disease gene. The method includes contacting a test compound with a cell containing one or more vectors that direct the expression of one or more CRISPR enzymes and a direct repeat sequence associated with a guide sequence; and detecting a change in readings that indicates a decrease or increase in a cell signal associated with, for example, a mutation in a disease gene contained in the cell.

[00838] A cell model or animal model can be created in conjunction with the method of the invention to diagnose changes in cellular function. Such a model can be used to study the effect of a genome sequence modified with the CRISPR complex of the invention on a cellular function of interest. For example, a cellular function model can be used to study the effect of a modified genome sequence on intracellular signaling or extracellular signaling. Alternatively, a cellular function model can be used to study the effects of a modified genome sequence on sensory perception. In some of these models, one or more genomic sequences associated with a signaling biochemical pathway in the model are modified.

[00839] Several disease models have been investigated. These include the de novo study of the autism risk genes CHD8, KATNAL2 and SCN2A; and the syndromic autism gene (Angelman syndrome) UBE3A. Of course, these genes and resulting models of autism are preferred, but serve to show the broad applicability of the invention to genes and related models.

[00840] Altered expression of one or more genomic sequences associated with a signaling biochemical pathway can be determined by analyzing the difference in mRNA levels of the respective genes between a test model cell and a control cell when they are in contact with a candidate. Alternatively, differential expression of sequences associated with the signaling biochemical pathway is determined by detecting differences in the amount of encoded polypeptide or gene product.

[00841] To analyze the agent-induced change in the level of mRNA transcripts or the corresponding polynucleotides, the nucleic acid contained in the sample is first extracted in accordance with standard methods in this field. For example, mRNA can be isolated using various lytic enzymes or chemical solutions according to the methods set forth in Sambrook et al. (1989) or extracted by binding the nucleic acid to resins according to the manufacturer's instructions. The mRNA contained in the extracted nucleic acid sample is then detected using amplification methods or conventional hybridization assays (eg Northern blotting) according to methods well known in the art or based on the methods described herein.

[00842] For the purposes of the present invention, amplification means any method using a primer and a polymerase capable of replicating the target sequence with sufficient fidelity. Amplification can be performed with natural or recombinant DNA polymerases such as TaqGold™ DNA polymerase, T7, the Klenow fragment of E. coli DNA polymerase, and reverse transcriptase. The preferred amplification method is PCR. In particular, isolated RNA can be analyzed using reverse transcription, which is coupled with a quantitative polymerase chain reaction (RT-PCR), to quantify the level of expression of a sequence associated with a signaling biochemical pathway.

[00843] Determining the level of gene expression can be done in real time in an amplification assay. In one aspect, the amplified products can be directly visualized with fluorescent DNA binding agents, including, but not limited to, DNA intercalators and DNA groove binding agents. Since the number of intercalators included in double-stranded DNA molecules is generally proportional to the number of amplified DNA products, it is convenient to quantify the amount of amplified products by quantifying the fluorescence of the intercalated dye using standard optical instruments used in the art. Suitable DNA-binding dyes for this purpose include SYBR green, SYBR blue, DAPI, propidium iodide, hoechst, SYBR gold, ethidium bromide, acridine dyes, daunomycin, chloroquine, distamycin D, chromomycin, mithramycin, ruthenium polypyridines, anthramycin etc.

[00844] In another aspect, other fluorescent labels, such as sequence-specific probes, can be used in the amplification reaction to facilitate detection and quantification of amplified products. Quantitative probe-based amplification depends on sequence-specific determination of the desired amplification product. It uses fluorescent target-specific probes (eg TaqMan® probes) resulting in increased specificity and sensitivity. Methods for performing quantitative probe-based amplification are well known in the art and are described in US Pat. No. 5,210,015.

[00845] In yet another aspect, conventional hybridization assays can be performed using hybridization probes homologous to sequences associated with the signaling pathway. Typically, probes are allowed to form stable complexes with sequences associated with the signal biochemical pathway contained in a biological sample obtained from the test individual during the hybridization reaction. One of skill in the art will appreciate that when an antisense sequence is used in a probe nucleic acid, the target polynucleotides present in the sample are chosen to be complementary to the antisense nucleic acid sequences. In contrast, when the nucleotide probe is a sense nucleic acid sequence, the sense nucleic acid sequence complementary to the sequences is selected as the target polynucleotide.

[00846] Hybridization can be performed under conditions varying in severity. Suitable hybridization conditions for the practice of the present invention are such that the interaction upon recognition by the probe of sequences associated with the signal biochemical pathway is sufficiently specific and sufficiently stable. Conditions that increase the stringency of the hybridization reaction conditions are widely known and published in the art. See, for example, (Sambrook, et al., (1989), Nonradioactive In Situ Hybridization Application Manual, Boehringer Mannheim, second edition). Hybridization assays can be performed using probes immobilized on any solid support including, but not limited to, nitrocellulose, glass, silicone, and a variety of gene arrays. It is preferred to carry out the hybridization assay on high density gene chips as described in US Pat. No. 5,445,934.

[00847] For convenient detection of probe-target complexes formed during hybridization assays, nucleotide probes can be conjugated to a detectable label. Detectable labels suitable for use in the present invention include any construct detectable by photochemical, biochemical, spectroscopic, immunochemical, electrical, optical, or chemical means. A large number of suitable detectable labels are known in the art, which include fluorescent or chemiluminescent labels, radioactive isotope labels, enzymatic or other ligands. In preferred embodiments, it may be desirable to use a fluorescent or enzyme label such as digoxigenin, β-galactosidase, urease, alkaline phosphatase or peroxidase, avidin/biotin complex.

[00848] The detection methods used to detect or quantify hybridization typically depend on the choice of label. For example, radioactive labels can be detected using photographic film or a Phosphoimager. Fluorescent markers can be detected and quantified using a photodetector to detect emitted light. Enzymatic labels are usually detected by providing the enzyme with a substrate and measuring the amount of the resulting reaction product created by exposing the enzyme to the substrate; and finally, colorimetric marks are detected by simply visualizing the color mark.

[00849] An agent-induced change in the expression of sequences associated with the signal biochemical pathway can also be determined by examining the corresponding gene products. Determining the level of a protein typically involves: a) reacting a protein contained in a biological sample with an agent that specifically binds to a protein associated with a signal biochemical pathway; and (b) identifying any agent-protein complex formed. In one aspect of this embodiment, the agent that specifically binds the protein associated with the signaling pathway is an antibody, preferably a monoclonal antibody.

[00850] The reaction is carried out by binding the agent to a sample of proteins associated with the signaling pathway obtained from the test samples under conditions that allow the formation of a complex between the agent and the proteins associated with the signaling pathway. Complex formation can be detected directly or indirectly according to standard techniques in the art. When using the direct detection method, agents are provided with a detectable label, and unreacted agents can be removed from the complex; the amount of unused label thereby allows the amount of complex formed to be determined. For such a method, it is preferable to choose labels that remain associated with the agent even under harsh washing conditions. Preferably, the label does not interfere with the coupling reaction. Alternatively, the indirect detection procedure may use an agent that is labeled either chemically or enzymatically. The desired label generally does not interfere with the binding or stability of the resulting polypeptide/agent complex. However, the label is usually designed to be available for effective binding by the antibody and hence generate a recognizable signal.

[00851] A large number of labels suitable for determining protein levels are known in the art. Non-limiting examples include radioisotopes, enzymes, colloidal metals, fluorescent compounds, bioluminescent compounds, and chemiluminescent compounds.

[00852] The amount of agent-polypeptide complexes formed during the binding reaction can be quantified by standard quantitative assay methods. As illustrated above, agent-polypeptide complex formation can be directly measured by the amount of label remaining at the binding site. Alternatively, the protein associated with the signaling biochemical pathway is tested for its ability to compete with the labeled counterpart for specific agent binding sites. In this competitive assay, the amount of label captured is inversely proportional to the amount of signal pathway-associated protein sequences present in the test sample.

[00853] A number of protein analysis methods based on the general principles outlined above are available in the art. These include, but are not limited to, radioimmunoassay methods, ELISA (enzyme-linked immunoradiometric assay), sandwich immunoassay, immunoradiometric assay, in situ immunoassay (using e.g. colloidal gold, enzyme or radioisotope label), assay using Western blotting, immunoprecipitation analysis, immunofluorescence analysis and SDS-PAGE.

[00854] Antibodies that specifically recognize or bind to proteins associated with the signaling pathway are preferred for the aforementioned protein assays. If desired, antibodies that recognize a particular type of post-translational modifications (eg, inducible modifications of the signaling biochemical pathway) can be used. Post-translational modifications include, but are not limited to, glycosylation, lipidation, acetylation, and phosphorylation. These antibodies can be purchased from commercial suppliers. For example, anti-phosphotyrosine antibodies that specifically recognize tyrosine-phosphorylated proteins are available from a number of vendors, including Invitrogen and Perkin Elmer. Antiphosphotyrosine antibodies are particularly useful for detecting proteins that are differentially phosphorylated at tyrosine residues in response to ER stress. Such proteins include, but are not limited to, eukaryotic translation initiation factor 2 alpha (eIF-2a). Alternatively, these antibodies can be generated using conventional polyclonal or monoclonal antibody technologies by immunizing a host animal or antibody-producing cell with a protein of interest that exhibits the desired post-translational modification.

[00855] When practicing a route of administration in an individual, it may be desirable to identify the expression pattern of a protein associated with a signaling biochemical pathway in different tissues of the body, in different cell types, and/or in different subcellular structures. These studies can be performed using tissue-specific, cell-specific or subcellular structures antibodies capable of binding to protein markers that are preferentially expressed in certain tissues, cell types or subcellular structures.

[00856] Altered expression of a gene associated with a signaling biochemical pathway can also be determined by examining the change in the activity of gene products compared to a control cell. The analysis of an agent-induced change in the activity of a protein associated with a signaling biochemical pathway will depend on the biological activity and/or signal transduction pathway that is being investigated. For example, when the protein is a kinase, a change in its ability to phosphorylate substrate(s) downstream in the same pathway can be determined using various assays known in the art. Typical assay methods include, but are not limited to, immunoblotting and immunoprecipitation with antibodies, such as anti-phosphotyrosine antibodies, that recognize phosphorylated proteins. In addition, kinase activity can be detected using high throughput chemiluminescent assay methods such as AlphaScreen™ (available from Perkin Elmer) and eTag™ (Chan-Hui, et al., (2003) Clinical Immunology 111: 162-174).

[00857] If the protein associated with the signaling biochemical pathway is part of a signaling cascade leading to fluctuations in intracellular pH, pH sensitive molecules such as fluorescent pH indicators can be used as reporter molecules. In another example, where the signaling pathway protein is an ion channel, fluctuations in membrane potential and/or intracellular ion concentration can be controlled. A number of commercial kits and high performance devices are particularly suitable for the fast and reliable screening of ion channel modulators. Typical devices include FLIPR™ (Molecular Devices, Inc.) and VIPR (Aurora Biosciences). These instruments are capable of simultaneously detecting reactions in more than 1000 microplate sample wells and provide real-time measurement and functional data within seconds or even milliseconds.

[00858] When using any of the methods described herein, a suitable vector can be introduced into a cell or embryo using one or more methods known in the art, including, but not limited to, microinjection, electroporation, pore formation using ultrasound, biolistics, calcium phosphate mediated transfection, cationic transfection, liposomal transfection, dendrimer transfection, heat shock transfection, transfection-nucleofection, magnetofection, lipofection, impalefection, optical transfection, proprietary agent capture of nucleic acids and delivery using liposomes, immunoliposomes, virus and artificial virions. In some methods, the vector is introduced into the embryo by microinjection. The vector or vectors can be introduced by microinjection into the nucleus or cytoplasm of the embryo. In some methods, the vector or vectors may be introduced into a cell by nucleofection.

[00859] The target polynucleotide of the CRISPR complex can be any polynucleotide, endogenous or exogenous to the eukaryotic cell. For example, the target polynucleotide may be a polynucleotide that resides in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence encoding a gene product (eg, a protein) or a non-coding sequence (eg, a regulatory polynucleotide or junk DNA).

[00860] Examples of target polynucleotides include a sequence associated with a signal biochemical pathway, for example, a signal biochemical pathway associated with a gene or polynucleotide. Examples of target polynucleotides include a disease-associated gene or polynucleotide. The term "disease-associated" gene or polynucleotide refers to any gene or polynucleotide that produces transcription or translation products at an abnormal level or in an abnormal form in cells derived from disease-affected tissues compared to unaffected control tissues or cells. disease. It may be a gene that is expressed at an abnormally high level; it may be a gene that is expressed at an abnormally low level, with changes in the level of expression correlated with the onset and/or progression of the disease. A disease-associated gene also refers to a gene having a mutation(s) or genetic variation that is directly responsible for, or in a disequilibrium relationship with, the gene(s) that is responsible for the etiology of the disease. Transcriptional or translational products may or may not be known and may be at normal or abnormal levels.

[00861] The target polynucleotide of the CRISPR complex can be any polynucleotide, endogenous or exogenous, in a eukaryotic cell. For example, the target polynucleotide may be a polynucleotide found in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence encoding a gene product (eg, a protein) or a non-coding sequence (eg, a regulatory polynucleotide or junk DNA). Without being limited by theory, it is believed that the target sequence should be associated with PAM (motif located near the protospecer); that is, a short sequence recognized by the CRISPR complex. The exact sequence and length requirements for a PAM sequence vary depending on the CRISPR enzyme used, but the PAM sequence is typically 2-5 bp from the protospacer (i.e., the target sequence). Examples of PAM sequences are provided in the examples section below, and one of skill in the art will be able to identify further PAM sequences for use with a given CRISPR enzyme.

[00862] The target polynucleotide of a CRISPR complex may include a number of disease-associated genes and polynucleotides, as well as genes and polynucleotides associated with biochemical signaling pathways, as described in U.S. Provisional Patent Applications 61/736527 and 61/748427, both titled "SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION", filed December 12, 2012 and January 2, 2013, respectively, and PCT Application PCT/US2013/074667 entitled "DELIVERY, ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION AND THERAPEUTIC APPLICATIONS" filed December 12, 2013, the contents of each of which are incorporated herein by reference in their entirety.

[00863] Examples of target polynucleotides include a sequence associated with a signaling pathway, for example, a gene or nucleotide associated with a signaling pathway. Examples of target polynucleotides include a disease-associated gene or polynucleotide. A gene or polynucleotide "associated with a disease" refers to any gene or polynucleotide that produces transcription or translation products at an abnormal level or in an abnormal form in cells derived from disease-affected tissues compared to unaffected control tissues or cells. disease. It may be a gene that is expressed at an abnormally high level; it may be a gene that is expressed at an abnormally low level, where the altered expression is correlated with the onset and/or progression of the disease. "Disease-associated gene" also refers to a gene having a mutation(s) or genetic variation that is directly responsible for, or in linkage disequilibrium with, the gene(s) responsible for the etiology of the disease. The transcribed or translated products may or may not be known and may be at normal or abnormal levels.

Whole genome screening by knockout

[00864] The experimental CRISPR protein complexes described herein can be used to conduct efficient and cost-effective functional genomic screening. Such screening can use genomic libraries based on the CRISPR effector protein. Such screens and libraries can provide insight into the functions of genes, the cellular pathways in which genes are involved, and how any change in gene expression can lead to a particular biological process. An advantage of the present invention is that the CRISPR system avoids off-target binding and resulting non-specific effects. This is achieved using systems organized with a high degree of sequence specificity for the target DNA. In preferred embodiments, the CRISPR effector protein complexes are C2c1 or C2c3 effector protein complexes.

[00865] In embodiments, a genome-wide library may contain multiple C2c1 or C2c3 guide RNAs as described herein, including guide sequences that are capable of targeting multiple target sequences at multiple genomic loci in a eukaryotic cell population. The cell population may be a population of embryonic stem (ES) cells. The target sequence at a genomic locus may be a non-coding sequence. The non-coding sequence may be an intron, a regulatory sequence, a splice site, a 3-'UTR, a 5'-UTR, or a polyadenylation signal. The function of one or more gene products can be changed by targeting. Targeting can result in gene function knockout. Targeting a gene product may include more than one guide RNA. Targeting a gene product can be done with 2, 3, 4, 5, 6, 7, 8, 9 or 10 guide RNAs, preferably 3 to 4 per gene. Off-target modifications can be minimized by using stepwise double strand breaks generated by C2c1 or C2c3 effector protein complexes, or by using methods similar to those used in CRISPR-Cas9 systems (see, for example, DNA targeting specificity of RNA- guided Cas9 nucleases Hsu, P., Scott, D., Weinstein, J., Ran, FA., Konermann, S., Agarwala, V., Li, Y., Fine, E., Wu, X., Shalem , O., Cradick, TX, Marraffmi, LA., Bao, G., & Zhang, F. Nat Biotechnol doi: 10.1038/nbt.2647 (2013)), incorporated herein by reference. Targeting may span approximately 100 or more sequences. Targeting may span approximately 1000 or more sequences. Targeting may be approximately 20,000 or more sequences. Targeting can span the entire genome. Targeting may encompass a number of target sequences targeted to the appropriate or desired process. The process may be an immune process. The process may be a process of cell division.

[00866] One embodiment of the invention encompasses a genomic library that may contain multiple guide RNAs for C2c1 or C2c3, which may contain guide sequences that can target multiple target sequences at multiple (genomic) loci, resulting in gene function knockout. This library could potentially contain guide RNAs that target every gene in an organism's genome.

[00867] In some embodiments, the organism or individual is a eukaryotic organism (including a mammal, including a human), or a non-human eukaryotic organism, or a non-human animal, or a non-human mammal. In some embodiments, the organism or individual is a non-human animal and may be an arthropod, such as an insect, or may be a nematode. In some methods of the invention, the organism or individual is a plant. In some methods of the invention, the organism or subject is a mammal or non-human mammal. A non-human mammal may be, for example, a rodent (preferably a mouse or rat), an ungulate, or a primate. In some methods of the invention, the organism or subject is an algae, including microalgae, or a fungus.

[00868] Gene function knockout may include introducing into each cell in a cell population a vector system of one or more vectors containing an engineered, non-naturally occurring C2c1 or C2c3 effector protein system containing I. a C2c1 or C2c3 effector protein, and II. one or more guide RNAs, where components I and II can be on the same or different vectors of the system, inserting components I and II into each cell, and the guide sequence is targeted to a unique gene in each cell, where the C2c1 or C2c3 effector protein is operably linked to a regulatory element, where, during transcription, the guide RNA containing the guide sequence provides sequence-specific binding of the C2c1 or C2c3 effector protein system to the target sequence at the genomic loci of the unique gene, causing cleavage of the genomic loci by the C2c1 or C2c3 effector protein, and confirmation of various cases of knockout during set of unique genes in each cell of the population, thereby obtaining a library of cells with a gene knockout. The invention contemplates that the cell population is a eukaryotic cell population, and in a preferred embodiment, the cell population is an embryonic stem (ES) cell population.

[00869] One or more of the vectors may be plasmid vectors. The vector may be a single vector containing a C2c1 or C2c3 effector protein, a single strand guide RNA, and optionally a selectable marker for target cells. Without wishing to be bound by theory, the simultaneous delivery of a C2c1 or C2c3 effector protein and a single-stranded guide RNA with a single vector allows this method to be applied to any type of target cell without the need to first generate cell lines that express the C2c1 or C2c3 effector protein. The regulatory element may be an inducible promoter. The inducible promoter may be a doxycycline-inducible promoter. In some methods of the invention, the expression of the targeting sequence is under the control of the T7 promoter and is directed by the expression of the T7 polymerase. Confirmation of various knockout-causing mutations can be accomplished by whole exome sequencing. A knockout mutation can be achieved in 100 or more unique genes. A knockout mutation can be achieved in 1000 or more unique genes. A knockout mutation can be achieved in 20,000 or more unique genes. A knockout mutation can also be achieved throughout the genome. Gene function knockout can be achieved in a variety of unique genes that function in a particular physiological process or state. The process or state may be an immune process or state. The process or state may be a cell division process or state.

[00870] The invention also relates to kits that include the extensive libraries of genomes mentioned in the present description. The kit may contain one container containing vectors or plasmids containing the library of the invention. The kit may also comprise a panel containing a selected set of unique guide RNAs of the C2c1 or C2c3 effector protein system containing guide sequences from a library of the invention, the selected set corresponding to a particular physiological condition. Within the scope of the invention, it is contemplated that approximately 100 or more sequences, approximately 1000 or more sequences, or approximately 20,000 or more sequences, or the entire genome are targeted. In addition, the target sequence panel may be focused on an appropriate or desired process, such as an immune process or cell division.

[00871] In a further aspect of the invention, the C2c1 or C2c3 effector protein may contain one or more mutations and may be used as a general DNA binding protein, with or without fusion to a functional domain. Mutations can be artificially introduced mutations or mutations of gain or loss of function. Mutations were characterized as described in the present description. In one aspect of the invention, the functional domain may be a transcriptional activation domain, which may be VP64. In other aspects of the invention, the functional domain may be a transcriptional repression domain, which may be KRAB or SID4X. Other aspects of the invention provide a mutant C2c1 or C2c3 effector protein fused to domains that include, but are not limited to: a transcription activator, repressor, recombinase, transposase, histone remodeling, demethylase, DNA methyltransferase, cryptochrome, light-induced/controlled domain, or chemically inducible/controlled domain. Some methods of the invention may include the induction of expression of target genes. In one embodiment, induction of expression by targeting multiple target sequences at multiple genomic loci in a population of eukaryotic cells is accomplished using a functional domain.

[00872] In the practice of the present invention using C2c1 or C2c3 effector protein complexes, methods using CRISPR-Cas9 systems may be used and reference may be made to:

[00873] Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem, O., Sanjana, NE., Hartenian, E., Shi, X., Scott, DA., Mikkelson, T., Heckl, D., Ebert, BE., Root, DE., Doench, JG., Zhang, F. Science Dec 12. (2013). Published in final edition as: Science. 2014 Jan 3; 343(6166): 84-87

[00874] Shalem et al. described a new way to study the function of the gene on a genome-wide scale. Their research showed that delivery of a genome-wide CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences allowed both negative and positive selection screening in human cells. First, the authors demonstrated the use of the GeCKO library to identify genes required for cell viability in malignant and pluripotent stem cells. Then, using a model of melanoma, the authors screened for genes whose loss is associated with resistance to vemurafenib, a drug that inhibits the BRAF mutant protein kinase. Their research has shown that the top ranked candidates include previously validated NF1 and MED 12 genes, as well as new high scores: NF2, CUL3, TADA2B and TADA1. The authors observed a high level of agreement between independent guide RNAs targeting the same gene and a high confirmation rate of high results, and thus demonstrated the promise of genome screening with Cas9.

[00875] See also US patent No. 20140357530; and PCT Patent Application Publication W02014093701, incorporated herein by reference. See also the October 22, 2015 NIH press release titled "Researchers identify potential alternative to CRISPR-Cas genome editing tools: New Cas enzymes shed light on evolution of CRISPR-Cas systems", incorporated herein by reference.

Functional changes and screening

[00 876] In another aspect of the implementation of the present invention relates to a method of functional evaluation and screening of genes. The use of the CRISPR system of the present invention to precisely deliver functional domains, to activate or silence genes, or to alter the epigenetic state by precisely altering the methylation site at a particular target locus, can be accomplished using one or more guide RNAs for a single cell, or cell population, or a library applied to the genome in a population of cellsex vivo orin vivo, including the introduction or expression of a library containing a plurality of RNA guide molecules (sg-RNA), and where the screening further includes the use of a C2c1 or C2c3 effector protein, and the CRISPR complex containing the C2c1 or C2c3 effector protein is modified to contain a heterologous functional domain . In one aspect, the invention provides a method for screening a genome, comprising administration to a host or expression in a host of a libraryin vivo. In one aspect, the invention relates to the method described in the present description, further comprising an activator administered to the host or expressed in the host. In one aspect, the invention relates to the method described in the present description, in which the activator is attached to the effector protein C2c1 or C2c3. In one aspect, the invention relates to the method described in the present description, in which the activator is attached to the N-terminus or C-terminus of the C2c1 or C2c3 effector protein. In one aspect, the invention relates to the method described in the present description, in which the activator is attached to the sg-RNA loop. In one aspect, the invention relates to the method described in the present description, further comprising a repressor administered to the host or expressed in the host. In one aspect, the invention relates to the method described in the present description, where the screening includes changing and detecting gene activation, gene inhibition or cleavage at a particular locus.

[00877] In one aspect, the invention provides an effective target activity and minimizes non-target activity. In one aspect, the invention provides efficient targeted cleavage by the C2c1 or C2c3 effector protein and minimizes non-targeted cleavage by the C2c1 or C2c3 effector protein. In one aspect, the invention provides target specific binding of a C2c1 or C2c3 effector protein at a gene locus without DNA cleavage. Accordingly, in one aspect, the invention provides for target-specific gene regulation. In one aspect, the invention provides target specific binding of a C2c1 or C2c3 effector protein at a gene locus without DNA cleavage. Accordingly, in one embodiment, the invention provides for cleavage at one gene locus and regulation of a gene at another gene locus using a single C2c1 or C2c3 effector protein. In one aspect, the invention provides orthogonal activation and/or inhibition and/or cleavage of multiple targets using one or more C2c1 or C2c3 effector proteins and/or enzymes.

[00878] In one aspect, the invention relates to the method described in the present description, in which the host cell is a eukaryotic cell. In one aspect, the invention relates to the method described in the present description, in which the host cell is a mammalian cell. In one aspect, the invention relates to the method described herein, wherein the host is a non-human eukaryotic organism. In one aspect, the invention relates to the method described herein, wherein the non-human eukaryotic organism is a non-human mammal. In one aspect, the invention relates to the method described herein, wherein the non-human mammal is a mouse. One aspect of the invention is the method described herein, comprising the delivery of complexes of C2c1 or C2c3 effector proteins or their component(s) or nucleic acid molecule(s) encoding them, wherein said nucleic acid molecule(s) is operably linked(s) to regulatory sequence(s) and expressed in vivo . In one aspect, the invention relates to the method described in the present description, in which the expression in vivo is carried out using a lentivirus, adenovirus or AAV. In one aspect, the invention relates to the method described in the present description, in which the delivery is through a particle, nanoparticle, lipid or cell penetrating peptide (CPP).

[00879] In one aspect, the invention relates to a pair of CRISPR complexes comprising a C2c1 or C2c3 effector protein, each containing a guide RNA molecule (sg-RNA) containing a guide sequence capable of hybridizing to a target sequence at a genomic target locus at cell, where at least one loop of each sg-RNA is modified by inserting a separate RNA sequence(s) that is associated with one or more adapter proteins, and where the adapter protein is associated with one or more functional domains, where each sg-RNA of each effector complex The C2c1 or C2c3 protein includes a functional domain having DNA cleavage activity. In one aspect, the invention relates to C2c1 or C2c3 effector protein paired complexes as described herein, wherein the DNA cleavage activity is mediated by the Fok1 nuclease.

[00880] In one aspect, the invention relates to a method of cutting a target sequence at a genomic locus of interest, comprising delivering to a cell complexes of a C2c1 or C2c3 effector protein, or a component thereof, or a nucleic acid molecule(s) encoding them, wherein the molecule(s) nucleic acid is operably linked to the regulatory sequence(s) and expressed in vivo . In one aspect, the invention relates to the method described in the present description, in which the delivery is made using a lentivirus, adenovirus or AAV. In one aspect, the invention provides a method as described herein, or a C2c1 or C2c3 effector protein pairing complex as described herein, wherein the target sequence for the first complex of the pair is on the first strand of the double-stranded DNA and the target sequence for the second complex The pair is located on the second strand of double-stranded DNA. In one aspect, the invention relates to the method described herein, or a C2c1 or C2c3 effector protein pairing complex, as described herein, wherein the target sequences of the first and second complexes are adjacent to each other such that the DNA is cut such that facilitate homology-guided reparation. In one aspect, the method of the present invention may further comprise introducing a DNA template into the cells. In one aspect, the method described herein or the C2c1 or C2c3 effector protein pair complexes described herein may involve cases where each C2c2 effector protein complex has a C2c1 or C2c3 effector enzyme that is mutated such that it has no more than approximately 5% nuclease activity of the effector enzyme C2c1 or C2c3 that does not have the mutation.

[00881] In one aspect, the invention relates to a library, method, or complex as described herein, wherein the single-stranded sgRNA guide is modified to have at least one non-coding functional hairpin, e.g., wherein the at least one non-coding functional hairpin is repressive; for example, in which at least one non-coding functional loop contains Alu.

[00882] In one aspect, the invention relates to a method for altering or modifying the expression of a gene product. Said method may include introducing into a cell containing and expressing a DNA molecule encoding a gene product, an engineered, non-naturally occurring CRISPR system containing a C2c1 or C2c3 effector protein and a guide RNA that targets the DNA molecule, whereby the guide RNA targets into a DNA molecule encoding a gene product and a C2c1 or C2c3 effector protein cleaves a DNA molecule encoding a gene product, resulting in an altered expression of the gene product, and where the C2c1 or C2c3 effector protein and the guide RNA do not naturally occur together. The invention encompasses a guide RNA containing a guide sequence linked to a direct repeat sequence. In addition, the invention contemplates that the C2c1 or C2c3 effector protein is codon-optimized for expression in a eukaryotic cell. In a preferred embodiment, the eukaryotic cell is a mammalian cell, and in a more preferred embodiment, the mammalian cell is a human cell. In a further embodiment of the invention, the expression of the gene product is reduced.

[00883] In some embodiments, one or more functional domains are associated with a C2c1 or C2c3 effector protein. In some embodiments, one or more functional domains are linked to an adapter protein, for example, using modified targeting molecules by Konnerman et al. (Nature 517, 583-588, January 29, 2015). In some embodiments, one or more functional domains are associated with a "dead" single-stranded guide (sg-RNA, d-RNA). In some embodiments, a d-RNA complex with an active C2c1 or C2c3 effector protein mediates gene regulation via a functional domain at the gene locus, while sgRNA mediates DNA cleavage with an active C2c1 or C2c3 effector protein at another locus, e.g., as described similarly for systems CRISPR-Cas9 in Dahlman et al., "Orthogonal gene control with a catalytically active Cas9 nuclease" (in press). In some embodiments, the dRNA is chosen to maximize the selectivity of regulation of the gene locus of interest over non-specific regulation. In some embodiments, the dRNA is chosen to maximize target gene regulation and minimize target cleavage.

[00884] For the purposes of the following discussion, a "functional domain" may be a functional domain associated with a C2c1 or C2c3 effector protein, or a functional domain associated with an adapter protein.

[00885] In some embodiments, one or more functional domains are NLS (Nuclear Localization Sequence) or NES (Nuclear Export Signal). In some embodiments, the one or more functional domains are a transcriptional activation domain, including VP64, p65, MyoD1, HSF1, RTA, SET7/9, and histone acetyltransfer. Other references herein to activation domains (or activator domains) in relation to domains that are associated with the CRISPR enzyme include any known transcriptional activation domain and in particular VP64, p65, MyoD1, HSF1, RTA, SET7/9, or histone acetyltransferase. .

[00886] In some embodiments, one or more functional domains are transcription suppression domains. In some embodiments, the transcription suppression domains are the KRAB domain. In some embodiments, the transcriptional repression domain is a NuE domain, an NcoR domain, a SID domain, or a SID4X domain.

[00887] In some embodiments, one or more functional domains have one or more functional activities, including methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription termination factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity , DNA integration activity or nucleic acid binding activity.

[00888] Histone modification domains are also preferred in some embodiments. Exemplary histone modification domains are discussed below. Also preferred as functional domains within the scope of the present invention are transposase domains, HR (homologous recombination) domains, recombinase and/or integrase domains. In some embodiments, the DNA integration activity includes HR domains, integrase domains, recombinase domains, and/or transposase domains. In some embodiments, histone acetyltransferase domains are preferred.

[00889] In some embodiments, the DNA cleavage activity is mediated by a nuclease. In some embodiments, the implementation of the nuclease contains the nuclease Fok1. See "Dimeric CRISPR RNA-guided Fok1 nucleases for highly specific genome editing", Shengdar Q. Tsai, Nicolas Wyvekens, Cyd Khayter, Jennifer A. Foden, Vishal Thapar, Deepak Reyon, Mathew J. Goodwin, Martin J. Aryee, J Keith Joung Nature Biotechnology 32(6): 569-77 (2014), which refers to RNA-guided dimeric Fok1 nucleases that recognize extended sequences and can edit endogenous genes with high efficiency in human cells.

[00890] In some embodiments, one or more functional domains are attached to a C2c1 or C2c3 effector protein such that, when bound to a single-stranded guide RNA (sgRNA) and target, the functional domain is in a spatial orientation that allows the functional domain to perform its proper function.

[00891] In some embodiments, one or more functional domains are attached to an adapter protein such that upon binding of the C2c1 or C2c3 effector protein to the single-stranded guide RNA (sgRNA) and target, the functional domain is in a spatial orientation allowing the functional domain to perform its inherent function.

[00892] In one aspect, the invention relates to a composition described herein, wherein one or more functional domains are attached to a C2c1 or C2c3 effector protein or adapter protein via a linker, optionally a GlySer linker, as described herein.

[00893] Endogenous transcriptional repression is often mediated by chromatin-modifying enzymes such as histone methyltransferases (HMTs) and deacetylases (HDACs). Histone repressive effector domains are known and an illustrative list of them is given below. In the table, proteins and functional truncations of small size are preferred to facilitate efficient packaging of viruses (eg, by AAV). In general, however, domains may include deacetylase (HDAC), histone methyltransferase (HMT), and histone acetyltransferase (HAT) inhibitors, as well as HDAC and HMT recruiting proteins. The functional domain can be or include, in some embodiments, HDAC effector domains recruiting HDAC effector domains, histone acetyltransferase (HMT) effector domains, recruiting histone methyltransferase (HMT) effector domains, recruiting histone acetyltransferase effector domains.

HDAC effector domains

Subtype/complex Name Substrate (if known) Modification (if known) organism Full size (a.k.) Selected shortening (a.k.) Final size (a.k.) catalytic domain HDAC I HDAC8 - - X. laevis 325 1-325 325 1-272: HDAC HDAC I RPD3 - - S.cerevisiae 433 19-340 322 (Vannier) 19-331: HDAC HDAC IV MesoLo4 - - M. loti 300 1-300
(Gregoretti) 300 - HDAC I HDAC11 - - H. sapiens 347 1-347 (Gao) 347 14-326: HDAC HD2 HDT1 - - A. thaliana 245 1-211(Wu) 211 - SIRT I SIRT3 H3K9Ac
H4K16Ac
H3K56Ac - H. sapiens 399 143-399 (Scher) 257 126-382: SIRT SIRT I HST2 - C. albicans 331 1-331
(Hnisz) 331 - SIRT I Cobb - E.coil (K12) 242 1-242
(Landry) 242 - SIRT I HST2 - S.cerevisiae 357 8-298
(Wilson) 291 - SIRT III SIRT5 H4K8Ac
H4K16Ac - H. sapiens 310 37-310
(Gertz) 274 41-309: SIRT SIRT III Sir2A - P. falciparum 273 1-273
(zhu) 273 19-273: SIRT SIRT IV SIRT6 H3K9Ac
H3K56Ac - H. sapiens 355 1-289
(Tennen) 289 35-274: SIRT

[894] Accordingly, the repressor domains of the present invention can be selected from histone methyltransferase (HMT), histone deacetylases (HDACs), histone acetyltransferase (HAT) inhibitors, and HDAC and HMT recruiting proteins.

[00895] The HDAC domain can be any of the following in the table above: HDAC8, RPD3, MesoLo4, HDAC11, HDT1, SIRT3, HST2, CobB, HST2, SIRT5, Sir2A, or SIRT6.

[00896] In some embodiment, the functional domain may be an HDCP recruiting effector domain. Preferred examples include those shown in the table below, namely MeCP2, MBD2b, Sin3a, NcoR, SALL1, RCOR1. NcoR is illustrated in the examples herein, and while the use of this protein is preferred, it is expected that other members of the class would also be useful.

HDAC table recruiting effector domains

Subtype/
complex Name Substrate (if known) Modification (if known) organism Full size (a.k.) Selected shortening (a.k.) Final size (a.k.) catalytic domain Sin3a MeCP2 - - R.norvegicus 492 207-492
(Nan) 286 - Sin3a MBD2b - - H. sapiens 262 45-262
(Bokee) 218 - Sin3a Sin3a - - H. sapiens 1273 524-851
(Laherty) 328 627-829:
interaction with HDAC1 NcoR NcoR - - H. sapiens 2440 420-488
(zhang) 69 - NuRD SALL1 - - M.musculus 1322 1-93
(Lauberth) 93 - CorEST RCOR1 - - H. sapiens 482 81-300
(Gu, Ouyang) 220 -

[897] In some embodiment, the functional domain may be a methyltransferase (HMT) effector domain. Preferred examples include those shown in the table below, namely NUE, vSET, EHMT2/G9A, SUV39H1, dim-5, KYP, SUVR4, SET4, SET1, SETD8 and TgSET8. The NUEs are illustrated in the examples herein and while the use of this protein is preferred, it is expected that other members of the class would also be useful.

Histone methyltransferase (HMT) effector domain table

Subtype/complex Name Substrate (if known) Modification (if known) organism Full size (a.k.) Selected shortening (a.k.) Final size (a.k.) catalytic domain SET NUE h2b,
H3, H4 - C. trachomatis 219 1-219
(Penny) 219 - SET vSET - H3K27me3 P. Bursaria chlorella virus 119 1-119 (Mujtaba) 119 4-112:
SET2 SUV39 family ENMT2/
G9A H14K2,
H3K9,
H3K27 H3K9me1/2,
H1K25me1 M.musculus 1263 969-1263
(Tachibana) 295 1025-1233:
Pre-SET
set,
Post-SET SUV39 SUV39H1 - H3K9me2/3 H. sapiens 412 79-412
(Snowden) 334 172-412:
Pre-SET
set,
Post-SET Suvar3-
9 dim-5 - H3K9me3 N. crassa 331 1-331
(Rathert) 3 3 1 77-331:
Pre-SET
set,
Post-SET Suvar3-9
(subfamily SUVH) KYP - H3K9me1/2 A. thaliana 624 335-601 267
(Jackson) - Suvar3-9
(subfamily SUVR) SUVR4 H3K9me1 H3K9me2/3 A. thaliana 492 180-492 313
(Thorstensen) 192-462:
Pre-SET
set,
Post-SET Suvar4-20 SET 4 - H4K20me3 C.elegans 288 1-288
(Vielle) 288 - SETS SET1 - H4K20me1 C.elegans 242 1-242
(Vielle) 242 - SETS SETD8 - H4K20me1 H. sapiens 393 185-393 209
(Couture) 256-382:
SET SETS TgSETS - H4K20me1/2/3 T. gondii 1893 1590-1893
(Sautel) 304 1749-1884:
SET

[898] In some embodiments, the functional domain may be a recruiting effector domain of histone methyltransferase (HMT). Preferred examples include those shown in the table below, namely Hp1a, PHF19 and NIPP1.

[899] Histone methyltransferase (HMT) recruiting effector domain potency table.

Subtype/
complex Name Substrate (if known) Modification (if known) organism Full size (a.k.) Selected shortening (a.k.) Final size (a.k.) catalytic domain - hpla - H3K9me3 M. muscle 19
one 73-191 119
(hathawa) 121-179:
chromoshad - PHF19 - H3K27me3 H. sapiens 58
0 (1-250)
Linker GGSG+(500-580) 335
(Ballaré) 163-250:
PHD2 - NIPP1 - H3K27me3 H. sapiens 35
one 1-329
(Jin) 329 310-329:
EED

[900] In some embodiments, the implementation of the functional domain may be an inhibitory effector domain of histone acetyltransferase. Preferred examples include SET/TAF-1β listed in the table below.

[901] Table of histone acetyltransferase inhibitory effector domains.

Subtype/
complex Name Substrate (if known) Modification (if known) organism Full size (a.k.) Selected shortening (a.k.) Final size (a.k.) catalytic domain - SET/TAF-1β - - M.
muscle 289 1-289
(Cervoni) 289 -

[902] It is also preferred to target endogenous (regulatory) control elements (such as enhancers and silencers), in addition to the promoter or elements located proximal to the promoter. Thus, the invention can also be used to target endogenous control elements (including enhancers and silencers) in addition to targeting the promoter. These control elements can be located upstream and downstream from the transcription start point (TSS) starting at 200 bp. from TSS to 100 kbp from it. Targeting known control elements can be used to activate or suppress the target gene. In some cases, a single control can influence the transcription of multiple target genes. Thus, targeting a single control element can be used to drive the transcription of multiple genes simultaneously.

[00903] Targeting putative controls on the other hand, (for example, by covering the area of the putative control, as well as from 200 bp to 100 kb around the element) can be used as a means of confirming such elements (by measuring transcription of a gene of interest) or to discover new control elements (eg, by spanning a region 100 kb upstream and downstream of the TSS of the gene of interest). In addition, targeting putative controls may be useful in the context of understanding the genetic causes of a disease. Many mutations and common SNP variants associated with disease phenotypes are located outside the coding regions. Once such regions have been targeted using the activation or silencing systems described herein, transcription data can be determined for either a) a set of putative targets (e.g., a set of genes located in close proximity to a control element) or b) the entire transcriptome eg by RNA sequencing or microarray. This would allow the identification of likely candidate genes involved in the disease phenotype. Such candidate genes may be useful as new drug targets.

[00904] Histone acetyltransferase (HAT) inhibitors are mentioned herein. However, in some embodiments, the implementation of the alternative is that one or more functional domains contain acetyltransferase, preferably histone acetyltransferase. They are useful in the field of epigenomics, for example, in methods for studying the epigenome. Methods for exploring the epigenome may include, for example, targeting epigenomic sequences. Targeting epigenomic sequences may include a guide molecule directed to the epigenomic target sequence. In some embodiments, the target epigenomic sequence may include a promoter, silencer, or enhancer sequence.

[00905] The use of a functional domain associated with a C2c1 or C2c3 effector protein as described herein, preferably a dead C2c1 or C2c3 effector protein, more preferably a dead AacC2c1 or C2c3 effector protein, to target epigenomic sequences is possible to activate or downregulate promoters, silencers or enhancers.

[00906] Examples of acetyltransferases are known, but may include, in some embodiments, histone acetyltransferases. In some embodiments, the histone acetyltransferase may comprise a human p300 acetyltransferase catalytic core (Gerbasch & Reddy, Nature Biotech, 6th April 2015).

[00907] In some preferred embodiments, the functional domain is linked to a dead C2c1 or C2c3 effector protein to target and activate epigenomic sequences such as promoters or enhancers. One or more targeting molecules that target such promoters or enhancers may also be provided to direct the binding of the CRISPR enzyme to such promoters or enhancers.

[00908] The term "associated with" is used herein to refer to the association of a functional domain with a C2c1 or C2c3 effector protein or adapter protein. It is used in relation to how one molecule "associates" (binds) to another, such as between an adapter protein and a functional domain, or between a C2c1 or C2c3 effector protein and a functional domain. In the case of such protein-protein interactions, this association can be considered in terms of recognition, similar to how an antibody recognizes an epitope. Alternatively, one protein can be linked to another protein by a fusion of two subunits, for example one subunit is fused to another subunit. Fusion usually occurs by adding one amino acid sequence to another, for example, by joint splicing of the nucleotide sequences that encode each protein or subunit. Alternatively, it can essentially be viewed as linking two molecules, or direct linkage, such as a fusion protein. In either case, the fusion protein may include a linker between the subunits of interest (ie, between the enzyme and the functional domain, or between the adapter protein and the functional domain). Thus, in some embodiments, the C2c1 or C2c3 effector protein or adapter protein is associated with the functional domain by binding to it. In other embodiments, the C2c1 or C2c3 effector protein or adapter protein is associated with a functional domain because both are fused together, optionally via an intermediate linker.

[00909] Attachment of a functional domain or fusion protein can be via a linker, such as a flexible glycine-serine (GlyGlyGlySer) or (GGGS) ₃ , or a rigid alpha-helical linker, such as (Ala(GluAlaAlaAlaLys)Ala). Preferably, linkers like (GGGGS) ₃ are used to separate domains of a protein or peptide within the scope of the present invention. (GGGGS) ₃ is preferred because it is a relatively long linker (15 amino acids). Glycine residues are the most flexible, serine residues increase the likelihood of the linker being outside the protein. As preferred alternatives, (GGGGS) ₆ (GGGGS) or (GGGGS) ₁₂ can be used. Other preferred options are (GGGGS) ₁ , (GGGGS) ₂ , (GGGGS) ₄ , (GGGGS) ₅ , (GGGGS) ₇ , (GGGGS) ₈ , (GGGGS) ₁₀ or (GGGGS) _n . Alternative linkers are also available, however, linkers with high flexibility are considered to function best to allow the 2 parts of C2c1 or C2c3 to come together and thus restore C2c1 or C2c3 activity. An alternative is, in particular, the use of NLS as a linker. For example, the linker may also be between C2c1 and any functional domain, or between C2c3 and any functional domain. Again, a (GGGGS) ₃ linker (or variants thereof containing 6, 9, or 12 repeats) or nucleoplasmin NLS can be used here as a linker between C2c1 and a functional domain, or between C2c3 and a functional domain.

Saturating mutagenesis

[00910] The C2c1 or C2c3 effector protein system(s) described herein can be used to perform saturation or deep scanning mutagenesis of genomic loci in combination with a cellular phenotype, for example, to determine critical minimum features and discrete vulnerabilities of functional elements required for gene expression, drug resistance and disease management. By "saturation" or "deep-scanning mutagenesis" is meant that each or substantially each DNA base is cut within genomic loci. A library of C2c1 or C2c3 effector protein guide RNAs can be introduced into the cell population. The library can be administered such that each cell receives one guide RNA molecule (sg-RNA). When the library is introduced by transducing a viral vector as described herein, a low multiplicity of infection is used. The library may include sgRNAs targeted to each sequence upstream from the sequence adjacent to the protospacer (PAM) at the genomic locus. The library may include at least 100 non-overlapping genomic sequences upstream from PAM for every 1000 bp at a genomic locus. The library may include sgRNAs targeting upstream sequences from at least one different PAM. C2c1 or C2c3 effector protein systems may include more than one C2c1 or C2c3 protein. Any C2c1 or C2c3 effector protein as described herein can be used, including orthologues or engineered C2c1 or C2c3 effector proteins that recognize different PAM sequences. The frequency of non-target sites for sg-RNA can be less than 500. The evaluation of off-target binding of sg-RNA is necessary to select the sg-RNA with the lowest level of possible binding to non-target sites. Any phenotype found to be associated with sgRNA target cleavage can be confirmed using sgRNAs targeting the same site in a single experiment. Target site validation can also be performed using a modified C2c1 or C2c3 effector protein as described herein and two sgRNAs targeting the target site in the genome. Without being bound by theory, it can be argued that a target site is indeed established if a change in the phenotype is observed in the validation experiments.

[00911] Genomic loci may include at least one contiguous genomic region. At least one contiguous genomic region may include the entire genome. At least one contiguous genomic region may contain a functional element of the genome. The functional element may be in a non-coding region, a coding gene, an intron region, a promoter, or an enhancer. At least one contiguous genomic region may contain at least 1 kb, preferably at least 50 kb. genomic DNA. At least one contiguous genomic region may contain a transcription factor binding site. The at least one contiguous genomic region may contain a region hypersensitive to DNase I cleavage. The at least one contiguous genomic region may contain a transcription-enhancing enhancer or a transcriptional repressor element. At least one contiguous genomic region may contain a site enriched in an epigenetic signature. At least one contiguous region of genomic DNA may contain an epigenetic insulator. At least one contiguous genomic region may contain two or more contiguous genomic regions that physically interact. The genomic regions that interact can be defined by "4C technology". 4C technology allows whole genome screening in an unbiased manner for DNA segments that physically interact with a selected DNA fragment, as described in Zhao et al. ((2006) Nat Genet 38, 1341-7) and US Pat. No. 8,642,295, which are incorporated herein by reference in their entirety. The epigenetic signature may be acetylation, methylation, ubiquitinylation, histone phosphorylation, DNA methylation, or the absence of all of the above.

[00912] The system(s) effector protein C2c1 or C2c3 for saturating or deep scanning mutagenesis can be used in a population of cells. The C2c1 or C2c3 effector protein system can be used in eukaryotic cells, including but not limited to mammalian and plant cells. The cell population may consist of prokaryotic cells. The eukaryotic cell population may be a population of embryonic stem (ES) cells, neurons, epithelial cells, immune cells, endocrine cells, muscle cells, erythrocytes, lymphocytes, plant cells, or yeast cells.

[00913] In one aspect, the present invention relates to a method for screening for functional elements associated with a change in phenotype. The library can be introduced into a population of cells that are adapted to contain a C2c1 or C2c3 effector protein. Cells can be sorted into at least two main groups based on phenotype. The phenotype may include gene expression, cell growth or viability. The relative abundance of guide RNAs present in each group can be determined, with genomic sites associated with phenotypic change determined by the abundance of guide RNAs present in each group. The change in phenotype may be a change in the expression of the target gene. Expression of the target gene can be increased, decreased, or completely turned off (gene knockout). Cells can be sorted into high and low expression groups. The cell population may include a reporter construct that determines the phenotype. The reporter construct may include a detectable marker. Cells can be sorted with a detectable marker.

[00914] In another aspect, the present invention relates to a method for screening genomic sites associated with resistance to a chemical compound. The chemical compound can be a drug or a pesticide. The library can be introduced into a population of cells adapted to contain the effector protein C2c1 or C2c3, where each cell of the population contains no more than one guide RNA; the cell population is treated with a chemical compound; and the presence of guide RNAs is determined after treatment with a chemical compound at a later time point compared to an early time point at which genomic regions associated with chemical compound resistance are determined by enrichment in guide RNAs. The representation of sgRNA can be determined by deep sequencing methods.

[00915] Useful in the practice of the present invention using C2c1 or C2c3 effector protein complexes are methods using CRISPR-Cas9 systems, and reference may be made to an article titled "BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis". Canver, M.C., Smith, E.C., Sher, F., Pinello, L., Sanjana, N.E., Shalem, O., Chen, D.D., Schupp, P.G., Vinjamur, D.S., Garcia, S.P., Luc, S., Kurita, R., Nakamura, Y., Fujiwara, Y., Maeda, T., Yuan, G., Zhang, F., Grkin, S.H., & Bauer, D.E. DOI:10.1038/naturel552I, published online September 16, 2015, which is incorporated herein by reference and discussed briefly below.

[00916] Canver et al. uses novel cr-RNA-Cas9 guide libraries to perform in situ saturation mutagenesis of human and mouse BCL11A erythroid enhancers, previously identified as a fetal hemoglobin (HbF)-associated enhancer and whose murine orthologue is required for BCL11A erythroid expression. This approach revealed critical minimum features and discrete vulnerabilities of these enhancers. Through editing of primary human progenitors and mouse transgenesis, the authors confirmed that the erythroid enhancer BCL11A can be used as a target for HbF reinduction. The authors have created a detailed enhancer map that can be used in therapeutic genome editing.

Ways to use the C2c1 or C2c3 systems to modify a cell or organism

[00917] In some embodiments, the invention relates to a method for modifying a cell or organism. The cell may be prokaryotic or eukaryotic. The cell may be a mammalian cell. The cell may be a non-human mammalian, primate, bovine, porcine, rodent or mouse cell. The cell may be a non-mammalian eukaryotic cell such as a poultry, fish or shrimp cell. The cell may also be a plant cell. The plant cell may be a cereal cell such as cassava, corn, sorghum, wheat, or rice. The plant cell may be an algae, tree or vegetable cell. The modification introduced into a cell according to the present invention may be such that the cell and the cell's progeny are modified to improve the production of biological products such as antibodies, starch, alcohol, or other desired cellular products. The modification introduced into a cell according to the present invention may be such that the cell and its descendants include a change that changes the biological product they produce.

[00918] The described system may contain one or more different vectors. In one aspect of the invention, the effector protein is codon-optimized for expression in the desired cell type, preferably eukaryotic cells, preferably mammalian or human cells.

[00919] Packaging cells are commonly used to produce viral particles that are capable of infecting a host cell. Such cells include 293 cells, which package adenoviruses, and ψ2 cells or PA317 cells, which package retroviruses. Viral vectors used in gene therapy are usually obtained by creating a cell line that packages nucleic acid vectors into viral particles. Vectors typically contain the minimum viral sequences necessary for packaging and subsequent integration into a host cell, with other viral sequences replaced by an expression cassette for the polynucleotide(s) to be expressed. Missing virus functions are usually supplied during delivery by the packaging cell line. For example, AAV vectors used in gene therapy typically only have the ITR sequences from the AAV genome that are required for packaging and integration into the host genome. Viral DNA is packaged with a cell line that contains an accessory plasmid encoding other AAV genes, namely rep and cap, but lacking the ITR sequence. The cell line can also be infected with adenovirus as a helper particle. The helper virus promotes replication of the AAV vector and expression of the AAV genes from the helper plasmid. The helper plasmid cannot be packaged in large quantities due to the lack of an ITR sequence. Adenovirus contamination can be reduced, for example, by heat treatment, to which adenovirus is more sensitive than AAV. Those skilled in the art are aware of additional methods for delivering nucleic acids to cells. See, for example, US20030087817, incorporated herein by reference.

Delivery

[00920] The invention encompasses at least one component of a CRISPR complex, such as RNA delivered via at least one nanoparticle complex. In some aspects, the invention relates to methods comprising delivering one or more polynucleotides, in particular one or more vectors as described herein, one or more transcripts thereof, and/or one or more proteins transcribed from them, into a host cell. . In some aspects, the invention also relates to cells obtained using such methods, and animals containing these cells or derived from these cells. In some embodiments of the invention, the CRISPR enzyme in combination (optionally as part of a complex) with a targeting sequence is delivered to the cell. Conventional gene transfer methods, whether or not based on viruses, can be used to introduce nucleic acids into mammalian cells or target tissues. Such methods can be used to introduce nucleic acids encoding components of the CRISPR system into cells in culture or the host. Non-viral delivery systems include DNA plasmids, RNA (eg, the transcript of the vector described in this specification), naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome. Viral vector-based delivery systems include DNA or RNA viruses that have episomal or integrated genomes upon delivery into a cell. For a review of gene therapy techniques, see Anderson, Science 256:808-813 (1992); Nabel & Feigner, TIBTECH 1.1:21.1-217 (1993); Mitam & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10): 1149-1154 (1988); Vigne, Restorati ve Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Bohm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

[00921] Methods for non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, gene guns, virosomes, liposomes, immunoliposomes, polycations or lipid-nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced DNA uptake. Lipofection is described, for example, in US patent No. 5049386, 4946787; and 4897355), and commercial lipofection reagents (eg Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient recognition of polynucleotide lipofection receptors include those of Feigner, WO 91/17424; WO 91/16024. Delivery can be to cells (eg, in vitro or ex vivo ) or target tissues (eg, in vivo ).

[00922] The preparation of complexes of lipids with nucleic acids, including targeted liposomes, such as immunolipid complexes, is well known to those skilled in the art (see, for example, Crystal, Science 270: 404-410 (1995), Blaese et al., Cancer Gene Ther 2: 291-297 (1995), Behr et at., Bioconjugate Chem., 5: 382-389 (1994), Remy et al., Bioconjugate Chem., 5: 647-654 (1994), Gao et al. ., Gene Therapy 2: 710-722 (1995), Ahmad et al., Cancer Res. 52: 4817-4820 (1992), US Pat. 4946787).

[00923] The use of RNA or DNA viral systems to deliver nucleic acids utilizes current techniques to target the virus to specific cells in the body and transfer the viral load to the nucleus. Viral vectors can be administered directly to patients ( in vivo ) or they can be used to treat cells in vitro and modified cells can be administered to patients ( ex vivo ). Typical viral systems may include retroviral, lentiviral, adenovirus, adeno-associated, and herpes simplex viruses for gene transfer. Integration into the host genome is possible using gene transfer techniques for retroviruses, lentiviruses, and adeno-associated viruses, often resulting in long-term expression of the introduced transgene. In addition, high transduction efficiency has been observed in many other types of target cells and tissues.

[00924] The tropism of a retrovirus can be altered by the incorporation of foreign envelope proteins, thereby expanding the potential population of target cells. Lentiviral vectors are retroviral vectors that are capable of transforming or infecting non-dividing cells and typically have high viral titers. Therefore, the choice of retroviral gene transfer system will depend on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats with a packaging capacity of up to 6-10 kb. foreign sequence. Minimal cis-acting LTRs are sufficient to replicate and package the vectors, which are then used to integrate the therapeutic gene into the target cell to ensure consistent transgene expression. Commonly used retroviral vectors include vectors based on murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol 66: 2731-2739 (1992), Johann et al., J. Virol 66: 1635-1640 (1992), Sommnerfelt et al., Virol 176: 58-59 (1990), Wilson et al. ., J. Virol, 63: 2374-2378 (1989), Miller et al., J. Virol 65: 2220-2224 (1991), PCT/US94/05700).

[00925] In another embodiment, cocal vesiculovirus pseudotyped retroviral vector particles are provided (see, for example, US Patent Publication No. 20120164118 registered with Fred Hutchinson Cancer Research Center). The Cocal virus belongs to the genus Vesiculovirus and is the causative agent of vesicular stomatitis in mammals. Cocal virus was originally isolated from ticks in Trinidad by Honkers et al. Am. J. Vet. Res. 25:236-242 (1964)), and infections have been reported in Trinidad, Brazil and Argentina in insects, cattle and horses. Many vesiculoviruses that infect mammals have been isolated from naturally infected arthropods, suggesting they are transmissible. Antibodies to vesiculoviruses are common in people living in industrial regions where these viruses are endemic and obtained in laboratories; infection in humans usually results in flu-like symptoms. The envelope glycoprotein of the Cocal virus at the amino acid level shares 71.5% identity with VSV-G Indiana, and phylogenetic comparison of the vesiculovirus envelope gene demonstrates that the Cocal virus is serologically distinct from, but most closely related to, VSV-G Indiana strains (Jonkers et al Am J Vet Res 25:236-242 (1964) and Travassos da Rosa et al Am J Tropical Med & Hygiene 33:999-1006 (1984)). Cocal vesiculovirus pseudo-typed retroviral vector particles may include, for example, lentiviral, alpharetroviral, betaretroviral, gammaretroviral and epsilon retroviral vector particles, which may contain retroviral Gag, Pol and/or one or more accessory protein(s), as well as the vesiculovirus envelope protein cocal. In some aspects of the invention, Gag, Pol, and accessory proteins are lentiviral and/or gammaretroviral. The invention encompasses AVVs containing or consisting for the most part of exogenous nucleic acid molecules encoding the CRISPR system, for example, a variety of cassettes comprising or consisting of a first cassette comprising or essentially consisting of a promoter, a nucleic acid, a molecule encoding a CRISPR-associated ( Cas) a protein (putative nuclease or helicase proteins), e.g. C2c1 or C2c3 and a terminator, and 2 or more, preferably up to the capacity limit of the package, e.g. a total of five (including the first cassette) cassettes containing or essentially consisting from a promoter, a nucleic acid molecule encoding a guide RNA (gRNA) and a terminator (for example, each cassette can be schematically represented as promoter-gRNA1-terminator, promoter-gRNA2-terminator ... promoter-gRNA(N)-terminator (where N is the number that can be inserted represents the upper limit of the vector packing), or two or more separate rAAVs, each containing contains one or more cassettes of the CRISPR system, for example, a first rAAV containing a first cassette containing or essentially consisting of a promoter, a nucleic acid molecule encoding Cas, for example, Cas (C2c1 or C2c3) and a terminator, and a second rAAV containing a variety of four cassettes containing or essentially consisting of a promoter, a nucleic acid molecule encoding a guide RNA (gRNA), and a terminator (e.g., each cassette can be schematically represented as promoter-gRNA1-terminator, promoter-gRNA2-terminator ... promoter-gRNA(N )-terminator (where N is the number that can be inserted, representing the upper limit of the vector packing). Since rAAV is a DNA-containing virus, the nucleic acid molecules described herein in relation to AAV or rAAV are preferably DNA. In some modifications, the promoter is preferably the synapsin I (hSyn) promoter. Additional methods for delivering nucleic acids into a cell are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference.

[00926] In some embodiments, the host cell is transiently or permanently transfected with one or more of the vectors described herein. In some embodiments, the cell is transfected as it is naturally present in the individual. In some embodiments, the cell to be transfected is derived from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art. Example cell lines include, but are not limited to: C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bc1-1, ВС-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS -1, COS-6, COS-M6A, monkey kidney epithelium BS-C-1, mouse embryonic fibroblasts BALB/3T3, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts, mouse fibroblasts 10.1, 293-T, 3T3 , 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1, BEAS-2B, bEnd.3, BHK-21, BR 293, VkhRSZ, C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr -/-, COR-L23, COR-L23/ CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10 .0, FM3, H1299, H69, HB54, H B55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38 cells , MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR/0.2R. MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN /OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2, Sf-9, SkBr3, T2, T-47D, T84, THP1, U373, U87, U937, VCaP cells , Vero, WM39, WT-49, X63, YAC-1, YAR cells and their transgenic varieties. Cell lines are available from a variety of sources known to those skilled in the art (see, for example, American Type Culture Collection (ATCC) (Manassus, Va.)). In some embodiments, a cell transfected with one or more of the vectors described herein is used to generate a new cell line containing one or more sequences derived from the vector. In some embodiments, a cell transiently transfected with components of a nucleic acid targeting system as described herein (e.g., by transient transfection of one or more vectors or RNA transfection) and modified with activity of the nucleic acid targeting complex is used to create a new cellular a line containing cells having the modification but not having any other exogenous sequence. In some embodiments, cells transiently or permanently transfected with one or more of the vectors described herein, or cell lines derived from such cells, are used to evaluate one or more test compounds.

[00927] In some embodiments, one or more of the vectors described herein are used to produce a transgenic non-human animal or transgenic plant. In some embodiments, the transgenic animal is a mammal, such as a mouse, rat, or rabbit. In some embodiments, the organism or subject is a plant. In some embodiments, the organism, subject, or plant is an algae. Methods for obtaining transgenic plants and animals are known in the art and typically start using a cell transfection method as described herein. In another embodiment, a needle array fluid delivery device (see, for example, Fred Hutchinson Cancer Research Center US Patent Publication No. 20110230839) can be considered as a means to deliver CRISPR Cas to hard tissues. A device according to US Patent Publication No. 20110230839 for delivering fluid to hard tissue may consist of a plurality of needles combined into a matrix; a number of reservoirs, each of which is connected by fluid flow with a corresponding needle from an array of needles, as well as a plurality of actuators, functionally associated with their respective reservoirs and configured in such a way as to control the pressure of the liquid in the reservoir. In some embodiments, each of the plurality of actuators may include one of the plurality of pistons, one end of each of the plurality of pistons is connected to its respective one of the plurality of reservoirs, and in some further embodiments, the pistons of the plurality of pistons are operatively connected together at a respective second end such that possible simultaneous pressure on them. Some further embodiments may include a piston trigger configured to actuate a plurality of pistons at a selectable varying speed. In other modifications, each of the plurality of drives may include one of the plurality of fluid transmission lines having first and second ends, the first end of each of the plurality of fluid transmission lines is associated with its corresponding one from the plurality of tanks. In other embodiments, such a device may include a fluid pressure source and each of the plurality of actuators includes a hydraulic transmission between the pressure source and its corresponding one of the plurality of reservoirs. In further embodiments, the fluid pressure source may comprise at least one of the following: a compressor, a vacuum accumulator, a peristaltic pump, a master cylinder, a microjet pump, and a valve. In another embodiment, each of the plurality of needles may comprise a plurality of holes distributed along its length.

[00928] In one aspect, the invention relates to methods for modifying a target polynucleotide in a eukaryotic cell. In some embodiments, such a method includes allowing a nucleic acid-targeted complex to bind to a target polynucleotide to cleave said target polynucleotide, thereby modifying the target polynucleotide, wherein the nucleic acid-targeted complex includes an effector protein targeted to the nucleic acid , and a guide RNA hybridized to a target sequence in said target polynucleotide.

[00929] In one aspect, the invention relates to a method for modifying the expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method includes allowing a complex targeted to a nucleic acid to bind to a polynucleotide such that said binding results in an increase or decrease in the expression of said polynucleotide; wherein the nucleic acid-targeting complex comprises a nucleic acid-targeting effector protein associated with a guide RNA that is hybridized to a target sequence in said polynucleotide.

[00930] The components of the CRISPR complex can be delivered in combination with transport parts (adapted, for example, from the approaches described in US Pat. Nos. 8,106,022, 8,313,772). Nucleic acid delivery strategies can, for example, be used to improve the delivery of guide RNAs, messenger RNAs, or coding DNAs that encode components of the CRISPR complex. For example, RNA molecules may include modified RNA nucleotides to increase stability, reduce immunostimulation, and/or improve specificity (see Deleavey, Glen F. et al., 2012, Chemistry & Biology, Volume 19, Issue 8, 937-954; Zalipsky, 1995, Advanced Drug Delivery Reviews 16. 157-182; Caliceti and Veronese, 2003, Advanced Drug Delivery Reviews 55: 1261-1277). Various structures have been described that can be used to modify nucleic acids such as gRNA for more efficient delivery, in particular reversible charge-neutralizing modifications of the phosphotriester backbone, which can be adapted to modify gRNA to be more hydrophobic and less anionic. facilitating cell entry (Meade BR et al., 2014, Nature Biotechnology 32,1256-1261). In further alternative embodiments, individual RNA motifs may be useful in mediating cell transfection (Magalhaes M, et al., Molecular Therapy (2012); 20 3, 616-624). Similarly, aptamers can be adapted to deliver components of the CRISPR complex, for example, by attaching aptamers to gRNA molecules (Tan W. et al., 2011, Trends in Biotechnology, December 2011, Vol. 29, No. 12).

[00931] In some embodiments, a triple-antennary N-acetylgalactosamine (GalNAc) conjugate with oligonucleotide components can be used to improve delivery, e.g., delivery to targeted cell types, e.g., hepatocytes (see WO2014118272, incorporated herein by reference; Nair , JK et al., 2014, Journal of the American Chemical Society 136(49), 16958-16961). It may be considered a sugar-based particle, and further details regarding other particle-based delivery systems and/or formulations are provided herein, thus GalNAc may be considered a particle as defined herein, so the main methods usage and other issues, such as those affecting the delivery of these particles, also apply to GalNAc particles. The strategy for obtaining conjugates in solution can be, for example, used to attach three-antenna clusters (with a molecular weight of ~2000) activated as esters of PFP (pentafluorophenyl) with 5'-hexamino-modified oligonucleotides (5'-HA ASO, molecular weight of ~8000 Yes; Ostergaard et al., Bioconjugate Chem., 2015, 26 (8), pp 1451-1455). Similarly, the use of poly(acrylate polymers for in vivo delivery of nucleic acids has been described (see WO2013158141 incorporated herein by reference). In the following alternative embodiments, the pre-addition of CRISPR nanoparticles (or protein complexes) to naturally occurring plasma proteins can be used to improve delivery methods (Akinc A et al, 2010, Molecular Therapy vol. 18 no. 7, 1357-1364).

[00932] Screening methods are available for use in identifying delivery enhancers, such as chemical library screening (Gilleron J. et al., 2015, Nucl. Acids Res. 43 (16): 7984-8001). Also described are approaches to quantify the effectiveness of delivery vehicles, in particular lipid nanoparticles, which can be used to identify effective delivery vehicles for CRISPR components (see Sahay G. et al., 2013, Nature Biotechnology 31, 653-658).

[00933] In some embodiments, delivery of CRISPR protein components can be facilitated by adding functional peptides to the protein, in particular hydrophobicity-altering peptides, for example, in order to improve in vivo functionality. Proteins that are components of CRISPR can similarly be modified to facilitate chemical reactions. For example, it is possible to add amino acids to a protein having click chemistry modified groups (Nikie I. et al., 2015, Nature Protocols 10,780-791). In such embodiments, the click group can further be used to add a wide range of alternative structures, in particular poly(ethylene glycol) to improve stability, cell-penetrating peptides, RNA aptamers, lipids and carbohydrates, in particular GalNAc . In further alternatives, the CRISPR protein component can be adapted for cell entry (see Svensen et al., 2012, Trends in Pharmacological Sciences, Vol. 33, No. 4), for example, by attaching cell entry peptides to the protein (see Kauffman, W. Berkeley et al., 2015, Trends in Biochemical Sciences, Volume 40, Issue 12, 749-764 Koren and Torchilin, 2012, Trends in Molecular Medicine, Vol 18, No. 7 In further alternative embodiments, compounds or formulations may be pre-administered to patients or individuals to facilitate subsequent delivery of CRISPR components.

Effector protein complexes C2c1 and C2c3 can be used in plants

[00934] The C2c1 or C2c3 effector protein system(s) (eg, single or multiplexed) can be used in conjunction with the latest advances in culture genomics. The system described herein can be used to perform efficient and cost-effective exploration or manipulation of a plant genome or genome, such as rapid exploration and/or selection and/or selection and/or comparison and/or manipulation and/ or transformation of plant genes or genomes, for example, to create, identify, develop, optimize or assign certain characteristic(s) or trait (set of traits) to plants or to transform the plant genome. Thus, the production of plants, namely new plants with new combinations of traits or characteristics, or new plants with improved traits, can be improved. The C2c1 or C2c3 effector protein system or systems can be used in plants using the following methods: site directed integration (SDI) or gene editing (GE), close backcross (NRB) or backcross (RB). Aspects of use of the C2c1 or C2c3 effector systems described herein may be similar to the use of CRISPR-Cas systems (eg, CRISPR-Cas9) in plants. In this regard, the University of Arizona website "CRISPR-PLANT" (http: /www.genome.arizona.edu/crispr/) (maintained by PennState and AGI) deserves mention. Embodiments of the invention may be used in genome editing in plants or where mRNA or similar genome editing methods have been used in the past; see, for example, Nekrasov, "Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR-Cas system," Plant Methods 2013, 9:39 (doi: 10.1186/1746-4811-9-39); Brooks, "Efficient gene editing in tomato in the first generation using the CRISPR-Cas9 system," Plant Physiology September 2014 pp 114.247577; Shan, "Targeted genome modification of crop plants using a CRISPR-Cas system," Nature Biotechnology 31, 686-688 (2013); Feng, "Efficient genome editing in plants using a CRISPR/Cas system," Cell Research (2013) 23:1229-1232 doi:10.1038/cr.2013.114; published online 20 August 2013; Xie, "RNA-guided genome editing in plants using a CRISPR-Cas system," Mol Plant. 2013 Nov;6(6):1975-83. doi: 10.1093/mp/sstl 19. Epub 2013 Aug 17; Xu, "Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice," Rice 2014, 7:5 (2014), Zhou et af., "Exploiting SNPs for biallelic CRISPR mutations in the outcrossing woody perennial Populus reveals 4- coumarate: CoA ligase specificity and Redundancy," New Phytologist (2015) (Forum) 1-4 (available online at wmv.newphytologist.com); Caliando et al, "Targeted DNA degradation using a CRISPR device stably carried in the host genome", NATURE COMMUNICATIONS 6:6989, DOI: 10.1038/ncomms7989, vvwvv.nature.com/naturecommunications DOI: 10.1038/ncomms7989; -Mediated Plant Transformation Method, US Patent No. 7868149 - Plant Genome Sequences and Uses Thereof, and US 2009/0100536 - Transgenic Plants with Enhanced Agronomic Traits, the contents of each of which are incorporated herein by reference in their entirety. , content and disclosure Morrell et al., "Crop genomics: advances and applications", NatRevGenet 2011 Dec 29; 13(21:85-96; incorporated herein by reference, including with respect to how the invention may be applied to plants. Accordingly, this reference may also apply to animal cells, subject to differences, to plant cells, unless otherwise indicated, in which case the enzymes possess having reduced non-specific effects and systems using such enzymes can be used in relation to plants, including those mentioned in this application.

Application of CRISPR C2c1 or C2c3 systems in plants and yeasts

Definition:

[00935] In general, the term "plant" refers to any variety of photosynthetic, eukaryotic, unicellular or multicellular organisms of the plant kingdom (Plantae) that are characterized by growth by cell division, the presence of chloroplasts, and cell walls composed of cellulose. The term "plant" includes monocots and dicots. More specifically, plants are intended to include, but are not limited to, angiosperms and gymnosperms such as acacia, alfalfa, amaranth, apple, apricot, artichoke, ash, asparagus, avocado, banana, barley, bean, beetroot, birch, beech , blackberries, blueberries, broccoli, brussels sprouts, cabbage, canola, cantaloupe, carrots, cassava, cauliflower, cedar, cereals, celery, chestnut, cherry, bok choy, citrus fruits, clementine, clover, coffee, corn, cotton, cowpea, cucumber, cypress, eggplant, elm, endive, eucalyptus, fennel, fig, spruce, geranium, grape, grapefruit, peanut, physalis, hemlock, hickory, kale, kiwi, kohlrabi, larch , lettuce, leek, lemon, lime, robinia, pine, maidenhair, maize, mango, maple, melon, millet, mushroom, mustard, nuts, oak, oats, oil palm, okra, onion, orange, ornamental plant or flower or tree, papaya, palm, parsley, parsnip, pea, peach, peanut, pear, sphagnum, feather c, persimmon, pigeon pea (kayan), pine, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, safflower, willow, soybean, spinach, spruce, squash, strawberry, sugar beet, sugarcane, sunflower, sweet potato, sweet corn, tangerine, tea, tobacco, tomato, trees, triticale, lawn grasses, turnip, vine, walnut, watercress, watermelon, wheat, yam, yew and zucchini . The term plant also covers algae (Algae), which are primarily photoautotrophs, united primarily by the absence of roots, leaves, and other organs that characterize higher plants.

[00936] Genome editing techniques using the C2c1 or C2c3 system, as described herein, can be used to confer desired properties on essentially any plant. A wide variety of plants and plant cell systems with the desired physiological and agronomic characteristics described herein can be developed using the nucleic acid construct described herein and the various transformation methods mentioned above. In preferred embodiments, the plants and plant cells to be engineered include, but are not limited to, monocots and dicots such as cereals including cereals (e.g. wheat, corn, rice, millet, barley), fruit crops (e.g. , tomato, apple, pear, strawberry, orange), fodder crops (eg alfalfa), vegetables (eg carrots, potatoes, sugar beets, yams), leafy vegetables (eg lettuce, spinach); flowering plants (eg petunia, rose, chrysanthemum), conifers and (eg pine, fir, spruce), plants used in phytoremediation (eg plants accumulating heavy metals); oilseeds (eg sunflower, rapeseed) and plants used for experimental purposes (eg Arabidopsis ). Thus, CRISPR-Cas methods and systems can be used in a wide range of plants, for example, in dicotyledonous plants belonging to the following orders: Magnoliales, llliciales, Laurales, Piperales, Aristochiales, Nymphaeales, Ranunculales, Papaverales, Sarraceniaceae, Trochodendrales, Hamamelidales, Eucomiales , Leitneriales, Myricales, Fagales, Casuarinales, Caryophyllales, Batales, Polygonales, Plumbaginales, Dilleniales, Theales, Malvales, Urticales, Lecythidales, Violales, Salicales, Capparales, Ericales, Diapensales, Ebenales, Primulales, Rosales, Fabales, Podostemales, Haloragales, Myrtales , Cornales, Proteales, Santales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales, Sapindales, Juglandales, Geraniales, Polygalales, Umbellales, Gentianales, Polemoniales, Lamiales, Plantaginales, Scrophulariales, Campanulales, Rubiales, Dipsacales and Asterales; CRISPR-Cas methods and systems can be used in monocots, such as plants belonging to the following orders: Alismatales, Hydrocharitales, Najadales, Triuridales, Commelinales, Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales, Bromeliales, Zingiberales, Arecales, Cyclanthales , Pandanales, Arales, Lilliales and Orchidales, or in plants belonging to the gymnosperms, such as those belonging to the orders: Pinales, Ginkgoales, Cycadales, Araucariales, Cupressales and Gnetales.

[00937] CRISPR C2c1 or C2c3 systems and methods of using them described herein can be used in a wide range of plant species included in an unlimited list of dicots, monocots or gymnosperms belonging to the following genera: Atropa, Alseodaphne, Anacardium, Arachis, Beilschmiedia, Brassica, Carthamus, Cocculus, Croton, Cucumis, Citrus, Citrullus, Capsicum, Catharanthus, Cocos, Coffea, Cucurbita, Daucus, Duguetia, Eschscholzia, Ficus, Fragaria, Glaucium, Glycine, Gossypium, Helianthus, Hevea, Hyoscyamus, Lactuca, Landolphia, Linum, Litsea, Lycopersicon, Lupinus, Manihot, Majorana, Malus, Medicago, Nicotiana, Olea, Parthenium, Papaver, Persea, Phaseolus, Pistacia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Senecio, Sinomenium, Stephania, Sinapis, Solanum, Theobroma, Trifolium, Trigonella, Vicia, Vinca, Vitis, and Vigna; and the genera Allium, Andropogon, Eragrostis, Asparagus, Avena, Cynodon, Elaeis, Festuca, Festulolium, Heterocallis, Hordeum, Lemna, Lolium, Musa, Oryza, Panicum, Pannesetum, Phleum, Poa, Secale, Sorghum, Triticum, Zea, Abies , Cunninghamia, Ephedra, Picea, Pirns and Pseudotsuga.

[00938] CRISPR C2c1 or C2c3 systems and methods of using them can also be used in a wide range of "algae" or "algae cells"; including, for example, algae selected from several eukaryotic phyla including the following: Rhodophyta (red algae), Chlorophyta (green algae), Phaeophyta (brown algae), Bacillariophyta (diatom), Eustigmatophyta and dinoflagellates, and the prokaryotic phylum Cyanobacteria (blue - green algae). The term "algae" includes, for example, algae selected from the following genera: Amphora, Anabaena, Ankistrodesmus, Botryococcus, Chaetoceros, Chlamydomonas, Chlorella, Chlorococcum, Cyclotella, Cylindrotheca, Dunaliella, Emiliana, Euglena, Hematococcus, Isochrysis, Monochrysis, Monoraphidium, Nannochloris , Nannochloropsis, Navicula, Nephrochloris, Nephroselmis, Nitzschia, Nodularia, Nostoc, Oochromonas, Oocystis, Oscillatoria, Pavlova, Phaeodactylum, Playtmonas, Pleurochrysis, Porphyra, Pseudoanabaena, Pyramimonas, Stichococcus, Synechococcus, Synechocystis, Tetraselmis, Thalassiosira and.

[00939] Part of a plant, i.e. "plant tissue" can be processed according to the methods of the present invention to obtain a plant with improved properties. The term "plant tissue" also includes plant cells. The term "plant cell", as used herein, refers to individual units of a living plant, either in intact whole plant or in isolated form grown in tissue culture in vitro , on nutrient media or agar, in suspension on a nutrient medium, or buffer or as part of higher organized units such as plant tissue, plant organ or whole plant.

[00940] The term "protoplast" refers to a plant cell, the protective cell wall of which has been completely or partially removed, for example, mechanically or enzymatically, resulting in an intact biochemically competent living plant unit that is able to re-form the cell wall, proliferate, regenerate and grow into a whole plant under proper growing conditions.

[00941] The term "transformation" broadly refers to the process of genetically modifying a host plant by introducing DNA through an Agrobacteria or by one of a number of chemical or physical methods. As used in the present description, the term "plant host" refers to plants, including any cells, tissues, organs or progeny of plants. Many suitable plant tissues or plant cells can be transformed and include, but are not limited to, the following: protoplasts, somatic embryos, pollen, leaves, seedlings, stems, calluses, stolons, microtubers and shoots. The term "plant tissue" also refers to any clone of such a plant, seed, progeny, cuttings resulting from sexual or vegetative propagation, and the descendants of any of them, such as cuttings or seeds.

[00942] The term "transformed" as used herein refers to a cell, tissue, organ, or organism into which a foreign construct, such as a DNA molecule, has been introduced. The introduced DNA molecule can be integrated into the DNA of the recipient cell, tissue, organ, or organism such that the introduced DNA molecule is passed on to subsequent offspring. In these embodiments, a "transformed" or "transgenic" cell or plant may also include progeny of the cell or plant and progeny obtained by artificial breeding using such a transformed plant as the parent in the cross and exhibiting an altered phenotype as a result of the presence of the introduced molecule. DNA. Preferably, the transgenic plant is fertile and capable of transferring the introduced DNA to offspring through sexual reproduction.

[00943] The term "progeny", such as the progeny of a transgenic plant, refers to that which is generated from, derived from, or derived from a plant or transgenic plant. The introduced DNA molecule may also be temporarily introduced into the recipient's cell such that subsequent progeny do not inherit the introduced DNA molecule and thus are not considered "transgenic". Accordingly, as used herein, a "non-transgenic" plant or plant cell is one that does not contain foreign DNA stably integrated into its genome.

[00944] The term "plant promoter", as used herein, is a promoter capable of initiating transcription in plant cells, whether or not it is derived from a plant cell. Illustrative suitable plant promoters include, but are not limited to, promoters derived from plants, plant viruses, and bacteria such as Agrobacterium or Rhizobium , which include genes expressed in plant cells.

[00945] As used herein, "fungal cell" refers to any organism from the fungal kingdom, including Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Glomeromycota, Microsporidia, and Neocallimastigomycota . Fungal cells may include yeasts, molds and filamentous fungi. In some modifications of the invention, the fungal cell is a yeast cell.

[00946] As used herein, the term "yeast cell" refers to any fungal cell in the genera Ascomycota and Basidiomycota. Yeast cells may include budding yeast cells, dividing yeast cells, and mold cells. While not limited to these organisms, many types of yeast used in laboratory and industrial settings are part of the genus Ascomycota. In some embodiments, the yeast cell is a S. cerevisiae , Kluyveromyces marxianus , or Issatchenkia orientalis cell. Other yeast cells may include, but are not limited to, Candida spp. (e.g. Candida albicans ), Yarrowia spp. (e.g. Yarrowia lipolytica ), Pichia spp. (e.g. Pichia pastoris ), Kluyveromyces spp. (e.g. Kluyveromyces lactis and Kluyveromyces marxianus ), Neurospora spp. (e.g. Neurospora crassa ), Fusarium spp. (e.g. Fusarium oxysporum ), and Issatchenkia spp. (e.g. Issatchenkia orientalis , also called Pichia kudriavzevii and Candida acidothermophilum ). In some embodiments, the fungal cell is a filamentous fungal cell. As used herein, the term "filamentous fungal cell" refers to any type of fungal cell that grows in the form of filaments, ie. hyphae or mycelium. Examples of fibrous fungal cells may include, but are not limited to, Aspergillus spp. (e.g. Aspergillus niger ), Trichoderma spp. (eg Trichoderma reesei ), Rhizopus spp., (eg Rhizopus oryzae ) and Mortierella spp. (e.g. Mortierella isabellina ).

[00947] In some embodiments, the fungal cell is an industrial strain. As used herein, "industrial strain" refers to any fungal cell strain used in or isolated from a manufacturing process, such as commercial or industrial production of a product. The term "industrial strain" may refer to a species of fungus that is typically used in a manufacturing process, or it may refer to an isolate of a species of fungus that may also be used for non-industrial purposes (eg laboratory research). Examples of manufacturing processes may include fermentation (eg, in the production of food or beverages), distillation, production of biofuels, production of various compounds and polypeptides. Examples of commercial strains may include, but are not limited to, JAY270 and ATCC4124.

[00948] In some embodiments, the fungal cell is a polyploid cell. As used herein, the term "polyploid" cell can refer to any cell in which the genome is present in more than one copy. The term "polyploid cell" may refer to a cell that naturally exists in a polyploid state, or a cell whose genome has been artificially induced into a polyploid state (for example, through specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or replication). DNA). "Polyploid cell" may refer to a cell whose entire genome is polyploid, or a cell that is a polyploid at a particular genomic target locus. Without being bound by theory, it is believed that the abundance of guide RNAs may limit the rate of genetic engineering of polyploid cells to a greater extent than haploid cells, and thus C2c1 or C2c2 protein CRISPR system methods described herein can use the benefits of a certain fungal cell type.

[00949] In some embodiments, the fungal cell is a diploid cell. As used herein, the term "diploid" cell can refer to any cell whose genome is present in two copies. A diploid cell may refer to a type of cell that naturally exists in a diploid state, a cell whose genome has been artificially induced into a diploid state (eg, through specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). For example, the S. cerevisiae strain, S228C, may be in a haploid or diploid state. A "diploid" cell may refer to a cell whose entire genome is diploid, or a cell that is diploid at a particular genomic target locus. In some embodiments, the fungal cell is a haploid cell. As used herein, a "haploid" cell may refer to any cell whose genome is present in one copy. A haploid cell may refer to a cell type that is naturally in a haploid state, or a cell whose genome has been artificially induced into a haploid state (eg, through specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). For example, the S. cerevisiae strain, S228C, may be in a haploid or diploid state. "Haploid" cell may refer to a cell whose entire genome is haploid, or a cell that is haploid at a particular genomic target locus.

[00950] As used herein, a "yeast expression vector" refers to a nucleic acid that contains one or more RNA and/or polypeptide coding sequences, and may further contain any desired elements that direct the expression of the nucleic acid(s), as well as any elements that ensure the replication and maintenance of the expression vector in the yeast cell. Many suitable yeast expression vectors and features thereof are known in the art; for example, various vectors and methods are illustrated in Yeast Protocols, 2nd edition, Xiao, W., ed. (Humana Press, New York, 2007) and Buckholz, R.G. and Gleeson, M.A. (1991) Biotechnology (NY) 9(11): 1067-72. Yeast vectors may contain, without limitation, a centromeric (CEN) sequence, an autonomously replicating sequence (ARS), a promoter such as an RNA polymerase III promoter operably linked to a target sequence or gene, a terminator such as an RNA polymerase III terminator, origin replication and marker gene (eg auxotrophy, antibiotic or other selected markers). Examples of expression vectors for use in yeast may include plasmids, yeast artificial chromosomes, 2μ plasmids, yeast integrative plasmids, yeast replication plasmids, shuttle vectors, and episomes.

Stable integration of the components of the CRISPR C2c1 or C2c3 system into the genome of plants and plant cells

[000001] In specific embodiments, the invention provides that polynucleotides encoding C2c1 or C2c3 components of the CRISPR system are introduced for stable integration into the plant cell genome. In these embodiments, the design of the transformation vector or expression system can be adjusted depending on when, where, and under what conditions the guide RNA and/or the C2c1 or C2c3 gene is expressed.

[000002] In specific embodiments, the invention provides for the introduction of components of the CRISPR C2c1 or C2c3 system stably into the genomic DNA of a plant cell. Additionally or alternatively, it is contemplated to introduce C2c1 or C2c3 components of the CRISPR system for stable integration into the DNA of plant organelles such as, but not limited to, plastids, mitochondria, or chloroplasts.

[000003] An expression system for stably integrating into the plant cell genome may comprise one or more of the following elements: a promoter element that can be used to express the RNA and/or C2c1 or C2c3 enzyme in the plant cell; 5'-untranslated regions to increase expression; an intron element to further increase expression in certain cells such as monocot cells, a multiple cloning site to provide convenient restriction sites for insertion of a guide RNA and/or a C2c1 or C2c3 gene sequence and other desired elements; and 3'-untranslated regions to ensure efficient termination of the expressed transcript.

[000004] The elements of the expression system can be on one or more expression constructs that are either circular, such as a plasmid or transformation vector, or linear, such as a DNA double helix.

[000005] In a particular embodiment, a C2c1 or C2c3 CRISPR expression system comprises at least:

(a) a nucleotide sequence encoding a guide RNA (gRNA) that hybridizes to a target sequence in a plant, and where the guide RNA includes a guide sequence and a direct repeat sequence;

(b) a nucleotide sequence encoding a C2c1 or C2c3 protein,

[00951] wherein components (a) or (b) are located on the same or different constructs, whereby different nucleotide sequences may be under the control of the same or different regulatory elements active in the plant cell.

[00952] The DNA construct(s) comprising the components of the C2c1 or C2c3 CRISPR system and, when applicable, the template sequence, can be introduced into the plant genome, plant part, or plant cell by a number of conventional methods. The process typically includes the steps of selecting a suitable cell or tissue, introducing the construct(s) into the cell or tissue, and regenerating the plant cells or plants from the cells or tissues.

[00953] In specific embodiments, the DNA construct may be introduced into a plant cell using methods such as, but not limited to, electroporation, microinjection, aerosol injection of plant cell protoplasts, or the DNA constructs may be introduced directly into plant tissue using biolistic methods such as DNA particle bombardment (see also Fu et al., Transgenic Res, 2000 Feb; 9(1):11-9). Particle bombardment of cells is based on the acceleration of particles coated with the target gene(s), resulting in particle penetration into the protoplasm and typically stable integration into the genome, (see, for example, Klein et al., Nature (1987), Klein et al., Bio/Technology (1992), Casas et al., Proc Natl Acad Sci USA (1993)).

[00954] In specific embodiments, DNA constructs containing components of the C2c1 or C2c3 CRISPR system can be introduced into a plant by agrobacterial transformation. The DNA constructs can be combined with suitable T-DNA flank regions and introduced into a conventional Agrobacterium tumefaciens vector. Foreign DNA can be incorporated into the plant genome by infecting plants or by incubating plant protoplasts with Agrobacterium bacteria containing one or more Ti-plasmids (tumor-inducing plasmid) (see, for example, Fraley et al., (1985), Rogers et al. , (1987) and US Pat. No. 5,563,055).

Plant promoters

[00955] To ensure suitable expression in a plant cell, components of the C2c1 or C2c3 CRISPR system described herein are typically placed under the control of a plant promoter, i. promoter in plant cells. The use of various types of promoters is contemplated.

[00956] A plant constitutive promoter is a promoter that is capable of expressing an open reading frame (ORF) that it controls in all or nearly all plant tissues during all or almost all stages of plant development (referred to as "constitutive expression"). One non-limiting example of a constitutive promoter is the cauliflower mosaic virus 35S promoter. The term "regulated promoter" refers to promoters that direct gene expression in a temporally and/or spatially regulated manner rather than constitutively, and includes tissue-specific, tissue-preferred, and inducible promoters. Different promoters may direct gene expression in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. In particular embodiments, one or more components of the C2c1 or C2c3 CRISPR system are expressed under the control of a constitutive promoter, such as the cauliflower mosaic virus 35S promoter. Tissue-preferred promoters can be used to target increased expression in certain cell types in certain plant tissues, such as vascular cells in leaves or roots, or in certain seed cells. Examples of specific promoters for use in a C2c1 or C2c3 CRISPR system can be found in Kawamata et al., (1997) Plant Cell Physiol 38:792-803; Yamamoto et al., (1997) Plant J 12:255-65; Hire et al., (1992) Plant Mol Biol 20:207-18, Kuster et al., (1995) Plant Mol Biol 29:759-72, and Capana et al., (1994) Plant Mol Biol 25:681- 91.

[00957] Exemplary inducible promoters that provide spatiotemporal control of gene editing or expression can use energy. The form of energy may include, but is not limited to, sound energy, electromagnetic radiation, chemical energy, and/or thermal energy. Examples of inducible systems include tetracycline-inducible promoters (Tet-On or Tet-Off), two-hybrid transcription activation systems using small molecules (FKBP, ABA, etc.), or light-inducible systems (phytochromes, LOV domains, or cryptochromes). ), such as the light-inducible transcription effector (LITE), which drive changes in transcriptional activity in a sequence-specific manner. Components of a light-inducible system may include a CRISPR C2c1 or C2c3 enzyme, a photosensitive cytochrome heterodimer (eg, from Arabidopsis thaliana ), and an activation/repression transcriptional domain. Other examples of inducible DNA binding proteins and methods for their use are given in US 61/736465 and US 61/721283, which are incorporated herein by reference in their entirety.

[00958] In certain embodiments of the invention, transient or inducible expression can be achieved using, for example, promoters regulated by chemical stimuli, i.e. those for which the use of an exogenous chemical agent induces gene expression. Modulation of gene expression can also be achieved using promoters repressed by chemical stimuli, i.e. those for which the use of a chemical agent suppresses gene expression. Such chemically inducible promoters include, but are not limited to, the ln2-2 maize promoter activated by benzenesulfonamide herbicide safeners (De Veylder et al., (1997) Plant Cell Physiol 38:568-77), the GST maize promoter (GST-lI-27 , W093/01294) activated by hydrophobic electrophilic compounds used in common pre-emergence herbicides, and the tobacco PR 1 promoter (Ono et al., (2004) Biosci Biotechnol Biochem 68:803-7) activated by salicylic acid. Promoters whose activity is regulated by antibiotics, such as tetracycline-inducible and tetracycline-repressed promoters (Gatz et al, (1991) Mol Gen Genet 227:229-37; US Pat.

Traversal to specific organelles and/or expression in specific plant organelles

[00959] The expression system may include elements for translocation to and/or expression in a particular plant organelle.

Targeting into chloroplasts

[00960] In specific embodiments, the invention provides for the use of a C2c1 or C2c3 CRISPR system in order to specifically alter chloroplast genes or provide expression in the chloroplast. For this purpose, methods of chloroplast transformation or compartmentalization of C2c1 or C2c3 components of the CRISPR system in the chloroplast are used. For example, the introduction of genetic modifications in the plastid genome can reduce biosecurity concerns such as gene transfer through pollen.

[00961] Methods for chloroplast transformation are known in the art and include particle bombardment ballistic transformation, PEG treatment, and microinjection. In addition, methods can be used that involve moving transformation cassettes from the nuclear genome to the plastid, as described in WO2010061186.

[00962] Alternatively, one or more of the C2c1 or C2c3 CRISPR components are targeted to the plant chloroplast. This is achieved by inserting into the expression construct a sequence encoding a claplast transit peptide (CTP) or a plastid transit peptide operably linked to the 5'-terminal region of the sequence encoding the C2c1 or C2c3 protein. CTP is removed during the processing step during transit to the chloroplast. The skilled artisan is aware of the targeting of expressed proteins to the chloroplast (see, for example, Protein Transport into Chloroplasts, 2010, Annual Review of Plant Biology, Vol. 61: 157-180). In such embodiments, it is also desirable to target the guide RNA to the plant chloroplast. Methods and constructs that can be used to translocate a guide RNA into a chloroplast due to a chloroplast localization sequence are described, for example, in US 20040142476 and incorporated herein by reference. Such design changes can be introduced into the expression systems of the present invention in order to efficiently move guide(s) RNA targeting C2c1 or C2c3.

Introduction of polynucleotides encoding the C2c1 or C2c3 CRISPR system into algal cells

[00963] Transgenic algae (or other plants such as rapeseed) may be particularly useful for the production of vegetable oils or biofuels such as alcohols (especially methanol and ethanol) or other products. They can be engineered to express or overexpress large volumes of oil or alcohols for use in petroleum or biofuel industries.

[00964] US 8945839 describes a method for modifying an engineered microalgae species ( Chlamydomonas reinhardtii cells) using Cas9. Using a similar technique, methods using the C2c1 or C2c3 CRISPR system described herein can be applied to species of the genus Chlamydomonas and other algae. In certain embodiments, the C2c1 or C2c3 and guide RNA are introduced into the algae in which they are expressed using a C2c1 or C2c3 expression vector under the control of a constitutive promoter such as Hsp70A-Rbc S2 or beta-2-tubulin. The guide RNA can optionally be delivered using a vector containing the T7 promoter. Alternatively, Cas9 RNA and an in vitro transcribed guide RNA can be delivered to algal cells. Electroporation protocols are available to the skilled person, including the standard recommended protocol from the GeneArt Chlamydomonas Engineering kit.

[00965] In specific embodiments, the endonuclease used in the present invention is a split C2c1 enzyme or a split C2c3 enzyme. Decoupled C2c1 or C2c3 enzymes are preferably used in algae for targeted genome modification as described for Cas9 in WO 2015086795. The use of decoupled C2c1 or C2c3 systems is particularly suited to the case of inducible genome targeting and avoids the potential toxic effect of C2c1 or C2c3 overexpression in algal cells. . In certain embodiments, said split C2c1 or C2c3 domains (RuvC and HNH domains) may be simultaneously or sequentially introduced into a cell such that said split C2c1 or C2c3 domain(s) processes the target nucleic acid sequence in the algal cell. The reduced size of the separated C2c1 or C2c3 compared to wild-type C2c1 or C2c3 allows other methods of delivery of the CRISPR system into cells, in particular the use of cell-penetrating peptides as described herein. This method is of particular interest for the production of genetically modified algae.

Introduction of Polynucleotides Encoding C2c1 or C2c3 Components into Yeast Cells

[00966] In certain embodiments, the invention relates to the use of a C2c1 or C2c3 CRISPR system for editing the genome of yeast cells. Yeast cell transformation methods that can be used to introduce polynucleotides encoding components of the C2c1 or C2c3 CRISPR system are known to the skilled artisan and discussed in Kawai et al., 2010, Bioeng Bugs. 2010 Nov-Dec, 1(6): 395-403). Non-limiting examples include transformation of yeast cells by treatment with lithium acetate (with the possibility of further treatment with transfer DNA and PEG), ballistic method or electroporation.

Transient expression of CRISPR system components C2c1 or C2c3 in plants and plant cell

[00967] In certain embodiments, the invention provides that the guide RNA and/or the C2c1 or C2c3 gene is expressed in the plant cell. In such embodiments, the CRISPR C2c1 or C2c3 system is only able to provide modification of the target gene when both the guide RNA and the C2c1 or C2c3 protein are present in the cell, with further control of the genome modification being possible. Since the expression of the C2c1 or C2c3 enzyme is transient, plants regenerating from such plant cells do not usually contain foreign DNA. In specific embodiments, the C2c1 or C2c3 enzyme is stably expressed by the plant cell and the guide sequence is transiently expressed.

[00968] In specific embodiments, components of the C2c1 or C2c3 CRISPR system can be introduced into plant cells using a plant viral vector (Scholthof et al. 1996, Annu Rev Pbytopathol. 1996;34:299-323). In further specific embodiments, said viral vector is a DNA virus vector. For example, a geminivirus (for example, cabbage leaf curl virus, bean yellow dwarf virus, wheat dwarf virus, tomato leaf curl virus, corn streak virus, tobacco leaf curl virus, or tomato golden mosaic virus), or nanovirus (nanovirus) (for example , legume yellow necrosis virus). In other specific embodiments, said viral vector is an RNA virus vector, e.g., tobravirus (e.g., tobacco rattle virus, tobacco mosaic virus), potexvirus (e.g., potato X-virus) or hordeivirus (e.g. hordeivirus) (e.g. , barley striated mosaic virus). Plant virus replication genomes are non-integral vectors.

[00969] In some embodiments, the vector used to transiently express the C2c1 or C2c3 CRISPR constructs may be, in particular, a pEAQ vector designed specifically for Agrobacterium mediated transient expression in the protoplast (Sainsbury F. et al., Plant Biotechnol J. 2009 Sep:7(7):682-93). Precise targeting of genome localization has been demonstrated using a modified cabbage leaf curl virus (CaLCuV) vector to express guide RNAs in stably transgenic plants expressing the CRISPR enzyme (Scientific Reports 5, Article number: 14926 (2015), doi: 10.1038/ srep14926).

[00970] In specific embodiments, double-stranded DNA fragments encoding a guide RNA and/or a C2c1 or C2c3 gene may be temporarily introduced into a plant cell. In such embodiments, the introduced double-stranded DNA fragments are present in amounts sufficient to modify the cell, but do not remain after the end of the time period in question or after one or more cell divisions. Methods for direct DNA transfer are known to the skilled artisan (see, for example, Davev et al. Plant Mol Biol 1989 Sep;13(3):273-85.)

[00971] In other embodiments of the invention, a polynucleotide encoding a C2c1 or C2c3 protein is introduced into a plant cell, followed by translation and processing in a cell that synthesizes this protein in an amount sufficient to change the cell (in the presence of at least one guide RNA molecule) , but which does not remain after the end of the considered period of time or after one or more cell divisions. Methods for introducing mRNA into the plant protoplast are known to the skilled artisan ((see, for example, Gallie, Plant Cell Reports (1993), 13; 119-122).

[00972] Combinations of the various methods described above are also contemplated.

Delivery of CRISPR components C2c1 or C2c3 into the plant cell

[00973] In particular embodiments, one or more components of the C2c1 or C2c3 CRISPR system are required to be delivered directly to a plant cell. This is of interest, inter alia, for the production of non-transgenic plants (see below). In some embodiments of the invention, one or more components of C2c1 or C2c3 received(s) outside the plant or plant cell and delivered(s) to the cell. For example, in some embodiments of the invention, the C2c1 or C2c3 protein is produced in vitro prior to introduction into the plant cell. Protein C2c1 or C2c3 can be obtained by various methods known to those skilled in the art, including recombinant production. After expression, the C2c1 or C2c3 protein is isolated, refolded if necessary, purified, and optionally de-purified, such as a His tag. When a crude, partially purified, or more fully purified C2c1 or C2c3 protein is obtained, it can be introduced into a plant cell.

[00974] In certain embodiments, the C2c1 or C2c3 protein is combined with a target gene targeting RNA molecule to form a preassembled ribonucleoprotein.

[00975] The individual components or the preassembled ribonucleoprotein itself can be introduced into the plant cell by electroporation, particle bombardment coated with a C2c1 or C2c3 associated gene product, chemical transfection, or some other means of transport across the cell membrane. For example, transfection of a plant protoplast with a pre-assembled CRISPR ribonucleoprotein has been demonstrated to confirm modification of the plant genome (as described in Woo et at. Nature Biotechnology, 2015; DQI: 1.0.1038/nbt.3389).

[00976] In specific embodiments, the C2c1 or C2c3 CRISPR system can be introduced into plant cells using nanoparticles. Components such as protein, nucleic acid, or a combination thereof can be loaded or packaged into nanoparticles and applied to plants (as described, for example, in WO 2008042156 and US 20130185823). In particular, embodiments of the invention include nanoparticles loaded with or containing DNA molecule(s) encoding C2c1 or C2c3 protein, DNA molecules encoding guide RNA, and/or isolated guide RNA, as described in WO2015089419.

[00977] A further means of introducing one or more components of the C2c1 or C2c3 CRISPR systems into a plant cell is through the use of cell penetrating peptides (CPPs). Accordingly, certain embodiments of the invention include compositions comprising cell-penetrating peptides linked to a C2c1 or C2c3 protein. In specific embodiments of the present invention, a C2c1 or C2c3 protein and/or guide RNA is coupled to one or more CPPs for their efficient delivery to the plant protoplast (also see Ramakrishna, Genome Res. 2014 Jun;24(6):1020-7, for Cas9 in human cells). In other embodiments, the RNA targeting protein gene and/or guide RNA(s) are encoded by one or more circular or non-circular molecule(s) coupled to one or more CPPs for delivery to the plant protoplast. Plant protoplasts are further regenerated into plant cells, and further into plants. CPPs in general are short peptides of less than 35 amino acids in length derived from either protein or chimeric sequences capable of transporting biomolecules across the cell membrane independent of receptors. CPPs can be cationic peptides, peptides having hydrophobic sequences, amphipathic peptides, proline-rich peptides and antimicrobial sequences, as well as chimeric or two-component peptides (Pooga and Langel, 2005). CPPs are able to penetrate biological membranes and, at the same time, trigger the movement of various biomolecules through cell membranes into the cytoplasm and improve their intracellular movement, thereby facilitating the interaction of the biomolecule with the target. Examples of CPP include, but are not limited to: Tat, a nuclear transcription activator protein required for HIV type I virus replication, penetratin, Kaposi's fibroblast growth factor (FGF) signal peptide, β3 integrin signal peptide; polyarginine peptide sequence Args, arginine-rich molecular transporters, "sweet arrow" peptide.

Use of CRISPR C2c1 or C2c3 systems to produce genetically modified non-transgenic plants

[00978] In certain embodiments, the methods described herein are used to modify endogenous genes or modify their expression without permanently introducing any foreign genes into the plant genome, including those encoding CRISPR components, to avoid the presence of foreign DNA in plant genome. This may be of interest due to the fact that the regulatory requirements for non-transgenic plants are less stringent.

[00979] In specific embodiments, this is provided by transient expression of the C2c1 or C2c3 CRISPR components. In some embodiments, one or more CRISPR components are expressed from one or more viral vectors that produce sufficient C2c1 or C2c3 protein and guide RNA to stably and continuously modify the gene of interest in accordance with the method described herein.

[00980] In specific embodiments, transient expression of the C2c1 or C2c3 CRISPR constructs occurs in plant protoplasts and thus does not integrate into the genome. Long-term expression may be sufficient for the C2c1 or C2c3 CRISPR system to be able to modify the target gene as described herein.

[00981] In some embodiments, the various components of the C2c1 or C2c3 CRISPR system are introduced into the plant cell, protoplast, or plant tissue individually or as a mixture using particulate delivery molecules, such as nanoparticles or CPP molecules, as described above.

[00982] Expression of the C2c1 or C2c3 CRISPR components can cause targeted modification of the genome either through the direct action of C2c1 or C2c3 nucleases and optionally introducing a template DNA, or through modification of target genes using the C2c1 or C2c3 CRISPR system as described herein. application. The various strategies described above enable C2c1 or C2c3 mediated genome editing without requiring the introduction of C2c1 or C2c3 CRISPR components into the plant genome. Components that are temporarily introduced into the plant cell usually do not persist after crossing over.

Detection of modifications in the plant genome - selective markers

[00983] In some embodiments, in which the method includes modifying an endogenous target gene in the genome of a plant, any suitable method can be used so that after the plant, plant part, or plant cell is infected or transfected with the C2c1 or C2c3 CRISPR system , to determine if gene targeting or site directed mutagenesis has taken place. Where the method includes introducing a transgene, the transformed plant cell, callus, or plant can be identified and isolated by selecting or screening the modified plant material for the presence of the transgene or the traits encoded by the transgene. Physical or biochemical methods can be used to identify plants or plant cell transformants containing inserted gene constructs or endogenous DNA modifications. Such methods include, but are not limited to: 1) Southern analysis or PCR amplification to detect and determine the structure of a recombinant DNA insert or modified endogenous genes; 2) Northern blot, RNase S1 protection, primer extension, or reverse transcriptase PCR amplification to detect and study RNA transcripts of gene constructs; 3) an enzymatic assay to determine the activity of enzymes or ribozymes, where such gene products are encoded by a gene construct or expression is affected by a genetic modification; 4) protein gel electrophoresis, Western blot techniques, immunoprecipitation, protein activity-related immunoassays when the gene constructs or endogenous gene products are proteins. Additional techniques such as in situ hybridization , enzyme staining, immunostaining can also be used to detect the presence or expression of recombinant constructs or detect endogenous gene modification in specific plant organs and tissues. The methods for carrying out all of these assay methods are well known to those skilled in the art.

[00984] Additionally (or alternatively) an expression system encoding C2c1 or C2c3 CRISPR components is typically designed to include one or more selectable markers that provide a tool for isolating or efficiently selecting cells containing and/or modified by the CRISPR C2c1 or C2c3 in the early stages and on a large scale.

[00985] In the case of Agrobacterium -mediated transformation, the marker cassette may be located near or between the borders of the flanking T-DNA and may be within the binary vector. In another embodiment, such a marker cassette may be outside the T-DNA. The selective marker cassette can also be located within or near the boundaries of the T-DNA, while the expression cassette can be located elsewhere within the second T-DNA on the binary vector (eg, the 2 T-DNA system).

[00986] For particle bombardment or protoplast transformation, the expression system may include one or more isolated linear fragments, or may be part of a larger construct that may contain bacterial replication elements, bacterial selectable markers, or other detectable elements. Such expression cassette(s) containing polynucleotides encoding a guide molecule and/or C2c1 or C2c3 may be physically linked to the marker cassette or may be mixed with a second nucleic acid molecule encoding the marker cassette. The marker cassette consists of elements necessary for the expression of a detectable or selectable marker, which enable efficient selection of transformed cells.

[00987] The cell selection procedure based on a selectable marker depends on the nature of the marker gene. In certain embodiments, a selectable marker is used, ie. a marker that allows direct selection of cells based on the expression of the marker. A selectable marker can provide positive or negative selection and be dependent or independent of the presence of external substrates (Miki et al. 2004, 107(3): 193-232). A more common case is when antibiotic or herbicide resistance genes are used as a marker, whereby selection is made by growing plant material on media containing inhibitory concentrations of the antibiotic or herbicide to which the marker gene confers resistance. Examples of such genes are genes that confer resistance to antibiotics such as hygromycin (hpt) and kanamycin (nptII) and genes that confer resistance to herbicides such as phosphinothricin (bar) and chlorosulforone (als).

[00988] Transformed plants and plant cells can also be identified by screening for the activity of a visible marker, usually an enzyme capable of processing a colored substrate (eg, β-glucuronidase, luciferase, B or Cl genes). Such a selection and screening methodology is well known to those skilled in the art.

Cultures and plant regeneration

[00989] In certain embodiments, a plant cell that has a modified genome, produced or obtained by any of the methods described in this specification, can be cultured to regenerate a whole plant having the modified genotype and, as a result, the desired phenotype. Conventional regeneration techniques are based on the manipulation of certain phytohormones in tissue culture growth media and are typically based on a biocidal and/or herbicidal marker that is introduced along with the desired nucleotide sequence. In certain further modifications of the invention, plant regeneration is achieved based on cultured protoplasts, plant calli, explants, organs, pollen, embryos, or parts thereof (see, for example, Evans et. al. (1983), Handbook of Plant Cell Culture, Klee et al. (1987) Ann. Rev. of Plant Phys. ) .

[00990] In certain embodiments, transformed or improved plants as described in this specification may be self-pollinating to provide seeds for homozygous improved plants of the present invention (homozygous for DNA modification) or may be crossed with non-transgenic plants or other improved plants in order to to provide seeds for heterozygous plants. If the recombinant DNA has been introduced into a plant cell, the resulting plant is heterozygous for the recombinant DNA molecule. Such plants, both homozygous and heterozygous, obtained by crossing improved plants and containing genetic modifications (which may be recombinant DNA) are referred to herein as "progeny". Progeny plants are plants derived from the original transgenic plants and containing genome modifications or introduced using the methods described in the present description, a recombinant DNA molecule. Alternatively, genetically modified plants can be obtained using one of the methods described above using the enzyme C2c1 or C2c3, whereby foreign DNA is not integrated into the genome. The progeny of such plants obtained through subsequent breeding work may also contain genetic modifications. Breeding work is carried out using any widely used breeding methods for various crops (for example, Allard, Principles of Plant Breeding, John Wiley & Sons, NY, U. of CA, Davis, CA, 50-98 (1960).

Obtaining plants with improved agronomic characteristics

[00991] The C2c1 or C2c3 based CRISPR systems described herein can be used to introduce targeted double-strand or single-strand breaks and/or introduce gene activator or repressor systems and, without limitation, can be used to target genes gene replacement, targeted mutagenesis, targeted deletions or insertions, targeted inversions and/or translocations. By co-expressing multiple targeting RNAs targeting multiple modifications in the same cell, it is possible to provide multiplexed genome modifications. This technology can be used to precision engineer plants with improved properties, including improved nutritional characteristics, increased resistance to diseases or stress of a biological or abiotic nature, and increased production of commercially important products or heterologous compounds.

[00992] In certain embodiments, C2c1 or C2c3 CRISPR systems, as described herein, are used to introduce directional double-strand breaks (DSBs) into an endogenous DNA sequence. DSBs activate DNA repair pathways in the cell, which can be used to achieve desired DNA sequence modifications near the break position. This is of interest if inactivation of endogenous genes can provide or contribute to the desired characteristic. In certain embodiments, homologous recombination with a template sequence may be performed at the DSB site in order to introduce a gene of interest.

[00993] In certain embodiments, the C2c1 or C2c3 CRISPR systems can be used as a nucleic acid binding protein generally fused or operably linked to a functional domain to activate and/or repress endogenous plant genes. Examples of conventionally used functional domains may include, but are not limited to, translation initiator, translation activator, translation repressor, nucleases such as ribonucleases, spliceosome, microbeads, light activated/controlled domain, or chemical stimulus activated/controlled domain. In the case of such modifications of the invention, the C2c1 or C2c3 protein usually contains at least one mutation, in particular such that it has no more than 5% of the activity of the C2c1 or C2c3 protein without such at least one mutation; the guide RNA includes a guide sequence capable of hybridizing to the target sequence.

[00994] The methods described herein generally provide improved plants having one or more desirable characteristics compared to wild-type plants. In certain embodiments, the resulting plants, plant cells, or plant parts are transgenic plants containing an exogenous DNA sequence inserted into the genome of all or part of the plant's cells. In specific embodiments, non-transgenic genetically modified plants, plant parts, or cells are obtained in the sense that no exogenous DNA sequence is included in the genome of any of the plant's cells. In such embodiments, the improved plant is non-transgenic. In the event that only the modification of some endogenous gene is provided and foreign genes are not introduced and maintained in the plant genome, the resulting genetically modified crops do not contain foreign genes and thus should be considered essentially non-transgenic. Various applications of the CRISPR C2c1 or C2c3 systems for editing plant genomes are described in more detail below:

Introduction of one or more foreign genes to communicate an agronomic characteristic of interest

[00995] The present invention relates to methods for editing the genome or modifying sequences associated with or located at target loci of interest, wherein such method comprises introducing a C2c1 or C2c3 effector protein complex into a plant cell, whereby the C2c1 or C2c3 effector protein complex effectively inserts a DNA insert, for example, encoding a foreign gene of interest into the genome of a plant cell. In preferred embodiments, such integration of the insert DNA is facilitated by HR with an exogenously introduced DNA template or repair template. Typically, the exogenously introduced DNA template or repair template is co-delivered with a C2c1 or C2c3 effector protein complex, or a single component or polynucleotide vector to express a component of the complex.

[00996] The C2c1 or C2c3 CRISPR systems contemplated by the present invention allow targeted gene delivery. It is becoming increasingly clear that the efficiency of expression of a gene of interest is largely determined by the location of the insertion in the genome. The methods of the present invention allow the targeted integration of a foreign gene into a desired position in the genome. Localization may be selected based on information from previously obtained inserts, or may be selected using the methods described herein.

[00997] In certain embodiments, the methods described herein comprise (a) introducing into a cell a CRISPR C2c1 or C2c3 complex comprising a guide RNA comprising a direct repeat and a guide sequence, wherein the guide sequence hybridizes to a target sequence that is endogenous to plant cell; (b) introducing into the plant cell a C2c1 or C2c3 effector molecule that forms a complex with the guide RNA, where the guide sequence hybridizes to the target sequence and causes a double-strand break at or near the sequence targeted by the guide; (c) introducing into the cell a nucleotide sequence encoding an HDR repair template that encodes a gene of interest and that is inserted at the location of the HDR double-strand break. In certain embodiments of the invention, the step of introducing may include delivering to the plant cell one or more polynucleotides encoding a C2c1 or C2c3 effector protein, a guide RNA, and a repair template. In certain embodiments, these polynucleotides are delivered to the cell by a DNA-containing virus (eg, geminivirus) or an RNA-containing virus (eg, tobravirus). In certain embodiments, the steps of administration include delivery to a plant cell of a T-DNA comprising one or more polynucleotide sequences encoding a C2c1 or C2c3 effector protein, a guide RNA, and a repair template, where delivery is mediated by Agrobacterium. A nucleic acid sequence encoding a C2c1 or C2c3 effector protein may be operably linked to a promoter, in particular a constitutive promoter (eg, the cauliflower mosaic virus 35S promoter) or a cell-specific or inducible promoter. In certain embodiments, the polynucleotide is introduced by microparticle bombardment. In certain embodiments, the method also includes screening the plant cell after the injection steps to determine if the repair template has been, i. the gene of interest has been successfully introduced. In certain embodiments, these methods include the step of regenerating a plant from a plant cell. In further embodiments, such methods include crossing a plant to produce offspring with the desired genetic properties. Examples of foreign genes encoding a characteristic of interest are listed below.

b) Editing endogenous genes to report an agronomic trait of interest

[00998] The present invention relates to methods for editing or modifying sequences associated with or located at a target locus of interest, wherein such method comprises introducing a C2c1 or C2c3 effector protein complex into a plant cell, whereby the C2c1 or C2c3 complex modifies the expression of an endogenous plant gene. This can be achieved in various ways. In certain embodiments, cessation of endogenous gene expression is desired and a C2c1 or C2c3 CRISPR complex is used to target and cleave the endogenous gene in order to modify gene expression. In such embodiments, the methods described herein comprise (a) introducing into a plant cell a C2c1 or C2c3 complex comprising a guide RNA containing a direct repeat and a guide sequence, wherein the guide sequence hybridizes to a target sequence within a gene of interest in the plant genome. cells; (b) introducing into the cell a C2c1 or C2c3 effector protein which, when bound to the guide RNA, includes a guide sequence that is hybridized to the target sequence and introduces a double-strand break at or near the sequence targeted by the guide sequence. In certain embodiments, the step of introducing may include delivering to the plant cell one or more polynucleotides encoding a C2c1 or C2c3 effector protein and a guide RNA.

[00999] In certain embodiments, the polynucleotides are delivered to the cell by a DNA-containing virus (eg, geminivirus) or an RNA-containing virus (eg, tobravirus). In certain embodiments, the steps of administration include delivery to a plant cell of a T-DNA comprising one or more polynucleotide sequences encoding a C2c1 or C2c3 effector protein and a guide RNA, where delivery is mediated by Agrobacterium. The nucleic acid sequence encoding components of the C2c1 or C2c3 effector protein system may be operably linked to a promoter, in particular a constitutive promoter (eg, the cauliflower mosaic virus 35S promoter) or a cell-specific or inducible promoter. In certain embodiments, the polynucleotide is introduced by microparticle bombardment. In certain embodiments, the method also includes screening the plant cell after the injection steps to determine if the repair template has been, i. the gene of interest has been successfully introduced. In certain embodiments, these methods include the step of regenerating a plant from a plant cell. In further embodiments, such methods include crossing a plant to produce offspring with the desired genetic properties.

[001000] In specific embodiments of the methods described above, disease-resistant crops are obtained by targeted mutation of disease susceptibility genes or genes encoding negative regulators (eg, the Mio gene) of plant defense genes. In a particular embodiment, herbicide-resistant crops are obtained by targeted substitution of specific nucleotides in plant genes, such as those encoding acetolactate synthase (ALS) and protoporphyrinogen oxidase (PPO). In specific embodiments, drought and salinity tolerant crops are obtained by targeted mutation in genes encoding negative regulators of abiotic stress tolerance, low amylose crops by targeted mutation of the Waxy gene, rice or other cereals with reduced rancidity by targeted mutation of the main lipase genes in the aleurone layer, etc. A more extensive list of endogenous genes encoding traits of interest is given below.

c) modulation of endogenous genes by CRISPR C2c1 or CRISPR C2c3 to obtain agricultural characteristics of interest

[001001] Also within the scope of the present invention are methods for modulating (ie, activating or suppressing) the expression of endogenous genes using the C2c1 or C2c3 protein described herein. Such methods use specific RNA sequence(s) that are inserted into the plant genome via a C2c1 or C2c3 complex. In more detail, a particular RNA sequence(s) bind to two or more adapter proteins (e.g., aptamers), whereby each adapter protein is associated with one or more functional domains, and where at least one of the one or more functional domains associated with the adapter protein, has one or more types of activity, including methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription termination factor activity, histone modification activity, DNA integration activity, RNA cleavage activity, DNA cleavage activity, or nucleic acid binding activity. Functional domains are used to modulate the expression of endogenous plant genes for desired characteristics. Typically, in these embodiments, the C2c1 or C2c3 effector protein has one or more mutations such that it has no more than 5% of the nuclease activity of the C2c1 or C2c3 effector protein without any of the mutations.

[001002] In specific embodiments, the methods described herein include the following steps: (a) introducing into the cell a CRISPR C2c1 or CRISPR C2c3 complex comprising a guide RNA containing a direct repeat and a guide sequence, where the guide sequence hybridizes to a sequence - a target that is endogenous to the plant cell; (b) introducing into the plant cell a C2c1 or C2c3 effector molecule that forms a complex with the guide RNA when the guide sequence hybridizes with the target sequence; and where either the guide RNA is modified to include a specific RNA sequence (aptamer) that binds to the functional domain and/or the C2c1 or C2c3 effector protein is modified to bind to the functional domain. In particular embodiments of the invention, the step of introducing may include delivering to the plant cell one or more polynucleotides encoding a (modified) C2c1 or C2c3 effector protein and a (modified) guide RNA. Details of the CRISPR C2c1 or C2c3 system components for use in these methods are described elsewhere in the present invention.

[001003] In specific embodiments, the polynucleotides are delivered to the cell via a DNA virus (eg, geminivirus) or RNA virus (eg, tobravirus). In specific embodiments, the introduction step comprises delivery to a plant cell of a T-DNA containing one or more polynucleotide sequences encoding a C2c1 or C2c3 effector protein and a guide RNA, where delivery is via Agrobacterium . A nucleic acid sequence encoding one or more components of the C2c1 or C2c3 CRISPR CRISPR system may be operably linked to a promoter, such as a constitutive promoter (eg, the 35S promoter of the cauliflower mosaic virus), or a cell-specific or inducible promoter. In specific embodiments, the implementation of the polynucleotide is introduced by bombardment with microparticles. In specific embodiments, the method further comprises screening the plant cell after administration to determine if the expression of the gene of interest has been modified. In specific embodiments, the methods include the step of regenerating a plant from a plant cell. In further embodiments, the methods include crossing plants to obtain a genetically desirable plant line. A more extensive list of endogenous genes encoding characteristics of interest is listed below.

Use of C2c1 or C2c3 to modify polyploid plants

[001004] Many plants are polyploids, which means that they contain duplicated copies of their genomes - sometimes as many as six, as in wheat. According to the present invention, methods using the C2c1 or C2c3 effector protein of the CRISPR system can be "multiplexed" to affect all copies of a gene or target dozens of genes at once. For example, in specific embodiments, the methods of the present invention are used to simultaneously introduce a loss-of-function mutation into various genes responsible for suppressing disease protection. In specific embodiments, the methods of this invention are used to simultaneously suppress the expression of the nucleic acid sequences TaMLO-A1, TaMLO-B1 and TaMLO-D1 in a wheat plant cell and regenerate a wheat plant from it to provide wheat resistance to powdery mildew (see also WO2015109752) .

Illustrative genes responsible for agronomic characteristics

[ 000006] As described herein above, in specific embodiments, the invention encompasses the use of a C2c1 or C2c3 CRISPR system, as described herein, to insert DNA of interest, including one or more expressible genes. In further specific embodiments, the invention encompasses methods and tools using the C2c1 or C2c3 system, as described herein, to partially or completely delete one or more plant-expressed genes. In other further specific embodiments, the invention encompasses methods and tools using the C2c1 or C2c3 system as described herein to effect modification of one or more plant-expressed genes by mutation, substitution, insertion, of one or more nucleotides. In other specific embodiments, the invention encompasses the use of a CRISPRC2c1 or C2c3 system, as described herein, to modify the expression of one or more plant-expressible genes by specifically modifying one or more regulatory elements that control the expression of said genes.

[000007] In specific embodiments, the invention encompasses methods that include introducing exogenous genes and/or targeting endogenous genes and their regulatory elements, such as those mentioned below:

[000008] 1. Genes conferring resistance to pests or diseases:

- Plant disease resistance genes. The plant can be transformed with cloned resistance genes to engineer plants that are resistant to specific pathogen strains. See, for example, Jones et al., Science 266:789 (1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum); Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistance to Pseudomonas syringae pv, tomato encodes a protein kinase); Mindrinos et al, Cell 78:1089 (1994) (Arabidopsmay be RSP2 gene for Pseudomonassyringae resistance).

- Genes conferring resistance to a pest such as the soybean cyst nematode. See, for example, PCT Application WO 96/30517, PCT Application WO 93/19181.

- Proteins of Bacillus thuringiensis see, for example, Geiser et al. Gene 48:109 (1986).

- Lectins, see, for example, VanDamme et al. Plant Molec. Biol. 24:25 (1994).

- Vitamin binding proteins such as avidin, see application PCTUS93/06487, which describes the use of avidin and avidin homologues as a larvicide against insect pests.

- Enzyme inhibitors such as protease or proteinase inhibitors or amylase inhibitors. See, for example, Abe et al., J. Biol. Chem. 262:16793 (1987), Huub et al. Plant Molec. Biol. 21:985 (1993), Sumitani et al., Biosci. Biotech. Biochem. 57:1243 (1993) and US Pat. No. 5,494,813.

- Insect-specific hormones or pheromones such as ecdysteroid or juvenile hormone, variants thereof, mimetics based on them, or antagonists or agonists thereof. See, for example, Nature 344:458 (1990).

- Insect-specific peptides or neuropeptides which, when expressed, disrupt the physiology of the affected pest. For example, Regan, J. Biol. Chem . 269:9 (1994) and Pratt et al., Biochem. Biophys. Res. Comm. 163:1243 (1989). See also U.S. Patent No. 5,266,317.

- Insect-specific poisons produced naturally by snakes, wasps or any other organism. For example, see Pang et al., Gene 116: 165 (1992).

- Enzymes responsible for the hyperaccumulation of a monoterpene, sesquiterpene, steroid, hydroxamic acid, phenylpropanoid derivative or other non-protein molecule with insecticidal activity.

- Enzymes involved in the modification, including post-translational modification, of a biologically active molecule, such as glycolytic enzyme, proteolytic enzyme, lipolytic enzyme, nuclease, cyclase, transaminase, esterase, hydrolase, phosphatase, kinase, phosphorylase, polymerase, elastase, chitinase and glucanase, natural or synthetic. See PCT application WO93/02197, Kramer et al., Insect Biochem. Molec. Biol. 23:691 (1993) and Kawalleck et al. , Plant Molec. Biol 21:673 (1993).

- Molecules that stimulate signal transduction. For example, see Botella et al., Plant Molec. Biol. 24:757 (1994), and Griess et al., Plant Physiol. 104:1467 (1994).

- Invasive viral proteins or complex toxin based on them. See Beachy et al., Ann. rev. Phytopathol. 28:451 (1990).

- Proteins that retard development produced naturally by a pathogen or parasite. See Lamb et al., Bio/Technology 10:1436 (1992) and Toubart. et al., Plant J. 2:3(57 (1992).

- Proteins that delay the development, produced in nature by the plant. For example, Logemann et al., Bio/Technology 10:305 (1992).

- In plants, pathogens are often host-specific. For example, some Fusarium species cause tomato wilt but only affect tomatoes, while other Fusarium species only affect wheat. Plants have innate (constitutive) and acquired (induced) phytoimmunity to protect against most pathogens. Mutations and recombination events in plant generations lead to genetic variation that leads to susceptibility, especially since pathogens reproduce at a higher frequency than plants. Plants may have non-host resistance, e.g. host and pathogen are incompatible, or partial resistance against all races of the pathogen, usually controlled by many genes, and/or also complete resistance to some races of pathogens but not other races. . Such resistance is usually controlled by several genes. Using the methods and components of the CRISPR-C2c1 or CRISPR-C2c3 system, there is now a new tool to induce specific, delayed and preventive mutations. Accordingly, the genome of sources of resistance genes can be analyzed and, in plants having the desired characteristics or properties, the method and components of the CRISPRC2c1 or C2c3 system can be used to induce increased expression of resistance genes. The present systems can do this with greater accuracy than previous mutagenic agents and therefore speed up and improve plant breeding programs.

[000009] 2. Genes involved in plant diseases, such as those listed in WO 2013046247:

- Rice diseases: Magnaporthe grisea, Cochliobolus miyabeanus, Rhizoctonia solani, Gibberella fujikuroi ; Wheat diseases: Erysiphe graminis, Fusarium graminearum, F. avenaceum, F. culmorum, Microdochium nivale, Puccinia striiformis, P. graminis, P. recondita, Micronectriella nivale, Typhula sp., Ustilago tritici, Tilletia caries, Pseudocercosporella herpotrichoides, Mycosphaerella graminicola, Stagonospora nodorum, Pyrenophora tritici-repentis ; Barley diseases: Erysiphe graminis, Fusarium graminearum, F. avenaceum, F. culmorum, Microdochium nivale, Puccinia striiformis, P. graminis, P. hordei, Ustilago nuda, Rhynchosporium secalis, Pyrenophora teres, Cochliobolus sativus, Pyrenophora graminea, Rhizoctonia solani ; Maize diseases: Ustilago maydis, Cochliobolus heterostrophus, Gloeocercospora sorghi, Puccinia polysora, Cercospora zeae-maydis, Rhizoctonia solani .

- Citrus diseases: Diaporthe citri, Elsinoe fawcetti, Penicillium digitatum, P. italicum, Phytophthora parasitica, Phytophthora citrophthora ; Apple diseases: Monilinia mali, Valsa ceratosperraa, Podosphaera leucotricha, Alternaria alternata apple pathotype, Venturia inaequalis, Colletotrichum acutatum, Phytophtora cactorum ;

- Pear diseases: Venturia nashicola, V. pirina, Alternaria alternata . Japanese pear pathotype, Gymnosporangium haraeanum, Phytophtora cactorum ;

- Peach diseases: Monilinia fructicola, Cladosporium carpophilum, Phomopsis sp .;

- Grape diseases: Elsinoe ampelina, Glomerella cingulata, Uninula necator, Phakopsora ampelopsidis, Guignardia bidwellii, Plasmopara viticola ;

- Persimmon diseases: Gloesporium kaki, Cercospora kaki, Mycosphaerela nawae ;

- Pumpkin diseases: Colletotrichum lagenarium, Sphaerotheca fuliginea, Mycosphaerella melonis, Fusarium oxysporum, Pseudoperonospora cubensis, Phytophthora sp., Pythium sp .;

- Tomato diseases: Alternaria solani, Cladosporium fulvum, Phytophthora infestans ;

- Eggplant diseases: Phomopsis vexans, Erysiphe cichoracearum ;

- Diseases of cruciferous vegetables: Alternaria japonica, Cercosporella brassicae, Plasmodiophora brassicae, Peronospora parasitica ;

- Diseases of onions: Puccinia allii, Peronospora destructor ;

- Soybean diseases: Cercospora kikuchii, Elsinoe glycines, Diaporthe phaseolorum var. sojae, Septoria glycines, Cercospora sojina, Phakopsora pachyrhizi, Phytophthora sojae, Rhizoctonia solani, Corynespora casiicola, Sclerotinia sclerotiorum ;

- Bean diseases: Colletrichum lindemthianum ;

- Peanut diseases: Cercospora personata, Cercospora arachidicola, Sclerotium rolfsii ;

- Diseases of peas: Erysiphe pisi ;

- Potato diseases: Alternaria solani, Phytophthora infestans, Phytophthora erythroseptica, Spongospora subterranea, f. sp. subterranea ;

- Strawberry diseases: Sphaerotheca humuli, Glomerella cingulata ;

- Diseases of tea: Exobasidium reticulatum, Elsinoe leucospila, Pestalotiopsis sp., Colletotrichum theae-sinensis ;

- Tobacco diseases: Alternaria longipes, Erysiphe cichoracearum, Colletotrichum tabacum, Peronospora. tabacina, Phytophthora nicotianae;

- Diseases of rapeseed: Sclerotinia sclerotiorum, Rhizoctonia solani ;

- Cotton diseases: Rhizoctonia solani ;

- Beet diseases: Cercospora beticola, Thanatephorus cucumeris, Aphanomyces cochlioides ;

- Rose diseases: Diplocarpon rosae, Sphaerotheca pannosa, Peronospora sparsa ;

- Diseases of chrysanthemums and the Asteraceae family: Bremia lactuca, Septoria chrysanthemi-indici, Puccinia horiana ;

- Diseases of various plants: Pythium. aphanidermatum, Pythium debarianum, Pythium graminicola, Pythium irregulare, Pythium ultimum, Botrytis cinerea, Sclerotinia sclerotiorum ;

- Radish diseases: Alternaria brassicicola ;

- Zoysia diseases: Sclerotinia homeocarpa, Rhizoctonia solani ;

- Diseases of bananas: Mycosphaerella fijiensis, Mycosphaerella musicola ;

- Sunflower diseases: Plasmopara halstedii ;

- Seed or early growth diseases of various plants caused by Aspergillus spp., Penicillium spp., Fusarium spp., Gibberella spp., Tricoderma spp., Thielaviopsis spp., Rhizopus spp., Mucor spp., Corticium spp., Rhoma spp. ., Rhizoctonia spp., Diplodia. spp ., etc.;

- Viral diseases of various plants mediated by Polymixa spp., Olpidium spp ., etc.

[000010] 3. Examples of genes conferring herbicide resistance;

- Resistance to herbicides that inhibit growth points or meristem, such as imidazolinone or sulfonylurea, eg Lee et al., EMBOJ. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449 (1990), respectively.

- Glyphosate resistance (resistance is conferred, for example, by mutant 5-enolpyruvylshikimate-3-phosphate synthase (EPSP), aroA and glyphosate acetyltransferase (GAT) genes, respectively), or resistance to other phosphono group compounds such as glufosinate (phosphinothricin acetyltransferase (PAT) genes ) from Streptomyces species, including Streptomyces hygroscopicus and Streptomyces viridichromogenes ), and to pyridineoxy- or phenoxy-propionic acids and cyclohexenes via genes encoding an ACCase inhibitor. See, for example, US Patent No. 4,940,835 and US Patent No. 6,248,876, US Patent No. 4,769,061, EP No. 0,333,033 and US Patent No. 4,975,374. See also No. 0242246, DeGreef et al., Bio/Technology 7:61 (1989 ), Marshall et al., Theor. Appl. Genet. 83:435 (1992), WO 2005012515 Castle et. al. and W02005107437.

- Resistance to photosynthesis-inhibiting herbicides such as triazine (psbA and .gs+ genes) or benzonitrile (gennitrilases), glutathione S-transferase in Przibila et al., Plant Cell 3:169 (1991), US Pat. No. 4,810,648 and Hayes et al., Biochem. J. 285: 173 (1992).

- Genes encoding herbicide detoxifying enzymes or mutant glutamine synthetase enzyme resistant to inhibition, such as in US Patent Application Serial No. 11/760602. Otherwise, the detoxifying enzyme is an enzyme encoding a phosphinothricin acetyltransferase (such as the bar or pat protein from Streptomyces species). Phosphinothricin acetyltransferases are described, for example, in US Pat. Nos. 5,561,236; 5648477, 5646024; 5273894; 5637489; 5276268; 5739082; 5908810 and 7112665.

- Hydroxyphenylpyruvate dioxygenase (HPPD) inhibitors, i.e. naturally occurring HPPD resistant enzymes or genes encoding a mutated or chimeric HPPD enzyme, as described in WO 96/38567, WO 99/24585, and WO 99/24586, WO 2009/144079, WO 2002/046387 or US Pat. No. 6,768,044.

[000011] 4. Examples of genes involved in resistance to abiotic stress:

- A transgene capable of reducing the expression and/or activity of the poly(ADP-ribose) polymerase (PARP) gene in plant cells or plants, as described in WO00/04173 or, WO/2006/045633.

- Transgenes capable of reducing the expression and/or activity of plant genes or plant cells encoding PARG, as described, for example; in WO2004/090140.

- Transgenes encoding a plant-functional nicotinamide adenine dinucleotide rescue pathway enzyme, including nicotinamidase, nicotinate phosphoribosyltransferase, nicotinic acid mononucleotide adenyltransferase, nicotinamide adenine dinucleotide synthetase or nicotinamide phosphoribosyl transferase, as described, for example, in EP 04077624.7, WO2006/13 EP07/002433, EP 1999263 or WO2007/107326.

- Enzymes involved in the biosynthesis of carbohydrates include those described, for example, in EP 0571427, WO 95/04826, EP 0719338, WO 96/15248, WO 96/19581, WO 96/27674, WO 97/11188, WO 97/ WO 97/32985, WO 97/42328, WO 97/44472, WO 97/45545, WO 98/27212, WO 98/40503, WO99/58688, WO 99/58690, WO 99/58654, WO 00/08184 WO 00/08185 WO 00/08175 WO 00/28052 WO 00/77229 WO 01/12782 WO 01/12826 WO 02/101059 WO 03/071860 WO 2004/056999 , Wo 2005/030941, Wo 2005/095632, Wo 2005/095617, Wo 2005/095619, Wo 2005/095618, Wo 2005/123927, Wo 2006/018319, Wo 2006/103107, Wo 2006/108702, Wo 2007/009823 , Wo 00/22140, Wo 2006/063862, Wo 2006/072603, Wo 02/034923, EP 06090134.5, EP 06090228.5, EP 06090227.7, EP 0709000.1, EP 0709000.7, Wo 01/14569, Wo 02/79410, Wo 02/79410, Wo 02/79410, Wo 02/79410, Wo 02/79410, Wo 02/79410, Wo 02/79410, Wo 02/79410, Wo 02/79410, Wo 02/79410, Wo 02/79410 , WO 2004/078983, WO 01/19975, WO 95/26407, WO 96/34968, WO 98/20145, WO 99/12950, WO 99/66050, WO 99/53072, US Pat. , WO 98/22604, WO 98/32326, WO 01/98509, WO 01/98509, WO 2005/00235 9, US Pat. No. 5,824,790, US Pat. No. 6,013,861, WO94/04693, WO94/09144, WO94/11520, WO95/35026 or WO97/20936 or enzymes involved in the synthesis of polyfructans, especially inulin and levan types, as described in EP 0663956 , WO 96/01904, WO 96/21023, WO 98/39460, and WO 99/24593, the synthesis of alpha-1,4-glucans as described in WO 95/31553, US 2002031826, US Pat. WO 97/47806, WO 97/47807, WO 97/47808 and WO 00/14249, synthesis of alpha-1,6-branched alpha-1,4-glucans as described in WO 00/73422, synthesis of alternan as described, for example, in WO 00/47727, WO 00/73422, EP 06077301.7, US Pat. 039316, JP 2006304779 and WO 2005/012529.

- Genes that increase resistance to drought. For example, WO 2013122472 describes that the absence or reduced level of a functional ubiquitin ligase (UPL) protein, more specifically UPL3, results in a reduced water requirement or improved drought tolerance of said plant. Other examples of transgenic plants with increased drought tolerance are described in, for example, US2009/0144850, US2007/0266453, and WO2002/083911. US2009/0144850 describes a plant having a drought tolerance phenotype due to altered expression of the DR02 nucleic acid. US2007/0266453 describes a plant having a drought tolerant phenotype due to altered expression of the DR03 nucleic acid and WO 2002/083911 describes a plant having increased drought tolerance due to reduced activity of the ABC transporter that is expressed in stomatal guard cells. Another example is the work of Kasuga et al. (1999), who described overexpression of complementary DNA (cDNA) encoding DREB1A in transgenic plants, which upregulated the expression of many stress tolerance genes under normal growing conditions and resulted in improved drought, salinity, and freeze tolerance. However, DREB1A expression also resulted in severe growth retardation under normal growing conditions (Kasuga (1999) NatBiotechnol 17(3) 287-291).

[000012] In the following specific embodiments, cereals can be improved by influencing specific plant characteristics. This can be done, for example, by developing pesticide-resistant plants, improving plant disease resistance, improving plant resistance to insects and nematodes, improving plant resistance to parasitic weeds, improving plant drought tolerance, improving plant nutritional value, improving plant resistance to stress, avoiding self-pollination, increasing the digestibility of plant food biomass, increasing grain yield, etc. Several specific non-limiting examples are provided herein below.

[001005] In addition to targeted mutation of single genes, CRISPR complexes with C2c1 or C2c3 can be designed for targeted mutation of multiple genes, deletion of chromosomal fragments, site-specific integration of transgenes, site-directed mutagenesis in vivo , and exact replacement gene or allele in plants. Therefore, the methods described herein are widely used for gene discovery and screening, mutational and cisgenic selection, and hybrid selection. These applications facilitate the production of a new generation of genetically modified crops with various improved agronomic characteristics such as herbicide tolerance, disease resistance, abiotic stress tolerance, high yield and superior quality.

Using the C2c1 or C2c3 Gene to Generate Male Sterile Plants

[001006] Hybrid plants generally have advantageous agronomic characteristics compared to inbred plants. However, for self-pollinating plants, creating hybrids can be difficult. In various types of plants, genes have been identified that are important for plant fertility, more specifically male fertility. For example, in maize, at least two genes have been determined to be important for fertility (Amitabh Mohanty International Conference on New Plant Breeding Molecular Technologies Technology Development And Regulation, Oct 9-10, 2014, Jaipur, India; Svitashev et al. Plant Physiol. 2015 Oct; 1.69(2):93 N45; Djukanovic et al. Plant J. 2013 Dee;76(5):888-99). The methods described herein can be used to target genes required for male fertility in order to produce male sterile plants that easily cross to form hybrids. In specific embodiments, the C2c1 or C2c3 CRISPRc system described herein is used to target mutagenesis of a cytochrome P450-like (MS26) or meganuclease (MS45) gene, thereby conferring male sterility on maize plants. Corn plants genetically modified in this way can be used in hybrid breeding programs.

Increasing the fertile stage in plants

[001007] In specific embodiments, the methods described herein are used to prolong the fertile stage of a plant, such as rice. For example, a rice fertile stage gene, such as Ehd3, can be targeted to introduce a mutation in the gene and seedlings can be selected for long-term fertile plant regeneration (as described in CN 104004782).

Use of C2c1 or C2c3 to create genetic variation in a culture of interest

[001008] The availability of wild germplasm and genetic variation in cereals is key to cereal improvement programs, but the diversity of cereal germplasm available is limited. The present invention relates to methods for generating genetic diversity in a germplasm of interest. This method of using the C2c1 or C2c3 CRISPR systems involves introducing into plant cells a library of guide RNAs targeted at various locations in the plant genome, along with the C2c1 or C2c3 effector protein. In this way, a collection of point mutations and gene knockouts across the scale of genome size can be created. In specific embodiments, the methods include creating a plant part or plant from the cells thus obtained and screening the cells for a trait of interest. Target genes can include both coding and non-coding regions. In specific embodiments, the trait is stress tolerance and the method is a method of creating stress resistant crop varieties.

Use of C2c1 or C2c3 to influence fruit ripening

[001009] Ripening is a normal phase in the ripening process of fruits and vegetables. Only a few days after the start of ripening, the fruit or vegetable becomes inedible. This process brings significant losses to farmers and consumers. In specific embodiments, the implementation of the methods of the present invention are used to reduce the synthesis of ethylene. This is provided in one or more of the following ways: a. suppression of ACC synthase gene expression. ACC (1-aminocyclopropane -l-carboxylic acid) synthase is the enzyme responsible for converting S-adenosylmethionine (SAM) to ACC; penultimate step in ethylene biosynthesis. Enzyme expression is hindered when an antisense ("mirror image") or truncated copy of the synthesized gene is inserted into the plant genome, b. Insertion of the ACC deaminase gene. The gene encoding the enzyme is derived from Pseudomonas chlororaphis , a common non-pathogenic soil bacterium. This enzyme converts ACC to another compound, thus reducing the amount of ACC available for ethylene synthesis; c. Insertion of the SAM hydrolase gene. This approach is similar to that with ACC deaminase, where ethylene synthesis stops when the amount of the metabolite that precedes it decreases; in this case, SAM is converted to homoserine. The gene encoding the enzyme is derived from the bacteriophage T3 E. coli, and d. Suppression of expression of the ACC oxidase gene. ACC oxidase is the enzyme that catalyzes the oxidation of ACC to ethylene, the last step in the ethylene biosynthetic pathway. Using the methods described herein, negative feedback regulation of the ACC oxidase gene leads to suppression of ethylene synthesis, thus delaying fruit ripening. In specific embodiments, in addition to or alternatively to the modifications described above, the methods described herein are used to modify ethylene receptors to interfere with fruit-derived ethylene signals. In specific embodiments, the expression of the ETR1 gene encoding the ethylene-binding protein is modified, more specifically, suppressed. In specific embodiments, in addition to or alternatively to the modifications described above, the methods described herein are used to modify the expression of a gene encoding polygalacturonase (PG), which is the enzyme responsible for the breakdown of pectin, a substance that maintains the integrity of the cell wall of a plant cell. The destruction of pectin occurs at the beginning of the ripening process, which leads to softening of the fruit. Accordingly, in specific embodiments, the methods described herein are used to mutate the PG gene or suppress the activation of the PG gene to reduce the amount of PG enzyme synthesized, thereby slowing down the degradation of pectin.

[001010] Thus, in specific embodiments, the methods include using a C2c1 or C2c3 CRISPR system to effect one or more plant cell genome modifications such as described above and regenerate a plant from those cells. In specific embodiments, the plant is a tomato.

Increase plant storage life

[001011] In specific embodiments, the methods of the invention are used to modify genes involved in the production of compounds that affect the shelf life of a plant or plant part. In more detail, a modification is made to a gene that prevents the accumulation of reducing sugars in potato tubers. After high temperature processing, these reducing sugars react with free amino acids, resulting in brown products with a bitter taste and increased levels of acrylamide, which is a potential carcinogen. In specific embodiments, the methods described herein are used to reduce or interfere with the expression of the vacuolar invertase (VInv) gene encoding a protein that breaks down sucrose to glucose and fructose (Clasen et al. DOI: 10 1111/pbi. 12370) .

Using the CRISPR C2c1 or C2c3 system to deliver value addition

[00 0013] In specific embodiments, the implementation of the C2c1 or C2c3 CRISPR system is used to produce agricultural crops with improved nutritional properties. In specific embodiments, the methods described herein are adapted to produce "functional products", i.e. modified foods or their ingredients that may be more beneficial to health. In addition to traditional nutrients, they contain "nutraceuticals", i.e. a substance that can be considered a food or part of a food that provides health benefits, including the prevention and treatment of disease. In specific embodiments, nutraceuticals are useful in the prevention and/or treatment of one or more types of cancer, diabetes, cardiovascular disease, and hypertension.

[000014] Examples of nutrient-enhanced cereals include (Newell-McGloughlin, Plant Physiology , July 2008, Vol. 147, pp. 939-953):

- modified protein quality, content and/or amino acid composition, which have been described for Bahia (Luciani et al. 2005, Florida Genetics Conference Poster), Canola (Roesler et al., 1997, Plant Physiol 113 75-81), Maize (Cromwell et al, 1967, 1969 JAnim Sci 26 1325-1331, O'Quin et al 2000 JAnim Sci 78 2144-2149, Yang et al 2002, Transgenic Res 11 11-20, Young et al 2004, Plant J 38 910 -922), potato (Yu J and Ao, 1997 Acta Bot Sin 39 329-334; Chakraborty et al. 2000, Proc Natl Acad Sci USA 97 3724-3729; Li et al., 2001 Chin Sci Bull 46 482-484) , rice (Katsube et al. 1999, Plant Physiol 120 1063-4074), soybean (Dinkins et al. 2001, Rapp 2002, In Vitro Cell Dev Biol Plant 37 742-747), sweet potato (Egnin. and Prakash 1997, In Vitro Cell Dev Biol 33 52A).

- content of essential amino acids, which has been described for canola (Falco et al. 1995, Bio/Technology 13 577-582), lupine (White et al. 2001, J Sci Food Agric 81 147-154), corn (Lai and Messing, 2002, Agbios 2008 GM crop database (March 11, 2008)), potato (Zeh et al. 2001, Plant Physiol 127 792-802), sorghum (Zhao et al. 2003, Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 413 -416), Soy (Falco et al. 1995 Bio/Technology 13 577-582; Galdi et al. 2002 Crit Rev Plant Sci 21:167-204).

- Oils and fatty acids such as for canola (Dehesh et al. (1996) Plant J 9 167-172 [PubMed]; Del Vecchio (1996) INFORM International News on Fats, Oils and Related Materials 7 230-243; Roesler et al (1997) Plant Physiol 113 75 81 [PMC free article] [PubMed] Froman and Ursin (2002, 2003) Abstracts of Papers of The American Chemical Society 223 U35 James et al (2003) Am J Clin Nutr 77 1140-1145 [PubMed]; Agbios (2008, supra), cotton (Chapman et al. (2001). J Am. OH Chem. Soc 78 941-947; Liu et al. (2002) J Am Coll Nutr 21 205S- 21 IS [PubMed] O'Neill (2007) Australian Life Scientist http://www.biotechnews.com.au/index.php/id;866694817;fp;4;fpid;2 (June 17, 2008), Flax (Abbadi et al., 2004, Plant Cell 16: 2734-2748), corn (Young et al., 2004, Plant J 38 910-922), oil palm (Jalani et al. 1997, J Am Oil Chem Soc 74 1451 1455; Parveez, 2003, AgBiotechNet 113 1-8), Rice (Anai et al., 2003, Plant Cell Rep 21 988-992), Soybean (Reddy and Thomas, 1996, Nat Biotechnol 14 639-642; K inney and Kwolton, T9Q8. Blackie Academic and Professional, London, pp 193-213), sunflower (Arcadia, Biosciences 2008).

- Carbohydrates such as fructans described for chicory (Smeekens (1997) Trends Plant Sci 2 286-287, Sprenger et al. (1997) FEBS Lett 400 355-358, Sévenier et al. (1998) Nat Biotechnol 16 843-846 ), maize (Caimi et al. (1996) Plant Physiol 110 355-363), potato (Hellwege et al., 1997 Plant J 12 1057-1065), sugar beet (Smeekens et al. 1997, above); inulin as described for potatoes (Hellewege et al. 2000, Proc Natl Acad Sci USA 97 8699-8704), starch as described for rice (Schwall et al. (2000) Nat Biotechnol 18 551-554, Chiang et al. ( 2005) Mol Breed 15 125-143).

- Vitamins and carotenoids as described for canola (Shintani and DellaPenna (1998) Science 282 2098-2100), maize (Rocheford et al. (2002) J Am Coll Nutr 21 191S-198S, Cahoon et al. (2003) Nat Biotechnol 21 1082-1087, Chen et al. (2003) Proc Natl Acad Sci USA 100 3525-3530), mustard seed (Shewmaker et al. (1999) Plant J 20 401-412), potato (Ducreux et al., 2005, J Exp Bot 56 81-89), rice (Ye et al. (2000) Science 287 303-305), strawberry (Agius et al. (2003), Nat Biotechnol 21 177-181), tomato (Rosati et al. ( 2000) Plant J 24 413-419, Fraser et al (2001) J Sci Food Agric 81 822-827, Mehta et al (2002) Nat Biotechnol 20 613-618, Diaz de la Garza et al (2004) Proc Natl Acad Sci USA 101 13720-13725, Enfissi et al (2005) Plant Biotechnol J 3 17-27, DellaPenna (2007) Proc Natl Acad Sci USA 104 3675-3676).

- Functional secondary metabolites such as those described for apple (stilbenes, Szankowski et al. (2003) Plant Cell Rep 22: 141-149), alfalfa (resveratrol, Hipskind and Paiva (2000) Mol Plant Microbe Interact 13 551-562), kiwi (resveratrol, Kobayashi et al. (2000) Plant Cell Rep 19 904-910), corn and soy (flavonoids, Yu et al. (2000) Plant Physiol 124 781-794), potato (anthocyanins, alkaloids and glycoids, Lukaszewicz et al. (2004) J Agric Food Chem. 52 1526-1533), rice (flavonoids and resveratrol, Stark-Lorenzen et al. (1997) Plant Cell Rep 16 668-673, Shin et al. (2006) Plant Biotechnol J 4 303-315), tomato (resveratrol, chlorogenic acid, flavonoids, stilbenes, Rosati. et al. (2000) above, Muir et al. (2001) Nature 19 470-474, Niggeweg et al. (2004) Nat Biotechnol 22 746-754, Giovinazzo et al (2005) Plant Biotechnol J 3 57-69), wheat (caffeic and ferulic acids, resveratrol, United Press International (2002)); and

- Minerals such as those described for alfalfa (phytase, Austin-Phillips et al. (1999) http://www.molecularfarming.com/nonmedical.html), lettuce (iron, Goto et al. (2000) TheorApplGenet 100 658-664), rice (iron, Lucca et al. (2002) JAmCollNutr 21 184S-190S), corn, soy and wheat (phytase, Drakakaki et al. (2005) Plant Mol Biol 59 869-880, Denbow et al (1998) Poult Sci 77 878-881, Brinch-Pedersen et al (2000) Mol Breed 6 195-206).

[000015] In specific embodiments, the value enhancement feature is related to the intended health benefit of the compounds present in the plant. For example, in specific embodiments, a value-added crop is obtained using the methods of the invention to modify or induce/increase the synthesis of one or more of the following compounds:

- Carotenoids such as α-carotene, found in carrots, which neutralizes free radicals that can damage cells, or β-carotene, found in various fruits and vegetables, which neutralizes free radicals

- Lutein, found in green vegetables, which contributes to healthy vision

- Lycopene, found in tomatoes and tomato products, which is thought to reduce the risk of prostate cancer

- Zeaxanthin, found in citrus fruits and corn, which contributes to healthy vision

- Dietary fiber, such as insoluble fiber found in wheat bran, which may reduce the risk of breast and/or colon cancer, and β-glucans found in oats, soluble fiber found in Psylium and whole grains, which may reduce the risk of cardiovascular disease (CVD)

- Fatty acids such as ω-3 fatty acids, which may reduce the risk of cardiovascular disease and improve mental and visual function, CLA, which may improve body shape, reduce the risk of certain cancers, and GLA, which may reduce risk of inflammation in cancer and cardiovascular disease (CVD), may improve body shape

- Flavonoids such as hydroxycinnamates found in wheat may have antioxidant activity, reduce the risk of degenerative diseases, flavonols, catechins and tannins found in fruits and vegetables neutralize free radicals and may reduce the risk of cancer

- Glucosinolates, indoles, isothiocyanates such as sulforaphane found in cruciferous vegetables (broccoli, kale), horseradish, which neutralize free radicals, may reduce cancer risk

- Phenols, such as stilbenes found in grapes, may reduce the risk of degenerative diseases, heart disease, and cancer, may have a positive effect on life expectancy, caffeic acid and ferulic acid, found in vegetables and citrus fruits, have similar antioxidant activity, may reduce risk of degenerative disease, heart disease, and eye disease and epicatechin found in cocoa has a similar antioxidant activity, may reduce the risk of degenerative disease and heart disease

- Plant stanols/sterols found in corn, soy, wheat and wood oils, which may reduce the risk of coronary heart disease by lowering blood cholesterol

- Fructans, inulins, fructooligosaccharides found in Jerusalem artichoke, shallot, onion powder, which may improve gastrointestinal health

- Saponins found in soy may lower LDL cholesterol

Soy protein found in soy may reduce the risk of heart disease

- Phytoestrogens such as the isoflavones found in soy may reduce menopausal symptoms such as hot flashes, may reduce the risk of osteoporosis and cardiovascular disease (CVD), and lignans found in flax, rye and vegetables may protect against heart disease and some cases of cancer, can lower LDL cholesterol, total cholesterol.

- Sulfides and thiols, such as diallyl sulfide found in onions, garlic, olives, leeks, and squalene and allyl methyl trisulfide, dithiolthiones found in cruciferous vegetables, which can lower LDL cholesterol levels, help support a healthy immune system

- Tannins, such as proanthocyanidins found in cranberries, cocoa, may improve urinary tract health, reduce the risk of developing cardiovascular disease (CVD) and high blood pressure

- etc.

[0016] In addition, the methods of the invention also include modification of protein/starch functionality, shelf life, taste/aesthetic characteristics, fiber and allergen quality, antinutrients, and toxin reduction features.

[000017] Accordingly, the invention encompasses methods for the production of plants with increased nutritional value, which methods include introducing into the plant cell a gene encoding an enzyme involved in the synthesis of a component of increased nutritional value, using the C2c1 or C2c3 CRISPR system, as described in the present description, and regenerating a plant from said plant cell, wherein said plant is characterized by increased expression of said increased nutritional value component. In specific embodiments, the CRISPRC2c1 or C2c3 system is used to modify the endogenous synthesis of these compounds indirectly, such as modifying one or more transcription factors that control the metabolism of the compound. Methods for introducing a gene of interest into a plant cell and/or modifying an endogenous gene using the CRISPRC2c1 or C2c3 system are described herein above.

[000018] Some specific examples of modifications in plants that have been modified to acquire an increased value trait include: plants with a modified fatty acid metabolism, for example, by transforming a plant with a stearyl ACP desaturase antisense gene to increase the plant's stearic acid content. See Knultzon et al., Proc. Natl. Acad. sci. USA 89:2624 (1992). Another example involves the reduction of phytate, for example by cloning and then re-introducing DNA associated with a single allele, which may be responsible for maize mutants characterized by low levels of phytic acid. See Raboy et al, Maydica 35:383 (1990).

[001012] Similarly, gene expression in maize (Zea mays) Tfs C1 and R, which regulate the production of flavonoids in the aleurone layers of corn under the control of a strong promoter, led to a high rate of accumulation of anthocyanins in Arabidopsis (Arabidopsisthaliana), presumably through activation of the entire pathway (Bruce et al., 2000,PlantCell 12:65-80). DellaPenna (Welsch et al., 2007)AnnaRevPlantBiol 57: 711-738) found that Tf RAP2.2 and its interacting SINAT2 increased carotenogenesis in Arabidopsis leaves. Expression of Tf Dof1 induced regulation of genes coding for carbon backbone synthesis, a marked increase in amino acid content, and a reduction in Glc levels in transgenic Arabidopsis (Yanagisawa, 2004PlansCellPhysiol 45: 386-391), and DOF Tf AtDof1.1 (OBP2) intensified all steps of the glucosinolate biosynthetic pathway in Arabidopsis (Skirycz et al., 2006Plant J47:10-24).

Reduction of allergen content in plants

[001013] In specific embodiments, the methods described herein are used to produce plants with reduced levels of allergens, making them safer for the consumer. In specific embodiments, the methods include modifying the expression of one or more genes responsible for the production of plant allergens. For example, in specific embodiments, the methods include down-regulating the expression of the Lolp5 gene in a plant cell, such as a ryegrass plant cell and a plant regenerating from it, to reduce pollen allergenicity of said plant (Bhalla et al. 1999, Proc. Natl. Acad. Sci. USA Vol.96: 1.1.676-11680).

[001014] Peanut allergy and legume allergy in general are a real and serious medical problem. The C2c1 or C2c3 effector protein system of the present invention can be used to identify and then edit or suppress genes encoding the allergenic proteins of such legumes. Without being limited to such genes and proteins, Nicolaou et al. identified allergenic proteins in peanuts, soybeans, lentils, peas, lupins, green beans and mung beans. See, Nicolaou et al., Current Opinion in Allergy and Clinical Immunology 2011; 11(3):222).

Screening methods for endogenous genes of interest

[001015] The methods described herein further allow for the identification of valuable genes encoding enzymes involved in the production of nutritionally enhanced components, or in general genes affecting agronomic traits of interest, across species, phyla and the entire plant kingdom. By selectively targeting, for example, genes encoding metabolic pathway enzymes in plants using the CRISPRC2c1 or C2c3 system as described herein, the genes responsible for certain nutritional aspects of the plant can be determined. Similarly, by selectively targeting genes that may affect a desired agronomic characteristic, the corresponding genes can be identified. Accordingly, the present invention encompasses methods for screening genes encoding enzymes involved in the synthesis of compounds with particular nutritional value and/or agronomic traits.

The following applications of the CRISPR C2c1 or C2c3 system in plants and yeasts

Use of the CRISPR C2c1 or C2c3 system in biofuel production

[001016] The term "biofuel" in the present description refers to an alternative fuel derived from plants and other plant resources. Renewable biofuels can be extracted from organic matter, the energy of which was stored during the process of carbon fixation or obtained through the use or conversion of biomass. This biomass can be used as a biofuel or can be converted into usable energy-containing substances through thermal, chemical or biochemical processing. Such processing will make it possible to obtain fuel in solid, liquid or gaseous form. There are two types of biofuels: bioethanol and biodiesel. Bioethanol is produced mainly by the fermentation of carbohydrates - cellulose (starch), obtained mainly from corn and sugar cane. Biodiesel, on the other hand, is mainly produced from oilseeds such as rapeseed, oil palm and soybeans. Biofuels are mainly used as fuel for vehicles.

Improving the properties of a plant for the production of biofuels

[001017] In specific embodiments, methods using the C2c1 or C2c3 CRISPR system as described herein are used to modify cell wall properties to facilitate access of key hydrolysis agents and more efficiently release sugars used for fermentation. In some embodiments, the biosynthesis of cellulose and/or lignin can be modified. Cellulose is the main component of the cell wall. The biosynthesis of cellulose and lignin are regulated in concert. By reducing the proportion of lignin in a plant, the proportion of cellulose can be increased. In some embodiments, the methods described herein are used to reduce the level of lignin biosynthesis in a plant in order to increase the proportion of fermentable carbohydrates. More specifically, the methods described herein are used to reduce the activity of at least a first lignin biosynthesis gene selected from the group consisting of 4-coumarate-3-hydroxylase (C3H), phenylalanine ammonium lyase (PAL), cinnamate-4-hydroxylase (C4H ), hydroxlicinnamoylttransferase (HCT), caffeic acid O-methyltransferase (COMT), coffeoyl-CoA-3-O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamyl alcohol dehydrogenase (CAD), cinnamoyl-CoA reductase (CCR), 4-coumaroate-CoA ligase (4CL), monolignol-lignin-specific glycosyltransferase and aldehyde dehydrogenase (ALDH), as described in WO 2008064289 A2.

[001018] In some embodiments, the methods described herein are used to produce plant biomass that produces lower levels of acetic acid upon fermentation (see also WO 2010096488). More specifically, the methods described herein are used to mutate CasIL homologues to reduce polysaccharide acetylation.

Yeast modification for biofuel production

[001019] In specific embodiments of the invention, the proposed enzyme C2c1 or C2c3 is used for the production of bioethanol in recombinant microorganisms. For example, C2c1 or C2c3 enzymes can be used to produce microorganisms such as yeast, to produce biofuels or biopolymers from fermentable sugars, and possibly to decompose lignocellulose derived from plants extracted from agricultural waste as a source of fermentable sugars. More specifically, the invention relates to methods in which a C2c1 or C2c3 CRISPR complex is used to introduce into microorganisms foreign genes required for biofuel synthesis and/or alter endogenous genes that may interfere with biofuel synthesis. More specifically, such methods involve introducing into a microorganism, such as yeast, one or more nucleotide sequences encoding enzymes involved in the conversion of pyruvate to ethanol or other target product. In certain embodiments, such methods provide stimulation of the expression of one or more enzymes that allow microorganisms to degrade cellulose, such as cellulase. In other further embodiments, an RNA-targeted CRISPR complex is used to suppress endogenous metabolic processes that compete with the biofuel production process.

[001020] Accordingly, in more specific embodiments, the methods described herein are used to modify a microorganism as follows:

[001021] introducing at least one heterologous nucleic acid or increasing the expression of at least one endogenous nucleic acid encoding a plant cell wall degrading enzyme such that said microorganism is capable of expressing said nucleic acid and of synthesizing and secreting said cell wall degrading enzyme. plant wall;

[001022] introducing at least one heterologous nucleic acid or increasing the expression of at least one endogenous nucleic acid encoding an enzyme that converts pyruvate to acetaldehyde, optionally combined with at least one heterologous nucleic acid encoding an enzyme that converts acetaldehyde to ethanol such that said host cell is capable of expressing said nucleic acid; and/or

[001023] modification of at least one nucleic acid encoding an enzyme in a metabolic pathway in said host cell, wherein said pathway produces a metabolite other than acetaldehyde from pyruvate or ethanol from acetaldehyde, and wherein said modification results in a decrease in the production of said metabolite, or administration by at least one nucleic acid encoding an inhibitor of said enzyme.

Modification of algae and plants to produce vegetable oils or biofuels

[1024] Transgenic algae or other plants, such as rapeseed, may be of particular use in the production of vegetable oils or biofuels, such as alcohols (especially methanol and ethanol). They can be purposefully designed to express or overexpress large volumes of oils or alcohols for use in petroleum or biofuel industries.

[001025] According to specific embodiments of the invention, the C2c1 or C2c3 CRISPR system is used to produce lipid-rich diatoms useful in biofuel production.

[001026] In specific embodiments, specific modifications are contemplated for genes that are involved in modifying the amount of lipids and/or the quality of lipids produced by the algal cell. Examples of genes encoding enzymes involved in fatty acid synthesis pathways can encode proteins having, for example, acetyl-CoA carboxylase, fatty acid synthase, 3-ketoacyl-acyl transfer protein synthase III, glycerol-3-phosphate dehydrogenase ( G3PDH), enoyl acyl transfer protein reductase, glycerol-3-phosphate acyltransferase, lysophosphatide acyltransferase or diacylglycerol acyltransferase, phospholipid:diacylglycerol acyltransferase, phosphatidate phosphatase, fatty acid thioesterase, such palmitoyl protein thioesterase or enzymatic activity for malic acid. In further embodiments of the invention, it is envisaged to create diatoms in which the accumulation of lipids is increased. This can be achieved by targeting genes that reduce lipid catabolism. Of particular interest for use in the methods of the present invention are genes involved in the activation of both triacylglycerol and free fatty acids, as well as genes directly involved in fatty acid β-oxidation, such as acyl-CoA synthetase, 3-ketoacyl-CoA. -thiolase, activity of acyl-CoA oxidase and phosphoglucomutase. The CRISPR C2c1 or C2c3 system and methods described herein can be used to specifically activate such genes in diatoms to increase their lipid content.

[001027] Organisms such as yeast and microalgae are widely used in synthetic biology. Stovicek et al. (Metab.Eng. Comm., 2015; 2:13) describe genome editing of commercial yeasts, such as Saccharomyces cerevisae , to efficiently create resistant strains for industrial production. Stovicek used a yeast codon-optimized CRISPR-Cas9 system to simultaneously produce both an endogenous gene allele break and a heterologous gene knockin. Cas9 and gRNA were expressed at genomic or episomal positions on the vector. The authors also showed that gene disruption efficiency could be improved by optimizing Cas9 and gRNA expression levels. Hlavova et al. (Biotechnol, Adv., 2015) have described the evolution of microalgae species or strains using methods such as CRISPR to target nuclear and chloroplast genes for insertional mutagenesis and screening. The same plasmids and vectors can be applied to the C2c1 or C2c3 systems of the present invention.

[001028] US 8945839 describes a method for modifying an engineered microalgae species ( Chlamydomonas reinhardtii cells) using Cas9. Using a similar technique, methods using the C2c1 or C2c3 CRISPR system described herein can be applied to species of the genus Chlamydomonas and other algae. In certain embodiments, the C2c1 or C2c3 protein and guide RNA(s) are introduced into the algae in which they are expressed using a C2c1 or C2c3 protein expression vector under the control of a constitutive promoter such as Hsp70A-Rbc S2 or beta-2-tubulin. The guide RNA can optionally be delivered using a vector containing the T7 promoter. Alternatively, C2c1 or C2c3 mRNA and an in vitro transcribed guide RNA can be delivered to algal cells. Electroporation protocols are available to the skilled person, including the standard recommended protocol from the GeneArt Chlamydomonas Engineering kit.

Use of C2c1 or C2c3 to create microorganisms capable of producing fatty acids

[001029] In specific embodiments, the methods of the invention are used to create genetically modified microorganisms capable of producing fatty acid esters such as fatty acid methyl esters ("FAME") and fatty acid ethyl esters ("FAEE").

[001030] Generally, host cells can be engineered to produce fatty acid esters from a carbon source, such as an alcohol, in the environment by expressing or overexpressing a gene encoding thioesterase, a gene encoding acyl-CoA synthase, and a gene coding for ester synthase. Accordingly, the methods described herein are used to modify microorganisms to overexpress or introduce a thioesterase gene, a gene encoding an acyl-CoA synthase, and a gene encoding an ester synthase. In specific embodiments, the thioesterase gene is selected from tesA, 'tesA, tesB, fatB, fatB2, fatB3, fatA1, or fatA. In specific embodiments, the gene encoding afil-CoA synthase is selected from fadDJadK, BH3103, pf1-4354, EAV15023, fadD1, fadD2, RPC_4074, fadDD35, fadDD22, faa39, or an identified gene encoding an enzyme having the same properties. In particular embodiments, the gene encoding the ester synthase is the gene encoding the synthase/acyl-CoA:diacylglyceryl acyltransferase from Simmondsiachinensis, Acinetobacter sp. ADP, Alcanivoraxborkumensis, Pseudomonasaeruginosa, Fundibacterjadensis, Arabidopsisthaliana, or Alkaligeneseutrophus or a variant thereof. Additionally or alternatively, the methods described herein are used to reduce the expression in said microorganism of at least one of a gene encoding an acyl-CoA dehydrogenase, a gene encoding an outer membrane protein receptor, and a gene encoding a transcriptional regulator of fatty acid biosynthesis. In specific embodiments, one or more of these genes are inactivated, such as by introducing a mutation. In specific embodiments, the gene encoding acyl-CoA dehydrogenase is fadE. In specific embodiments, a gene encoding a transcriptional regulator of fatty acid biosynthesis encodes a DNA transcriptional repressor, eg, fabR.

[001031] Additionally or alternatively, said microorganism is modified to reduce the expression of at least one gene encoding pyruvate formate lyase, a gene encoding lactate dehydrogenase, or both. In specific embodiments, the gene encoding pyruvate formate lyase is pflB. In specific embodiments, the gene encoding lactate dehydrogenase is IdhA. In specific embodiments, one or more of these genes are inactivated, such as by introducing a mutation into them.

[001032] In specific embodiments, the microorganism is selected from the following genera: Escherichia , Bacillus , Lactobacillus , Rhodococcus , Synechococcus , Synechoystis , Pseudomonas , Aspergillus , Trichoderma , Neurospora , Fusarium , Humicola , Rhizomucor , Kluyveromyhances , Pichia , Mutorium , Penerocha , Myceliophyle , Pleurotus , Trameles , Chrysosporium , Saccharomyces , Stenotrophamonas , Schizosaccharomyces , Yarrowia , or Streptomyces .

Use of C2c1 or C2c3 to create microorganisms capable of producing organic acids

[001033] The methods described herein are further used to engineer microorganisms capable of producing organic acids, more specifically from pentose or hexose sugars. In specific embodiments, the methods include introducing an exogenous LDH gene into the microorganism. In specific embodiments, organic acid production in said microorganisms is further or alternatively increased by inactivating endogenous genes encoding proteins involved in an endogenous metabolic pathway that produces a metabolite other than the organic acid of interest, and/or where the endogenous metabolic pathway utilizes the organic acid. In particular embodiments, the modification ensures that the production of a metabolite other than the organic acid of interest is reduced. In specific embodiments, the methods are used to introduce at least one engineered deletion into a gene and/or inactivate an endogenous pathway in which an organic acid is used or a gene encodes a product involved in an endogenous pathway producing a metabolite other than the organic acid of interest. In specific embodiments, the at least one engineered gene deletion or inactivation is in one or more genes encoding an enzyme selected from the group consisting of pyruvate decarboxylase (pdc), fumarate reductase, alcohol dehydrogenase (adh), acetaldehyde dehydrogenase, phosphoenolpyruvate carboxylase (ppc), D-lactate dehydrogenase (d-ldh), L-lactate dehydrogenase (l-ldh), lactate-2-monooxygenase. In further embodiments, the at least one engineered deletion and/or inactivation of the gene is in an endogenous gene encoding pyruvate decarboxylase (pdc).

[001034] In further embodiments, the microorganism is engineered to produce lactic acid and at least one engineered gene deletion and/or inactivation is in an endogenous gene encoding lactate dehydrogenase. Additionally or alternatively, the microorganism includes at least one engineered deletion or inactivation of a gene in an endogenous gene encoding a cytochrome-dependent lactate dehydrogenase, such as cytochrome B2-dependent L-lactate dehydrogenase.

Use of C2c1 or C2c3 to create improved xylose or cellobiose yeast strains

[001035] In specific embodiments, a C2c1 or C2c3 CRISPR system can be used to select improved xylose or cellobiose yeast strains. Reduced precision PCR can be used to amplify one (or more) genes involved in xylose utilization pathways or cellobiose utilization pathways. Examples of genes involved in xylose utilization pathways or cellobiose utilization pathways may include, but are not limited to, the pathways described in Ha, SJ et al. (2011) Proc. Natl Acad. sci. USA 108(2):504-9 and Galazka, JM, et al. (2010) Science 330(6000):84-6. The resulting libraries of double-stranded DNA molecules, each containing a random mutation in such a selected gene, can be co-transformed with CRISPR system components C2c1 or C2c3 into a yeast strain (e.g., S288C), and strains with increased ability to utilize xylose or cellobiose can be selected. as described in WO2015138855.

Use of C2c1 or C2c3 in the creation of improved yeast strains for isoprenoid biosynthesis

[001036] Tadas Jakociunas et al. described the successful use of the CRISPR/Cas9 multiplex system to genomically engineer up to 5 different genomic loci in a single transformation step in baker's yeastSaccharomyces cerevisiae (Metabolic Engineering Volume 28, March 2015, Pages 213-222), resulting in strains with high production of mevalonate, a key intermediate in an industrially important isoprenoid biosynthetic pathway. In specific embodiments, a C2c1 or C2c3 CRISPR system can be used in a multiplex genomic engineering method, as described herein, to identify additional high-yielding yeast strains for use in isoprenoid synthesis.

Using C2c1 or C2c3 to Create Lactic Acid-Producing Yeast Strains

[001037] Another embodiment of the invention encompasses the successful use of a C2c1 or C2c3 CRISPR multiplex system. By analogy with Vratislav Stovicek et al. (Metabolic Engineering Communications, Volume 2, December 2015, Pages 13-22), improved lactic acid producing strains can be developed and produced in a single transformational event. In a specific embodiment, the C2c1 or C2c3 CRISPR system is used to simultaneously insert a heterologous lactate dehydrogenase gene and disrupt two endogenous PDC1 and PDC5 genes.

The following applications of the CRISPR C2c1 or C2c3 system in plants

[001038] In specific embodiments, the CRISPR system, and preferably the C2c1 or C2c3 CRISPR system described herein, can be used to visualize the dynamics of genetic elements. For example, CRISPR can be used to visualize both repetitive and non-repeated genomic sequences, changes in telomere length and telomere movement, and track the dynamics of gene loci throughout the cell cycle (Chen et al., Cell, 2013). These methods can also be applied to plants.

[001039] Other applications of the CRISPR system, and preferably the C2c1 or C2c3 CRISPR system described herein, are in vitro and in vivo screening for gene disruption targets (Malina et al. ., Genes and Development, 2013). These methods can also be applied to plants.

[001040] In specific embodiments, fusion of inactivated C2c1 or C2c3 endonucleases with histone modification enzymes can introduce targeted changes to the complex epigenome (Rusk et al., Nature Methods, 2014). These methods can also be applied to plants.

[001041] In specific embodiments, the CRISPR system, and preferably the C2c1 or C2c3 CRISPR system described herein, can be used to purify a specific portion of chromatin and identify associated proteins to elucidate their regulatory roles in transcription (Waldrip et al., Epigenetics, 2014). These methods can also be applied to plants.

[001042] In particular embodiments, the invention is intended to be used as a therapy to remove viruses from plant systems, as it allows the cleavage of both viral DNA and viral RNA. Previous studies in human systems have demonstrated the successful use of CRISPR to target HCV containing single-stranded RNA (A. Price, et al., Proc. Natl. Acad. Sci, 2015) as well as HBV containing double-stranded DNA (V. Ramanan, et al., Sci. Rep, 2015). These methods can also be adapted to use the RNA-targeted CRISPR system in plants.

[001043] In specific embodiments, the implementation of the present invention can be used to change the complexity of the genome. In a further specific embodiment, the CRISPR system, and preferably the C2c1 or C2c3 CRISPR system described herein, can be used to destroy or change the number of chromosomes and create haploid plants that contain chromosomes from only one parent. In such plants, chromosome duplication can be induced with transformation into diploid plants containing only homozygous alleles (Karimi-Ashtiyani et al., PNAS, 2015; Anton et al. Nucleus, 2014). These methods can also be applied to plants.

[001044] In specific embodiments, the C2c1 or C2c3 CRISPR system described herein can be used for self-cleavage. In these embodiments, the promoter of the C2c1 or C2c3 enzyme and the gRNA may be a constitutive promoter and the second gRNA is introduced in the same transformation cassette but driven by an inducible promoter. This second gRNA can be engineered to cause a specific cleavage in the C2c1 or C2c3 gene to produce non-functional C2c1 or C2c3. In a further specific embodiment, the second gRNA causes cleavage at both ends of the transformation cassette, resulting in the removal of the cassette from the host genome. This system offers a controlled duration of exposure of the Cas enzyme to the cell and further minimizes off-target editing. In addition, cleavage of both ends of the CRISPR/Cas cassette can be used to produce transgene-free T0 plants with biallelic mutations (as described for Cas9, e.g., Moore et al., NucleicAcidsResearch, 2014; Schaeffer et al., PlantScience, 2015). The methods of Moore et al. can be applied to the C2c1 or C2c3 CRISPR systems described herein.

[001045] Sugano et al. (Plant Cell Physiol. 2014 Mar;55(3):475-81. doi: 10.1093/pcp/pcu014. Epub 2014 Jan 18) reports the use of CRISPR-Cas9 for site-directed mutagenesis of the liverwort Marchantiapolymorpha L., which has come to be used as a model for experiments to study the evolution of higher plants. The M. polymorpha U6 promoter was identified and cloned for gRNA expression. The gRNA target sequence was designed to disrupt the gene encoding auxin factor 1 (ARF1) in M. polymorpha . Using transformation with Agrobacterium , Sugano et al. obtained resistant mutants in the gametophyte phase for M. polymorpha . Using CRISPR-Cas9, site-directed mutagenesis in vivo was obtained using the 35S cauliflower mosaic promoter or the M. polymorpha EF1α promoter to express Cas9. Isolated mutant individuals showing an auxin-resistant phenotype were not chimeras. Moreover, stable mutants were obtained by asexual reproduction of T1 plants. Numerous arf1 alleles were easily generated using site-directed mutagenesis based on the CRIPSR/Cas9 system. The methods of Sugano et al. can be used for the C2c1 or C2c3 effector protein system of the present invention.

[001046] Kabadi et al. (Nucleic Acids Res. 2014 Oct 29;42(19):e147. doi: 10.1093/nar/gku749. Epub 2014 Aug 13) developed a single lentiviral system to express the Cas9 variant, a reporter gene, and up to four sgRNAs from independent promoters of RNA polymerase III, which are included in the vector using a fairly convenient GoldenGate cloning method. Each sgRNA has been efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells. The methods of Kabadi et al. can be used for the C2c1 or C2c3 effector protein system of the present invention.

[001047] Ling et al. (BMC Plant Biology 2014, 14:327) developed a CRISPR-Cas9 binary vector kit based on the pGreen or pCAMBIA scaffold and gRNA. This toolkit does not require any additional restriction enzymes besides BsaI to generate high efficiency final constructs in just one cloning step. This instrumentation has been validated using maize protoplasts, transgenic maize lines and transgenic Arabidopsis lines and has been shown to exhibit high efficiency and specificity. However, what seems to be even more important, using this toolkit, targeted mutations of three Arabidopsis genes were detected in transgenic T1 generation shoots. Moreover, mutations with multiple genes can be inherited by the next generation. A set of vector modules (guide RNA) was used as a tool for multiplex genome editing in plants. Toolkit Ling et al. can be used for the C2c1 or C2c3 effector protein system of the present invention.

[001048] Protocols for targeted plant genome editing using CRISPR-Cas9 are also available in Methods in Molecular Biology Volume 1284 pp. 239-255, February 10, 2015. A detailed methodology for designing, constructing, and evaluating double gRNAs for codon-optimized Cas9-mediated genomic editing for plant expression (pcoCas9) using protoplasts is described.Arabidopsis thaliana andNicotiana benthamiana as model cell systems. Strategies for using the CRISPR-Cas9 system to introduce targeted modifications to the whole plant genome are also discussed. The protocols described in this chapter can be applied to the C2c1 or C2c3 effector protein system of the present invention.

[001049] Ma et al. (Mol Plant, 2015 Aug 3, 8 (8): 1274-84. Doi: 10.1016./j.molp.2015.04.007) report a robust CRISPR-Cas9 vector system using a plant codon-optimized Cas9 gene for convenient and of highly efficient multiplex genome editing in monocots and dicots. Ma et al. developed specific PCR-based procedures to rapidly generate multiple sgRNA expression cassettes that can be assembled into CRISPR-Cas9 binary vectors in a single round of cloning by GoldenGate ligation or Gibbs assembly (GibsonAssembly). In this system, Ma et al. edited 46 target rice sites with an average mutation rate of 85.4%, mostly in the biallelic and homozygous state. Ma et al. give examples of modeling loss of function gene mutation in T0 and Arabidopsis T1 rice plants by simultaneously targeting multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. The methods of Ma et al. can be used for the C2c1 or C2c3 effector protein system of the present invention.

[001050] Lowder et al. (PlantPhysiol. 2015 Aug 21. doi: pp.00636.2015) also developed the CRISPR-Cas9 toolkit, which allows multiplex genome editing and transcriptional regulation of expressed genes, genes with suppressed expression or non-coding genes in plants. This toolkit provides researchers with a protocol and reagents for the rapid and efficient assembly of functional CRISPR-Cas9 T-DNA constructs for monocots and dicots using the GoldenGate and Gateway cloning methods. It comes with a full range of possible tools, including multiplexed gene editing and transcriptional activation or repression of endogenous plant genes. T-DNA-based transformation technology is of fundamental importance for modern plant biotechnology, genetics, molecular biology and physiology. Therefore, the inventors have developed a method for assembling Cas9 (WT, nickase or dCas9) and one or more gRNAs into a final T-DNA vector of interest. The build method is based on both GoldenGate build type and MultiSite Gateway recombination. Three modules are required for assembly. The first module is the original Cas9 vector, which contains Cas9 genes without promoters or genes derived from it, flanked by the attL1 and attR5 sites. The second module is the gRNA entry vector, which contains the input gRNA. Expression cassettes are flanked by attL5 and attL2 sites. The third module includes attR1-attR2 containing terminal T-DNA vectors that provide the promoters of choice for Cas9 expression. Instrumentation of Lowder et al. can be applied to the C2c1 or C2c3 effector protein system of the present invention.

[001051] In a preferred embodiment, the plant may be a tree. The present invention can also use the herbal CRISPR-Cas system described herein (see, for example, Belhaj et al., Plant Methods 9: 39 and Harrison et al., Genes & Development, 28: 1859-1872). In a particularly preferred embodiment, the CRISPR-Cas system of the present invention can target a single nucleotide polymorphism (SNP) in trees (see, e.g., Zhou et al., NewPhytologist, Volume 208, Issue 2, pages 298-301, October 2015) . Zhou et al. applied the CRISPR-Cas system on the arboreal perennial Populus using the 4-coumarate:CoA ligase (4CL) gene family as an example and achieved 100% mutation efficiency for the two thus transformed 4CL genes, with each transformed organism being examined for transfer biallelic modifications. In a study by Zhou et al. the CRISPR-Cas9 system was very sensitive to single nucleotide polymorphisms (SNPs) because the cleavage of the third 4CL gene was abolished due to the SNP in the target sequence. These methods can be applied to the C2c1 or C2c3 effector protein system of the present invention.

[001052] The methods of Zhou et al. (New Phytologist, Volume 208, Issue 2, pages 298-301, October 2015) can be applied to the present invention as follows. Two genes 4CL, 4CL1 and 4CL2, associated with the biosynthesis of lignin and flavonoids, respectively, are targets for CRISPR-Cas9 editing. Populustremula x alba clone 717-1B4, commonly used for transformation, differs from the Populus trichocarpa genotype. Thus, 4CL1 and 4CL2 gRNAs constructed from a reference genome can be analyzed using the 717 RNA sequencing data itself, to ensure that there are no SNPs that may limit the effectiveness of Cas. A third gRNA is also provided for 4CL5, a duplicate of 4CL1. The corresponding 717 sequences contain one SNP in each allele near/within PAM, both of which are expected to abolish targeting by 4CL5-gRNA. All three gRNA target sites are located within the first exon. For 717 transformations, gRNA is expressed from the Medicago U6.6 promoter along with codon-optimized human Cas under the control of the CaMV 35S promoter in a binary vector. Transformation using only the Cas vector can serve as a control. Randomly selected 4CL1 and 4CL2 lines are subjected to amplicon sequencing. The data is then processed and biallelic mutations are confirmed in all cases. These methods can be applied to the C2c1 or C2c3 effector protein system of the present invention.

[001053] In plants, pathogens are often host specific. For example, Fusarium oxysporum f. sp. lycopersici affects only tomato, while F. oxysporum f. dianthii Pucciniagraminis f. sp. tritici only wheat. Plants have natural and inducible defenses to resist most pathogens. Mutations and recombination phenomena between plant generations lead to genetic variation that results in disease susceptibility, especially when pathogens reproduce at a higher frequency than plants. Non-host resistance can be observed in plants, for example, when the host and pathogen are not compatible. There can also be horizontal resistance, such as partial resistance to pathogen race alleles, usually controlled by many genes, and vertical resistance, such as complete resistance to some races of a pathogen but not to other races, usually controlled by a few genes. At the level of "gene against gene" relationship, plants and pathogens develop together, there is an eternal disturbance and restoration of balance: genetic changes in one lead to disturbances in the other. Accordingly, using the natural variability of the genome, breeders combine in one plant the most useful genes for yield, quality, uniformity, endurance, and resistance to various diseases. Sources of resistance genes are local or foreign varieties, old varieties of non-hybrid origin, relatives of wild plants and varieties with induced mutation, for example, after treatment of plant material with mutagenic agents. Thus, in accordance with the present invention, breeders are offered a new tool for stimulating mutations. Accordingly, a person skilled in the art will be able to analyze the genomes of different varieties - possible sources of resistance genes having the desired characteristics or traits, and will be able to use the present invention to induce the emergence of resistance genes with significantly greater accuracy than previous mutagenic agents, which, therefore, will lead to acceleration and improvement of plant breeding.

Improving the properties of plants and yeasts

[001054] The present invention also relates to plants and yeast cells, which can be obtained using the methods described in the present description. Plants with improved properties obtained by the methods described herein may be useful for food or feed production through the expression of genes providing, in particular, resistance to pests, herbicides, drought, low or high temperatures, excessive moisture, etc.

[001055] Plants with improved properties obtained by the methods described herein, especially crops and algae, can be produced as food and feed to provide, for example, a higher content of proteins, carbohydrates, nutrients or vitamins compared to those for the wild type. In this regard, plants with improved properties are preferred, especially legumes and tuberous plants.

[001056] Improved algae or other plants, such as rapeseed, may be particularly useful for the production of vegetable oils or biofuels, such as alcohols (especially methanol and ethanol). They can be engineered to express or overexpress high levels of oils or alcohols for use in petroleum or biofuel industries.

[1057] The invention also relates to the improvement of constituent parts of plants. Such plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, eggs, and pollen. Such plant parts, as contemplated within the scope of the present invention, may be viable, non-viable, regenerable and/or non-regenerable.

[001058] The present invention also encompasses the production of plant cells and plants obtained according to the methods of the invention. Gametes, seeds, embryos, either zygotic or somatic, progeny or hybrids of plants bearing the genetic modification, which are obtained by conventional breeding methods, are also within the scope of the present invention. Such plants may contain a heterologous or foreign DNA sequence incorporated into or in replacement of the target sequence. Alternatively, such plants may contain only one modification (mutation, deletion, insertion, substitution) in one or more nucleotides. Essentially, such plants may differ from parent plants only by the presence of a particular modification.

[001059] Thus, the invention relates to a plant, animal or cell obtained by the methods of the present invention, or their progeny. The offspring may be a clone of the resulting plant or animal, or may be the result of sexual reproduction by crossing with other individuals of the same species to further introgress the desired traits to offspring. The cell may exist in vivo or ex vivo in the case of multicellular organisms, in particular animals and plants.

[001060] The present invention can also be applied to other agricultural fields, such as, for example, farming and animal husbandry. For example, pigs have many features that make them attractive biomedical models, especially in regenerative medicine. In particular, pigs with severe combined immunodeficiency (SCID) may serve as suitable models for regenerative medicine, xenotransplantation, and tumor development and will help in the development of treatments for patients with human SCID. Lee et al., (Proc Natl Acad Sci USA. 2014 May 20, 11 ((20): 7260-5) used nucleases with transcription activator analog domains (TALEN) to generate targeted modifications of the recombination activating gene (RAG) 2, including those , which acted on both alleles, in somatic cells with high efficiency In such a system, the effector protein C2c1 or C2c3 can be used.

[001061] The methods of Lee et al. (Proc Natl Acad Set USA. 2014 May 20, 111 (20): 7260-5) can be applied to the present invention as follows. Pigs with the mutation are generated by targeted modification of RAG2 in fetal fibroblast cells, followed by SCNT and embryo transfer. The CRISPR-Cas-encoding constructs and the reporter are introduced by electroporation into fetal fibroblast cells. After 48 hours, transfected cells expressing green fluorescent protein are sorted into individual wells of a 96-well plate at an intended concentration of one cell per well. Screening for the target modification of RAG2 is carried out by amplification of a genomic DNA fragment flanking any CRISPR cleavage sites, followed by sequencing of the PCR products. After screening and checking for the absence of off-target mutations, cells with a targeted RAG2 modification are used for SCNT. The polar body, together with a part of the adjacent oocyte cytoplasm, presumably containing the metaphase II plate, is removed and the donor cell is placed in the perivitellin. The recovered embryos are then electroporated to connect the donor cell to the oocyte and then chemically activated. Activated embryos are incubated in Porcine Zygote Medium 3 (PZM3) with 0.5 Scriptaid (S7817, Sigma-Aldrich) for 14-16 hours. The embryos are then washed to remove Scriptaid and cultured in PZM3 prior to transfer to the oviducts of surrogate pigs.

[001062] The present invention is also applicable to modification by SNPs of other animals such as cows. Tan et al, (Proc Natl Acad Sci USA, 2013 Oct 8, 110 (41): 16526-16531) have expanded the toolbox for gene editing in animal husbandry by incorporating nucleases with transcription activator analog domains and clustered regular clustered short palindromic repeats (CRISPR)/Cas9-stimulated homologous repair ( HDR) using plasmids, rAAV and oligonucleotide templates. The gene-specific gRNA sequences were cloned into the Church lab gRNA vector (Addgene ID: 41824) according to their methods (Mali P, et al., (2013) RNA-Guided Human Genome Engineering via Cas9.Science 339(6121):823-826). The Cas9 nuclease was obtained either by co-transfection with the hCas9 plasmid (Addenene ID: 41815) or by mRNA synthesized with RCIScript-hCas9. This RCIScript-hCas9 was constructed by subcloning the XbaI-AgeI fragment from the hCas9 plasmid (spanning the hCas9 cDNA) into the RCIScript plasmid.

[001063] Heo et al.(Stem Cells Dev. 2015 Feb 1;24(3):393-402. doi: 10.1089/scd.2014.0278. Epub 2014 Nov 3) reported highly efficient targeting of the bovine genome using bovine pluripotent cells and clustered short palindromic repeats arranged in regular groups. First, Heo et al. obtained induced pluripotent stem cells ((iPSC) from bovine somatic fibroblasts by ectopic expression of yamanaka factors and treatment with GSK3p and MEK inhibitor (2i). Heo et al saw that bovine iPSCs are similar to naïve pluripotent stem cells in terms of gene expression and developmental potential in Moreover, the CRISPR-Cas9 nuclease, specific for the bovine NANOG locus, has been shown to be highly efficient in editing the bovine genome in bovine iPSCs and embryos.

[001064] Igenity® profiles animals, such as cows, to achieve and convey traits of commercial importance such as carcass composition, carcass quality, maternal and reproductive characteristics, and average weight gain per day. Igenity® Comprehensive Profile Analysis begins with the identification of DNA markers (most commonly single nucleotide polymorphisms or SNPs). All DNA markers in the Igenity® profile have been determined by independent scientists at research institutes, including universities, research organizations, and government agencies such as the USDA. The markers were then verified on the Igenity® sample. Igenity® uses a variety of sampling sources that represent a variety of production conditions and biological types, often collaborating with industry partners such as the cow-calf, feed, and packaging segment of the beef industry to collect rare phenotypes that are not normally available. Cattle genome databases are widely available, see for example the NAGRP Cattle Genome Coordination Program (http://www.anirnaigenorne.org/cattle/rnaps/db.htnai). Thus, the present invention can be applied to target bovine SNPs. One of skill in the art can use the above protocols for targeting SNPs and apply them to bovine SNPs as described, for example, by Tan et al. or Heo et al.

[001065] Qingjian Zou et al. (Journal of Molecular Cell Biology, Advance Access published October 12, 2015) demonstrated an increase in canine muscle mass by targeting the first exon of the canine myostatin (MSTN) gene, a negative regulator of muscle and skeletal mass. First, the efficacy of sgRNA was confirmed by co-transfection of MSTN-targeted sgRNA and Cas9 vector into canine fetal fibroblasts. MSTN KO dogs were then obtained by microinjection of Cas9 mRNA and sg-RNA of MSTN into embryos with normal morphology and autotransplantation of zygotes into the oviduct of the same female dog. Knockout puppies showed a distinct muscular phenotype in the thigh region compared to wild-type littermates. A similar result can also be obtained using CRISPR C2c1 or C2c3 described in the present description.

Livestock - pigs

[001066] Virus targets in livestock, in some embodiments, may include porcine CD163; for example, in porcine macrophages, CD163 is associated with infection (which is thought to be associated with viral cell entry) by PRRSv (porcine reproductive and respiratory virus, arterivirus). Infection with PRRSv, especially porcine alveolar macrophages (found in the lungs), results in a previously incurable porcine syndrome ("Mystery Pig Disease" or "Blue Ear Disease") that causes symptoms including reproductive failure, weight loss, and high mortality rates in domestic pigs . Infections caused by opportunistic pathogens, as well as favorable conditions, such as enzootic pneumonia, meningitis, and ear edema, are considered to be caused by immunodeficiency in the background of loss of macrophage activity. They also have significant financial and environmental impacts due to increased antibiotic use and financial losses ($660 million per year).

[001067] As reported by Kristin M Whitworth and Dr Randall Prather et al. (Nature Biotech 3434 published online 07 December 2015) at the University of Missouri and in collaboration with Genus Pic, CD163 was targeted using CRISPR-Cas9 and genome-edited pig progeny were resistant to PRRSv. One male and one female with exon 7 mutations were bred to produce offspring. The founder male had an 11 bp deletion. in exon 7 on one allele, resulting in a frame mutation and skipping of amino acid 45 in region 5 and hence a premature stop codon at amino acid 64. The other allele had 2 bp. in exon 7 and deletion 377 b.p. in the preceding intron, which were predicted to result in the expression of the first 49 amino acids of domain 5, followed by a premature stop codon at amino acid 85. The sow had 7 bp. on one allele, which was predicted to result in the expression of the first 48 amino acids of domain 5 on transcription, followed by a premature stop codon at amino acid 70. The other porcine allele was non-amplifiable. It was predicted that after selection, the offspring would be a null animal (CD163 -/-) that would have a CD163 knockout.

[001068] Accordingly, in some embodiments, CRISPR proteins can be targeted to porcine alveolar macrophages. In some embodiments, the CRISPR proteins can be targeted to porcine CD163. In some embodiments, porcine CD163 can be knocked out by introducing DSNB or insertions and deletions, such as a targeted deletion or modification of exon 7, including one or more of those described above, or in other regions of the gene, such as a deletion or modification of exon 5.

[001069] Edited pigs and their offspring are contemplated, such as a CD163 knockout pig. This may be done for animal husbandry, breeding or modulation purposes (eg pig model). It also provides sperm with a knocked-out gene.

[001070] CD163 is a member of the cysteine-rich phagocytic receptor (SRCR) superfamily. As shown by in vitro studies , domain 5 of the SRCR protein is the domain responsible for the unpacking and release of the viral genome. Therefore, other members of the SRCR superfamily can also be targeted to increase resistance to other viruses. PRRSV is also a member of the mammalian arterivirus group, which also includes mouse lactate dehydrogenase growth virus, simian hemorrhagic fever virus, and equine arteritis virus. Arteriviruses have important pathogenetic properties, including macrophage tropism and the ability to cause both severe disease and persistent infection. Accordingly, arteriviruses, and in particular murine lactate dehydrogenase elevated virus, simian hemorrhagic fever virus, and equine arteritis virus, can be targeted, for example, via porcine CD163 or its homologues in other species, as well as in mouse, monkey, and horse models. , and a knockout is also provided.

[001071] Indeed, this approach can be extended to viruses or bacteria that cause other livestock diseases that can be transmitted to humans, such as swine influenza virus (SIV) strains, which include influenza C and influenza A subtypes known as H1N1, H1N2 , H2N1, H3N1, H3N2, and H2N3, as well as the pneumonia, meningitis, and edema mentioned above.

Therapeutic targeting via RNA-guided C2c1 or C2c3 effector protein complex

[001072] As will be appreciated, it is contemplated that the system of the present invention can be used to target any polynucleotide sequence of interest. The present invention relates to a non-naturally occurring or engineered composition, or one or more polynucleotides encoding components of said composition, or to vector systems or delivery systems containing one or more polynucleotides encoding components of said composition, for use in cell modification - targets in vivo, ex vivo, or in vitro, and can be carried out in a manner that alters the cell such that, after modification of the progeny or cell line, the CRISPR-modified cell retains the altered phenotype. The modified cells and progeny can be part of a multicellular organism such as a plant or animal using an ex vivo or in vivo CRISPR system for the desired cell type. The CRISPR of the invention may be a therapeutic method of treatment. Therapeutic treatment may include gene or genome editing, or gene therapy.

Treatment for pathogens such as bacterial, fungal and parasitic pathogens

[001073] The present invention can also be used to treat bacterial, fungal and parasitic pathogens. Most research efforts are focused on developing new antibiotics that, once developed, will nevertheless be subject to the same drug resistance problems. The invention relates to new CRISPR-based alternatives that overcome these difficulties. In addition, unlike existing antibiotics, CRISPR-based treatments can be pathogen-specific, inducing the death of the target pathogen's bacterial cells while avoiding the death of beneficial bacteria.

[001074] Jiang et al. ("RNA-guided editing of bacterial genomes using CRISPR-Cas systems,"Nature Biotechnology vol. 31, p. 233-9, March 2013) used the CRISPR-Cas9 system to mutate or killS. pneumoniae andE. coli. This work, which represents precise mutations in genomes, is based on a double cleavage of the RNA- and Cas9-targeted region of the genome to ensure non-mutant cell death and bypass the need for choice of markers or anti-selection systems. CRISPR systems can be used to promote the regression of drug resistance and eliminate the transfer of resistance between strains. Bickard et al. showed that Cas9, reprogrammed to target virulence genes, kills the virulent but not the avirulent,S. aureus. By reprogramming the nuclease for the target antibiotic resistance genes, the disruption of staphylococcal plasmids that contained antibiotic resistance genes and immunized against the propagation of plasmid resistance genes was carried out (see Bikardet al. "Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials,"Nature Biotechnology vol. 32, 1146-1150, doi: 1Q.1038/nbt.3043, published online 05 October 2014). Bikard has shown that CRISPR-Cas9-based antimicrobials function to kill S. aureus in a mouse skin colonization model. Similarly, Yosef et al. used CRISPR to target genes that code for enzymes) conferring resistance to β-lactam antibiotics (see Yousef et al., "Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria,"Proc. Nail Acad. sci. usa,vol. 112, p. 72.67-72.72, doi: 10.1073/pnas. 1500107112 published online on May 18, 2015).

[001075] CRISPR systems can be used to edit the genomes of parasites that are resistant to other genetic approaches. For example, the CRISPR-Cas9 system has been shown to introduce a double-strand break into the genomePlasmodium yoelii (See, Zhang et al., "Efficient Editing of Malaria Parasite Genome Using the CRISPR/Cas9 System," mBio. vol. 5, e01414-14, Jul-Aug 2014). Gorbalet al. ("Genome editing in the human malaria parasitePlasmodium falciparum using the CRISPR-Cas9 system,"Nature Biotechnology, vol. 32., p. 819-821, doi: 10.1038/nbt.2925, published online June 1, 2014) modified the sequences of two genes, orc1 and kelch13, which are thought to be involved in gene silencing and emerging artemisinin resistance, respectively. Parasites that were modified at the appropriate sites recovered with very high efficiency, despite the lack of direct selection for modification, indicating that neutral or even deleterious mutations can be introduced using this system. CRISPR-Cas9 is also used to modify the genomes of other pathogenic parasites, includingToxoplasma gondii (See Shenet al., "Efficient gene disruption in diverse strains of Toxoplasma gondii, using CRISPR/CAS9," mBio vol. 5:e01114-14, 2014; and SidiJket al., "Efficient Genome Engineering ofToxoplasma gondii Using CRISPR/Cas9," PLoS One vol. 9, el00450, doi: 10.1371/joumal.pone.0100450, published online June 27, 2014).

[001076] Vyas et al (" A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families," Science Advances, vol. 1, el500248, DOI: 10.1126/seiadv.1500248, April 3, 2015) used the CRISPR system to overcoming long standing hurdles in C. albicans genetic engineering and effectively mutated both copies of several different genes in one experiment. In an organism where multiple mechanisms contribute to drug resistance, Vyas generated homozygous double mutants that no longer exhibited the fluconazole or cycloheximide hyperresistance exhibited by the Can90 parent clinical isolate. Vyas has also generated homozygous loss-of-function mutations in major C. albicans genes by creating conditional alleles. Null alleles of DCR1, which is required for ribosomal RNA processing, are lethal at low temperature but viable at high temperature. Vyas used a recovery template that introduced a nonsense mutation and isolated the dcr1/dcr1 mutants that did not grow at 16°C.

[001077] The CRISPR system of the present invention was used in P. falciparum by destroying the chromosomal locus. Gorbal et al. ("Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR.-Cas9 system.". Nature Biotechnology, 32, 81.9-821 (2014), DOI: 10.1038/nbt.2925, June 1, 2014) used the CRISPR system to introduction of certain gene knockouts and single nucleotide substitutions in the malaria genome. To adapt the CRISPR-Cas9 system to P. falciparum , Ghorbal et al. constructed an expression vector to control plasmodium regulatory elements in the pUFl-Cas9 episome, which also carries the ydhodh selectable marker, which confers resistance to DSM1, an inhibitor of P. falciparum dihydrogenate dehydrogenase (PfDHODH), and small nuclear-based regulatory elements ( sn) P. facciparum U6 RNA by placing the guide RNA and the donor DNA template for homologous recombination repair on the same plasmid, pL7. See also Zhang C. V al. ("Efficient editing of malaria parasite genome using the CRISPR/Cas9 system", MBio, 2014 Jul 1, 5(4):E01414-I4, doi: 10. EI.28/MbKO.O1.414-M) and Wagner et al. ("Efficient CRISPR-Cas9-mediated genome editing in Plasmodium falciparum, Nature Methods 11, 915-918 (2014), DOI: 10.1038/nmeth.3063).

Treatment for pathogens such as viral pathogens such as HIV

[001078] Cas-mediated genome editing can be used to introduce protective mutations in somatic tissues to combat non-genetic or complex diseases. For example, NHEJ-mediated inactivation of the CCR5 receptor in lymphocytes (Lombardo et al.,Nat Biotechnol. Nov 2007; 25(11): 1298-306) may be a fruitful strategy in the fight against HIV infection, and deletion of PC5K9 (Cohen et al.,Nat Genet. February 2005; 37(2): 1(51-5) or angiopoietin (Musunuru et al., NEngl J Med. 2010 Dec 2; 363(231:2220-7) may have a therapeutic effect in statin-resistant hypercholesterolemia or hyperlipidemia. Although these targets can be removed by sgRNA-mediated protein knockout, the unique advantage of NHEJ-mediated inactivation is the ability to achieve permanent therapeutic benefit without the need for continued treatment. As with all gene therapy modalities, it is certainly important to ensure that each proposed therapeutic use has a favorable benefit/risk ratio.

[001079] It was shown that hydrodynamic delivery of plasmid DNA encoding Cas9 and guide RNA along with a repair template to the liver in an adult mouse model of tyrosinemia corrected the Fah gene mutation and restored wild-type Fah protein expression in ~1 of 250 cells(Nat Biotechnol. Jun 2014; 32(6):551-3). In addition, zinc finger nucleases have been successfully used in clinical trials to fight HIV infection by knocking out the CCR5 receptor.ex vivo. All patients had reduced levels of HIV DNA, and in one in four patients, HIV RNA became undetectable (Tebas et al.,N Engl J Med. 2014 Mar 6; 370(10):901-10). Both results show promise for using programmable nucleases as a new therapeutic platform.

[001080] In another embodiment, self-inactivating lentiviral vectors with siRNA, a targeted exon common to HIV tat/rev genes, a nucleolar-localized TAR decoy, and an anti-CCR5-specific "hammerhead" ribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) can be used and/or adapted for use in the nucleic acid targeting system of the present invention. A minimum of 2.5x10 ⁶ CD34 ⁺ cells per kilogram of patient body weight can be harvested and prestimulated for 16-20 hours in X-VIVO 15 culture medium (Lonza) containing 2 μM/L glutamine, a stem cell factor ( 100 ng/ml), Flt-3 ligand (Flt-3L) (100 ng/ml) and thrombopoietin (10 ng/ml) (CellGenix) at a density of 2×10 ⁶ cells/ml. Prestimulated cells can be transduced with a lentiviral vector at a MOI of 5 for 16-24 hours in 75 cm ² tissue culture flasks coated with fibronectin (25 mg/cm ² ) (RetroNectin, Takara Bio Inc.).

[01081] Those skilled in the art, using the information and ideas available in the art in accordance with the present invention, can correct an HSC for an immunodeficiency condition such as HIV/AIDS by bringing the HSC into contact with a CRISPR-Cas9 system that targets and knocks out CCR5. Guide RNA (and preferably a dual guide molecule approach, e.g. a pair of different guide RNAs, e.g. guide RNAs targeting two clinically relevant B2M and CCR5 genes in primary CD4+ T cells and CD34+ hematopoietic stem and progenitor cells ( human HSPC) that targets and knocks out CCR5, and particles containing C2c1 or C2c3 proteins are brought into contact with the HSC. Such contacted cells can be introduced and, if necessary, processed/upscaled (see Cartier., also see Kiem, "Hematopoietic stem cell-based gene therapy for HIV disease", Cell Stem Cell Feb 3, 2012; 10 (2): 137-147 incorporated herein by reference together with the documents cited therein Mandal et al., "Efficient Ablation of Genes in Human Hematopoietic Stem and Effector Cells using CRISPR/Cas9," Cell Stem Cell, Volume 15 , Issue 5, p643--652, 6 November 2014, incorporated herein by reference along with the documents cited therein. Reference may also be made to Ebina, "CRlSPR7Cas9 system, to suppress HIV-1 expression by editing HIV-1 integrated proviral DNA" SCIENTIFIC REPORTS | 3: 2510 | DOI: 10.1038/srep02510, incorporated herein by reference along with the documents cited therein, as another means of combating HIV/AIDS using the C2c1 or C2c3 CRISPR system.

[001082] The rationale for genome editing for HIV treatment is the observation that homozygous individuals for mutations in loss-of-function CCR5, the cellular co-receptor for the virus, are highly resistant to infection and are otherwise healthy, indicating that attempting to recreate this mutation may be safe. and effective treatment [Liu, R., et al.cell 86, 367-377 (1996)]. This idea was supported by clinical trials when an allogeneic bone marrow transplant from a donor homozygous for a loss-of-function CCR5 mutation was performed on an HIV-infected patient, resulting in undetectable levels of HIV and restoration of normal CD4 T cell counts [Flutter, G ., et al.The New England Journal Of Medicine 360, 692-698 (2009)]. Although bone marrow transplantation is not a realistic treatment option for most HIV patients due to cost and potential graft response, HIV therapy that converts the patient's own T cells into CCR5 mutants is desirable.

[001083] Previous studies using ZFN and HNEI to knock out CCR5 in a humanized mouse model of HIV showed that transplantation of CCR5-edited CD4 T cells reduced viral load and CD4 T cell count [Perez, E. E., et al. Nature Biotechnology 26, 808-816 (2008)]. Importantly, these models also showed that HIV infection led to selection of CCR5 null cells, indicating that editing conferred a tolerance advantage and potentially allowing a small number of edited cells to provide a therapeutic effect.

[001084] As a result of this and other promising preclinical studies, genome editing therapy that knocks out CCR5 in patient T cells has been further tested in humans [Holt, N., et al.Nature Biotechnology 28, 839-847 (2010); Li, L., et al.Molecular therapy: the journal of the American Society of Gene Therapy 21, 1259-1269 (2013)]. In a recent phase I clinical trial, CD4+ T cells from HIV patients were isolated, edited with ZFNs designed to knock out the CCR5 gene, and autologously transplanted back into patients [Tebas, P., et al.The New England Journal Of Medicine 370, 901-910 (2014)].

[001085] In another study by Mandal et al.,cell. Stem. cell, Volume 15, Issue 5, p643-652, 6 November 2014), CRISPR-Cas9 was targeted to two clinically relevant genes, B2M and CCR5, in human CD4+ T cells and CD34+ human hematopoietic stem and progenitor cells (HSPC). The use of single guide RNAs resulted in highly efficient mutagenesis in HSPC but no effect on T cells. The dual guide RNA approach increased the efficiency of gene deletion in both cell types. HSPCs genome-edited with CRISPR-Cas9 retained their multi-lineage potential. Predictive on- and off-target mutations were examined by capture sequencing in HSPC, and low levels of off-target mutagenesis were observed in only one site. These results demonstrate that CRISPR-Cas9 can effectively eliminate genes in HSPC with minimal off-target mutagenesis, which has broad applicability in hematopoietic cell-based therapy.

[001086] Wang et al. ((PLoS One, 2014 Dec 26;9(12):el 15987. doi: 10.1371/joumal.pone.Ol 15987) silenced CCR5 via CRISPR-associated protein 9 (Cas9) and single guide RNA(s) using lentiviral vectors expressing Cas9 and targeting anti-CCR5 RNA Wang et al showed that single cyclic transduction of lentiviral vectors expressing Cas 9 and targeting anti-CCR5 RNA in HIV-1 susceptible human CD4+ cells results in high frequencies of CCR5 gene disruption. Cells with a disrupted CCR5 gene are not only resistant to R5-tropical HIV-1, including transmissible/primary isolates (T/F) of HIV-1, but also have a selective advantage over cells with intact CCR5 during R5-tropical infection HIV-1 Genomic mutations at potential off-target sites that are highly homologous to these anti-CCR5 guide RNAs in stably transduced cells, even 84 days after transduction, were not detected. were analyzed using T7 endonuclease I assay.

[001087] Fine et al. (Sci Rep. 2015 Jui 1;5:10777. doi: 10.1038/'srep10777) identified a two-cassette system expressing fragments of the Cas9 proteinS. pyogenes (SpCas9), which join together in the cell to form a functional protein capable of site-specific DNA cleavage. Using specific CRISPR guide chains, Fine et al. demonstrated the efficiency of this system in cleaving the HBB and CCR5 genes in human HEK-293T cells as a single Cas9 and as a pair of Cas9 nickases. Trans-spliced SpCas9 (tsSpCas9) showed ~35% nuclease activity compared to wild-type SpCas9 (wtSpCas9) at standard transfection doses, but had significantly reduced activity at lower dosage levels. The significantly reduced open reading frame length of tsSpCas9 relative to wtSpCas9 potentially allows more complex and longer genetic elements to be packaged into an AAV vector, including tissue-specific promoters, guide RNA multiplex expression, and effector domain fusion to SpCas9.

[001088] Li et al.(J Gen Virol. 2015 Aug;96(8):2381-93. doi: 10.1099/vir.0.000139. Epub 2015 Apr 8) demonstrated that CRISPR-Cas9 can efficiently mediate editing of the CCR5 locus in cell lines, resulting in the induction of CCR5 expression on the cell surface. Next generation sequencing revealed that various mutations were introduced around the predicted CCR5 cleavage site. For each of the three most effective guide RNAs that were analyzed, no significant off-target effects were found in the 15 most promising sites. By constructing Ad5F35 chimeric adenoviruses bearing CRISPR-Cas9 components, Li et al. efficiently transduced primary CD4+ T lymphocytes and disrupted CCR5 expression, and positively transduced cells acquired resistance to HIV-1.

[001089] One of skill in the art can use any of the methods set forth above, for example, Holt, N, et al.nature biotechnology 28, 839-847 (2010), Li, L., et al.Molecular therapy: the journal of the American Society of Gene Therapy 21, 1259-1269 (2013), Mandal et al.,Cell Stem Cell, Volume 15, Issue 5, p643 65g, 6 November 2014, Wang et al. (PLoS One. 2014 Dec 26;9(12):e115987. doi: 10.1371/joumal.pone.0115987), Fine et al. (Sci Rep. 2015 Jul 1.5:10777. doi: 10.1038/srep 10777) and Li et al. (J Gen Virol. 2015 Aug;96(8): 23 81-93. doi: 10.1099/vir.0.000139. Epub 2015 Apr 8) to target CCR5 using the CRISPR cas system of the present invention.

Targeting pathogens, e.g. viral pathogens such as HBV

[001090] The present invention can also be used to treat hepatitis B virus (HBV). However, the CRISPR Cas system must be adapted to avoid the disadvantages of using RNAi, such as the risk of oversaturating endogenous small RNA pathways, for example, by optimizing dose and sequence (see, Grimm et al., Nature vol. 441, 26 May 2006). For example, low doses are contemplated, such as about 1-10×10^fourteen particles per person. In another embodiment, the HBV-targeted CRISPR Cas system can be administered in liposomes, such as a stable nucleic acid-lipid particle (SNALP) (see, e.g., Morrissey et al., Nature Biotechnology, Vol. 23, No, 8, August 2005). Daily intravenous injections of approximately 1, 3, or 5 mg/kg/day of CRISPR Cas targeting HBV RNA in SNALP are contemplated. Daily treatment may last more than three days and then continue weekly for about five weeks. In another embodiment, the system of Chen et al. (Gene Therapy (2007) 14, 11-19) can be used and/or adapted for the CRISPR Cas system of the present invention. Chen et al. used a double-stranded pseudotyped vector based on adeno-associated virus 8 fdsAAV2/8) for shRNA delivery. A single introduction of the dsAAV2/8 vector (1×10¹² vector genomes per mouse), carrying HBV-specific shRNA, effectively suppressed sustained levels of HBV protein, mRNA, and replicative DNA in the liver of HBV transgenic mice, resulting in a decrease in HBV load up to 2–3 Log_ten in the bloodstream. Significant suppression of HBV was maintained for at least 120 days after the introduction of the vector. The therapeutic effect of shRNA was dependent on the target sequence and did not include interferon activation. For the present invention, an HBV-targeted CRISPR Cas system can be cloned into an AAV vector, such as the dsAAV2/8 vector, and can be administered to a human, for example, at a dose of about 1 x 10^fifteen vector genomes up to about 1x10⁶ vector genomes per person. In another embodiment, the method of Wooddell et al. (Molecular Therapy, vol. 21, no 5, 973-985, May 2013) can be used and/or adapted to the CRISPR Cas system of the present invention, Woodell et al. show that a simple co-injection of a hepatocyte-targeted N-acetylgalactosamine-linked melittin-like peptide (NAG-MLP) with a liver-tropic cholesterol-conjugated siRNA (chol-siRNA) targeting coagulation factor VII (F7) results in effective knockdown F7 in mice and non-human primates with no change in clinical chemistry or cytokine induction. Using transient and transgenic mouse models for HBV infection, Wooddell et al. showed that a single co-injection of NAG-MLP with potent chol siRNAs targeting conserved HBV sequences resulted in long-term repression of viral RNA, proteins, and viral DNA of several orders of magnitude. In the present invention, intracerebral co-injections of, for example, about 6 mg/kg of NAG-MLP and 6 mg/kg of HBV-specific CRISPR Cas can be contemplated. Alternatively, about 3mg/kg NAG-MLP and 3mg/kg HBV-specific CRISPR Cas can be delivered on the first day, followed by about 2-3mg/kg NAG-MLP and 2-3mg/kg HBV-specific CRISPR Cas in two weeks.

[001091] Lin et al.(Mol Ther Nucleic Acids. 2014 Aug 19;3:el86. doi: 10.1Q38/mtna.2014.38) developed eight anti-HBV genotype A gRNAs. Using HBV-specific gRNAs, the CRISPR-Cas9 system significantly reduced the production of HBV core and surface proteins in Huh-7 cells transfected with an HBV expression vector. Among the eight screened gRNAs, two effective ones were identified. One gRNA targeting a conserved HBV sequence acted against different genotypes. Using a model of HBV hydrodynamic persistence in mice, Lin et al. further demonstrated that this system can cleave an intrahepatic HBV genome-containing plasmid and promote its purificationin vivo, resulting in a decrease in serum surface antigen levels. These data suggest that the CRISPR-Cas9 system can disrupt HBV-expressing matrices asin vitro, andin vivo, indicating its potential to completely cure persistent HBV infection.

[001092] Dong et al. (Antiviral Res., 2015, 118: 110-7. Doi: 10.1016/j.antiviral.2015.03.015, Epub 2015 Apr 3) used the CRISPR-Cas9 system to target the HBV genome and effectively inhibit HBV infection. Dong et al. synthesized four single guide RNAs directed to conserved regions of HBV. Expression of these guide RNAs from Cas9 reduced viral production in Huh7 cells as well as in the HBV replication cell HepG2.2.15. Dong et al. also demonstrated that direct cleavage by CRISPR-Cas9 and cleavage-mediated mutagenesis occurred in HBV ccc-DNA of transfected cells. In a mouse model carrying HBV cccDNA, tail vein injection of RNA-Cas9 targeting plasmids resulted in low levels of HBV cccDNA and protein.

[001093] Liu et al. (J Gen Virol, 2015 Aug 96 (8): 2252-61. Doi: 10.1099/vir.0.000159. Epub 2015 Apr 22) developed eight guide RNAs (gRNAs) that targeted conserved regions of different HBV genotypes, which could significantly inhibit HBV replication asin vitro, andin vivoto explore the possibility of using the CRISPR-Cas9 system to disrupt HBV DNA templates. The HBV-specific gRNA/C2c1 or /C2c3 system could inhibit the replication of HBV of different genotypes in cells, and the level of viral DNA was significantly reduced by the gRNA/C2c1 or /C2c3 system alone and eliminated by the combination of different gRNA/C2c1 or /C2c3 systems.

[001094] Wang et al. (World J Gastroenterol, 2015, Aug 28, 21 (32): 9554-65. Doi: 10.3748/wjg.v21.i32.9554) developed 15 anti-HBV genotypes A-D gRNA. Were selected 11 combinations of the two above gRNA (double gRNA), covering the regulatory region of HBV. The efficacy of each gRNA and 11 double gRNAs in inhibiting HBV replication (genotypes A-D) was examined by quantitation of HBV surface antigen (HBsAg) or antigen (HBeAg) in culture supernatant. HBV expression vector degradation was examined in HuH7 cells co-transfected with double gRNA and HBV expression vector using polymerase chain reaction (PCR) and sequencing method, and cccDNA destruction was examined in HepAD38 cells using KCl precipitation, safe for plasmids ATP-dependent DNase digestion (PSAD), rolling ring amplification, and a combined quantitative PCR method. The cytotoxicity of these gRNAs was assessed by mitochondrial tetrazolium assay. All gRNAs could significantly reduce the synthesis of HBsAg or HBeAg in the culture supernatant, which depends on the region targeted by the gRNA. All double gRNAs could effectively suppress the production of HBsAg and/or HBeAg of HBV genotypes A-D, and the effectiveness of double gRNAs in suppressing the production of HBsAg and/or HBeAg was significantly increased compared to a single gRNA. In addition, by direct PCR sequencing, the inventors confirmed that these double gRNAs can specifically destroy the HBV expression template by removing a fragment between the cleavage sites of the two gRNAs used. Most importantly, quantitative gRNA-5 and gRNA-12 could not only effectively suppress the production of HBsAg and/or HBeAg, but also destroy ccc-DNA reservoirs in HepAD38 cells.

[001095] Karimova et al.(Sci Rep. 2015 Sep 3;5:13734. doi: 10.1038/srepl3734) identified cross-genotypic HBV conserved sequences in the S and X regions of the HBV genome that were specifically and efficiently targeted for nickase Cas9 cleavage. This approach disrupted not only episomal ccc-DNA and chromosomal-integrated HBV target regions in reporter cell lines, but also HBV replication in chronically andde novo infected hepatoma cell lines.

[001096] A person skilled in the art can use any of the above studies, for example, Lin et al.(Mol Ther Nucleic Acids, 2014 Aug 19;3:e186, doi: 10.1038/mtna.2014.38), Dong et al. (Antiviral Res. 2015 Jun;l 18:110-7. doi: 10.1016/j.antiviral.2015.03.015. Epub 2015 Apr 3), Liu et al. (J Gen Virol. 2015 Aug;96(8):2252-61. doi: 10.1099/vir.0.000159. Epub 2015 Apr 22), Wang et al. (World J Gastroenterol. 2015 Aug 28;21(32):9554-65. doi: 10.3748/wig.v21.132.9554) and Karimova et al. (Sci Rep. 2015 Sep 3;5:13734. doi: 10.1038/srepl3734) to target HBV with the CRISPR cas system of the present invention.

[001097] Chronic infection with hepatitis B virus (HBV) is common, fatal, and rarely cured due to the persistence of viral episomal DNA (cccDNA) in infected cells. Ramanan et al. (Ramanan V, Shiomai A, Cox DB, Schwartz RE, Michailidis E, Bhatta A, Scott IDA, Zhang F, Rice CM, Bhatia SN, .Sci Rep. 2015 Jun 2, 5:10833. doi: 10.1038/srep10833, published online on June 2, 2015) showed that the CRISPR/Cas9 system can specifically target and cleave conserved regions in the HBV genome, resulting in robust downregulation of viral gene expression and replication. With stable expression of Cas9 and appropriately selected guide RNAs, they demonstrated cleavage of cccDNA by Cas9 and a sharp decrease in both the level of cccDNA and other expression and replication parameters of viral genes. Thus, they showed that direct targeting of viral episomal DNA is a new therapeutic approach to control the virus and possibly cure patients. This is also described in WO2015089465 Al, from Tire Broad Institute et al., the contents of which are incorporated herein by reference.

[001098] Thus, targeting viral DNA in HBV is preferred in some embodiments of the invention.

[001099] The present invention can also be applied to the treatment of pathogens, such as bacterial, fungal and parasitic pathogens. Most research efforts have been focused on the development of new antibiotics that, once developed, are nonetheless subject to the same resistance development problems. The invention provides new CRISPR-based alternatives that overcome these difficulties. In addition, unlike existing antibiotics, CRISPR-based treatments can be modeled to be pathogen-specific and induce bacterial cell death in the target pathogen, but have no effect on beneficial bacteria.

[001100] The present invention can also be used to treat hepatitis C virus (HCV). The methods of Roelvinki et al. can be applied to the CRISPR-Cas system. (Molecular Therapy vol. 20 no. 9, 1737-1749 Sep 2012). For example, an AAV vector such as AAV8 may be a putative vector and a dosage of about 1.25×10 ¹¹ -1.25×10 ¹³ vector genomes per kilogram of body weight may be provided. The present invention can also be applied to the treatment of pathogenic microorganisms, such as bacteria, fungi and parasites. Most research efforts have been focused on the development of new antibiotics that, once developed, are nonetheless subject to the same resistance development problems. The invention provides new CRISPR-based alternatives that overcome these difficulties. In addition, unlike existing antibiotics, CRISPR-based treatments can be modeled to be pathogen-specific and induce bacterial cell death in the target pathogen, but have no effect on beneficial bacteria.

Jiang et al. ("RNA-guided editing of bacterial genomes using CRISPR-Cas systems," Nature Biotechnology vol. 31, p. 233-9, March 2013) used the CRISPR-Cas9 system to mutate or kill S. pneumoniae and E. coli. This work, which represents precise mutations in genomes, is based on a double cleavage of the RNA- and Cas9-targeted region of the genome to ensure non-mutant cell death and bypass the need for choice of markers or anti-selection systems. CRISPR systems can be used to promote the regression of drug resistance and eliminate the transfer of resistance between strains. Bickard et al. showed that Cas9, reprogrammed to target virulence genes, killed virulent, but not avirulent, S. aureus . By reprogramming the nuclease for the target antibiotic resistance genes, disruption of staphylococcal plasmids that contained antibiotic resistance genes and immunized against the spread of plasmid resistance genes was carried out (see Bikard et al, "Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials," Nature Biotechnology vol 32, 1146-1150, doi: 1Q.1038/nbt.3043, published online 05 October 2014). Bikard has shown that CRISPR-Cas9-based antimicrobials function to kill S. aureus in a mouse skin colonization model. Similarly, Yosef et al. used CRISPR to target genes that code for enzymes) conferring resistance to β-lactam antibiotics (see Yousef et al., "Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria," Proc. Nail Acad. Sci. USA, vol. 112, pp. 72.67-72.72, doi: 10.1073/pnas. 1500107112, published online May 18, 2015).

[001075] CRISPR systems can be used to edit the genomes of parasites that are resistant to other genetic approaches. For example, the CRISPR-Cas9 system has been shown to introduce a double-strand break into the Plasmodium yoelii genome (see, Zhang et al., "Efficient Editing of Malaria Parasite Genome Using the CRISPR/Cas9 System," mBio. vol. 5, e01414-14 , Jul-Aug 2014). Gorbal et al. ("Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system," Nature Biotechnology, vol. 32., p. 819-821, doi: 10.1038/nbt.2925, published online June 1, 2014) was modified sequences of two genes, orc1 and kelch13, which are thought to be involved in gene silencing and emerging artemisinin resistance, respectively. Parasites that were modified at the appropriate sites recovered with very high efficiency, despite the lack of direct selection for modification, indicating that neutral or even deleterious mutations can be introduced using this system. CRISPR-Cas9 is also used to modify the genomes of other pathogenic parasites, including Toxoplasma gondii (see Shen et al., "Efficient gene disruption in diverse strains of Toxoplasma gondii, using CRISPR/CAS9," mBio vol. 5:e01114-14, 2014 and SidiJk et al., "Efficient Genome Engineering of Toxoplasma, gondii Using CRISPR/Cas9," PLoS One vol. 9, el00450, doi: 10.1371/joumal.pone.0100450, published online June 27, 2014).

[001076] Vyas et al (" A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families," Science Advances, vol. 1, el500248, DOI: 10.1126/seiadv.1500248, April 3, 2015) used the CRISPR system to overcoming long standing hurdles in C. albicans genetic engineering and effectively mutated both copies of several different genes in one experiment. In an organism where multiple mechanisms contribute to drug resistance, Vyas generated homozygous double mutants that no longer exhibited the fluconazole or cycloheximide hyperresistance exhibited by the Can90 parent clinical isolate. Vyas has also generated homozygous loss-of-function mutations in major C. albicans genes by creating conditional alleles. Null alleles of DOR1, which is required for ribosomal RNA processing, are lethal at low temperature but viable at high temperature. Vyas used a recovery template that introduced a nonsense mutation and isolated the dcr1/dcr1 mutants that did not grow at 16°C.

Treatment of diseases with genetic or epigenetic aspects

[001104] The CRISPR-Cas systems of the present invention can be used to correct genetic mutations that have previously been attempted with limited success using TALEN and ZFN and have been identified as potential targets for Cas9 systems, including, as in published applications by EditasMedicine, describing ways to use Cas9 to target loci to cure diseases with gene therapy, including WO 2015/048577 CRISPR-RELATED METHODS AND COMPOSITIONS Gluckmannet al, WO 2015/070083 CRISPR-RELATED METHODS AND COMPOSITIONS WITH GOVERNING gRNAS, Glucksmann et al. In some embodiments, the treatment, prevention, or diagnosis of primary open glaucoma (POAG) is contemplated. The target is preferably the MYOC gene. This is described in WO2015153780, the contents of which are incorporated herein by reference.

[001105] Mentioned in WO2015/134812 CRISPR/CAS-RELATED METHODS AND COMPOSITIONS FOR TREATING USHER SYNDROME AND RETINITIS PIGMENTOSA Maeder et al. In accordance with the ideas described in the present description, the invention covers the methods and materials of these documents, used in combination with the ideas described in the present description. In the aspect of ocular and auditory gene therapy, methods and constructs for the treatment of Usher's syndrome and retinitis pigmentosa can be adapted to the CRISPR-Cas system of the present invention (see, for example, WO02015/134812). In one embodiment, the invention of WO 2015/134812 includes the treatment or delay of the onset or progression of Usher syndrome type IIA (USH2A, USHIIA) and retinitis pigmentosa 39 (RP39) by gene editing, for example, using CRISPR-Cas9-mediated methods to correct the deletion of guanine at position 2299 in the USH2A gene (eg, replacement of a deleted guanine residue at position 2299 of the USH2A gene). A similar effect can be achieved with C2c1 or C2c3. In a related aspect, mutation targeting is by cleavage with one or more nucleases, one or more nicases, or a combination thereof, e.g., to induce HDR with a donor template that corrects for a point mutation (e.g., a single nucleotide, e.g., a deletion of guanine). Alteration or correction of the mutant USH2A gene may be mediated by any mechanism. Exemplary mechanisms that may be associated with altering (e.g., correcting) a mutant USH2A gene include, but are not limited to, non-homologous end-junction, microhomology-mediated end-junction (MMEJ), homology-related repair (e.g., mediated by endogenous donor matrix) , SDSA (synthesis dependent strand annealing), single strand annealing or single strand invasion. In one embodiment, the method used to treat Usher syndrome and retinitis pigmentosa may include collecting data on the mutation present in the individual, for example, by sequencing the appropriate portion of the USH2A gene.

[001106] WO 2015/138510 is also referenced and in accordance with the ideas described herein, the invention (using the CRISPR-Cas9 system) provides for the treatment or delay of the onset or progression of Leber congenital amarose 10 (LCA 10). LCA 10 is caused by a mutation in the CEP290 gene, for example, c.2991+1655, an adenine to guanine substitution in the CEP290 gene, which leads to the introduction of a hidden splicing site in intron 26. It is a mutation at nucleotide 1655 of CEP290 intron 26, for example, substitution A on G. CEP290 also known as: CT87; MKS4; POC3; rd16, BBS14; JBTS5; LCAJO; NPHP6; SLSN6 and 3H11Ag (see, for example, WO 2015/138510). In an aspect of gene therapy, the invention includes introducing one or more breaks near the location of the LCA target (eg, c.2991+1655, A to G change) in at least one allele of the CEP290 gene. A change in LCA10 target position means (1) gap-induced insertion-deletion insertion (also referred to herein as NHEJ-mediated insertion-deletion insertion) in the immediate vicinity or at a given LCA10 position (e.g., C.2991+1655A-G) or (2 ) a break-induced deletion (also referred to herein as an NHEJ-mediated deletion) of a genomic sequence involving a mutation at a given position in LCA10 (eg, C.2991+1655A-G). Both approaches result in the loss or destruction of the hidden splicing site as a result of mutation at a given position of LCA 10.

[001107] Researchers are considering using gene therapy to treat a wide range of diseases. CRISPR systems of the present invention based on C2c1 or C2c3 effector protein are provided for such therapeutic purposes, including, but not limited to, additional illustrative target regions and delivery methods as shown below. Some examples of conditions or diseases that can be effectively treated using the present system are included in the exemplary genes, and references are also included herein and currently associated with these conditions. The genes and conditions presented are not exhaustive.

Treatment of diseases of the circulatory system

[001108] The present invention also provides for the delivery of the CRISPR-Cas system, in particular the novel CRISPR protein systems described herein, to blood cells or hematopoietic stem cells. Plasma exosomes according to Wahlgren et al. (Nucleic Acids Research, 2012, Vol. 40, No. 17 e130) have been described previously and can be used to deliver the CRISPR Cas system to the blood. The nucleic acid targeting system of the present invention is also envisaged for the treatment of hemoglobinopathies such as thalassemia and sickle cell anemia. See, for example, International Patent Publication No. WO 2013/126794 for potential targets that can be targeted by the CRISPR Cas system of the present invention.

[001109] In Drakopoulou, "Review Article, The Ongoing Challenge of Hematopoietic Stem Cell-Based Gene Therapy for β-Thalassemia", Stem Cells International, Volume 2011, Article ID 987980, 10 pages, doi: 10.4061/2011/987980, included herein, by reference, together with the documents cited therein, as if they were set forth in their entirety, HSC modification using a lentivirus that delivers a gene for β-globin or γ-globin is described. In contrast to using a lentivirus, using the knowledge in the art and the ideas described herein, the skilled person can correct the HSC for β-thalassemia using a CRISPR-Cas system that targets and corrects the mutation (e.g., with a suitable HDR matrix , which delivers the coding sequence for β-globin or γ-globin, preferably non-sickle β-globin or γ-globin); in particular, a guide RNA can target a mutation that results in β-thalassemia, and HDR can provide coding for proper expression of β-globin or γ-globin. The guide RNA that targets the mutation and the particle containing the Cas protein is brought into contact with the HSC carrying the mutation. The particle may also contain an appropriate HDR template to correct mutations for correct expression of β-globin or γ-globin; or the HSC may be contacted with a second particle or vector that contains or delivers an HDR array. Cells brought into contact in this manner can be administered and optionally processed/upscaled, see Cartier. Mentioned in this connection is Cavazzana, "Outcomes of Gene Therapy for β-Thalassemia Major via Transplantation of Autologous Hematopoietic Stem Cells Transduced Ex Vivo with a Lentiviral β ^A-T87Q -Globin Vector." tif2014.org/abstractFiles/Jean%20Antoine%20Ribeil. abstract.pdf; Cavazzana-Calvo, "Transfusion independence and HMGA2 activation after gene therapy of human β-thalassemia", Nature 467, 318-322 (16 September 2010) doi:10.1038/n.nature09328; Nienhuis, "Development of Gene Therapy for Thalassemia, Cold Spring Harbor Perpsectives in Medicine, doi: 10.n01/cshperspect.a011833 (2012), LentiGlobin BB305, a lentiviral vector containing an engineered β-globin gene (PA-T87Q); and Xie et al., "Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyback" Genome Research gr.1713427.114 (2014) http://www.genome.org/cgi/doi/10.1101/ gr.173427.114 (Cold Spring Harbor Laboratory Press), which are the subject of Cavazzana's work on human β-thalassemia and the subject of Xie's work, and are incorporated herein by reference with all documents cited therein or related thereto. Within the scope of the present invention, an HDR template can be provided by an HSC to express an engineered β-globin gene (eg, βA-T87Q) or β-globin according to Xie.

[001110] Xu et al. (SciRep. 2015 Jul9;5:12065. doi: 10.1038/srepl2065) developed TALEN and CRISPR-Cas9 to directly target the IVS2-654 intron 2 mutation site in the globin gene. Xu et al. observed different frequencies of double-strand breaks (DSBs) at the IVS2-654 loci using TALEN and CRISPR-Cas9, and TALEN mediated higher targeting efficiency for homologous genes compared to CRISPR-Cas9 in combination with the transposon donor piggyBac. In addition, more off-target events were observed for CRISPR-Cas9 compared to TALEN. Finally, TALEN-corrected iPSC clones were selected for erythroblast differentiation using an OP9 co-culture system and found relatively higher HBB transcription than uncorrected cells.

[001111] Song et al. (Stem Cells Dev. 2015 May 1;24(9): 1053-65. doi: 10, 1089/scd.2014.0347. Epub 2015 Feb 5) used CRISPR/Cas9 to patch β-Thal iPSC; gene-corrected cells showed normal karyotypes and full pluripotency because human embryonic stem cells (hESCs) showed no off-target effects. Then Song et al. evaluated the differentiation efficiency of genetically adjusted P-Thal iPSCs. Song et al. found that at hematopoietic differentiation, genetically adjusted P-Thal iPSCs showed an increased proportion of embryoid bodies and different percentages of hematopoietic progenitors. More importantly, the genetically corrected β-Thal iPSC lines restored HBB expression and reduced reactive oxygen species production compared to the uncorrected group. In a study by Song et al. it was shown that the efficiency of hematopoietic differentiation of β-Thal iPSC improved significantly after adjustment with the CRISPR-Cas9 system. Similar methods can be applied using the CRISPR-Cas systems described herein, for example, systems containing C2c1 or C2c3 effector proteins.

[001112] Sickle cell anemia is an autosomal recessive genetic disease in which red blood cells take the form of a sickle. This is caused by a single base change in the β-globin gene, which is located on the short arm of chromosome 11. As a result, valine is formed instead of glutamic acid, causing the formation of sickle-shaped hemoglobin (HbS). This leads to the formation of a distorted form of red blood cells. Because of this abnormal shape, small blood vessels can become blocked, causing serious damage to the tissues of the bone, spleen, and skin. This can lead to pain episodes, frequent infections, hand-foot syndrome, or even multiple organ failure. Distorted red blood cells are also more susceptible to hemolysis, leading to severe anemia. As with β-thalassemia, sickle cell anemia can be corrected by changing the HSC using the CRISPR-Cas system. The system allows you to specifically edit a cell's genome by cutting its DNA and then allowing it to repair itself. The Cas protein is injected and directed by the RNA to the point of mutation and then cuts the DNA at that point. Simultaneously, the normal version of the sequence is entered. This sequence is used by the proprietary repair system to close the induced incision. Thus, CRISPR-Cas makes it possible to correct the mutation in previously obtained stem cells. With knowledge of the art and the ideas described herein, the skilled artisan can correct HSC for sickle cell anemia using a CRISPR-Cas system that targets and corrects the mutation (e.g., with a suitable HDR template that provides the coding sequence for β-globin , predominantly non-sickle β-globin); in particular, the guide RNA can target a mutation that causes sickle cell anemia, and HDR can provide coding for proper expression of β-globin. A guide RNA that targets the mutation and a particle containing the Cas protein is brought into contact with the HSC carrying the mutation. The particle may also contain an appropriate HDR template to correct mutations for proper expression of β-globin; or the HSC may be contacted with a second particle or vector that contains or delivers an HDR array. Thus brought into contact cells can be introduced or optionally processed/increased in number. The HDR template can provide expression in the HSC of an engineered β-globin gene (eg, βA-T87Q) or β-globin, as in Xie.

[001113] In Williams, "Broadening the Indications for Hematopoietic Stem Cell Genetic Therapies," Cell Stem Cell 13:263-264 (2013), incorporated herein by reference with the documents cited therein, as if they were set forth fully described lentivirus-mediated gene transfer into HSC/P cells in patients with metachromatic leukodystrophy (MLD), a genetic disorder caused by arylsulfatase A deficiency (ARSA), resulting in nerve demyelination; and lentivirus-mediated gene transfer into HSCs in patients with Wiskott-Aldrich Syndrome (WAS) (patients with a defective WAS protein, an effector of the CDC42 small GTPase, which regulates cytoskeletal function in blood cell lines and thus are immunodeficient with recurrent infections, autoimmune symptoms, and thrombocytopenia with abnormally small and dysfunctional platelets leading to excessive bleeding and an increased risk of leukemia and lymphoma). Unlike using a lentivirus, using the knowledge in the art and the ideas described herein, a qualified person can correct HSC for MLD (arylsulfatase A deficiency (ARSA)) using the CRISPR-Cas system, which targets and corrects a mutation (deficiency arylsulfatase A (ARSA)) (eg, with a suitable HDR template that provides the coding sequence for ARSA); in particular, the guide RNA can target the mutation that results in MLD, and the HDR can provide coding for proper ARSA expression. A guide RNA that targets the mutation and a particle containing the Cas protein is brought into contact with the HSC carrying the mutation. The particle may also contain an appropriate HDR template to correct mutations for proper ARSA expression; or the HSC may be contacted with a second particle or vector that contains or delivers an HDR array. Thus brought into contact cells can be introduced or optionally processed/increased in number. Unlike using a lentivirus, using the knowledge in the art and the ideas described herein, the skilled artisan can correct the HSC for WAS using a CRISPR-Cas system that targets and corrects a mutation (WAS protein deficiency) (e.g., with a suitable an HDR matrix that provides the coding sequence for WAS); in particular, a guide RNA can target a mutation that results in WAS deficiency, and HDR can provide coding for proper WAS expression. A guide RNA that targets the mutation and a particle containing the Cas protein is brought into contact with the HSC carrying the mutation. The particle may also contain an appropriate HDR template to correct mutations for proper WAS expression; or the HSC may be contacted with a second particle or vector that contains or delivers an HDR array. Thus brought into contact cells can be introduced or optionally processed/increased in number.

[001114] In Watts, "Hematopoietic Stem Cell Expansion and Gene Therapy" Cytotherapy 13(10): 1164-1171. doi: 10.3109/14653249.2011.620748 (2011), incorporated herein by reference together with the documents cited therein as if they were set forth in their entirety, describes gene therapy for hematopoietic stem cells (HSCs), such as virus-mediated HSC gene therapy , as a highly attractive treatment option for many disorders, including hematological conditions, immunodeficiencies such as HIV/AIDS, and other genetic disorders such as lysosomal storage diseases, including SCID-X1, ADA-SCID, β-thalassemia, X- associated CGD, Wiskott-Aldrich syndrome, Fanconi anemia, adrenoleukodystrophy (ALD), and metachromatic leukodystrophy (MLD).

[001115] US Patent Publication Nos. 20110225664, 20110091441, 20100229252, 20090271881, and 20090222937 assigned to Cellectis refer to CREI variants wherein at least one of the two I-CreI monomers has at least two substituents, one in each of two functional subdomains of the central domain LAGLIDADG (SEQ ID NO: 26) located at positions 26-40 and 44-77 of I-CreI, respectively, said variant being capable of cleaving a target DNA sequence from the human receptor gamma chain gene (IL2RG) human, also referred to as the common cytokine receptor gamma chain or gamma C gene.

[001116] Severe combined immunodeficiency (SCID) results from a defect in T lymphocyte maturation, always associated with a functional defect in B lymphocytes (Cavazzana-Calvo et al., Annu. Rev. Med., 2005, 56, 585-602; Fischer et al. ., Immunol. Rev., 2005, 203, 98-109). The overall incidence of the disease is estimated at 1 in 75,000 births. Patients with untreated SCID who have multiple opportunistic infections with microorganisms and typically do not survive more than one year, SCID can be treated with allogeneic donation of hematopoietic stem cells from a familial donor. Donor histocompatibility can vary widely. In the case of adenosine deaminase (ADA) deficiency, a form of SCID, patients can be treated by injection of the recombinant adenosine deaminase enzyme.

[001117] Since the ADA gene was shown to be mutated in SCID patients (Giblettetah, Lancet, 1972, 2, 1067-1069), several other genes involved in SCID have been identified (Cavazzana-Calvo et al., Annu Rev Med 2005 56 585-602 Fischer et al Immunol Rev 2005 203 98-109 There are four main causes for SCID: (i) The most common form of SCID, SCID-X1 (X-linked SCID or X-SCID), is caused by a mutation in the IL2RG gene resulting in the absence of mature T lymphocytes and NK cells. IL2RG encodes the γ-C protein (Noguchi, et al., Cell, 1.993, 73, 147-157), a common component of at least five interleukin receptor complexes. These receptors activate multiple targets via the JAK3 kinase (Macchietah, Nature, 1995, 377, 65-68), inactivation of which results in the same syndrome as gamma-C inactivation; (ii) a mutation in the ADA gene results in a defect in purine metabolism that is lethal to progenitor lymphocytes, which in turn results in the near absence of B, T, and NK cells; (iii) V(D)J recombination is an essential step in the maturation of immunoglobulins and T-lymphocyte receptors (TCRs). Mutations in recombination-activating gene 1 and 2 (RAG1 and RAG2) and Artemis, three genes involved in this process, result in the absence of mature T-lymphocytes; and (iv) mutations in other genes such as CD45 involved in T-cell cascades have also been reported, although these represent a minority of cases (Cavazzana-Calvo et al., Ann. Rev. Med., 2005, 56, 585- 602, Fischer et al., Immunol, Rev., 2005, 203, 98-109). Since their genetic basis was identified, the various forms of SCID have become the paradigm for gene therapy approaches (Fischer et al., Immunol. Rev., 2005, 203, 98-109) for two main reasons. First, as with all blood diseases, ex vivo treatment may be envisaged. Hematopoietic stem cells (HSC) can be isolated from the bone marrow and can retain their pluripotent properties over several cell divisions. Therefore, they can be processed in vitro , and then reintroduced into the patient's body, where they colonize the bone marrow. Second, because lymphocyte maturation is impaired in SCID patients, the corrected cells have a selective advantage. Therefore, a small number of corrected cells can restore a functional immune system. This hypothesis has been supported several times by: (i) partial restoration of immune functions associated with mutation reversal in SCID patients (Hirschhom et al., Nat. Genet., 1996, 13, 290-295; Stephan et al., N. Engl J Med., 1996, 335, 1563-1567, Bousso et al., Proc. Natl., Acad. Set. USA, 2000, 97, 274-278, Wada et al., Proc. Natl. Acad. Set., 2001, 98, 8697-8702, Nishikomori et al., Blood, 2004, 103, 4565-4572), (ii) correcting SCTD-X1 deficiency in vitro in hematopoietic cells (Candotti et. Ah, Blood, 1996, 87, 3097 -3102, Cavazzana-Calvo et al., Blood, 1996, Blood, 88, 3901-3909; Taylor et al., Blood, 1996, 87, 3103-3107; Hacein-Bey et al., Blood, 1998, 92, 4090-4097), (iii) correction of SCID-X1 deficiency (Soudais et al., Blood, 2000, 95, 3071-3077; Tsai et al., Blood, 2002, 100, 72-79), JAK-3 (Bunting et ah, Nat. Med., 1998, 4, 58-64, Bunting et al., Hum. Gene Ther., 2000, 11, 2353-2364) and RAG2 (Yates et al., Blood, 2002, 100, 3942 -3949) in vivo animal models, and (iv) res the culmination of gene therapy clinical trials (Cavazzana-Calvo et al., Science, 2000, 288, 669-672; Aiuti et al., Nat. Med., 2002; 8, 423-425, Gaspar et al., Lancet. 2004, 364, 2181-2187).

[001118] U.S. Patent Publication No. 20110182867, assigned to the Children's Medical Center Corporation and the President and Fellows of Harvard College, relates to methods and uses for modulating fetal hemoglobin expression (HbF) in hematopoietic progenitor cells using inhibitors of BCL11A expression or activity, such as like RNAi and antibodies. Targets disclosed in US Patent Publication No. 20110182867, such as BCL11A, can be targeted by the CRISPR Cas system of the present invention to modulate fetal hemoglobin expression. See also Bauer et al. (Science 11 October 2013: vol. 342, no. 6155, pp. 253-257) and Xu et al. (Science Nov. 18, 2011: vol. 334, no. 6058 pp. 993-996) for additional BCL11A targets.

[001119] Using the knowledge in this field and the ideas described in the present description, a qualified specialist can correct HSC in relation to hematological disorders such as β-thalassemia, hemophilia or genetic lysosomal disease.

HSC delivery and editing of hematopoietic stem cells; and specific states

[001120] The term "hematopoietic stem cell" or "HSC" includes in a broad sense those cells that are considered HSC, for example, blood cells that give rise to all other blood cells and are derived from the mesoderm; located in the red marrow, which is found in the medulla of most bones, the HSCs of the invention include cells having a hematopoietic stem cell phenotype identified by small size, absence of lineage markers (lin) and markers that belong to differentiation clusters, for example: CD34, CD38 , CD90, CD133, CD105, CD45, and e-kit, the stem cell factor receptor. Hematopoietic stem cells are negative for markers that are used to detect lineage adherence and are thus called Lin- and contain up to 14 different mature blood markers during FACS purification, e.g. CD13 and CD33 for myeloid cells, CD71 for erythroid cells , CD19 for B cells, CD61 for megakaryocytes, etc. for humans, and B220 (mouse CD45) for B cells, Mac-1 (CD11b/CD18) for monocytes, Gr-1 for granulocytes, Ter119 for erythroids, IL7Ra, CD3, CD4, CD5, CD8 for T cells, etc. Mouse HSC markers: CD34lo/-, SCA-1+, Thy1.1+/lo, CD38+, C-kit+, lin- and human HSC markers : CD34+, CD59+, Thyl/CD90+, CD38lo/-, C-kit/CD117+ and lin-. HSCs are identified by markers. Therefore, in the embodiments discussed herein, the HSCs may be CD34+ cells. HSCs can also be hematopoietic stem cells that are CD34-/CD38-. Stem cells that may lack c-kit on the cell surface, which are considered in this field as HSC, are included in the scope of the invention, as well as CD133+ cells, which are similarly considered as HSC in this field.

[001121] The CRISPR-Cas system (eg, C2c1 or C2c3) can be designed to target the genetic locus or loci in the HSC. It is possible to obtain a Cas protein (e.g. C2c1 or C2c3) predominantly codon-optimized for a eukaryotic cell and especially a mammalian cell, e.g. a human cell, HSC, and an sgRNA targeting a locus or loci in the HSC, e.g. the EMX1 gene. They can be delivered via particles. The particles may be formed by a Cas protein (eg C2c1 or C2c3) mixed with gRNA. A mixture of gRNA and Cas protein can be mixed, for example, with a mixture containing or essentially consisting of a surfactant, a phospholipid, a biodegradable polymer, a lipoprotein and an alcohol, whereby particles containing gRNA and Cas (for example, C2c1 or C2c3) can be formed. . The invention provides for obtaining particles in this way and particles obtained in this way, as well as their use.

[001122] More generally, particles can be formed using an efficient process. First, the Cas protein (e.g., C2c1 or C2c3) and the gRNA targeting the EMX1 gene or the LacZ control gene can be mixed together in a suitable molar ratio, e.g., 3:1 to 1:3 or 2:1 to 1:2 or 1:1, at a suitable temperature, e.g. 15-30°C, 20-25°C, room temperature, for a suitable time period, e.g. 15-45, 30 minutes, preferably in sterile, nuclease-free buffer, eg 1X PBS. Separately, particle components such as or containing: a surfactant, eg a cationic lipid, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); a phospholipid such as dimyristoylphosphatidylcholine (DMPC); a biodegradable polymer such as an ethylene glycol or PEG polymer and a lipoprotein such as low density lipoprotein such as cholesterol can be dissolved in an alcohol, preferably a C1-6 alkyl alcohol such as methanol, ethanol, isopropanol, such as 100% ethanol. These two solutions can be mixed together to form particles containing Cas (eg C2c1 or C2c3)-gRNA complexes. In some embodiments, the particle may comprise an HDR array. This may be a particle co-administered with a protein-containing gRNA+Cas particle (eg, C2c1 or C2c3) or, in addition to contacting the HSC with a protein-containing gRNA+Cas particle (eg, C2c1 or C2c3), the HSC is contacted with a particle containing an HDR template; or the HSC is contacted with a particle containing all of the gRNA, Cas (eg C2c1 or C2c3) and HDR template. The HDR template can be introduced with a separate vector, as a result of which, in the first case, the particle enters the HSC cell, and the separate vector also enters the cell where the HSC genome is modified by gRNA + Cas (for example, C2c1 or C2c3) and the HDR template is also present, in which, for example, genomic loci are modified by HDR, which can result in the mutation being corrected.

[001123] Once formed, HSC particles in 96-well plates can be transfected with 15 μg of Cas protein (eg, C2c1 or C2c3) per well. HSCs can be harvested three days after transfection and the number of insertions and deletions (insertions-deletions) at the EMX1 locus can be determined.

[001124] This illustrates how HSCs can be modified using CRlSPR-Cas (eg, C2c1 or C2c3) targeted at the genomic locus or loci of interest in the HSC. The HSCs to be modified can be in vivo , i.e. in the body of e.g. humans or other eukaryotes, e.g. animals such as fish (zebrafish), mammals, in particular primates (chimpanzees, macaques), rodents (mice, rabbits, rats) dogs, livestock, birds. The HSCs to be modified may be in vitro , that is, outside of such an organism. Further, modified HSCs can be used ex vivo , i.e. one or more HSCs of such an organism can be obtained or isolated from the body, possibly the HSCs can be increased in number, the HSCs are modified with a construct including CRISPR-Cas (e.g. C2c1 or C2c3), that target the genetic locus or loci in the HSC, for example, by contacting the HSC with a construct, for example, where the latter contains a particle containing the CRISPR enzyme and one or more gRNA that targets the genetic locus or loci in the HSC, such as the particle obtained or obtained by mixing a mixture of gRNA and Cas protein (e.g., C2c1 or C2C3) with a mixture containing or essentially consisting of a surfactant, a phospholipid, a biodegradable polymer, a lipoprotein, and an alcohol (wherein one or more gRNAs are targeted to a genetic locus or loci into HSCs), optionally increasing the amount of modified HSCs produced and introducing the resulting modified HSCs into the body. In some cases, the isolated or derived HSCs may be from a first organism, such as an organism of the same species as the second organism, and the second organism may be an organism to which the resulting modified HSCs are administered, e.g., the first organism may be a donor (such as a relative (parent or sibling)) for the second organism. The modified HSCs may be genetically modified to eliminate or alleviate or reduce the symptoms of a disease or condition in an individual, subject or patient. Modified HSCs, such as in the case of donation of a first organism to a second one, may have genetic modifications so that the HSCs have one or more proteins, such as surface markers or proteins more similar to those of the second organism. Modified HSCs may have genetic modifications to mimic a disease or condition in an individual or subject or patient, and may be re-introduced into a non-human organism to produce animal models. Increasing the amount of HSC is within the skill of the skilled artisan based on the present description and knowledge in the art, see, for example, Lee, "Improved ex vivo expansion of adult hematopoietic stem cells by overcoming CUL4-mediated degradation of HOXB4." Blood. 2013 May 16;121(20):4082-9. doi: 10.1182/blood-2012-09-455204. Epub 2013 Mar 21.

[001125] As indicated, to improve activity, the gRNA can be pre-complexed with the Cas protein (eg, C2c1 or C2c3) before being fully complexed in the particle. Formulations can be formulated from various molar ratios of various components known to assist in the delivery of nucleic acids to cells (e.g., 1,2-dioleyl-3-trimethylammonium-propane (DOTAP) or 1,2-ditradecanoyl- sn -3-phosphocholine (DMPC), and/or in which the hydrophilic polymer contains ethylene glycol or polyethylene glycol (PEG) and/or in which the particle further contains cholesterol). For example, a particle of the composition DOTAP 100, DMPC 0, PEG 0, cholesterol 0; or DOTAP 90, DMPC 0, PEG 10, cholesterol 0; or DOTAP 90, DMPC 0, PEG 5, cholesterol 5, or DOTAP 100, DMPC 0, PEG 0, cholesterol 0. Accordingly, the invention provides for the addition of gRNA, Cas protein (e.g., C2c1 or C2c3), and particle-forming components, and particles resulting from this addition.

[001126] In a preferred embodiment, particles containing Cas (e.g., C2c1 or C2c3) gRNA complexes can be formed by mixing a Cas protein (e.g., C2c1 or C2c3) and one or more gRNAs, preferably in a molar ratio of enzyme and guide RNA equal to 1:1. Separately, the various components known to aid delivery of nucleic acids (eg DOTAP, DMPC, PEG and cholesterol) are dissolved, preferably in ethanol. The two solutions are mixed together to form particles containing Cas complexes (eg C2c1 or C2c3) with gRNA. After particle formation, Cas complexes (eg C2c1 or C2c3) with gRNA can be transfected into cells (eg HSC). Barcoding may be applied. Particles, Cas- and/or gRNA can be barcoded.

[001127] The invention in one of the embodiments provides a method for obtaining a particle containing a Cas protein (for example, C2c1 or C2c3) and gRNA, including mixing a mixture of gRNA and Cas protein (for example, C2c1 or C2c3) with a mixture containing or essentially consisting of surfactants, phospholipids, biodegradable lipoprotein and alcohol. The invention relates to a particle containing a Cas protein (eg C2c1 or C2c3) and gRNA according to this method. The invention in one embodiment provides for the use of a particle in a method of modifying a genomic target locus, or a non-human organism, by manipulating a target sequence at the genomic target locus, including bringing a cell containing the genomic target locus to particle contact, where the gRNA targets the genomic target locus; or a method of modifying a genomic target locus, or an organism, or a non-human organism by manipulating a target sequence at the genomic target locus, including bringing a cell containing the genomic target locus into contact with a particle wherein the gRNA is targeted to the genomic target locus. In these embodiments, the target genomic locus is predominantly the genomic locus in the HSC.

[001128] Therapeutic considerations: A consideration in gene editing therapy is the choice of a sequence-specific nuclease, such as a C2c1 or C2c3 nuclease variant. Each nuclease variant may have its own unique set of advantages and disadvantages, many of which must be balanced in the context of treatment to maximize therapeutic efficacy. So far, two therapeutic approaches to nuclease editing have shown significant promise: gene disruption and gene editing. Gene disruption involves stimulating non-homologous end joining (NHEJ) repair to create directed insertions-deletions in genetic elements, often resulting in loss-of-function mutations that are beneficial to patients. In contrast, gene repair uses homology-directed repair (HDR) to directly reverse the disease-causing mutation to restore function while maintaining the physiological regulation of the corrected element. Homology-guided repair can also be used to insert a therapeutic transgene at a specific safe harbor locus in the genome to restore lost gene function. For specific editing therapy to be effective, a sufficiently high level of modification must be achieved in the target cell population to reverse the symptoms of the disease. This "threshold" of therapeutic modification is determined by the survival of the edited cells after treatment and the amount of gene product needed to reverse the symptoms. Considering cell survival, editing creates three potential outcomes for treated cells relative to their unedited counterparts: increased survival, reduced survival, and no effect on survival. In the case of increased survival, for example in the treatment of X-linked severe immunodeficiency, modified hematopoietic progenitor cells are selectively increased in number compared to their unedited counterparts. X-linked severe immunodeficiency is a disease caused by mutations in the IL2RG gene, the functioning of which is necessary for the proper development of the hematopoietic lymphocytic lineage [Leonard, WJ, et al. Immunological reviews 138, 61-86 (1994); Kaushansky, K. & Williams, WJ Williams hematology, (McGraw-Hill Medical, New York, 2010)]. In clinical trials, patients treated with viral gene therapy for X-linked severe immunodeficiency in a rare instance of spontaneously corrected mutation associated with X-linked severe immunodeficiency, corrected hematopoietic progenitor cells can overcome this developmental block and proliferate, compared to their unedited counterparts. , contributing to the treatment [Bousso, P., et al. Proceedings of the National Academy of Sciences of the United States of America 97, 274-278 (2000); Hacein-Bey-Abina, S, et al. The New England journal of medicine 346, 1185-1193 (2002); Gaspar, HB, et al. Lancet 364, 2181-2187 (2004)]. In this case, when the edited cell has a selective advantage, even a low number of edited cells can be increased in number, providing a therapeutic benefit to the patient. In contrast, editing other hematopoietic diseases, like granulomatous disorder, may not alter the survival of edited hematopoietic progenitors, increasing the threshold for therapeutic modification. Granulomatous disorder is caused by mutations in genes encoding phagocytic oxidase proteins that are normally used by neutrophils to generate reactive oxygen species to kill pathogens [Mukherjee, S. & Thrasher, AJ Gene 525, 17-4-181 (2013)]. Since dysfunction of these genes does not affect the survival or development of hematopoietic progenitor cells, but only the ability of mature hematopoietic cells to fight infection, it is unlikely that there would be preferential expansion of edited cells in this disease. Indeed, no selective advantage was observed in edited hematopoietic progenitor cells in granulomatous disorder during gene therapy trials, leading to difficulties in long-term implantation of cells [Malech, HL, et al. Proceedings of the National Academy of the United States of America 94, 12133-12138 (1997); Kang, HJ, et al. Molecular therapy: the journal of the American Society of Gene Therapy 19, 2092-2101 (2011)]. As such, a significantly higher level of editing may be required to treat diseases such as granulomatous disorder, in which editing does not provide a survival benefit, relative to diseases in which editing provides survival benefits to target cells. If editing deprives cells of their survival advantage, as would be the case if the tumor suppressor gene was restored to function in the tumor cell, the modified cells would be crowded out by unedited cells, causing the therapeutic benefits to be lower relative to the level of editing. This last class of diseases could be particularly difficult to treat with genomic editing therapy.

[001129] In addition to cell survival, the minimum level of therapeutic genome editing that must be performed to reverse symptoms is also affected by the amount of gene product required to treat a disease. Hemophilia B is one of the diseases in which a small change in the amount of a gene product can lead to significant changes in the clinical outcome. This disease is caused by mutations in the gene encoding factor IX, a protein that is normally secreted from the liver into the blood, where it functions as a component of the blood clotting cascade. The clinical severity of hemophilia B is related to the level of factor IX activity. While disease severity is associated with activity below 1% of normal activity, milder forms of the disease are associated with activity in excess of 1% of normal factor IX activity [Kaushansky, K. & Williams, WJ Williams hematology, (McGraw-Hill Medical, New York, 2010), Lofqvist, T,, et al. Journal Of Internal Medicine 241, 395-400 (1997)]. This suggests that editing therapy, which can restore factor IX expression in even a small percentage of liver cells, could have a significant impact on clinical outcome. A study using ZFNs to correct a model of hemophilia B in mice immediately after birth showed that 3-7% is sufficient to reverse the symptoms of the disease, providing preclinical evidence for this hypothesis [Li, H., et al. Nature Alb, 217-221 (2011)].

[001130] Disorders in which a small change in the level of a gene product can affect clinical outcome, and diseases in which edited cells have a survival advantage, are ideal targets for genome editing therapy because the threshold for therapeutic modification is sufficient. low to ensure a high probability of success in today's technology environment. The use of these diseases as targets has now led to success in editing therapy at the level of preclinical studies and phase I clinical trials. Manipulations to improve double-strand break repair and nuclease delivery are needed to extrapolate these promising results to diseases in which edited cells do not have a survival advantage or in which a higher level of gene product is needed for treatment. The table below shows some examples of the application of genome editing for therapeutic models, and the references in the table below and the documents cited in these references are incorporated herein as if they were set forth in their entirety.

Type of disease Nuclease used as a platform Therapeutic strategy Links Hemophilia B ZFN Correct Sequence Insertion Mediated by HDR Li, H., et al. Nature 475, 217-221 (2011) Severe combined immunodeficiency ZFN Correct Sequence Insertion Mediated by HDR Genovese, P., et al. Nature
510, 235-240 (2014) hereditary
tyrosinemia CRISPR HDR-mediated Liver Mutation Correction Yin, H., et al. Nature biotechnology 32, 551-553 (2014)

[001131] Treatment of each of the conditions in the table using the CRISPR-Cas system (e.g., C2c1 or C2c3) with HDR-mediated correction or insertion of the correct HDR-mediated gene sequence, advantageously with the delivery system of the present invention, e.g. , particle delivery systems, is within the purview of the skilled artisan on the basis of this description and knowledge in the art. Thus, one possible embodiment involves the interaction of HSCs carrying mutations associated with hemophilia B, severe combined immunodeficiency, or hereditary tyrosinemia with a particle carrying gRNA and a Cas protein (for example, C2c1 or C2c3) directed to a genomic target locus in hemophilia B, severe combined immunodeficiency (eg, X-linked severe combined immunodeficiency, severe combined immunodeficiency associated with adenosine deaminase deficiency) or hereditary tyrosinemia (eg, as in the case of Li, Genovese or Yin). The particle may also contain a suitable HDR matrix for mutation correction; or the HSCs may interact with a second particle or vector containing or delivering a homology-directed repair template. In this regard, it is mentioned that hemophilia B is an X-linked recessive disease caused by loss-of-function mutations in the gene encoding factor IX, a key component of the blood coagulation cascade. Restoration of factor IX activity above 1% of its level in seriously ill patients can transform the disease into its much milder form, since prophylactic administration of recombinant factor IX into the body of such patients from an early age to achieve this level of activity greatly alleviates clinical complications. With the knowledge in the art and the ideas described in this application, a qualified person can correct HSC as in the case of hemophilia B using the CRISPR-Cas system (for example, C2c1 or C2c3), which directionally corrects the mutation (X-linked recessive disease caused by the causing loss of function by a mutation in the gene encoding factor IX) (e.g., using an appropriate HDR template that delivers the factor IX coding sequence), in particular, the gRNA can be directed to a mutation that causes the development of hemophilia B, and the HDR can encode the correct expression of the factor IX. The particle containing the gRNA directed to the mutation and the Cas protein (for example, C2c1 or C2c3) interacts with the HSC carrying the mutation. The particle may also contain an appropriate HDR template to correct the mutation for correct expression of factor IX, or the HSC can interact with a second particle that contains or delivers the HDR template. Cells that have interacted in this way can be injected; and optionally processed/enhanced (see the Cartier article discussed herein).

[001132] Cartier, "MINI-SYMPOSIUM: X-Linked Adrenoleukodystrophypa, Hematopoietic Stem. Cell Transplantation and Hematopoietic Stem Cell Gene Therapy in X-Linked Adrenoleukodystrophy," Brain Pathology 20 (2010) 857-862, incorporated herein in as a reference in its entirety with the documents cited therein, it states that allogeneic hematopoietic stem cell (HSC) transplantation is used to deliver a normal lysosomal enzyme to the brain of a patient with Hurler's disease, and discusses HSC-assisted gene therapy for the treatment of adrenoleukodystrophy. In two patients, peripheral CD34+ cells were harvested after mobilization with granulocyte-macrophage colony-stimulating factor (G-CSF) and transduced with a myeloproliferative sarcoma virus enhancer, the downregulation site was removed, and the dl587rev primer-binding site was replaced in the (MND)-ALD lentiviral vector. The patients' CD34+ cells were transduced with a (MND)-ALD vector for 16 hours in the presence of a low concentration of cytokines. The transduced CD34+ cells were frozen after transduction to allow various safety tests to be carried out on 5% of the cells, which included, in particular, three assays for lentivirus replication competence. The transduction efficiency of CD34+ cells ranged from 35% to 50% with an average number of integrated lentivirus copies between 0.65 and 0.7. After thawing of transduced CD34+ cells, patients were injected with more than 4.106 transduced CD34+ cells/kg, followed by myeloablation using bisulfan or cyclophosphamide. The patient's HSCs were removed to improve the engraftment of gene corrected HSCs. Hematological recovery in two patients occurred between 13 and 15 days. Almost complete immunological recovery occurred after 12 months in the case of the first patient and after 9 months in the case of the second patient. In contrast to the use of a lentivirus, the skilled artisan, based on the knowledge in the art and the teachings described herein, can correct HSCs in adrenoleukodystrophy using a CRISPR-Cas system (eg, C2c1 or C2c3) that specifically corrects the mutation (eg, with a suitable homology-guided reparation matrix); in particular, gRNA can target mutations in the ABCD1 gene located on the X chromosome, which encodes ALD, a lysosomal membrane transport protein, and the HDR template can encode correct protein expression. A particle containing the gRNA targeted for mutation and a Cas protein (eg C2c1 or C2c3) interacts with HSCs carrying the mutation, eg CD34+ cells carrying the mutation, according to Cartier. The particle may also contain an appropriate HDR template to correct the mutation for correct expression of the lysosomal membrane transport protein, or the HSC may interact with a second particle or vector that either contains or delivers the HDR template. Cells thus interacted can be processed according to Cartier. Cells thus interacted can be injected according to Cartier.

[001133] WO 2015/148860 is referenced, and through the teachings described herein, the invention relates to the methods and materials of this document applicable in conjunction with the teachings described herein. In the aspect of gene therapy for blood disease, methods and compositions for the treatment of β-thalassemia can be adapted to the CRISPR-Cas systems of the present invention (see, for example, WO 2015/148860). In one possible embodiment, WO 2015/148860 includes the treatment or prevention of β-thalassemia or its symptoms, for example, by altering the B-cell CLL/lymphoma 11A (BCL11A) gene. The BCL11A gene is also known as the B-cell CLL/lymphoma 11A gene, BCLI1A-L, BCLI1A-S, BCL11AXL, CTIP1, HBFQTL5 and ZNF. BCLI1A encodes a zinc finger motif protein that is involved in the regulation of globin gene expression. By changing the BCL11A gene (for example, one or both alleles of the BCL11A gene), it is possible to increase the level of γ-globin. γ-globin can replace β-globin in the hemoglobin complex and effectively deliver oxygen to tissues, thus improving the phenotype characteristic of β-thalassemia.

[001134] WO 2015/148863 is also referenced, and through the teachings described herein, the methods and materials of this document are provided that can be adapted to the CRISPR-Cas systems of the present invention. In one of the possible aspects of the treatment and prevention of sickle cell anemia, which is an inherited blood disease, WO 2015/148863 provides for a change in the BCL11A gene. By changing the BCL11A gene (for example, one or both alleles of the BCLI 1A gene), the level of γ-globin can be increased. γ-globin can replace β-globin in the hemoglobin complex and effectively deliver oxygen to tissues, thus improving the sickle cell phenotype.

[001135] In one possible aspect of the invention, methods and compositions are provided, comprising editing a target nucleic acid sequence or modulating the expression of a target nucleic acid sequence, and using them in connection with cancer immunotherapy by adapting the CRISPR-Cas system of the present invention. Reference is made to the use of gene therapy in WO 2015/161276, which includes methods and compositions that can be used to affect T cell proliferation, survival and/or function by altering one or more genes expressed in T cells, for example, CBLB and/or PTPN6 genes, FAS and/or BID, CTLA4 and/or PDCDI and/or TRAC and/or TRBC genes.

[001136] Chimeric antigen receptor 19 (CAR19) T cells exhibit anti-leukemic effects in cancer patients. However, leukemia patients often do not have enough T cells to harvest, which means that treatment must involve modified donor T cells. Accordingly, it is of interest to create a bank of donor T-cells. Qasim et al. ("First Clinical Application of Talen Engineered Universal CAR19 T Cells in B-ALL" ASH 57th Annual Meeting and Exposition, Dec. 5-8, 2015, Abstract 2046 (https://ash.confex.com/ash/2015/webprogram /Paper81653.html published online November 2015) discusses the modification of CAR19 T cells to prevent the risk of graft versus host disease by disrupting T cell receptor expression and targeting CD52. insensitive to alemtuzumab, and this allowed alemtuzumab to prevent host rejection of CAR19 cells with human leukocyte antigen (HLA) mismatch.The investigators used a self-inactivating 3rd generation lentiviral vector encoding RQR8, then cells were injected by electroporation with two pairs of TALEN mRNA for multiple targeting at both loci: the locus of the α-constant chain of T-cell re receptors (TCR) and the CD52 gene locus. A pool of cells that continued to express TCR after ex vivo expansion was depleted using the CliniMacs α/β TCR depletion technique to produce a product consisting of T cells (UCART19) with TCR expression <1% TCR, 85% of which expressed CAR19 and 64% became CD52 negative. CAR19 modified T cells were administered to treat a patient with a relapse of acute lymphoblastic leukemia. The teachings described herein provide efficient methods for providing modified HSCs and their progeny, including but not limited to cells of myeloid and lymphoid blood cell lines, including T cells, B cells, monocytes, macrophages, neutrophils, basophils, eosinophils , erythrocytes, dendritic cells and megakaryocytes or platelets or natural killer cells and their precursors and derivatives. Such cells can be modified by knockin, knockout, or other modulating agents, for example, to remove or modulate CD52, as described above, or other targets such as, but not limited to, CXCR4 and PD-1. Thus, the compositions, cells and methods of the invention can be used to modulate immune responses and to treat, but not limited to, malignant diseases, viral infections and immune diseases, in combination with modification of the administration of T cells and other cells to a patient.

[001137] Patent WO 2015/148670 is referenced, and through the teachings described herein, the invention provides the methods and materials of this document applicable in conjunction with the teachings set forth herein. In the aspect of gene therapy, methods and compositions are provided for editing a target sequence related to or associated with human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS). In a related aspect, the invention described herein provides for the prevention and treatment of HIV infection and AIDS by introducing one or more mutations in the C-C chemokine receptor type 5 (CCR5) gene. CCR5 is also known as CKR5, CCR-5, CD195, CKR-5, CCCKR5, CMKBR5, 1DDM22 and CC-CKR-5. In a further aspect, the invention described herein provides for preventing or reducing HIV infection or reducing the ability of HIV to enter a host cell, for example, in already infected individuals. Exemplary HIV host cells include, but are not limited to, CD4 cells, T cells, gut-associated lymphoid tissue (GALT) cells, macrophages, dendritic cells, myeloid progenitor cells, and microglia. Entry of the virus into the cell requires the interaction of viral glycoproteins gp41 and gpl20 simultaneously with the CD4 receptor and co-receptor, for example, CCR5. If a co-receptor, such as CCR5, is not present on the surface of the host cells, the virus cannot bind to and enter the host cells. The development of the disease is thus slowed down. By knocking out or knocking down CCR5 in host cells, for example by introducing a protective mutation (such as the Δ32 mutation of CCR5), HIV is prevented from entering the host cells.

[001138] X-linked chronic granulomatous disease is an inherited disease of the host defense system due to the absence or decrease in the activity of phagocytic NADPH oxidase. The use of a CR1SPR-Cas system (C2c1 or C2c3) is contemplated, which specifically corrects for a mutation (missing or reduced phagocytic NADPH oxidase activity) (eg, with an HDR template that delivers a coding sequence for phagocytic NADPH oxidase); in particular, gRNA can be directed to a mutation that gives rise to X-linked granulomatous disease (deficiency of phagocytic NADPH oxidase), and HDR repair can provide coding for the correct expression of phagocytic NADPH oxidase. The particle containing the gRNA to be mutated and the Cas protein (eg C2c1 or C2c3) is brought into contact with the HSCs carrying the mutation. The particle may also contain an appropriate HDR template to correct the mutation for correct expression of the phagocytic NADPH oxidase, or the HSC may interact with a second particle or vector that contains or delivers the HDR template. Cells that have interacted in this way can be injected; and optionally processed/enlarged (see Cartier).

[001139] Fanconi anemia: mutations in at least 15 genes (FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ/BACTI1/BRIP1, FANCL/PHF9/POG, FANCM, FANCN/PALB2 , FANCO/RadSlC and FANCP/SLX4/BTBD12) can cause Fanconi anemia. The proteins produced by these genes are involved in a cellular process known as the Fanconi anemia signaling pathway. The Fanconi anemia signaling pathway is turned on (activated) when the process of making new copies of DNA, called DNA replication, is blocked by DNA damage. The Fanconi anemia signaling pathway sends certain proteins to the damaged site, triggering DNA repair so that DNA replication can continue. The Fanconi anemia signaling pathway is particularly sensitive to specific types of DNA damage known as interstrand cross-links. Interstrand crosslinks occur when two DNA building blocks (nucleotides) on opposite strands of DNA are abnormally attached to each other or joined together, which stops the process of DNA replication. Interchain crosslinks can be caused by the incorporation of toxic compounds formed in the body, or by treatment with certain anticancer compounds. The eight proteins associated with Fanconi anemia cluster together to form a complex known as the core FA complex. The core FA complex activates two proteins called FANCD2 and FANCF. Activation of these two proteins attracts DNA repair proteins to the interstrand junction site so that the junction can be removed and DNA replication can continue. Specifically, the core FA complex is a nuclear multiprotein complex composed of FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM, functioning as a ubiquitin ligase and mediating activation of the ID complex, which is a heterodimer composed of FANCD2 and FANCI. Being monoubiquitinylated, it interacts with classical tumor suppressors downstream of the Fanconi anemia signaling pathway, including the FANCD1/BRCA2, FANCN/PALB2, FANCJ/BRIP1, and FANCO/Rad51C interactions, and thus contributes to DNA repair through homologous recombination. 80-90% of cases of Fanconi anemia are due to mutations in one to three genes: FANCA, FANCC and FANCG. These genes provide instructions for the formation of the components of the core FA complex. Mutations in such genes associated with the core FA complex would render the complex nonfunctional and disrupt the entire Fanconi anemia signaling pathway. As a result, DNA damage will not be repaired efficiently and interstrand crosslinks will accumulate over time. Geiselhart, "Review Article, Disrupted Signaling through the Fanconi Anemia Pathway Leads to Dysfunctional Hematopoietic Stem Cell Biology: Underlying Mechanisms and Potential Therapeutic Strategies," Anemia Volume 2012 (2012), Article ID 265790, http://dx.doi. org/10. 1155/2012/265790 discusses Fanconi's anemia and animal experiments involving intrafemoral injection of a lentivirus encoding the FANCC gene resulting in in vivo correction of HSC. The use of a CRISPR-Cas system (C2c1 or C2c3) that targets one or more mutations associated with Fanconi anemia is contemplated, e.g. a CRISPR-Cas system (C2c1 or C2c3) containing gRNA or HDR template(s) that are respectively targeted one or more FANCA, FANCC or FANCG mutations that cause Fanconi's anemia and ensures the correct expression of one or more FANCA, FANCC or FANCG; for example, gRNA can be directed to mutate into FANCC, and HDR repair (homology directed repair) can encode the correct expression of FANCC. A particle containing an gRNA directed to the mutation(s) (for example, one or more involved in the development of Fanconi anemia, such as mutation(s) of one or more of FANCA, FANCC or FANCG) and a Cas protein (C2c1 or C2c3) are brought into contact with HSCs carrying the mutation(s). The particle may also contain an appropriate HDR template to correct the mutation for the correct expression of one or more proteins involved in Fanconi anemia, or the HSC can interact with a second particle or vector that contains or delivers the HDR template. Cells thus interacted can be introduced and optionally processed/upscaled (see Cartier).

[001140] The particle referred to herein (e.g. containing gRNA and Cas (e.g. C2c1 or C2c3), optionally HDR template(s); e.g. for diseases such as hemophilia B, severe combined immunodeficiency (SCID), X-linked severe combined immunodeficiency, severe combined immunodeficiency associated with adenosine deaminase deficiency, hereditary tyrosinemia, β-thalassemia, X-linked granulomatous disease, Wiskott-Aldrich syndrome, Fanconi anemia, adrenoleukodystrophy, metachromatic leukodystrophy, HIV infection/AIDS, immunodeficiency syndrome, hematological diseases or genetic lysosomal storage disease) are advantageously made or can be made by adding a mixture of gRNA and Cas (C2c1 or C2c3) protein (optionally containing HDR template(s) or such a mixture containing only HDR template(s) if a single particle is desired for matrix(es)) to a mixture containing or essentially consisting of or consisting of surfactants stv, phospholipids, biodegradable lipoprotein and alcohol (with one or more gRNAs targeting genomic(s) locus(s) in neuronal stem cells).

[001141] Indeed, the invention is particularly suited to the treatment of genetic hematopoietic diseases by genome editing and immunodeficiency syndromes such as genetic immunodeficiency syndromes, especially through the use of the particle technologies described herein. Genetic immunodeficiencies are diseases for which genome editing interventions according to the present invention may be successful. Reasons include: hematopoietic cells, of which immune cells are a subset, are therapeutically available. They can be removed from the body and transplanted autologously or allogeneically. Further, specific genetic immunodeficiencies, such as severe combined immunodeficiency, are proliferatively disadvantageous to immune cells. Correction of genetic damage causing severe combined immunodeficiency by rare spontaneous "back" mutations indicates that the correction of even a single lymphocyte progenitor may be sufficient to restore immune function in patients (see Bousso, P., et al. Diversity, functionality, and stability of the T cell repertoire derived in vivo from a single human T cell precursor Proceedings of the National Academy of Sciences of the United States of America 97, 274-278 (2000)). The selective advantage of the edited cells ensures that even a low level of editing results in a therapeutic effect. This effect of the present invention can be seen in severe combined immunodeficiency, Wiskott-Aldrich syndrome and other conditions mentioned here, including other genetic diseases of the hematopoietic system, such as α- and β-thalassemia, in which hemoglobin deficiency adversely affects the survival of red blood cell precursors.

[001142] NHEJ and HDR repair activity varies greatly depending on cell type and cell condition. NHEJ repair is not highly regulated by the cell cycle and is efficient in a variety of cell types, resulting in a high level of gene disruption in available target cell populations. In contrast, HDR repair occurs primarily during the S/G2 phase and is therefore limited to cells that are actively dividing, which limits treatment targets requiring precise genomic modifications to mitotic cells [Ciccia, A. & Elledge, SJ Molecular cell 40 , 179-204 (2010); Chapman, JR, et al. Molecular cell 47, 497-510 (2012)].

[001143] The efficiency of correction in HDR repair can be regulated by the epigenetic state or sequence of the target locus, or the configuration of the specific repair template used (single or double stranded, with long or short homologous arms). [Hacein-Bey-Abina, S., et al. The New England journal of medicine 346, 1185-1193 (2002); Gaspar, HB, et al. Lancet 364, 2181-2187 (2004); Beumer, KJ, et al. G3 (2013)]. The relative activity of the NHEJ repair and HDR repair machinery in target cells may also influence the efficiency of gene repair, as these pathways may compete for repair of double-strand breaks Beumer, KJ, et al. Proceedings of the National Academy of Sciences of the United Slates of America 105, 19821-19826 (2008)], HDR repair also imposes delivery restrictions that are not present with NHEJ repair strategies because it requires simultaneous delivery of nucleases and repair templates. . In practical application, these limitations have so far resulted in low levels of HDR repair in therapeutically relevant cell types. Clinical applications are therefore largely focused on NHEJ repair strategies for disease management, although preclinical studies to test the concept of HDR repair are now described for a mouse model of hemophilia B and hereditary tyrosinemia [Li, H., et al. Nature 475, 217-221 (2011); Yin, H, et al. Nature biotechnology 32, 551-553 (2014)].

[001144] Any given application of genomic editing may involve combinations of proteins, small RNA molecules, and/or repair templates, making delivery of many of these parts much more difficult than small molecule therapy. Two main strategies have been developed for the delivery of ex vivo and in vivo genome editing tools. In ex vivo treatments, disease-associated cells are removed from the body, edited, and transplanted back into the patient. Ex vivo editing has the advantage of allowing a clear definition of the target cell population and determining the specific dosage of therapeutic molecules to be delivered to the cells. The latter consideration may be especially important when there is a threat of off-target modifications, since titration of the amount of nuclease can reduce the level of such mutations (Hsu et al., 2013). Another advantage of ex vivo approaches is the generally high level of editing that can be achieved by developing efficient systems for delivering proteins and nucleic acids to cells in culture for gene therapy research and applications.

[001145] Approachesex vivo there may be disadvantages that limit the use to a small number of diseases. For example, target cells must be able to survive manipulation outside the body. In the case of many tissues, such as brain tissues, culturing cells outside the body is a major problem, as the cells either do not survive or lose the properties necessary for their functions.in vivo. Thus, in light of the present invention and knowledge in the art, therapy is possibleex vivo in the case of tissues with populations of adult stem cells, such as the hematopoietic system, amenable to cultivation and manipulationex vivothrough the system CRISPR-Cas (C2c1 or C2c3). [Bunn, H.F. &Aster,J. Pathophysiology Of Blood Disorders, (McGraw-Hill, New York, 2011)].

[001146] In vivo genomic editing involves the direct delivery of editing systems to cell types in their tissues. In vivo editing allows the treatment of diseases in which the affected cell population is not amenable to ex vivo manipulation. Moreover, in situ delivery of nucleases into cells allows for the treatment of many tissues and cell types at once. These properties likely allow in vivo treatments to be applied to a wider range of diseases than ex vivo therapy approaches.

[001147] To date, in vivo editing has been largely achieved through the use of viral vectors with specific tissue-specific tropism. Such vectors are currently limited in terms of cargo size and tropism, limiting this type of therapy to organ systems where transduction with clinically effective vectors is effective, such as the liver, muscles, and eyes [Kotterman, MA & Schaffer, DV Nature reviews. Genetics 15, 445-451 (2014); Nguyen, TH & Ferry, N. Gene therapy 11 Suppl 1, S76-84 (2004), Boye, SE, et al. Molecular therapy: the journal of the American Society of Gene Therapy 21, 509-519 (2013)].

[001148] A potential barrier to in vivo delivery is the immune response that can be elicited in response to a high amount of virus required for treatment, but this phenomenon is not unique to genomic editing and is often observed in other virus-based gene therapy methods [Bessis, N., et al. Gene therapy 11 Suppl 1, S10-17 (2004)]. It is also possible that peptides from editing nucleases are themselves presented on MHC class I molecules, stimulating an immune response, although there is little evidence for this in preclinical studies. Another significant problem with this therapy is the control of the spread and subsequently dosage of genome-editing nucleases in vivo , which leads to off-target mutation profiles that can be difficult to predict. However, in view of the present invention and knowledge in the art, the use of virus- and particle-based therapies for use in the treatment of cancer, in vivo modification of HSCs, such as by particle or virus delivery, is within the purview of the skilled artisan.

[001149] Ex vivo Editing Therapy: Long-term clinical studies on hematopoietic cell purification, culture, and transplantation have brought blood diseases such as AIDS, Fanconi anemia, Wiskott-Aldrich syndrome, and sickle cell anemia into the focus of ex vivo editing therapy. Another reason to focus on HSCs is the existence of relatively highly efficient delivery systems due to previous attempts to develop gene therapy for blood disorders. In view of these advantages, this method of therapy can be applied to diseases in which the edited cells have a survival advantage, so that a small number of edited cells grafted can proliferate and treat the disease. One such disease is HIV, in which infection results in a survival disadvantage for CD4+ T cells.

[001150] The scope of ex vivo -based editing therapy has recently expanded to include gene editing strategies. Ex vivo interfering HDR factors were overcome in a recent paper by Genovese et al., who managed to gene-correct a mutated IL2RG gene in hematopoietic stem cells (HSC) derived from a patient with X-linked severe combined immunodeficiency (SCID-X1) [Genovese, P ., et al. Nature 510, 235240 (2014)]. Genovese et. al successfully performed gene editing in HSCs using a multimodal strategy. First, HSCs were transduced using a reduced integration lentivirus containing an HDR template encoding a therapeutic cDNA for IL2RG. After transduction, cells were electroporated with mRNA encoding ZFN targeting the "hot spot" of mutations in IL2RG in order to stimulate HDR-based gene repair. In order to increase the HDR rate, optimized culture conditions are used to stimulate HSC division. Under optimized culture conditions, nucleases and HDR templates carrying the corrected HSC genes from a patient with SCID-X1 were obtained in culture at a rate of therapeutic relevance. HSCs from diseased individuals treated with the same gene-correction procedure can maintain long-term hematopoiesis in mice, the gold standard of HSC function, HSCs are capable of producing all types of hematopoietic cells and can be autologously transplanted, making them an exceptionally valuable cell population for use in all hematopoietic genetic diseases [Weissman, IL & Shizuru, JA, Blood 112, 3543-3553 (2008)]. Gene-corrected HSCs could, in principle, be used to treat a wide range of genetic blood disorders, making this study an exciting breakthrough in therapeutic genome editing.

[001151] In vivo editing therapy: in vivo editing can be successfully applied, as follows from the present Application and the submissions in the field. In the case of organ systems for which delivery is successful, a number of promising preclinical successes have been achieved to date. The first example of successful therapy based on in vivo editing was shown in a mouse model of hemophilia B [Li, H., et al. Nature 475, 217-221 (2011)]. As noted earlier, hemophilia B is an X-linked recessive disorder caused by a loss-of-function mutation in the gene encoding factor IX, which is a key component of the blood coagulation cascade. Returning the level of Factor IX activity to values above 1% of that in individuals with severe pathology can bring the disease to a much milder form, the introduction of recombinant Factor IX in such patients as a prophylaxis from an early age in order to achieve such levels to a large extent reduces the severity of clinical complications [Lofqvist, T. et al. Journal of internal medicine 241, 395-400 (1997)]. Thus, only low levels of HDR gene correction are needed to change the clinical outcomes of patients. In addition, factor IX is synthesized and secreted by the liver, an organ that can be efficiently transduced by viral vectors encoding editing systems.

[001152] Using hepatotropic adeno-associated viral (AAV) serotypes encoding ZFN and HDR correction matrix, it is possible to achieve gene correction of the mutated humanized Factor IX gene in mouse liver up to 7% [Li, H., et al. Nature 475, 217-221 (2011)]. The result was an improvement in thrombus kinetics, a measure of the function of the coagulation cascade, demonstrating for the first time that in vivo edit-based therapy is not only possible, but also effective. As discussed herein, the skilled artisan should be guided by the ideas and knowledge of the art set forth in this specification, such as the work of Li, to address the treatment of hemophilia B with an HDR particle array and a CRISPR-Cas (C2c1 or C2c3) system that targets to a mutation in an X-linked recessive disease in order to reverse the loss-of-function mutation.

[001153] Building on this study, other teams have recently used CRlSPR-Cas in vivo genome editing in the liver to successfully treat hereditary tyrosinemia in a mouse model and generate mutations conferring protection against cardiovascular disease. These two different applications show the versatility of this approach for diseases involving liver dysfunction. [Yin, H., et al. Nature biotechnology, 32, 551-553 (2014); Ding, Q., et al. Circulation research 115, 488-492 (2014)]. The application of in vivo editing to other organ systems is necessary to justify that this strategy is widely applicable. Efforts are underway to optimize both viral and non-viral vectors in order to expand the range of diseases that can be treated using this approach [Kotterman, MA & Schaffer, DV Nature reviews. Genetics 15, 445-451 (2014); Yin, H., et al. nature reviews. Genetics 15, 541-555 (2014)]. According to the present disclosure, the skilled artisan should use the insights and knowledge of the art provided herein, for example, the work of Yin, in order to use, in the treatment of hereditary tyrosinemia, an HDR particulate matrix and a CRISPR-Cas system (C2c1 or C2c3) targeting this mutation.

[001154] Targeted deletion, therapeutic applications: Targeted gene deletion may be preferred. Therefore, genes involved in immunodeficiency states, hematologic disorders, or genetic lysosomal storage diseases are preferred, e.g., hemophilia B, severe combined immunodeficiency (SCID), X-linked severe combined immunodeficiency (SCID-X1), severe combined immunodeficiency with adenosine deaminase deficiency ( ADA-SCID), hereditary tyrosinemia, β-thalassemia, X-linked chronic granulomatosis (CGD), Wiskott-Aldrich syndrome, Fanconi anemia, adrenoleukodystrophy (ALD), metachromatic leukodystrophy (MLD), HIV/AIDS, other metabolic diseases, genes, encoding misfolded proteins involved in diseases, genes leading to loss of function involved in diseases; in general, mutations that can be targeted in an HSC using any of the delivery systems described herein in a particle-based system are considered to be preferred.

[001155] In the present invention, the immunogenicity of the CRISPR enzyme can be reduced, in particular using the approach first described by Tangri et al. in relation to erythropoietin and received further development. Accordingly, directed evolution or rational design can be used to reduce the immunogenicity of a CRISPR enzyme (eg, C2c1 or C2c3) in the host (human or other species).

[001156] Genome Editing: The CRISPR/Cas (C2c1 or C2c3) systems of the present invention can be used to correct genetic mutations that have been previously attempted with limited success with TALEN, ZFN and lentiviruses, including those discussed herein; see also WO2013163628.

Treatment of diseases of the brain, central nervous and immune systems

[001157] The present invention also provides for the delivery of CRISPR-Cas systems to the brain or neurons. For example, RNA interference (RNAi) provides therapies for these disorders by reducing the expression of HTT, the gene that causes Huntington's disease (see, for example, McBride et al. Molecular Therapy vol. 19 no. 12 Dec. 2011, pp. 2152-2162), therefore, Applicants claim that it can be used in conjunction with and/or adapted to the CRISPR-Cas system. A CRISPR-Cas system can be generated using an off-target potential reduction algorithm for antisense sequences. The CRISPR-Cas sequences can be targeted to the mouse exon 512 sequence, Rhesus or human huntingin and be expressed using a viral vector, in particular AAV. Animals, including humans, can receive approximately three microinjections per hemisphere (six injections in total): the first 1 mm rostral to the anterior commissure (12 µl) and the remaining two injections (12 and 10 µl, respectively) located 3 and 6 mm caudal to the first injection containing 1x12 mg/ml AAV at a rate of approximately 1 µl/min, with the needle held in place for an additional 5 minutes to allow the injected solution to spread from the needle tip.

[001158] DiFiglia et al. (PNAS, October 23, 2007, vol. 104, no. 43, 17204-17209) made the observation that a single injection into the adult striatum of HTT-targeting siRNAs can cause mutant HTT silencing, attenuate neuronal pathology, and delay the onset of a pathological behavioral phenotype. observed in the rapidly evolving form of a virus-derived transgenic model of HD. In the work of DiFiglia, 2 µl of Cy3-labeled cc-siRNA-HTT or unconjugated siRNA-HTT at a concentration of 10 µM was injected intrastrially into mice. A similar dosage of HTT-targeting CRISPR Cas can be contemplated within the scope of the present invention in humans, eg, approximately 5-10 ml of 10 μM HTT-targeting CRISPR Cas can be administered intrastrially.

[001159] In another case, Boudreau et al. (Molecular Therapy vol. 17 no. 6 June 2009) injected 5 μl of recombinant AAV serotype 2/1 vectors expressing virus with HTT-specific mRNA (at a concentration of 4×10 ¹² virus genomes/ml) into the striatum. A similar dosage of HTT-targeted CRISPR Cas can be contemplated in the present invention for humans, for example, approximately 10-20 ml of an HTT-targeted CRISPR Cas solution at a concentration of 4 x 10 ¹² viral genomes per ml can be administered intrastrially.

[001160] Another example is CRISPR Cas, which targets HTT and is administered continuously (see, for example, Yu et al., Cell 150, 895-908, August 31, 2012). Yu et al. used osmotic pumps infusing 0.25 µl/h (Model 2004) to deliver ss-miRNA or phosphate buffered saline at a rate of 300 mg/day (PBS) (Sigma Aldrich) for 28 days and pumps designed to deliver 0 .5 µl/hour (Model 2002) was used to infuse 75 mg/day positive control MOE ASO for 14 days. Pumps (Durect Corporation) were filled with ss-miRNA or MOE dissolved in sterile PBS and further incubated at 37° C. for 24 or 48 hours (Model 2004) prior to implantation. Mice were anesthetized with 2.5% isofluorane, and a median incision was made at the base of the skull. Stereotactically guided implantation of the cannula served to introduce it into the right lateral ventricle, then it was hermetically fixed with Loctite adhesive. A catheter attached to a mini osmotic pump was connected to a cannula; the pump was placed subcutaneously in the region of the middle scapula. The incision was closed with 5.0 nylon sutures. A similar dosage of HTT-targeting CRISPR Cas can be contemplated within the scope of the present invention in humans, eg, about 500 to 1000 g of HTT-targeting CRISPR Cas daily may be administered.

[001161] In another example of continuous administration, Stiles et al. (Experimental Neurology 233 (2012) 463-471) implanted a catheter with a titanium needle tip into the right putamen inside the parenchyma. The catheter was connected to a SynchroMed® II Pump (Medtronic Neurological, Minneapolis, MN) implanted subcutaneously in the abdominal cavity. After 7 days of phosphate buffered saline infusion at a rate of 6 μl/day, the pumps were refilled with test sample and programmed for continuous infusion for 7 days. About 2.3 to 11.52 mg of siRNA per day were infused at a varying infusion rate of about 0.1 to 0.5 µl/min. A similar dosage of HTT-targeting CRISPR Cas may be contemplated in the present invention for humans, eg, about 20 to 200 mg of HTT-targeting CRISPR Cas may be administered. In another example, the methods of US Patent Publication No. 20130253040 registered to Sangamo can also be adapted based on TALES for the nucleic acid targeting system of the present invention for the treatment of Huntington's disease.

[001162] Another example is the methods of US Patent Publication No. 20130253040 (WO2013130824) registered to Sangamo, which can also be adapted from TALES to the CRISPR Cas system of the present invention for the treatment of Huntington's disease.

[001163] WO2015089354 A1, registered by Broad Institute et al., incorporated herein by reference, describes targets for Huntington's disease (HD). Possible target genes of the CRISPR complex in relation to Huntington's disease: PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4 and TGM2. Accordingly, in some embodiments of the present invention, one or more of the PRKCE list; IGF1; EP300; RCOR1; PRKCZ; HDAC4 and TGM2 can be selected as targets for Huntington's disease.

[001164] Other diseases associated with trinucleotide repeats. These may include any of the following: category I includes Huntington's disease (HD) and spinocerebellar ataxias; category II expansions differ phenotypically from heterogeneous expansions, which are usually short in extent but also found in exons of genes; category III includes fragile X syndrome, myotonic dystrophy, two spinocerebellar ataxias, juvenile myoclonic epilepsy, and Friedreich's ataxia.

[001165] A further aspect of the invention relates to the use of the CRISPR-Cas system to correct defects in the EMP2A and EMP2B genes that have been identified as being associated with Lafora's disease. Lafora disease is an autosomal recessive disorder characterized by progressive myoclonal epilepsy that may begin with epileptic seizures during adolescence. Rare cases of this disease may be caused by mutations in genes that have not yet been identified. The disease causes seizures, muscle spasms, walking problems, dementia, and eventually death. There are currently no treatments that have been shown to be effective in the progression of the disease. The CRISPR-Cas system can also target other genetic abnormalities associated with epilepsy, and the underlying genetic mechanisms are described in Genetics of Epilepsy and Genetic Epilepsies, edited by Giuliano Avanzini, Jeffrey L. Noebels, Mariani Foundation Pediatric Neurology: 20; 2009).

[001166] The methods of US Patent Publication No. 20110158957 registered with Sangamo BioSciences, Inc. involved in the inactivation of T cell receptor (TCR) genes can also be modified for the CRISPR-Cas system of the present invention. In another example, the methods of U.S. Patent Publication No. 20100311124 to Sangamo BioSciences, Inc. and the methods of U.S. Patent Publication No. 20110225664 to Cellectis, which in both cases are involved in the inactivation of glutamine synthetase gene expression, can also be modified for the CRISPR-Cas system of the present invention.

[001167] Choice of brain delivery methods include encapsulating the CRISPR enzyme and guide RNA in the form of DNA or RNA in liposomes and conjugating them with molecular Trojan horses for delivery across the blood-brain barrier (BBB). Molecular Trojan horses have been shown to be effective in delivering B-gal expression vectors to the brains of non-human primates. The same approach can be used for delivery using vectors containing the CRISPR enzyme and guide RNA. For example, Xia CF and Boado RJ, Pardridge WM ("Antibody-mediated targeting of siRNA via the human insulin receptor using avidin-biotin technology." Mol Pharm. 2009 May-Jun;6(3):747-51. doi: 10.1021 /mp800194) describes how delivery of small interfering RNAs (siRNAs) into cultured and in vivo cells can be possible with the combined use of receptor-specific monoclonal antibodies (mAbs) and avidin and biotin technology. The authors also reported that, because the association between targeting mAb and siRNA is stable using avidin-biotin technology, effects of RNAi at distant sites, particularly in the brain, are observed in vivo after intravenous administration of targeted siRNA. .

[001168] Zhang et al. (Mol Ther. 2003 Jan;7(l):11-8.)) describe expression plasmids encoding reporter constructs such as luciferase encapsulated within a virus containing 85 nm PEGylated immunoliposomes that target the brain of the rhesus monkey in vivo, via monoclonal antibodies (MAb) to the human insulin receptor (HIR). HIRMAb enables liposome transfer of an exogenous gene for its subsequent transcytosis across the blood-brain barrier and endocytosis across the plasma membrane of a neuron after intravenous administration. The expression level of the luciferase gene in the brain was 50 times higher in the case of the rhesus monkey compared to the mouse. The course of neuronal expression of the beta-galactosidase gene in a large part of the brain was demonstrated using histochemistry and confocal microscopy. The authors indicate that this approach allows reversible transgenesis in adults within 24 hours. Accordingly, the use of immunoliposomes is preferred. They can be used in combination with antibodies to target specific tissues or cell surface proteins.

[001169] US Patent Publication No. 20110023153 describes the use of zinc finger nucleases to genetically modify cells, animals and proteins associated with Alzheimer's disease (AD). Once modified, cells and animals can then be tested using known methods to investigate the effects of targeted mutations on the development and/or progression of AD using widely used measures in the study of AD, such as, but not limited to, learning and memory, anxiety, depression. , addiction, sensory and motor functions, as well as analysis methods that evaluate behavioral, functional, pathological, metabolic and biochemical functions.

[001170] This application covers editing any chromosomal sequences encoding proteins associated with AD. AD-associated proteins are typically selected based on experimental association of AD-associated proteins with AD disease. For example, the rate of formation or circulating concentration of an AD-associated protein may be increased or decreased in a population with AD-related disorders compared to a population without AD. Differences in protein levels can be assessed using proteomics techniques including, but not limited to, Western blotting, immunohistochemical staining, ELISA, and mass spectrometry. Alternatively, AD-associated proteins can be identified by obtaining gene expression profiles for protein-coding genes using genomic techniques, including, but not limited to, DNA microarray analysis, serial gene expression analysis (SAGE), and quantitative real-time polymerase chain reaction. time (qPCR).

[001171] Examples of Alzheimer's associated proteins are the very low density lipoprotein receptor (VLDLR) encoded by the VLDLR gene, the ubiquitin-like modifying activating protein 1 (UBA1) encoded by the UBA1 gene, or the NEDD8-activating catalytic subunit E1 enzyme (UBE1C) encoded, for example, by the UBA3 gene.

[001172] As a non-limiting example, AD-associated proteins may include, but are not limited to, the following: chromosomally encoded ALAS2 protein, delta-aminolevulenate synthase 2 (ALAS2), ABCA1 ATP-binding cassette transporter (ABCA1), angiotensin converting enzyme I ACE (ACE), apolipoprotein E precursor APOE (APOE), amyloid precursor protein APP (APP), aquaporin protein I AQP1 (AQP1), Myc-box dependent interacting protein I BIN1 or fusion integrating protein 1 (BIN1), neurotrophic brain factor BDNF (BDNF), butyrophilin-like protein 8 BTNL8 (BTNL8), chromosome 1 open reading frame 49 C10RF49, cadherin-4 CDH4, acetylcholine receptor beta-2 subunit CHRNB2, CKLF-like protein 2 CKLFSF2 containing MARVEL transmembrane domain (CKLFSF2 ), type C lectin domain, CLEC4E family member 4 (CLEC4E), clusterin protein CLU (also called J), erythrocyte complement receptor 1 CR1 (CR1, also on called CD35, C3b/C4b receptor and immune adhesion receptor), erythrocyte complement receptor CR1L (CR1L), granulocyte colony stimulating factor receptor 3 CSF3R (CSF3R), cystatin C or cystanine 3 CST3, cytochrome P450 2C CYP2C, death-associated protein kinase 1 DAPK1 ( DAPK1), Estrogen receptor 1 ESR1, IgA receptor FCAR Fc fragment (FCAR, also called CD89), IgG Fc fragment, FCGR3B low density receptor IIIb (FCGR3B or CD16b), FFA2 free fatty acid receptor 2 (FFA2), FGA fibrinogen (factor I), GRB2-associated GAB2 binding protein 2 (GAB2), GRB2-associated GAB2 binding protein 2 (GRB2), galanin-like peptide GALP, spermatogenic glyceraldehyde-3-phosphate dehydrogenase GAPDHS (GAPDHS), GMPB - GMBP, haptoglobin HP (HP ), 5-hydroxytryptophan (serotonin) receptor 7 (adenylate cyclase dependent) FITR7, insulin degradation enzyme IDE, IF127 - IF127, interferon, alpha inducible protein 6 IFI6 (IFI6), interferon inducible protein with repeat ami tetratricopeptide 1 IFIT2 (IFIT2), IL1RN - interleukin-1 receptor antagonist (IL-IRA), interleukin 8 receptor ILSRA (ILSRA), receptor alpha (IL8RA or CD181), interleukin 8 receptor IL8RB (IL8RB), beta (IL8RB), Jagged I JAG1 (JAG1) forward-rectifying potassium channel, subfamily J, member 15 KCNJ15 (KCNJ15), low-density lipoprotein 6 receptor-associated protein LRP6 (LRP6), microtubule-associated protein tau MART (MART), regulating MAP/microtubule affinity MARK4 kinase 4 (MARK4) phase 1 phosphoprotein M MPHOSPH1, 5,10-methylenetetrahydrofolate reductase MTHFR, interferon-induced GTP binding protein MX2, nibrin NBN, also referred to as NBN, nicastrin NCSTN, niacin receptor 2 NIACR2 (NIACR2, also referred to as GPR109B), nicotinamide nucleotide adenylyltransferase 3 NMNAT3, NTM neurotrimin (HINT), ORM1 oroscumoid 1 (ORM1) or alpha-1-acid glycoprotein 1, P2Y13 purinoreceptor P2RY13 (P2RY13), nicotinamide phosphoribosyltransferase PBEF1 (NAmPRTase or Nampt ), also called progenitor colony-enhancing factor 1 (PBEF1) or visfatin, phosphoenolpyruvate carboxylase PCK1, phosphatidylinositol binding clathrin involved in association (PICALM), urokinase-type plasminogen activator PLAU (PLAU), plexin PLXNC1 (PLXNC1), prion protein PRNP, PSEN1 presenilin 1 protein (PSEN1), PSEN2 presenilin 2 protein (PSEN2), PTPRA protein tyrosine phosphatase type A receptor protein (PTPRA), Ral GEF with PH domain and SH3 binding motif 2 RALGPS2 (RALGPS2), signaling regulator-like protein with G-protein signaling 2, G-protein signaling-associated regulator 2 RGSL2 (RGSL2), selenium-binding protein 1 SELENBP1 (SELNBP1), mitoferrin 1 SLC25A37, sortilin-associated receptor L (class DLR), repeat A-containing protein SORL1 ( SORL1), transferrin TF, mitochondrial transcription factor A TFAM, tumor necrosis factor TNF, tumor necrosis factor receptor superfamily, member 10C TNFRSF10C (TNFRSF10C), superce tumor necrosis factor receptor agent (TRAIL), representative 10a TNFSF10 (TNFSF10), ubiquitin-like activating modifier protein 1 UBA1 (UBA1), UBA3, NEDD8-activating catalytic subunit of enzyme E1 (UBE1C), ubiquitin B UBB (UBB), ubiquitin- 1 UBQLN1, ubiquitin carboxylterminal esterase of protein L1 UCHL1 (UCHL1), ubiquitin carboxylterminal hydrolyase of protein L3 isozyme UCHL3 (UCHL3), very low density lipoprotein receptor protein VLDLR (VLDLR).

[001173] In exemplary embodiments, the AD-associated proteins whose chromosomal sequence is being edited may belong to a very low density lipoprotein receptor (VLDLR) protein encoded by a gene similar to activating modifier enzyme 1 (UBA1) encoded by the UBA1 gene NEDD8- catalytic subunit activating protein E1 encoded by the UBA3 gene; aquaporin 1 (AQP1) protein encoded by the AQP1 gene; ubiquitin carboxyl terminal esterase L1 (UCHL1) protein encoded by the UCHL1 gene; , ubiquitin B protein (UBB) encoded by the UBB gene, microtubule-associated tau protein (MART) encoded by the MART gene, tyrosine phosphatase type A receptor protein (PTPRA) encoded by the PTPRA gene, phosphatidylinositol binding protein involved in clathrin assembly (P1CALM), encoded by the P1CALM gene, clusterin protein (also called apo lipoprotein J) encoded by the CLU gene, presenelin 1 protein encoded by the PSEN1 gene, presenelin 2 protein encoded by the PSEN2 gene, sortilin-associated receptor L (class DLR), repeat-containing A protein (SORL1) encoded by the SORL1 gene, amyloid precursor protein (APP) encoded by the APP gene, apolipoprotein E (APOE) precursor encoded by the APOE gene, or brain-derived neurotrophic factor (BDNF) encoded by the BDNF gene. In exemplary embodiments, the genetically modified animals are rats and the edited chromosomal sequence encoding the AD associated protein may be: APP - amyloid precursor protein NM_019288, AQP1 - aquaporin 1 (AQP1) protein NM_012778, BDNF - brain-derived neurotrophic factor NM_012513 , CLU - clusterin protein (so-called NM_053021 - apolipoprotein J), MART - microtubule-associated tau protein NM_017212 (MART), PICALM - phosphatidylinositol binding protein involved in clathrin assembly NM_053554 (P1CALM), PSEN1 - presenelin 1 protein (PSEN1) NM_019163 , PSEN2 - presenelin 2 protein (PSEN2) NM_031087, PTPRA - tyrosine phosphatase type A receptor protein NM_012763 (PTPRA), SORL1 - sortilin-associated receptor L (class DLR, NM_053519) containing repeats A SORL1 protein XM_001065506, XM_217115 protein, UBA1 - similar ubiquitin-activating protein-modifier 1 (UBA1) NM_001014080, UBA3 - protein, NEDD8-activating th catalytic subunit of the enzyme E1 (NEDD8- activating enzyme E1 catalytic subunit protein, UBE1C, NM_057205), UBB - protein ubiquitin B (UBB, NM_138895), UCHL1 - ubiquitin carboxylterminal esterase of protein L1 (UCHL1), UCHL3 ubiquitin carboxylterminal hydrolysis of protein L3 isozyme ( UCHL3) NM_001110165, VLDLR - very low density lipoprotein receptor protein (VLDLR) NM_013155.

[001174] An animal or cell may contain 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15 or more interrupted chromosomal sequences encoding an AD associated protein, and zero, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more chromosomally integrated sequences encoding an AD-associated protein.

[001175] The edited or integrated chromosomal sequence can be modified to encode an altered AD-associated protein. A number of mutations in AD-related chromosomal sequences have been associated with AD. For example, the V717I missense mutation (ie, a change from valine at position 717 to isoleucine) in APP causes a familial form of AD. Several mutations in the presenilin-1 protein, in particular, H163R (i.e. the replacement of histidine at position 163 with arginine), A246E (i.e. the replacement of alanine at position 246 with glutamate), L286V (i.e. the replacement of leucine at position 286 to valine) and C410Y (i.e. the replacement of cysteine at position 410 with tyrosine) cause a familial form of type 3 Alzheimer's disease. Mutations in the presenelin-2 protein, specifically N141I (i.e. asparagine at position 141 is replaced by isoleucine), M239V (i.e. methionine 239 is replaced by valine) and D439A (i.e. aspartate 439 is replaced by alanine) cause the familial form of Alzheimer's disease type 4. Other associations of genetic variants associated with AD genes and disease are known in the present field. See, for example, Waring et al. (2008) Arch. Neurol. 65:329-334, the contents of which are incorporated herein by reference.

Secretase related disorders

[001176] US Patent Publication No. 20110023146 describes the use of zinc finger nucleases to genetically modify cells, animals, or proteins associated with diseases associated with secretases. Secretases are required for the processing of protein precursors to their biologically active forms. Defects in various components of the secretase metabolic pathways contribute to various diseases, in particular those that have the hallmarks of amyloidogenesis and amyloid plaques, in particular Alzheimer's disease (AD).

[001177] Secretase-related disorders and proteins associated with such diseases are a diverse set of proteins that affect the susceptibility to numerous diseases, the presence of such diseases, their severity, or a combination of them. The present invention includes editing any chromosomal sequences encoding proteins associated with disorders associated with secretases. The proteins associated with disorders of secretase function are selected based on the experimental association of proteins associated with secretase disorders with secretase-related disease. For example, the rate of formation or circulating concentration of a protein associated with a secretase-related disorder may be increased or decreased in a population with a secretase-related disorder compared to a population without a secretase-related disorder. Differences in protein levels can be assessed using proteomics techniques, including, but not limited to, Western blotting, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, proteins associated with secretase-associated disorders can be identified by obtaining gene expression profiles for protein-coding genes using genomics techniques including, but not limited to, DNA microarray analysis, serial gene expression analysis (SAGE), and quantitative polymerase chain analysis. real-time reaction (qPCR).

[001178] As a non-limiting example, proteins associated with secretase-related disorders include PSENEN ( C. elegans presenelin 2 enhancer homolog), CTSB (cathepsin B), PSEN1 (presenelin 1), APP (beta amyloid (A4)), APH1B (anterior pharynx defective 1 homolog B, C. elegans) homolog B, PSEN2 (presenelin 2 (Alzheimer's disease 4)), BACE1 (APP beta site cleavage enzyme 1) , ITM2B (integral membrane protein 2B), CTSD (cathepsin D), NOTCH1 (translocation-associated Notch 1 homologue (Drosophila)), TNF (tumor necrosis factor (TNF superfamily, member 2)), INS (insulin), DYT10 ( dystonia 10), ADAM 17 (metal peptidase domain ADAM 17), APOE (apolipoprotein E), ACE (angiotensin converting enzyme I (peptidyl dipeptidase A) 1), STN (statin), TP53 (tumor protein p53), IL6 (interleukin 6 (interferon beta 2)), NGFR (nerve growth factor receptor (TNFR superfamily, member 16)), IL I B (interleukin I, beta), ACHE (acetylcholinesterase (Yt blood group)), CTNNB1 (catenin (cadherin-associated protein), beta 1, 88 kDa), IGF1 (insulin-like growth factor 1 (somatomedin C)), IFNG (interferon , gamma), NRG1 (neuregulin 1), CASP3 (caspase 3, apoptosis-associated cysteine peptidase), MAPK 1 (mitogen-activating protein kinase 11), CDH1 (caderin 1, type 1, E-coderin (epithelial)), APBB1 (binding protein- amyloid beta precursor (A4), family B, member 1 (Fe65)), HMGCR (3-hydroxy-3-methylglutaryl coenzyme A reductase), CREB1 (cAMP-dependent element-binding protein 1), PTGS2 (prostaglandin endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)), HES1 (hairy gene and splt 1 enhancer, (Drosophila)), CAT (catalase), TGFB1 (transforming growth factor beta 1), ENO2 (enolase 2 (gamma, neuronal)) , ERBB4 (v-erb-a homologue of erythroblastic leukemia viral oncogene 4 (avian)), TRAPPC10 (protein transport particle complex 10), MAOB (monoa minoxidase B), NGF (nerve growth factor (beta-polympeptide)), MMP12 (matrix metallopeptidase 12 (macrophage elastase)), JAG1 (jagged 1 (Alagille syndrome)), CD40LG (CD40 ligand), PPARG (peroxisome proliferator-activated receptor gamma ), FGF2 (fibroblast growth factor 2 (major)), IL3 (interleukin 3 (colony stimulating factor, multiple), LRP1 (low density lipoprotein receptor-associated protein 1), NOTCH4 (Notch homologue 4 (Drosophila)), MAPK8 (mitogen-activated protein kinase 8), PREP (prolyl endopeptidase), NOTCH3 (Notch 3 homolog 3 (Drosophila)), PRNP (prion protein), CTSG (cathepsin G), EGF (epidermal growth factor (beta-urogastron)), REN (renin), CD44 (CD44 molecule (Indian blood group classification)), SEEP (selectin P (140-kDa membrane granule protein, CD62 antigen)), GHR (growth hormone receptor), ADCYAP1 (adenylate cyclase activating polypeptide 1 (pituitary)), INSR (receptor insulin), GFAP (glial fibrillar acidic protein), MMP3 (matrix metallopeptidase 3 (stromelysin 1, progelatinase)), MAPK10 (mitogen-activated protein canase 10), SP1 (transcription factor Sp1), MYC (homologous y-myc myelocytomatous viral oncogene (avian)), CTSE (cathepsin E), PPARA (profilator-activated peroxisome receptor alpha), JUN (jun oncogene), TEMPI (metallopeptidase inhibitor TEMP 1), ILS (interleukin 5 (colony stimulating factor, eosinophil)), ILIA (interleukin 1, alpha), MMP9 (matrix metallopeptidase 9 (gelatinase B, 92-kDa gelatinase , 92-kDa type IV collagenase)), HTR4 (5-hydroxytryptamine (serotonin) receptor 4), HSPG2 (heparan sulfate proteoglycan 2), KRAS (v-Ki-ras2 homologue of rat Kirsten's sarcoma viral oncogene), CYCS (cytochrome c, somatic ), SMG1 (SMG1 homologue of phosphatidylinositol-related 3-kinase kinase (C. elegans)), IL1R1 (interleukin 1 receptor type I), PROK1 (prokineticin 1), MAPK3 (mitogen-activated protein kanase 3), NTRK1 (neurotorphic tyrosine kinase receptor type 1), IL13 (interleukin 13), MME (membrane metalloendopeptidase), TKT ( transketolase), CXCR2 (chemokine receptor 2 (CXC motif)), IGF1R (insulin-like growth factor 1 receptor), KARA (retinoic acid receptor alpha), CREBBP (CREB binding protein), PTGS1 (prostaglandin endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase)), GALT (galactose-1-phosphate uridyl transferase), CHRM1 (cholinergic receptor, muscarinic 1), ATXN1 (atkasin 1), PAWR (PRKC, apoptosis, WT1, regulator), NOTCH2 (Notch (Drosophila) homolog 2) , M6PR (mannose-6-phosphate receptor (cation-dependent)), CYP46A1 (cytochrome P450, family 46, subfamily A, polypeptide 1), CSNK1 D (casein kinase 1, delta), MAPK14 (mitogen-activated protein canase 14), PRG2 (proteoglycan 2, bone marrow (natural killer activator, the main main b trees)), PRKCA (protein kinase C, alpha), LI CAM (cell adhesion molecule LI), CD40 (CD40 molecules, TNF superfamily receptor, member 5), NRH2 (subfamily nuclear receptor 1, group I, member 2), JAG2 ( jagged 2), CTNND1 (catenin (cadherin-associated protein), delta 1), CDH2 (caderin 2, type 1, N-caderin (neuronal)), CMA1 (chymase 1, mast cells), SORT (sortilin 1), DLK1 (delta-like homolog 1 (Drosophila)), THEM4 (thioesterase superfamily, member 4), JUP (junction site placoglobin), CD46 (D46 molecule, complement regulatory protein), CCL11 (chemokine ligand (CC motif)11), CAV3 (caveolin 3), RNASES (ribonuclease, RNase A family, 3 (eosinophil cationic protein)), HSPA8 (70 kDa heat shock protein 8), CASP9 (caspase 9, apoptosis-associated cysteine peptidase), CYP3A4 (cytochrome P450, family 3, subfamily A , polypeptide 4), CCR3 (chemokine receptor (CC motif) 3), TFAP2A (transcription factor AP-2 alpha (activating enhancer protein 2 alpha)), SCP2 (styrene transfer protein 2), CDK4 (cyclin-dependent kinase 4), HIF1A (hypoxia inducible alkaline transcription factor helix-loop-helix factor 1), TCF7L2 (7- transcription factor-like 2 (T-cell specific, HMG box)), IL1R2 (interleukin 1 receptor, type II), B3GALTL (1,3-galactosyltransferase-like), MDM2 (Mdm2 homologue of p53-binding protein (mouse)), RELA ( v-rel homologue of reticuloendotheliosis virus (avian) oncogene A), CASP7 (caspase 7, apoptosis-associated cysteine peptidase), IDE (insulin degrading enzyme), FABP4 (fatty acid binding protein 4, adipocyte), CASK (calcium/calmodulin-dependent serine protein kinase (MAGUK family)), ADCYAP1R1 (type I type I adenylaticlase-activating polypeptide 1 (pituitary) receptor), ATF4 (activating transcription factor 4 (tax-responsive enhancer element B67)), PDGFA (platelet growth factor alpha polypeptide ), C21 or 0 3 (open reading frame 33 chromosome 21), SCG5 (secretogranin V (protein 7B2)), RNF123 (zinc finger protein 123), NFKB1 (nuclear factor enhancer gene for kappa light polypeptide in B cells 1), ERBB2 (v-erb -b2 erythroblastic leukemia viral oncogene homologue 2, neuro/glioblastoma derived oncogene homologue (avian)), CAV1 (caveolin 1, pit protein, 22 kDa), MMP7 (matrix metallopeptidase 7 (matrilysin, uterin)), TGFA (transforming growth factor , alpha), RXRA (retinoid receptor X, alpha), STX1A (syntaxin 1A (brain)), PSMC4 (proteasome 26S ATPase 4 subunit (prosome, multicatalytic protease), P2RY2 (P2Y purinergic receptor coupled to G-protein, 2), TNFRSF21 (tumor necrosis factor family member 21), DLG1 (disks, large homolog 1 (Drosophila)), NUMBL (numb gene-like homolog (Drosophila)), SPN (sialophorin), PLSCR1 (phospholipid scramblase 1), UBQLN2 (ubiquitin 2), UBQLN1 (ubiquitin 1), PCSK7 (subtilisin/kexin convertase precursor) type 7), SPON1 (spondin 1, extracellular matrix protein), SILV (silver homologue (mouse)), QPCT (glutamyl peptide cyclotransferase), HESS (hairy and split 5 enhancer (Drosophila)), GCC1 (GRIP and coiled-coil containing domain 1) or any combination of them.

[001179] A genetically modified animal or cell may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more discontinuous chromosomal sequences encoding a protein associated with diseases associated with secretases, and zero, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chromosomally integrated sequences encoding an interrupt protein associated with secretase-associated diseases.

ALS

[001180] US Patent Publication No. 20110023144 describes the application of zinc finger nucleases to genetically modified cells, animals and proteins associated with amyotrophic lateral sclerosis (ALS). ALS is characterized by gradual, stable degeneration of certain nerve cells in the cerebral cortex, brainstem, and spinal cord that are involved in voluntary movement.

[001181] Motor neuron diseases and the proteins associated with such diseases are a diverse set of proteins that cause motor neuron disease susceptibility, motor neuron disease severity, or any combination thereof. The present invention encompasses the editing of any chromosomal sequences encoding proteins associated with ALS, a specific motor neuron disease. ALS-associated proteins are usually identified based on the experimental association of ALS-associated proteins with ALS. For example, the rate of formation or circulating concentration of an ALS-associated protein may be increased or decreased in a population with ALS compared to a population without ALS. Differences in protein levels can be assessed using proteomics techniques, including, but not limited to, Western blotting, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, ALS-associated proteins can be identified by obtaining gene expression profiles for protein-coding genes using genomic techniques, including, but not limited to, DNA microarray analysis, serial gene expression analysis (SAGE), and quantitative real-time polymerase chain reaction. time (qPCR).

[001182] As a non-limiting example, ALS-associated proteins include, but are not limited to, the following: SOD1 superoxide dismutase 1, ALS3 soluble senataxin amyotrophic lateral sclerosis 3 SETX, ALS5 5 FUS amyotrophic lateral sclerosis, as a chimera in sarcoma, ALS7 - amyotrophic lateral sclerosis 7, ALS2 amyotrophic lateral sclerosis, DPP6 dipeptidyl peptidase 6 sclerosis 2, prostaglandin polypeptide endoperoxide synthase 1 of heavy PTGS1 neurofilament synthase NEFH, SLC1A2 soluble family transporter 1, TNFRSF10B tumor necrosis factor (high affinity glial receptor subfamily, glutamate transporter), member 10b , PRPH peripherin, HSP90AA1 90-kDA heat shock protein alpha (cytosolic), class A, representative 1, GRIA2 glutamate receptor, IFNG interferon, gamma ionotropic, AMPA 2 S100B S100 calcium-binding, FGF2 fibroblast growth factor B protein 2, A0X1 aldehyde oxidase 1, CS citrate synthase, TARDBP TAR DNA-binding protein, TXN thioredoxin, RAPH1 Ras association, MAP3K5 mitogen-activating protein (RaIGDS/AF-6) and plextrin kinase 5 homology domains 1, NBEAL1 neurobichin-like 1, GPX1 glutathione peroxidase 1, ICA1L islet cell autoantigen, RAC1 substrate bound with ras C3 like 1,69-kDa botulinum toxin toxin 1, MART microtubule-associated, ITPR2 triphosphate receptor inositol-1,4,5-tau protein type 2, ALS2CR4 amyotrophic lateral sclerosis, GLS glutaminase sclerosis 2 (juvenile) chromosomal region, candidate 4, ALS2CR8 amyotrophic lateral, CNTFR - ciliary neurotrophic sclerosis factor chromosomal region receptor (juvenile), candidate 8, ALS2CR11 amyotrophic lateral sclerosis, FOLH1 folate hydrolase 1 chromosome region of sclerosis 2 (juvenile), candidate 11, FAM117B family with sequence P4HB prolyl-4 -hydroxylases, similarity 117, representative B beta polypeptide, ciliary CNTF, neurotrophic factor, SQSTM1 seq esthosome 1, STRADB STE20-associated kinase, NAIP family NLR, inhibitory beta apoptosis adapter protein, YWHAQ tyrosine 3-SLC33A1 soluble monooxygenase/tryptophan family 33 transporter (acetyl-CoA transporter), representative 1 5-monooxygenase activating protein, theta polypeptide, TRAK2 transport protein, FIG. 4 FIG. 4 homologue, SACI lipid phosphatase domain, kinesin 2 binding, containing NIF3L1, NIF3, NGGI, interacting INA, intemexin, type 1 neuronal factor 3, intermediate filament protein, alpha PARD3B par-3 (separating) COX8A defective cytochrome oxidase B homologue c, VIHA subunit , CDK15 cyclin-dependent kinase, HECW1, HECT, C2 and WW 15 ubiquitin domain containing E3 protein ligase 1, NOS1 nitric oxide synthase 1, MET proto-cancer met, SOD2 superoxide dismutase 2, HSPB1 mitochondrial heat shock protein 1, 27-kDa, NEFL neurofilament, light, CTSB cathepsin B polypeptide, ANG angiogenin, HSPA8 heat shock ribonuclease, 70-kDa, RNase A protein 8 family 5, 5 VAPB, VAMP (vesicular-ESR1 estrogen receptor 1 membrane protein)-associated protein B and C, SNCA synuclein, alpha, HGF hepatocyte growth factor, CAT catalase, ACTB actin, beta, NEFM neurofilament, medium, TH tyrosine hydroxylase polypeptide, B-cell BCL2, CLL/lymphoma 2, FAS Fas (superfamily receptor tva TNF, representative 6), CASP3 caspase 3, apoptosis-associated CLU cluster cysteine peptidase, SMN1 motor neuron survival, G6PD glucose-6-phosphate 1, telomeric dehydrogenase, BAX associated with BCL2 X, HSF1 heat shock transcription factor 1, RNF19A AA zinc finger protein, JUN jun oncogene, ALS2CR12 amyotrophic lateral sclerosis, HSPA5 70 kDa sclerosis heat shock protein 2 (juvenile), chromosome region 5 protein candidate 12, MAPK14 mitogen-activated protein, IL10 interleukin 10 kinase 14, APEX1 APEX nuclease, TXNRD1 thioredoxin reductase 1 (DNA multifunctional repair domain) 1, NOS2 nitric oxide synthase 2, TIMP1 TIMP inducible metallopeptidase inhibitor 1, CASP9 caspase 9, apoptosis-XIAP X-linked cysteine-linked apoptosis inhibitor, GLG1 Golgi complex glycoprotein 1, EPOEGFA, Verythropoietin vascular endothelial growth factor factor A, elastin ELN factor A, GDNF nuclear factor NFE2L2 derived from glial cells, gr uppa (erythroid derived neurotrophic factor 2), 2 SLC6A3 soluble transporter, family 6, HSPA4 70-kDa heat shock protein (neurotransmitter transport protein 4, dopamine), representative 3, APOE apolipoprotein E, PSMB8 beta proteasome subunit (prosoma, multicatalytic protease ), type 8, DCTN1 dynactin 1, TIMP A3 TIMP metallopeptidase inhibitor 3, KIFAP3 kinesin-associated SLC1A1 soluble family transporter 1, protein 3 (high affinity neuronal/epithelial glutamate transporter, Xag system), representative 1, SMN2 cyclin C2 motor neuron survival CCNC, membrane protein centromere MPP4, STUB1, STIP1 homology and U-palmitoleated box 4 containing protein 1, ALS2 amyloid beta (A4), PRDX6 peroxiredoxin precursor protein 6, SYP synaptophysin, CABIN1 calcineurin binding protein 1, CASP1 caspase 1, apoptosis-GART phosphoribosylglycinamine -linked cysteine deformyltransferase, peptidase phosphoribosylglycineamine desynthetase, phosphoribosyl aminoimidase olsynthetase, CDK5 cyclin-dependent kinase 5, ATXN3 ataxin 3, RTN4 reticulon 4, C1QB complement component 1, q subcomponent, Nerve growth factor chain VEGFC, HTT huntingin receptor, Parkinson's disease PARK7 7, XDH xanthine dehydrogenase, GFAP glial fibrillary acid protein, MAP2 associated with microtubules protein 2, CYCS cytochrome c, somatic, FCGR3B Fc fragment of IgG, low affinity IIIb, CCS copper chaperone of ubiquitin-like superoxide dismutase 5 UBL5, MMP9 matrix metallopeptidase 9, SLC18A3 soluble family transporter 18 ((vesicular aetylcholine), representative 3, TRPM7 transient receptor , HSPB2 27-kDa heat shock cation channel, M subfamily protein 2, member 7, AKT1 v-akt murine thymoma, DERL1 family of Der1-like domains, viral oncogene homologue 1, member 1, CCL2 chemokine (motif C--C), NGRN neugrin, neuritic ligand 2, budding-associated GSR gutation reductase, TPPP3 tubulin polymerization-stimulating protein family, p representative 3, APAF1 apoptosis peptidase, BTBD10 BTB domain activating factor (POZ) factor 1 containing 10 GLUD1 glutamate, CXCR4 chemokine (C--X--C motif), dehydrogenase receptor 4 1, SLC1A3 family 1 soluble transporter, FLT1 associated with fms tyrosine (high affinity glial glutamate transporter), representative 3, PON1 paraoxonase 1, androgen receptor AR, L1P leukemia inhibitory factor, ERBB3 v-erb-b2 homologue 3 of the erythroblat leukemia viral oncogene, LGALS1 lectin, galactoside, CD44 binding CD44, soluble protein 1 , TP53 p53 tumor protein, TLR3 toll-like receptor 3, GR1A1 glutamate receptor, GAPDH glyceraldehyde-3 ionotropic, AMPA 1 phosphate dehydrogenase, GR1K1 glutamate receptor, DES desmin, ionotropic, kainate 1, CHAT choline acetyltransferase, FLT4 fms-related triosine kinase 4, CHMP2B chromatin-modifying, BAG1 BCL2-associated protein, 2B athanogen, MT3 metallothionenin 3, CHRNA4 cholinergic receptor, nicotinic alpha 4, GSS - glutathione synthetase BAK1, BCL2/killer 1 antagonist, KDR kinase insertion domain, GSTP1 glutathione S-transferase receptor (type III tyrosine kinase receptor pi 1), OGG1 8-oxoguanine DNA, IL6 interleukin 6 (interferon, glycosylase beta 2).

[001183] An animal or cell may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more discontinuous chromosome sequences encoding an ALS-associated protein and null, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chromosomally integrated sequences encoding an ALS-associated interrupt protein. Preferred proteins associated with ALS include SOD1 (superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis protein 2), FUS (sarcoma fusion), TARDBP (TAR DNA binding protein), VAGFA (vascular endothelial growth factor A), VAGFB (factor vascular endothelial growth factor B) and VAGFC (vascular endothelial growth factor C) and any combination thereof.

Autism

[001184] US Patent Publication No. 20110023145 describes the application of zinc finger nucleases to genetically modified cells, animals, or proteins associated with autism spectrum disorder (ASD). Autism Spectrum Disorders (ASD) are a group of disorders characterized by a quantitative deterioration in social interactions and communication, and fixed repetitive and stereotypical patterns of behavior, interests and activities. The three diseases: autism, Asperger's Syndrome (AS), and Pervasive Developmental Disorder Not Otherwise Specified (PDD-NOS) represent a spectrum of a single disorder with varying severity associated with intellectual functioning and health status. ASDs are predominantly genetic diseases with a heritability of about 90%.

[001185] US Patent Publication No. 20110023145 describes the editing of any chromosomal sequences encoding ASD associated proteins to which the CRISPR-Cas systems of the present invention can be applied. ASD-associated proteins are usually established on the basis of experimental association of ASD-associated proteins with the occurrence or manifestation of ASD. For example, the rate of formation or circulating concentration of an ASD-associated protein may be increased or decreased in a population with ASD compared to a population without ASD. Differences in protein levels can be assessed using proteomics techniques including, but not limited to, Western blotting, immunohistochemical staining, ELISA, and mass spectrometry. Alternatively, ASD-associated proteins can be identified by obtaining gene expression profiles for protein-coding genes using genomic techniques, including, but not limited to, DNA microarray analysis, serial gene expression analysis (SAGE), and quantitative real-time polymerase chain reaction. time (qPCR).

[001186] Non-limiting examples of pathological conditions or disorders that may be associated with ASD-associated proteins include autism, Asperger's syndrome (AS), pervasive developmental disorder not otherwise specified (PDD-NOS), Rett syndrome, tuberous sclerosis, phenylketonuria , Smith-Lemli-Opitz syndrome and fragile X syndrome. As a non-limiting example, proteins associated with ASD include, but are not limited to, the following: ATP10C aminophospholipid, MET receptor ATPase transporting tyrosine kinase (ATP10C), BZRAP1 MGLUR5 (GRM5) metabotropic glutamate receptor 5, (MGLUR5) CDH10 cadherin-10, MGLUR6 (GRM6) metabotropic glutamate receptor 6 (MGLUR6), CDH9 cadherin-9 NLGN1, neuroligin-1, CNTN4 contactin-4, NLGN2 neuroligin-2, CNTNAP2 contact-associated, SEMA5A neuroligin-3 protein type 2 (CNTNAP2), DHCR7 7 -dehydrocholesterol, NLGN4X X-reductase-related neuroligin-4 (DHCR7) DOC2A, dual C2-like domain, NLGN4Y neuroligin-4, Y-containing protein-alpha-related, DPP6 dipepidyl, NLGN5 neuroligin-5, aminopeptidase-like protein 6, EN2 engrailed 2 (EN2), NRCAM neuronal cell adhesion molecule (NRCAM), MDGA2 mental retardation with fragile X chromosome, NRXN1 - neurexin-1 1 (MDGA2), FMR2 (AFF2), AF4/FMR2 family representative 2 OR4M2 olfactory receptor ( AFF2), 4M2 FOXP2 protein with box Forkhead P2, OR4N4 olfactory receptor (FOXP2), 4N4 FXR1 mental retardation with X fragility, OXTR oxytocin receptor, autosomal (OXTR) homologue 1, (FXR1) FXR2 mental retardation with fragile X, PAH phenylalanine hydroxylase (PAH) ), homologue 2, (FXR2) GABRA1 gamma-aminobutyric acid, PTEN phosphatase and receptor, tensin homolog, alpha-1 subunit (GABRA1) (PTEN), GABRA5 GABAA (aminobutyric acid receptor PTPRZ1) receptor alpha 5 tyrosine protein subunit, ( GABRA5) phosphatase zeta (PTPRZ1), GABRBI reelin aminobutyric acid receptor RELN, subunit beta-1 (GABRB1), GABRB3 GABAA receptor beta 3 subunit protein L10 (gamma-aminobutyric acid RPL10 ribosome 60S), L10 (GABRB3), GABRG1 gamma-aminobutyric acid, SEMA5A semaphorin-5A receptor, gamma-1 subunit, (SEMA5A) (GABRG1) HIRIP3 HIRA-interacting protein 3, SEZ6L2 seizure-associated homologue group 6 (mouse), 2 HOXA1 Hox-A1 homeobox protein, SHANKS SH3 and (HOXA1 ) domains multiple ankyrin repeat 3 (SHANKS), 1L6 interleukin-6, SHBZRAP1 SH3 and multiple ankyrin repeat 3 domains (SHBZRAP1), LAMB1 laminin, beta-1 subunit, SLC6A4 serotonin transporter (LAMB1) (SERT), MAPK3 mitogen-activated protein, TAS2R1 kinase taste receptor 3, type 2, representative 1, TAS2R1 MAZ Myc-associated zinc finger, TSC1 tuberous sclerosis protein 1, MDGA2 MAM domain containing TSC2, glycosylphosphatidylinositol protein 2, anchor 2 (MDGA2), MECP2 methyl binding CpG, UBE3A ubiquitin protein, 2, (MECP2) E3A ligase (UBE3A), MECP2 methyl binding CpG WNT2, Wingless type protein 2 (MECP2), MMTV integration site family member 2 (WNT2).

[001187] The type of ASD-associated protein whose chromosomal sequence is being edited can and will vary. In preferred embodiments, ASD-associated proteins whose chromosomal sequences can be edited can be: Benzodiazepine receptor-related (peripheral) protein 1 (BZRAP1), encoded by the BZRAP1 gene, AF4/FMR2 family member protein 2 (AFF2) , encoded by the AMA gene (also called MFR2), autosomal fragile X mental retardation homologue protein 1 (FXR1), encoded by the FXR1 gene, autosomal fragile X mental retardation homologue protein 2 (FXR2), encoded by the FXR2 gene, MAXI domain containing glycosylphosphatidylinositol anchor protein 2 (MDGA2) encoded by the MDGA2 gene, methyl-CpG binding protein 2 (XIECP2) encoded by the MECP2 gene, metabotropic glutamate receptor 5 (MGLUR5) encoded by the MGLUR5-1 gene (also called GRM5), protein neurexin 1 encoded by the NRXN1 gene or semaphorin-5A (SEMA5A) protein encoded by the SEMA5A gene. In an exemplary embodiment, the genetically modified animal is a rat, the edited chromosomal sequence encoding an ASD-associated protein is in the following list: BZRAP1 benzodiazepine receptor XM_002727789, (peripheral) associated XM_001081125, AFF2 (FMR2), member 2 of the AF4/FMR2 family XM_219832 , (AFF2) XM__001054673, FXR1 умствеенной отсталости при хрупкой X-хромосоме NM_001012179, аутосомальный гомолог 1 (FXR1) FXR2 умственной отсталости при хрупкой X-хромосоме NX_001100647, аутосомальный гомолог 2 (FXR2) MDGA2, домен MAXI, содержащий якорь 2 гликозилфосфатидилинозитола NM_199269, ( MDGA2) MECP2 methyl-CpG binding protein 2 NM_022673, (MECP2) MGLUR5 metabotropic glutamate receptor 5 NM_017012 (GRM5) (MGLUR5), NRXN1 neurexin-1 NM_021767 SEMA5A, semaphorin-5A (SEMA5A) NM_001107659.

Trinucleotide repeat expansion disorders

[001188] US Patent Publication No. 20110016540 describes the use of zinc finger nucleases to genetically modify cells, animals, and proteins associated with trinucleotide repeat expansion diseases. Trinucleotide repeat expansion diseases are complex, progressive diseases that involve developmental neuroscience and often affect cognition and sensorimotor function.

[001189] Trinucleotide repeat expansion proteins are a diverse set of proteins associated with susceptibility to developing trinucleotide repeat expansion disease, the presence of trinucleotide repeat expansion disease, or a combination thereof. Trinucleotide repeat expansion diseases are divided into 2 categories depending on the type of repeats. The most common repeat is the CAG triplet, which, when present in the coding region of the gene, codes for the amino acid glutamine (Q). Therefore, such diseases are referred to as polyglumatin (polyQ) diseases and include the following diseases: Huntington's disease (HD); spinobulbar muscular atrophy (SBMA), spinocerebellar ataxias (SCA, types 1, 2, 3, 6, 7, and 17), dental rubro-pallidoluis atrophy (DRPLA). The rest of the trinucleotide repeat diseases either do not imply the presence of the CAG triplet, or the CAG triplet is not located in the coding region of the gene and, as a result, are called non-polyglutamine diseases. Non-polyglutamine disorders include fragile X syndrome (FRAXA); mental retardation with a fragile X chromosome (FRAXE); Friedreich's ataxia (FRDA); myotonic dystrophy (DM); and spinocerebellar ataxia (SCA, types 8 and 12).

[001190] Disease-associated trinucleotide repeat expansion proteins are generally established based on the experimental association of disease-associated trinucleotide repeat expansion proteins with trinucleotide repeat expansion disease. For example, the rate of formation or circulating concentration of a disease-associated trinucleotide repeat expansion disease may be increased or decreased in a population with trinucleotide repeat expansion disease compared to a population without trinucleotide repeat expansion disease. Differences in protein levels can be assessed using proteomics techniques including, but not limited to, Western blotting, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, disease-associated trinucleotide repeat expansion proteins can be identified by obtaining gene expression profiles for protein-coding genes using genomics techniques including, but not limited to, DNA microarray analysis, gene expression serial analysis (SAGE), and quantitative polymerase chain analysis. real-time reaction (qPCR).

[001191] Non-limiting examples of proteins associated with trinucleotide repeat expansion diseases are: AR (Androgen receptor), FMR1 (Fragile X-chromosome mental retardation1), HTT (Huntingin), DMPK (Myotonic dystrophy protein kinase), FXN (Frataxin) , ATXN2 (ataxin 2), ATN1 (atrophin 1), FENl (structure-specific flap 1 endonuclease), TNRC6A (containing trinucleotide repeat 6A), PABPN1 (poly(A) binding nuclear protein 1), JPH3 (junctophilin 3), MED15 (subunit 15 transmitter complex), ATXN1 (ataxin 1), ATXN3 (ataxin 3), TBP (TATA box binding protein), CACNA1A (calcium channel, voltage gated, P/Q type, alpha 1A subunit), ATXN80S (ATXN8 reverse chain (non-coding )), PPP2R2B (protein phosphatase 2, regulatory subunit B, beta), ATXN7 (ataxin 7), TNRC6B (containing trinucleotide repeat 6B), TNRC6C (containing trinucleotide repeat 6C), CELF3 (CUGBP, Elav-like family, representative 3), MAB21L1 (mab-21-like I ( C. elega ns )), MSH2 (mutS 2 homolog, rectal cancer, non-polyposis type 1 ( E. coli )), TMEM185A (185A transmembrane protein), SIX5 (SIX homeobox 5), CNPY3 (canopy 3 (zebrafish) homolog), FRAXE ( fragile site, folic acid type, rare, fra(X)(q28) E), GNB2 (nucleoetide-binding guanine protein (G protein), beta polypeptide 2), RPL14 (ribosomal protein L14), ATXN8 (ataxin 8), INSR (insulin receptor), TTR (transthyretin), EP400 (E1A binding protein p400), GIGYF2 (GRB10 interacting GYT protein 2), OGGI (8-oxoguanine DNA glycosylase), STC1 (stanniocalcin 1), CNDPI (carnosine dipeptidase 1 ( metallopeptidase family M20)), C10orf2 (open reading frame 2 chromosome 10), MAML3 like mastermind 3 ( Drosophila ), DKC1 (dyskerin congenita 1), PAXIP1 PAX interacting (with transcription activation domain) protein 1, CASK (calcium/calmodulin -dependent protein kinase (MAGUK family)), MART (microtubule-associated protein tau), SP1 (transcription factor Sp 1), POLG (DNA-dependent RNA polymerase gamma), AFF2 (AF4/FMR2 family, member 2), THBS1 (thrombospondin 1), TP53 (p53 tumor protein), ESR1 (estrogen receptor 1), CGGBP1 (triplet repeat binding CGG protein 1), ABT1 (basal transcription activator 1), KLK3 (calicrein-related peptidase 3), PRNP (prion protein), JUN (jun oncogene), KCNN3 (medium/low conductance calcium-activated potassium channel, subfamily N, member 3 ), BAX (BCL2-associated protein X), FRAXA (fragile site, folic acid type, rare, fra(X)(q27.3), A (mental retardation macroorchidism)), KBTBD10 (kelch repeat and containing BTB domain ( POZ) 10), MBNL1 (muscleblind-like ( Drosophila )), RAD51 (RAD51 homolog (RecA, E. coli homolog) ( S. cerevisiae )), NCOA3 (nuclear receptor coactivator 3), ERDA1 (expanded repeat domain, CAG/CTG 1), TSC1 (tuberous sclerosis 1), COMIP (oligomeric cartilage matrix protein), GCLC (glutamate cysteine ligase, catalytic RRAD (coupling Ras associated with diabetes), MSH3 (mutS 3 homologue ( E. coli )), DRD2 (dopamine receptor D2), CD44 (CD44 molecule (Indian blood group system)), CTCF (CCCTC binding factor (zinc finger protein )), CCND1 (cyclin DI), CLSPN (claspin homologue (Xenopus laevis)), MEF2A (myocyte enhancer factor 2A), PTPRU (protein tyrosine phosphatase, receptor type U), GAPDH (glyceraldehyde 3-phosphate dehydrogenase), TRIM22 (tripartite containing motif 22), WT1 (Wilms tumors 1), AHR (aryl hydrocarbon receptor), GPX1 (glutathione peroxidase 1), TPMT (thiopurine-S-methyltransferase), NDP (Norrie's disease (pseudoglioma)), ARX (aristaless homeobox-associated), MUS81 (MUS81 endonuclease homologue ( S. cerevisiae )), TYR (tyrosinase (LA oculocutaneous albinism)), EGR1 (early growth response 1), UNG (uracil-DNA glycosylase), NUMBL (numb homologue ( Drosophila ) similar), FABP2 ( fatty acid binding protein 2, intestinal), EN2 (engrailed, gene containing homeobox 2), CRYGC (crystalin ha mma C), SRP14 (14-kDa recognition signal particle (homologous Alu RNA-binding protein)), CRYGB (crystallin, gamma B), PDCD1 (programmed cell death 1), HOXA1 (homeobox Al), ATXN2L (ataxin-like 2) , PMS2 (PMS2 postmeiotic segregation enhancement 2 ( S. cerevisiae )), GLA (galactosidase, alpha), CBL (Cas-Br-M (mouse) ecotropic retroviral transforming sequence), FTH1 (ferritin, heavy polypeptide 1), IL12RB2 (receptor interleukin 12, beta 2), OTX2 (homeobox orthodenticle 2), HOXA5 (homeobox A5), POLG2 (DNA-dependent RNA polymerase, gamma 2, additional subunit), DLX2 (homeobox distal-less 2), SIRPA. (alpha signaling regulatory protein), OTX1 (orthodenticle homeobox 1), AHRR (aryl hydrocarbon receptor repressor), MANF (mesencephalic astrocyte-derived neurotrophic factor), TMEM158 (transmembrane protein 158 (gene/pseudogene)) and ENSG00000078687.

[001192] Preferred proteins associated with diseases associated with the expansion of trinucleotide repeats include HTT (huntingin), AR (androgen receptor), FXN (frataxin), Atxn3 (ataxin), Atxn1 (ataxin), Atxn2 (ataxin), Atxn7 ( ataxin), Atxn10 (ataxin), DMPK (myotonic dystrophy protein kinase), Atn1 (utrophin 1), CBP (creb binding protein), VLDLR (very low density lipoprotein receptor), and combinations thereof.

Hearing disease treatment

[001193] The present invention also provides for delivery of the CRISPR-Cas system to one or both ears.

[001194] Attention of researchers is focused on the question of whether gene therapy can be used to improve existing methods of treating deafness, namely cochlear implants. A common cause of deafness is the loss or damage of hair cells, which causes impaired signal transmission to auditory neurons. In such cases, cochlear implants can be used to provide sound response and electrical signals to nerve cells. However, such neurons often degenerate and are retracted from the cochlea when there is a decrease in the amount of growth factors released by damaged hair cells.

[001195] U.S. Patent Application 20120328580 describes injecting a pharmacological mixture into the ear (e.g., ear administration), such as injecting into the cochlear lumen (e.g., scala media, scala vestibulae), and scala tympani (Scala tympani)), for example, using a syringe, such as a disposable syringe. For example, one or more of the compounds described in this specification may be administered by intratympanic injection (particularly into the middle ear), and/or by injection into the outer, middle, and/or inner ear. Such methods are widely used in this field, for example, for the introduction of steroids and antibiotics into the human ear. The injection can be made, in particular, through a round window in the ear or a cochlear capsule. Other routes of administration to the inner ear are also known in this art (see, for example, Salt and Plontke, Drug Discovery Today, 10:1299-1306, 2005).

[001196] When using another method of administration, the pharmacological mixture can be administered in situ using a catheter or pump. The catheter or pump may, for example, direct the pharmacological mixture into the cochlear cavity or round window in the ear and/or rectal lumen. Commonly used drug delivery devices and methods that are suitable for delivering one or more of the compounds described in this specification to the ear, such as the human ear, as described in McKenna et al., (US Publication No. 2006/0030837) and Jacobsen et al. . (US patent No. 7206639). In some embodiments, a catheter or pump may be placed, for example, in the ear (eg, outer, middle, and/or inner ear) of a patient during surgery. In some embodiments of the invention, a catheter or pump may be placed, for example, in the ear (eg, outer, middle, and/or inner ear) of a patient without the need for surgery.

[001197] Alternatively, or in addition, one or more of the components described in this specification may be administered in conjunction with a mechanical device, such as a cochlear implant and a hearing aid, that is placed in the outer ear. A commonly used cochlear implant suitable for use with the present invention is described by Edge et al. (US Publication No. 2007/0093878).

[001198] In some embodiments, the methods of administration described above may be used in any order, simultaneously or alternately.

[001199] Alternatively or in addition, the present invention may be carried out in accordance with any Food and Drug Administration approved methods, such as those described in the Data Standards Manual. CDER version number 004 (which is available at fda.give/cd.er/dsmZDRG/drg003 01. htm).

[001200] In general, the cell therapy methods described in U.S. Patent Application 20120328580 can be applied to result in complete or partial cell differentiation, or applied to mature cell types, e.g., inner ear (e.g., hair cells ) in vitro . Cells resulting from such methods can then be transplanted or implanted into a patient in need of such treatment. The cell culture methods necessary to carry out these methods, including methods for identifying and selecting appropriate cell types, methods for promoting complete or partial differentiation of selected cells, methods for identifying complete or partially differentiated cell types, and methods for implanting complete or partially differentiated cells are described below.

[001201] Cells suitable for use in the present invention include, but are not limited to, cells capable of fully or partially differentiating into a mature inner ear cell, e.g., a hair cell (e.g., inner and/or outer hair cell) upon contact, e.g. , in vitro, with one or more of the compounds described in this application. Examples of cells capable of differentiating into hair cells include, but are not limited to, stem cells (e.g., inner ear stem cells, adult stem cells, bone marrow stem cells, embryonic stem cells, mesenchymal stem cells, skin stem cells, iPS induced stem cells). and adipose tissue-derived stem cells), progenitor cells (eg, inner ear progenitor cells), accessory cells (eg, Deiters cells, columnar cells, internal phalangeal cells, tectum cells, and Hansen cells), and/or germ cells. The use of stem cells to replace inner ear sensory cells is described in Li et al. (US Publication No. 2005/0287127) and Li et al. (U.S. Patent No. 11/953,797). The use of bone marrow-derived stem cells to replace inner ear sensory cells is described in Edge et al., PCT/US2007/084654. The use of inducible stem cell (iPS) cells is described, for example, in Takahashi et al. Cell, Volume 131, Issue 5, Pages 861-872 (2007); Takahashi and Yamanaka, Cell 126, 663-76 (2006); Okita et al., Nature 448, 260-262 (2007); Yu, J. et al., Science 318(5858):1917-1920 (2007), Nakagawa et al., Nat. Biotechnol. 26:101-106 (2008); and Zaehres and Scholer, Cell 131(5):834-835 (2007). Such suitable cells can be identified by analyzing (eg, qualitatively or quantitatively) the presence of one or more tissue-specific genes. For example, gene expression can be detected by detecting the protein product of one or more tissue-specific genes. Methods for detecting a protein include staining proteins (eg using cell or whole cell extracts) using antibodies against the appropriate antigen. In this case, the appropriate antigen is the protein product of tissue-specific expression of the gene. Although, in general, the primary antibody (ie, the antibody that binds the antigen) may be labeled, it is more common (and improves visualization) to use a second antibody to the first (eg, anti-IgG). This second antibody is conjugated to either fluorochromes or appropriate enzymes for colorimetric reactions, or gold beads (for electron microscopy) or a biotin-avidin system, so that the location of the primary antibody and hence the antigen can be determined.

[001202] The CRISPR Cas molecules of the present invention can be delivered to the ear by direct application of the pharmaceutical composition to the outer ear with compositions modified from US Published Application 20110142917. In some embodiments, the pharmaceutical composition is delivered to the ear canal. Delivery to the ear may also be referred to as otic or ear-guided delivery.

[001203] In some embodiments, the RNA molecules of the invention are provided in liposome or lipofectin and the like formulations and can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in US patent No. 5599772, 5589466 and 5580859, which are included in the present description by reference.

[001204] Delivery systems specifically targeting enhanced and improved siRNA delivery to mammalian cells have been developed (see, for example, Shen et al. FEBS Let. 2003, 539:111-114; Xia et al., Nat. Biotech. 2002, 20:1006-1010; Reich et al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol. Biol. 2003, 327: 761-766; Lewis et al., Nat Gen. 2002, 32: 107-108 and Simeoni et al., NAR 2003, 31, 11: 2717-2724) and can be applied to the present invention. Recently, siRNA has been successfully used to inhibit gene expression in primates (see, for example, Tolentino et al., Retina 24(4):660) and can also be applied to this invention.

[001205] Qi et al. describes methods for efficient transfection of siRNA into the inner ear through an intact round hole according to a new proteoid delivery technology that can be applied to the nucleic acid targeting system of the present invention (see, for example, Qi et al., Gene Therapy (12013), 1- 9). In particular, the delivery of TAT double-stranded RNA-binding domains (TAT-DRBD), which can transfect Cy3-labeled siRNA into cells of the inner ear, including inner and outer hair cells, semicircular canals (crista ampullaris), auditory spots: oval sac (macula utriculi) and the macula sacculi spot, through intact penetration into the foramen magnum, has been successful for double-stranded siRNAs in vivo to treat various inner ear diseases and preserve auditory function. Approximately 40 µl of 10 mM RNA can be considered as the optimal dosage for administration to the ear.

[001206] According to Rejali et al. (Hear Res, 2007 Jun, 228 (l-2): 180-7), cochlear implant function can be improved by good preservation of helical ganglion neurons that are the object of electrical stimulation by the implant. Brain-derived neurotropic factor (BDNF) has previously been shown to increase spiral ganglion survival in experimentally stunned ears. Rejali et al. tested a modified cochlear implant electrode design that included a coating of fibroblast cells transduced with a viral vector with a BDNF gene insert. To perform this type of ex vivo gene transfer, Rejali et al. transduced adenovirus guinea pig fibroblasts with a BDNF cassette insert and verified that these cells secrete BDNF, then attached the BDNF-secreting cells to a cochlear implant with agarose gel and implanted an electrode in the scala tympani (scala tympani). Rejali et al. determined that BDNF-expressing electrodes were able to preserve significantly more helical ganglion neurons in the basal coils of the cochlea after 48 days of implantation compared with control electrodes and demonstrated the possibility of combining cochlear implant treatment with ex vivo gene transfer to enhance the survival of helical ganglion neurons. ganglion. Such a system can be applied to the nucleic acid targeting system of the invention for delivery to the ear.

[001207] Mukherjea et al. (Antioxidants & Redox Signaling Volume 13, Number 5, 2010), demonstrated that N0X3 knockdown using short interfering (si) RNA blocked cisplatin ototoxicity, as evidenced by protecting outer hair cells (OHC) from damage and reducing threshold shifts in auditory response. brain stem (ABR). Various doses of siNOX3 (0.3, 0.6 and 0.9 μg) were administered to rats and NOX3 expression was assessed by real-time RT-PCR. The lowest dose of NOX3 siRNA used (0.3 µg) showed no inhibition of NOX3 mRNA compared to transtympanic administration of scrambled siRNA or untreated cochleas. However, administration of higher doses of N0X3 siRNA (0.6 and 0.9 μg) reduced the expression of N0X3 compared to control scrambled siRNA. Such a system can be applied to the CRISPR Cas system of the present invention for transtympanic administration at a dose of about 2 mg to about 4 mg CRISPR Cas for administration to a human.

[001208] Jung et al. (Molecular Therapy, vol. 21 no. 4, 834-841 apr. 2013) demonstrate that the level of Hes5 in the elliptical pouch decreased after siRNA application and that the number of hair cells in said elliptical pouch was significantly greater than in the control. The data indicate that siRNA technology may be useful for inducing repair and regeneration in the inner ear and that the Notch signaling pathway is a potentially useful target for specific inhibition of gene expression. Jung et al. 8 µg of Hes5 siRNA was injected in a volume of 2 µl obtained by adding sterile normal saline to lyophilized siRNA to the vestibular epithelium of the ear. Such a system can be applied to the nucleic acid targeting system of the present invention for administration to the vestibular epithelium of the ear at a dose of about 1 to about 30 mg of CRISPR Cas for administration to a human.

Targeting genes in non-dividing cells (neurons and muscles)

[001209] Non-dividing (especially non-dividing, fully differentiated) cell types present problems for gene targeting or genetic engineering, for example, because homologous recombination (HR) is typically downregulated in the G1 phase of the cell cycle. However, by studying the mechanisms by which cells control normal DNA repair systems, Durocher discovered a previously unknown switch that keeps HR "off" in non-dividing cells and developed a strategy to flip that switch back. Orthwein et al. (Daniel Durocher lab at Mount Sinai Hospital in Ottawa, Canada) recently published (Nature 16142, published online on December 9, 2015) that HR suppression can be reversed, and thus targeting genes in both kidney cells (293T), and in osteosarcoma cells (U2OS) was successfully carried out. The tumor suppressors BRCA1, PALB2, and BRAC2 are known to promote repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). They found that the formation of the BRCA1 complex with PALB2-BRAC2 is regulated by the ubiquitin site on PALB2, such that such an action is regulated at this site by the E3 ubiquitin ligase. This E3 ubiquitin ligase consists of KEAP1 (PALB2 interacting protein) in complex with Cullin-3 (CUL3)-RBX1. Ubiquitylation of PALB2 inhibits its interaction with BRCA1 and counteracts deubiquitinase USP11, which itself is controlled by the cell cycle. Restoration of the BRCA1-PALB2 interaction, combined with activation of resection (removal) of DNA ends, is sufficient to induce homologous recombination at G1, which can be measured in a number of ways, including a CRISPR-Cas9-based gene targeting assay directed to USP11 or KEAP1 (expressed from a vector pX459). However, when BRCA1-PALB2 interaction was restored in Gram-negative G1 cells using either KEAP1 depletion or PALB2-KR mutant expression, a significant increase in gene targeting was found.

[001210] Thus, in some embodiments, HR reactivation is preferred, especially in non-dividing cells of fully differentiated cell types. In some embodiments, the implementation of the interaction BRCA1-PALB2 is preferred. In some embodiments, the target cell is non-dividing. In some embodiments, the target cell is a neuron or a muscle cell. In some embodiments, target cell targeting occurs in vivo . In some embodiments, the cell is in G1 and the HR is suppressed. In some embodiments, the use of KEAP1 depletion, such as inhibition of the expression of KEAP1 activity, is contemplated, which is preferred. KEAP1 depletion can be achieved by siRNA, for example, as shown in Orthwein et al. Alternatively, expression of the PALB2-KR mutant (minus all eight Lys residues in the BRCA1 interaction domain) is preferred and is used either alone or in combination with KEAP1 depletion. PALB2-KR interacts with BRCA1 regardless of cell cycle time, thus promoting or restoring BRCA1-PALB2 interaction, especially in G1 cells, is preferred in some embodiments, especially where target cells are not dividing, or where deletion and return ( ex vivo gene targeting) is problematic, for example, in neurons and muscle cells. KEAP1 siRNA is available from ThermoFischer. In some embodiments, the BRCA1-PALB2 complex can be delivered to the G1 cell. In some embodiments, PALB2 deubiquitination can be stimulated, for example, by increasing the expression of USP11 deubiquitinase, so it is contemplated that a construct can be provided to stimulate or upregulate USP11 deubiquitinase expression or activity.

Treatment of eye diseases

[001211] The present invention also contemplates delivery of the CRISPR-Cas system to one or both eyes.

[001212] In yet another embodiment, the CRISPR-Cas system can be used to correct eye defects that arise from several genetic mutations, described in detail below in Genetic Diseases of the Eye, Second Edition, edited by Elias I. Traboulsi, Oxford University Press, 2012.

[001213] For ocular administration, lentiviral vectors, in particular equine infectious anemia viruses (EIAV), are especially preferred.

[001214] In one embodiment, Minimal Equine Infectious Anemia Virus (EIAV) lentiviral vectors are also contemplated, especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med 2006; 8: 275-285, Published online 21 November 2005 in Wiley InterScience (www.interscience.wiley.com) DOF 10.1002/jgm.845). The vectors are expected to have a cytomegalovirus (CMV) promoter that induces expression of the target gene. Intracameral, subretinal, intraocular, and intravitreal injections (see, for example, Balagaan, J Gene Med 2006; 8: 275-285, Published online 21 November 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm .845). Intraocular injections can be performed using an operating microscope. For subretinal and intravitreal injections, the eyes can be prolapsed under slight pressure and under observation and inspection, which are visualized using a contact lens system consisting of a drop of a solution of the connecting medium on the cornea covered with a microscope coverslip. For subretinal injections, the tip of a 10 mm 34 gauge needle mounted on a 5 µl halide syringe can be advanced under direct visualization through the superior equatorial sclera tangentially to the posterior pole until the needle opening is visible into the subretinal space. Then 2 µl of the vector suspension can be injected to obtain a prominent bullous retinal detachment, which confirms the introduction of the subretinal vector. This approach creates a self-sealing sclerotomy that allows the vector suspension to be retained in the subretinal space until it is taken up by the retinal pigment epithelium (RPE), usually within 48 hours after the procedure. This procedure can be repeated in the lower hemisphere to produce a retinal detachment there. This method results in approximately 70% of the neurosensory retina and retinal pigment epithelium (RPE) being exposed to the vector suspension. For intravitreal injections, the tip of the needle can be advanced through the sclera 1 mm posteriorly to the corneoscleral limbus and 2 µl of the vector suspension can be injected into the vitreous cavity. For intracameral injections, the tip of the needle can be advanced through the corneoscleral limbal paracentesis towards the central cornea and 2 µl of the vector suspension can be injected. These vectors can be injected in titers of 1.0-1.4×10 ¹⁰ or 1.0-1.4×10 ⁹ transducing units (TU)/ml.

[001215] In another embodiment, RetinoStat®, an equine infectious anemia virus-based lentiviral gene therapy uses a vector expressing the angiostatic proteins endostatin and angiostatin delivered by subretinal injection to treat the reticular form of age-related macular degeneration (see, e.g., Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)). Such a vector can be modified for use in the nucleic acid targeting system of the present invention. Each eye can be treated with RetinoStat® at a dosage of 1.1×10 ⁵ transducing units per eye (TU/eye) in a total volume of 100 μl.

[001216] In another embodiment, an E1-, partial E3-, E4-deleted adenoviral vector can be provided for delivery to the eye. Twenty-eight patients with advanced neovascular age-related macular degeneration (AMD) received one intravitreal injection of an E1-, partial E3-, E4-deleted adenoviral vector expressing epithelial pigment factor II (AdPEDF II) (see, for example, Campochiaro et al., Human Gene Therapy 17:167-176 (February 2006) Doses from 10 ⁶ to 10 ^9.5 units (PU) have been studied and no serious side effects associated with AdPEDF II have been shown and no dose-limiting toxicity has been shown (see ., e.g., Campochiaro et al., Human Gene Therapy 17:167-176 (February 2006)). .

[001217] In another embodiment, the sd-rxRNA® RXi Pharmaceuticals system can be used and/or adapted to deliver CRISPR-Cas to the eye. This system involves a single intravitreal injection of 3 µg of sd-rxRNA, which results in a consistent decrease in PPIB mRNA levels over 14 days. The sd-rxRNA® system can be applied to the nucleic acid targeting system of the present invention, assuming a dose of approximately 3 to 20 mg of CRISPR administered to a human.

[001218] Millington-Ward et al. (Molecular Therapy, vol. 19 no. 4, 642-649 Apr. 2011) describe the use of adeno-associated viruses (AAVs) to deliver an RNA interference (RNA-i) suppressor of rhodopsin and a codon-modified gene of rhodopsin resistant to suppression due to nucleotide changes in degenerate positions in the target site of RNA interference. Injection into the eye of a dose of 6.0×10 ⁸ virus particles (vp) or 1.8×10 ¹⁰ hv AAV was performed subretinally by Millington-Ward et al. AAV vectors Millington-Ward et al. can be applied to the CRISPR Cas system of the present invention, with an intended dose of about 2×10 ¹¹ to about 6×10 ¹³ viral particles (vp) administered to a human.

[001219] Dalkara et al. (Sci Transl Med 5, 189ra76 (2013)) also turns to directed evolution in vivo to create an AAV vector that delivers wild-type versions of the defective genes throughout the retina after non-invasive injection into the vitreous body of the eye. Dalkara describes a 7-mer peptide library and an AAV library constructed by DNA shuffling of the cap genes from AAV1, 2, 4, 5, 6, 8, and 9. rcAAV libraries and rAAV vectors expressing GFP under the influence of a CAG or Rho promoter were packaged and deoxyribonuclease-resistant genomic titers were obtained by quantitative PCR. The libraries were pooled and two rounds of evolution were conducted, each consisting of an initial diversification of the libraries followed by three rounds of in vivo selection. At each such step, P30 rho-GFP mice were injected intravitreally with 2 ml of iodixanol-purified phosphate buffered saline (PBS) dialyzed library with a genomic titer of about 1×10 ¹² viral genomes (vg)/ml. AAV vectors Dalkara et al. can be applied to the nucleic acid targeting system of the present invention, providing a dose of from about 1x10 ¹⁵ to about 1x10 ¹⁶ viral genomes (vg)/ml administered to a human.

[001220] Alternatively, the rhodopsin gene can be targeted for the treatment of retinitis pigmentosa (RP), in this embodiment the system of US Patent Publication No. 20120204282 assigned to Sangamo BioSciences Inc. can be modified according to the CRISPR-Cas system of the present invention.

[001221] In one embodiment, the methods of U.S. Patent Publication No. 20130183282 assigned to Cellectis, which are methods for cleaving a target sequence from the human rhodopsin gene, can also be modified for the nucleic acid targeting system of the present invention.

[001222] U.S. Patent Publication No. 20130202678 assigned to Academia Sinica relates to methods of treating retinopathy and vision-threatening ophthalmological disorders associated with the delivery of the Puf-A gene (which is expressed in the retinal ganglion and pigmented cells of the tissues of the eye and shows a unique anti-apoptotic activity) in subretinal or intravitreal space of the eye. In particular, desirable targets are zgc: 193933, prdm1a, spata2, tex10, rbb4, ddx3, zp2.2, Blimp-1 and HtrA2, all of which can be targeted by the nucleic acid targeting system of the present invention.

[001223] Wu (Cell Stem Cell, 13: 659-62, 2013) developed a guide RNA that directed Cas9 to a single base pair mutation that caused cataracts in mice, where it induced DNA cleavage. Then, using either a different wild-type allele or oligonucleotides introduced into the zygotes, repair mechanisms corrected the sequence of the broken allele and repaired the genetic defect causing cataracts in the mutant mouse.

[001224] US Patent Publication No. 20120159653 describes the use of zinc finger nucleases to genetically modify cells, animals, and proteins associated with macular degeneration (MD). Macular degeneration (MD) is a major cause of visual impairment in the elderly, but is also a defining symptom of childhood diseases such as Stargardt's disease, Sorsby fundus dystrophy, and fatal neurodegenerative diseases in children, with first signs of the disease in infancy. Macular (macula) degeneration results in loss of vision in the center of the visual field (macula) because the retina is damaged. Currently, existing animal models do not replicate the main features of the disease in a manner that is observed in humans. Available animal models containing mutant genes encoding MD-associated proteins also result in a highly diverse phenotype, which complicates the transition to the study of the disease in humans and creates problems for the development of therapy.

[001225] In one aspect, US Patent Publication No. 20120159653 relates to editing any chromosomal sequences that encode proteins associated with the development of MD, which can be applied to the nucleic acid targeting system of the present invention. MD-associated proteins are usually chosen experimentally by linking the MD-associated protein to the MD disorder. For example, the rate of synthesis or circulating concentration of an MD-associated protein may be increased or decreased in a population with an MD disorder relative to a population without an MD disorder. Differences in protein levels can be assessed using proteomics methods including, but not limited to, Western blotting, immunohistochemical staining, ELISA, and mass spectrometry. Alternatively, MD-associated proteins can be identified by obtaining gene expression profiles of protein-coding genes using genomic methods, including, but not limited to, DNA microarray analysis, sequential gene expression analysis (SAGE), and quantitative polymerase chain reaction. in real time (qPCR).

[001226] As a non-limiting example, MD-associated proteins include, but are not limited to: ATP binding cassette, subfamily A member 4 (ABCA4) (ABC1), ACHM1 achromatopsia member 4 (rod monochromasia), 1 ApoE apolipoprotein E (ApoE), C1QTNF5 (CTRP5) Clq and tumor necrosis factor-associated protein 5 (C1QTNF5), C2 complement component 2 (C2), complement components C3 (C3), CCL2 chemokine ligand (CC motif) 2 (CCL2), CCR2 chemokine receptor 2 (CC motif) (CCR2), CD36 cluster of differentiation 36, CFB complement factor B, CFH complement factor CFH, complement factor 1 associated with H CFHR1, complement factor 3 associated with H CFHR3, cyclic gated nucleotide beta 3 CNGB3, CP-celluloplasmin (CP), CRP C-reactive protein (CRP), CST3 cystatin C or cystatin 3 (CST3), CTSD cathepsin D (CTSD), CX3CR1 chemokine receptor 1 (C-X3-C motif), ELOVL4 very long chain fatty acid elongation protein 4, DNA excision repair ERCC6, cross-complement deficiency protein rodent, complement group 6, fibulin-5 FBLN5, fibulin 6 FBLN6, FSCN2 fascin (FSCN2), HMCN1 hemicentrin 1, HTRA1 HTRA serine peptidase 1 (HTRA1), IL-6 interleukin 6, IL-8 interleukin 8, LOC387715, hypothetical protein PLEKHA1, member 1 of family A containing a plextrin homology domain (PLEKHA1), PROM1 prominin 1 (PROM1 or CD133), PRPH2 peripherin-2, RPGR regulator of retinitis pigmentosa GTPase, SERPING1 inhibitor of serpin peptidase, member 1 of clade G (Cl-inhibitor ), TCOF1, Treacle TIMP3 inhibitor of metalloproteinase 3 (TIMP3), Toll-like receptor 3 TLR3.

[001227] The type of MD-associated protein whose chromosomal sequence has been edited can and will change. In preferred embodiments, the MD-associated proteins whose chromosomal sequence is being edited may be ATP-binding cassette, subfamily A member 4 (ABCA4) (ABC1) encoded by the ABCR gene, apolipoprotein E (APOE) encoded by the APOE gene, chemokine ligand ( C-C motif) 2 (CCL2) encoded by the CCL2 gene, chemokine receptor 2 (C-C motif) (CCR2) encoded by the CCR2 gene, ceruloplasmin (CP) encoded by the CP gene, cathepsin D (CTSD) encoded by the CTSD gene, or metalloproteinase inhibitor 3 (TIMP3), encoded by the TIMP3 gene. In an exemplary embodiment, the genetically modified animal is a rat and the edited chromosomal sequence encoding the MD-related protein may be: (ABCA4) ATP binding cassette, subgroup NM_000350 of subfamily A (ABC1), APOE member 4 apolipoprotein E NM138828 (APOE), CCL2 chemokine ligand 2 (C-C NM_031530 motif) (CCL2), CCR2 chemokine (C-C NM_021866 motif) receptor 2 (CCR2), CP ceruloplasmin (CP) NM_012532, CTSD cathepsin D (CTSD) NM_134334, TIMP3 metalloproteinase inhibitor NM_012886 3. An animal or cell may contain 1, 2, 3, 4, 5, 6, 7 or more abnormal chromosomal sequences encoding an MD-associated protein, as well as zero, 1, 2, 3, 4, 5, 6, 7 or more chromosomally integrated sequences encoding a disrupted MD-associated protein.

[001228] The altered or integrated chromosomal sequence may be modified to encode an altered MD-associated protein. Several mutations in chromosomal sequences have been associated with MD. Non-limiting examples of mutations in chromosomal sequences associated with MD include those that can cause MD, including in the ABCR protein, E471K (i.e., glutamate at position 471 is replaced by lysine), R1129L (i.e., arginine at position 11129 is replaced to leucine), T1428M (i.e., threonine at position 1428 is replaced by methionine), R1517S (i.e., arginine at position 1517 is replaced by serine), I1562T (i.e., isoleucine at position 1562 is replaced by threonine), and G1578R (i.e. glycine at position 1578 changed to arginine); in the CCR2 V64I protein (that is, the valine at position 192 is replaced by isoleucine); in the CP protein G969B (i.e. glycine at position 969 is replaced by asparagine or aspartate); in the TIMP3 protein S156C (i.e. serine at position 156 is replaced by cysteine), G166C (i.e. glycine at position 166 is replaced by cysteine), G167C (i.e. glycine at position 167 is replaced by cysteine), Y168C ( i.e. tyrosine at position 168 is changed to cysteine), S17C (i.e. serine at position 170 is changed to cysteine), Y172C (i.e. tyrosine at position 172 is changed to cysteine) and S181C (i.e. serine changed to cysteine at position 181). Also, other associations of genetic variants in MD-associated genes and diseases are known in the art.

Treatment of diseases of the circulatory and muscular systems

[001229] The present invention also provides for delivery of the CRISPR-Cas system described herein, for example, C2c1 or C2c3 effector protein systems to the heart. For delivery to the heart, myocardial adeno-associated virus (AAVM) is preferred, in particular AAVM41, which has shown preferential gene transfer to the heart (see, e.g., Lin-Yanga et al., PNAS, March 10, 2009, vol, 106, no. ten). Delivery can be systemic or local. For systemic administration, a dosage of about 1-10×10 ¹⁴ vector genomes is envisaged. See, for example, Eulalio et al. (2012) Nature 492: 376 and Somasuntharam et al. (20013) Biomaterials 34:7790.

[001230] For example, US Patent Publication No. 20110023139 describes the use of zinc finger nucleases to genetically modify cells, animals, and proteins associated with cardiovascular disease. Cardiovascular disease typically includes high blood pressure, heart attacks, heart failure and stroke, and TIA. Any chromosomal sequence involved in cardiovascular disease or protein encoded by any chromosomal sequence involved in cardiovascular disease can be used in the methods described herein. Proteins associated with cardiovascular disease are usually selected based on the experimental association of a protein associated with cardiovascular disease with the development of cardiovascular disease. For example, the rate of synthesis or circulating concentration of a protein associated with cardiovascular disease may be increased or decreased in a population with a cardiovascular disorder relative to a population without a cardiovascular disorder. Differences in protein levels can be assessed using proteomics methods including, but not limited to, Western blotting, immunohistochemical staining, ELISA, and mass spectrometry. Alternatively, proteins associated with CVD can be identified by profiling the expression of protein-coding genes using genomic methods including, but not limited to, DNA microarray analysis, Sequential Gene Expression Analysis (SAGE), and quantitative polymerase assay. real-time chain reaction (qPCR).

[001231] By way of example, the chromosomal sequence may include, but is not limited to, the following: IL1B (interleukin 1, beta), XDH (xanthine dehydrogenase), TP53 (p53 tumor protein), PTGIS (prostaglandin 12 (prostacyclin) synthase), MB (myoglobin ), IL4 (interleukin 4), ANGPT1 (angiopoietin 1), ABCG8 (ATP-binding cassette, subfamily G (WHITE), member 8), CTSK (cathepsin K), PTGIR (prostaglandin 12 (prostacyclin) receptor (IP)), KCNJ11 (internal rectifier potassium channel, subfamily J, member 11), INS (insulin), CRP (C-reactive protein related to pentraxin), PDGFRB (platelet growth factor receptor beta polypeptide), CCNA2 (cyclin A2), PDGFB (platelet growth factor beta polypeptide (viral oncogenic homologue of simian sarcoma (v-sis)), KCNJ5 (intrinsic rectifier potassium channel, subfamily J, member 5), KCNN3 (medium/low conduction calcium-activated potassium channels, subfamily N, member 3), CAPN10 (calpain 10), PTGES (prostaglandin E synthase), ADRA2B (a drenergic, alpha-2B-, receptor), ABCG5 (ATP-binding cassette, subgroup G (WHITE), element 5), PRDX2 (peroxiredoxin 2), CAPN5 (calpain 5), PARP14 (poly(ADP-ribose) family of polymerases , member 14), MEX3C (mex-3 homolog C (C. elegans)), ACE angiotensin converting enzyme I (peptidyl dipeptidase A) 1, TNF (tumor necrosis factor (TNF superfamily, member 2)), IL6 (interleukin 6 (interferon, beta 2)), STN (statin), SERPINE1 (serpinate inhibitor peptidases, clade E (nexin, type 1 plasminogen activator inhibitor), member 1), ALB (albumin), ADIPOQ (adiponectin, C1Q and containing collagen domain), APOB (apolipoprotein B (including Ag(x) antigen)), APOE ( apolipoprotein E) LEP (leptin), MTHFR (5,10-methylenetetrahydrofolate reductase (NADPH)), APOA1 (apolipoprotein A-I), EDN1 (endothelin I), NPPB (natriuretic peptide B precursor), NOS3 (nitric oxide synthase 3 (endothelial cell) ), PPARG (peroxisome proliferator-activated receptor gamma), PLAT (plasminogen activator, tissue), PTGS2 (prostaglandin endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)), CETP (cholesterol ester transfer protein, plasma) , AGTR1 (angiotensin II receptor type 1), HMGCR (3-hydroxy-3-4-methylglutaryl-cofer ment A reductase), IG F1 (insulin-like growth factor 1 (somatomedin C)), SELE selectin E E), REN (renin), PPARA (peroxisome proliferator-activated receptor alpha), PON1 (paraoxonase 1), KNG1 (kininogen 1) , CCL2 (chemokine (CC motif) ligand 2), LPL (lipoprotein lipase), VWF (von Willebrand factor), F2 (coagulation factor II (thrombin)), ICAM1 (intercellular adhesion molecule 1), TGFB1 (transforming growth factor beta 1 ), NPPA (natriuretic peptide A precursor), IL10 (interleukin 10), EPO (erythropoietin), SOD1 (superoxide dismutase 1, soluble), VCAM1 (vascular cell adhesion molecule 1), IFNG (interferon, gamma), LPA (lipoprotein, Lp (a)), MPO (myeloperoxidase), ESR1 (estrogen receptor 1), MAPK1 (mitogen-activated protein kinase 1), HP (haptoglobin), F3 (coagulation factor III (thromboplastin, tissue factor)), CST3 (cystatin C), COG2 (component of the oligomeric Golgi complex 2), MMP9 (matrix metallopeptidase 9 (gelatinase B, 92-kDa gelatinase, collagen aza type IV), SERPINC1 (serpin peptidase inhibitor, clade C (antithrombin), member 1), F8 (coagulation factor VIII, procoagulant component), HMOX1 (hemoxygenase (decycling) 1), APOC3 (apolipoprotein C-III), IL8 ( interleukin 8), PROK1 (prokineticin 1), CBS (cystathionine beta synthase), NOS2 (nitric oxide synthase 2, inducible), TLR4 (toll-like receptor 4), SELF (selectin P (granular membrane protein 140 kDa, antigen CD62)), ABCA1 (ATP-binding cassette, subfamily A (ABC 1), member 1), AGT (angiotensinogen (serpin peptidase inhibitor, clade A, member 8)), LDLR (low-density lipoprotein receptor), GPT (glutamate- pyruvate transaminase (alanine aminotransferase)), VEGFA (vascular endothelial growth factor A), NR3C2 (nuclear receptor subfamily 3, group C, member 2), IL18 (interleukin 18 (interferon-gamma-inducing factor)), NOS1 (nitric oxide synthase 1 (neuronal)), NR3C1 (nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)), FGB (beta-chain fibrinogen), HGF (hepatocyte growth factor (hepapoetin A, scatter factor)), IL1A (interleukin 1, alpha), RETN (resistin), AKT1 (murine thymoma viral oncogenic homologue (v-akt) 1), LIPC (lipase, hepatic), HSPD1 (60 kDa heat shock protein 1 (chaperonin)), MAPK14 (mitogen-activated protein kinase 14), SPP1 (secreted phosphoprotein 1), ITGB3 (integrin, beta 3 (platelet glycoprotein 111a, CD61 antigen)), CAT ( catalase), UTS2 (urotensin 2), THBD (thrombomodulin), F10 (coagulation factor X), CP (ceruloplasmin (ferroxidase)), TNFRSF11B (tumor necrosis factor receptor superfamily member 11b), EDNRA (endothelin type A receptor), EGFR (epidermal growth factor receptor (viral oncogenic homologue of erythroblastic leukemia (v-erb-b), avian)), MMP2 (matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 12 kDa type IV collagenase), PLG (plasminogen), NPY ( neuropeptide Y), RHOD (ras gene family member D), MAPK8 (mitogen-activated protein kinase 8), MYC (v- myc myelocytomatosis viral oncogenic homologue of myelocytomatosis (v-myc) (avian)), FN1 (fibronectin 1), CMA1 (chymase 1, mast cell), PLAU (plasminogen activator, urokinase), GNB3 (guanine nucleotide binding protein (G-protein ), beta polypeptide 3), ADRB2 beta-2-adrenergic receptor, surface), APOA5 (apolipoprotein A-V), SOD2 (superoxide dismutase 2, mitochondrial), F5 (coagulation factor V (proaccelerin, labile factor)), VDR (vitamin D receptor (1,25-dihydroxyvitamin D3)), ALOX5 (arachidonate 5-lipoxygenase), HLA-DRB1 (major histocompatibility complex class II, DR beta I), PARP1 (poly (ADP-ribose) polymerase 1), CD40LG (CD40 ligand ), PON2 (paraoxonase 2), AGER (glycosylation end product receptor), IRS1 (insulin receptor substrate 1), PTGS1 (prostaglandin endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase)), ECE1 (endothelin converting enzyme 1), F7 (blood clotting factor VII (serum prothrombin conversion accelerator)), URN (antagonist interleukin 1 receptor), EPHX2 (epoxide hydrolase 2, cytoplasmic), IGFBP1 (insulin-like growth factor binding protein 1), MAPK 10 (mitogen-activated protein kinase 10), FAS (Fas (TNF receptor superfamily member 6)), ABCB1 (ATP -binding cassette, subfamily (MDR/TAP), member 1), JUN (jun-oncogene), IGFBP3 (insulin-like growth factor binding protein 3), CD14 (CD14 molecule), PDE5A (phosphodiesterase 5A, cGMP-specific), AGTR2 (angiotensin II receptor type 2), CD40 (CD40 molecule, member of the TNF receptor superfamily 5), LCAT (lecithincholesterol acyltransferase), CCR5 (chemokine receptor (C-C motif) 5), MMP1 (matrix metallopeptidase 1 (interstitial collagenase)), TIMP1 (TIMP 1 metallopeptidase inhibitor), ADM (adrenomedullin), DYT10 (dystonia 10), STAT3 (signal protein and transcription activator 3 (acute phase response factor), MMP3 (matrix metallopeptidase 3 (stromelysin 1, progelatinase)), ELN (elastin) , USF1 (upstream transcription factor 1), CFH (facto p complement H), HSPA4 (70-kDa heat shock protein 4), MMP12 (matrix metallopeptidase 12 (macrophage elastase)), MME (membrane metalloendopeptidase), F2R (coagulation factor II (thrombin) receptor), SELL (selectin L) , ANXA5 (annexin A5), ADRB1 (beta-1 adrenoreceptor), CYBA (cytochrome b-245, alpha polypeptide), FGA (fibrinogen alpha chain), GGT1 (gamma-glutamyltransferase 1), LIPG (lipase, endothelial) , HIF1A (hypoxia inducible factor 1, alpha subunit (basic transcription factor with helix-loop-helix domain)), CXCR4 (chemokine receptor (C-X-C motif)), PROC (protein C (inactivator of coagulation factors Va and VIIIa )), SCARB1 (class B scavenger receptor member 1), CD79A (CD79a molecule associated with immunoglobulin alpha), PLTP (phospholipid transfer protein), ADD1 (adductin 1 (alpha)), FGG (fibrinogen gamma chain), SAA1 (serum amyloid Al), KCNH2 (voltage-gated potassium channel, subfamily H (eag-related), member 2), DPP4 (dipeptide ylpeptidase 4), G6PD (glucose-6-phosphate dehydrogenase), NPR1 (natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide A receptor)), VTN (vitronectin), KIAA0101 (KIAA0101), FOS (homologue of mouse osteosarcoma viral oncogene FBJ), TLR2 (toll-like receptor 2), PPIG (peptidyl prolyl isomerase G (cyclophilin G)), IL1R1 (interleukin 1 receptor, type I), AR (androgen receptor), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), SERPINA1 (serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1), MTR (5-methyltetrahydrofolate homocysteine methyltransferase), RBP4 (retinol-binding protein 4, plasma), APOA4 (apolipoprotein A -IV), CDKN2A (inhibitor of cyclin-dependent kinase 2A (melanoma, p16, inhibits CDK4)), FGF2 (fibroblast growth factor 2 (major)), EDNRB (endothelin type B receptor), ITGA2 (integrin, alpha 2 (CD49B, alpha- 2 subunit of VLA-2 receptor)), CABIN1 (calcineurin binding protein 1), SHBG (globule sex hormone binding protein), HMGB1 (high mobility group protein 1), HSP90B2P (90 kDa heat shock protein beta (Grp94), member 2 (pseudogene)), CYP3A4 (cytochrome P450, family 3, subfamily A, polypeptide 4 ), GJA1 (gap junction protein, alpha 1, 43 kDa), CAV1 (caveolin 1, caveola protein, 22 kDa), ESR2 (estrogen receptor 2 (ER beta)), LTA (lymphotoxin alpha (TNF superfamily, member 1)) , GDF15 (growth differentiation factor 15), BDNF (brain-derived neurotrophic factor), CYP2D6 (cytochrome P450, family 2, subfamily D, polypeptide 6), NGF (nerve growth factor (beta polypeptide)), SP1 (Spl transcription factor), TGIF1 (homeobox factor 1 induced by TGFB), SRC (viral sarcoma oncogene homologue (Schmidt-Ruppin A-2) v-src (avian)), EGF (epidermal growth factor (beta-urogastron)), PIK3CG (phosphoinositide-3- kinase, catalytic, gamma polypeptide), HLA-A (major histocompatibility complex, class I, A), KCNQ1 (voltage-gated potassium channel, KQT-like subfamily, member 1), CNR1 (cannabinoid receptor 1 (brain)), FBN1 (fibrillin 1), CHKA (choline kinase alpha), BEST1 (bestrophin 1), APP (beta amyloid (A4) precursor protein), CTNNB1 (catenin (associated with cadherin protein), beta 1, 88 kDa), IL2 (interleukin 2), CD36 (CD36 molecule (thrombospondin receptor)), PRKAB1 (protein kinase, AMP-activated, non-catalytic beta 1 subunit), TPO (thyroid peroxidase), ALDH7A1 ( aldehyde dehydrogenase family 7, member A1), CX3CR1 (chemokine receptor (C-X3-C motif) 1), TH (tyrosine hydroxylase), F9 (clotting factor IX), GH1 (growth hormone 1), TF (transferrin), HFE ( hemochromatosis), IL17A (interleukin 17A), PTEN (phosphatase and tensin homologue), GSTM1 (glutathione S-transferase mu 1), DMD (dystrophin), GATA4 (GATA-binding protein 4), F13A1 (clotting factor XIII, polypeptide A1 ), TTR (transthyretin), FABP4 (fatty acid binding protein 4, adipocyte), PON3 (paraoxonase 3), APOC1 (apolipoprotein C-I), INSR (insulin receptor), TNFRSF1 B (tumor necrosis factor receptor family member 1B), HTR2A (5-hydroxytryptamine (serotonin) 2A receptor), CSF3 (colony stimulating factor 3 (granulocyte)), CYP2C9 (cytochrome P450 family 2 subfamily C, polypeptide 9), TXN (thioredoxin), CYP11B2 (cytochrome P450 family 11, subfamily B, polypeptide 2), PTH (parathyroid hormone), CSF2 (colony stimulating factor 2 (granulocyte macrophage)), KDR (kinase insertion domain receptor (type III tyrosine kinase receptor), PLA2G2A (phospholipase A2, group IIA (platelets, synovial fluid)), B2M (beta-2-microglobulin), THBS1 (thrombospondin 1), GCG (glucagon), RHOA (ras homology gene family member A), ALDH2 (aldehyde dehydrogenase family 2 (mitochondrial)), TCF7L2 (transcription factor 7-like 2 (T-cell specific, high mobility gene group - HMG-box)), BDKRB2 (bradykinin B2 receptor), NFE2L2 (nuclear factor 2 like erythroid derivative) , NOTCH1 (notch 1 homologue associated with translocation eu (Drosophila)), UGT1A1 (UDP-glucuronosyltransferase 1 family, Al polypeptide), IFNA1 (interferon, alpha I), PPARD (peroxisome proliferator-activated delta receptor), SIRT1 (sirtuin (silent mating type information regulation 2 homologue) 1 (S. cerevisiae)), GNRH1 (gonadotropin-releasing hormone 1 (luteinizing hormone)), PAPPA (plasma pregnancy-associated protein-A, pappalysin 1), ARR3 (arrestin 3, retina (X-arrestin)), NPPC (natriuretic peptide precursor C), AHSP (alpha hemoglobin stabilizing protein), PTK2 (PTK2 protein tyrosine kinase 2), IL13 (interleukin 13), MTOR (mechanical target of rapamycin (serine/threonine kinase)), ITGB2 (integrin, beta 2 (complementary component 3 of receptor 3 and subunits 4)), GSTT1 (glutathione S-transferase theta 1), IL6ST (interleukin 6 signaling protein (gpl30, oncostatin M receptor)), CPB2 (carboxypeptidase B2 (plasma)), CYP1A2 (cytochrome P450, family 1, subfamily A , polypeptide 2), HNF4A (hepatocyte nuclear factor 4, alpha), SLC6A4 (solute transporter family 6 (neurotransmitter transporter, serotonin), member 4), PLA2G6 (phospholipase A2, group VI (cytosolic, calcium independent)), TNFSF11 (tumor necrosis factor superfamily (ligand ), member 11), SLC8A1 (solute transporter family 8 (sodium/calcium exchanger), member 1), F2RL1 (coagulation factor II (thrombin) receptor-like 1), AKR1A1 (aldokete reductase family 1, member Al (aldehyde reductase) , ALDH9A1 (aldehyde dehydrogenase family 9, member Al), BGLAP (bone gamma carboxyglutamate (gla) protein), MTTP (microsomal triglyceride transfer protein), MTRR (5-methyltetrahydrofolate homocysteine methyltransferase reductase), SULT1A3 (sulfotransferase family, cytosolic, 1A, phenol-preferred, member 3), RAGE (renal tumor antigen), C4B (complement component 4B (Chido blood group), P2RY12 (P2Y purinergic receptor, G protein-coupled, 112.), RNLS (renalase, FAD-dependent amine oxidase) , CREB1 (cAMP-responsive element binding protein 1), POMC (pro-opiomelanocortin), RAC1 (ras-associated substrate of botulinum toxin C3 1 (rho family, small GTP-binding protein Racl)), LMNA (lamin NC), CD59 (CD59 molecule , regulatory white to complement), SCN5A (voltage-gated sodium channel type V, alpha subunit), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), MIF (macrophage migration inhibitor) (glycosylation inhibitory factor)), MMP13 ( matrix metallopeptidase 13 (collagenase 3)), TIMP2 (metallopeptidase inhibitor TIMP 2 inhibitor), CYP19A1 (cytochrome P450, family 19, subfamily A, polypeptide 1), CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2), PTPN22 ( protein tyrosine phosphatase, non-receptor type 22 (lymphoid)), MYH14 (myosin, heavy chain 14, non-muscle), MBL2 (mannose-binding lectin (protein C) 2, soluble (opsonic defect)), SELPLG (P-selectin ligand), AOC3 ( copper amine oxidase 3 (vascular adhesion protein 1)), CTSL1 (cathepsin L1), PCNA (proliferating cell nuclear antigen), IGF2 (insulin-like growth factor 2 (somatomedin A)), ITGB1 (integrin, beta 1 (fibronectin receptor, beta -polypeptide, CD29 antigen includes MDF2, M SK12)), CAST (calpastatin), CXCL12 (chemokine (C-X-C motif) ligand 12 (stromal cell factor 1)), IGHE (heavy constant chain epsilon immunoglobulin), KCNE1 (voltage-gated potassium channel, Isk-related family, member 1), TFRC (transferrin receptor (p90, CD71)), COL1A1 (collagen, type I, alpha 1), COL1A2 (collagen, type I, alpha 2), IL2RB (interleukin 2 receptor, beta), PLA2G10 (phospholipase A2, group X), ANGPT2 (angiopoietin 2), PROCR (protein C receptor, endothelial (EPCR)), NOX4 (-NADPH oxidase 4), HAMP (antimicrobial peptide hepcidin), PTPN11 (protein tyrosine phosphatase, non-receptor type 11), SLC2A1 (solute transporter family 2 (facilitated glucose transporter), member 1), IL2RA (interleukin 2 receptor, alpha), CCL5 (chemokine (C-C motif) ligand 5), IRF1 (interferon regulatory factor 1), CFLAR (CASP8- and FADD -like regulators of apoptosis), CALCA (calcitonin-associated alpha polypeptide), EIF4E (eukaryotic translation initiation factor 4E), GSTP1 ( glutathione S-transferase pi 1), JAK2 (Janus kinase2), CYP3A5 (cytochrome P450, family 3, subfamily A, polypeptide 5), HSPG2 (heparan sulfate proteoglycan 2), CCL3 (chemokine (C-C motif) ligand 3), MYD88 (myeloid differentiation primary response gene (88)), VIP (vasoactive intestinal peptide), SOAT1 (sterol-O-acyltransferase 1), ADRBK1 (beta-adrenergic receptor kinase 1), NR4A2 (nuclear receptor subfamily 4, group A, member 2) , MMP8 (matrix metallopeptidase 8 (neutrophil collagenase)), NPR2 (natriuretic peptide receptor B/guanylate cyclase B (atrionatriuretic peptide receptor B)), GCH1 (GTP cyclohydrolase 1), EPRS (glutamyl prolyl tRNA synthetase), PPARGC1A ( peroxisome proliferator-activated gamma activator, coactivator 1 alpha), F12 (coagulation factor XII (Hageman factor)), PECAM1 (platelet/endothelial cell adhesion molecule), CCL4 (chemokine (C-C motif) ligand 4), SERPINA3 (serpin peptidases, clade A (alpha-1 antiproteinase, and nittrypsin), member 3), CASR (calcium sensitive receptor), GJA5 (gap junction protein, alpha 5, 40 kDa), FABP2 (fatty acid binding protein 2, intestinal), TTF2 (transcription termination factor, RNA polymerase II ), PROS1 (protein S (alpha)), CTF1 (cardiotrophin 1), SGCB (sarcoglycan, beta (43 kDa, dystrophin-associated glycoprotein)), YME1L1 (YME1-like 1 (S. cerevisiae)), CAMP (antimicrobial peptide cathelicidin), ZC3H12A (CCCH-type zinc finger containing 12A), AKR1B1 (aldokete reductase 1 family, member B1 (aldose reductase)), DES (desmin), MMP7 (matrix metallopeptidase 7 (matrilysin, uterine ), AHR (aryl hydrocarbon receptor), CSF1 (colony stimulating factor 1 (macrophage)), HDAC9 (histone deacetylase 9), CTGF (connective tissue growth factor), KCNMA1 (high conductance calcium-activated potassium channel, subfamily M, member alpha 1) , UGT1A (UDP family UDP-glucuronosyltransferase 1 family, polypeptide A complex locus), PRKCA (protein kinase C, alpha), COMT (catechol beta methyltransferase), S100B (calcium binding protein S100 B), EGR1 (early growth factor 1 ), PRL (prolactin), IL15 (interleukin 15), DRD4 (dopamine receptor D4), CAMK2G (calcium/calmodulin dependent protein kinase II gamma), SLC22A2 (solute transporter family 22 (organic cation transporter) member 2), CCL11 (chemokine (C-C motif) ligand 11), PGF (placental growth factor B321), THPO (thrombopoetin), GP6 (glycoprotein VI (platelet)), TACR1 (tachykinin receptor 1), NTS (neurotensin), HNF1A (HNF1 homeobox A), SST (somatostatin), KCND1 (voltage-gated potassium channel of the Shal-related subfamily, member 1), LOC646627 (phospholipase inhibitor), TBXAS1 (thromboxane A synthase 1 (platelet)), CYP2J2 (cytochrome P450, family 2, subfamily J, polypeptide 2), TBXA2R (thromboxane A2 receptor), ADH1C (alcohol dehydrogenase 1C(class I), gamma polypeptide), ALOX12 (arachidonate-12-lipoxygenase), AHSG (alpha-2-HS-glycoprotein), BHMT (betaine homocysteine -methyltransferase), GJA4 (gap junction protein, alpha 4, 37 kDa), SLC25A4 (solute transporter family 25 (mitochondrial transporter; adenine nucleotide translocator), member 4), ACLY (ATP-citrate lyase), ALOX5AP (arachidonate-5-lipoxygenase-activating protein), NUMA1 (nuclear mitotic apparatus protein 1), CYP27B1 (cytochrome P450, family 27, subfamily B, polypeptide 1), CYSLTR2 (cysteinyl leukotriene 2 receptor), SOD3 (superoxide dismutase 3, extracellular), LTC4S (leukotriene C4 synthase), UCN (urocortin), GHRL (ghrelin/obestatin prepropeptide), APOC2 (apolipoprotein C-II), CLEC4A (C-type lectin family 4, member A), KBTBD10 (protein containing kelch repeat and BTB domain (POZ) 10), TNC (tenascin C), TYMS (thymidylate synthetase), SHC1 (SHC (containing src homology domain) -transforming protein 1), LRP1 (low-density lipoprotein receptor-associated protein 1), SOCS3 (cytokine signal suppressor 3), ADH1B (alcohol dehydrogenase 1B (class I), beta polypeptide), KLK3 (kallikrein-related peptidase 3), HSD11B1 (11 beta-hydroxysteroid--dehydrogenase 1), VKORC1 (vitamin K epoxide reductase complex, subed origin 1), SERPINB2 (serpin peptidase inhibitor, clade B (ovalbumin), member 2), TNS1 (tensin 1), RNF19A (ring protein 19A), EPOR (erythropoietin receptor), ITGAM (integrin, alpha M (complement component receptor 3 subunit 3)), PITX2 (pair-like homeodomain 2), MAPK7 (mitogen-activated protein kinase 7), FCGR3A (IgG Fc fragment, low affinity 111a, receptor (CD16a)), LEPR (leptin receptor), ENG (endoglin), GPX1 (glutathione peroxidase 1), GOT2 (glutamate oxaloacetate transaminase 2, mitochondrial (aspartate aminotransferase 2)), HRH1 (histamine H1 receptor), NR112 (nuclear receptor subfamily 1, group I, member 2), CRH (corticotropin releasing hormone) , HTR1A (5-hydroxytryptamine (serotonin) receptor lA), VDAC1 (voltage-gated anion channel 1), HPSE (heparanase), SFTPD (protein D), TAP2 (transporter 2, ATP-binding cassette, subgroup B (MDR/TAP )), RNF123 (ring protein 123), PTK2B (PTK2B protein tyrosine kinase 2-beta), NTRK2 (neurotrophic tyrosine kinase, receptor, type 2), IL6R (interleukin 6 receptor), ACHE (acetylcholinesterase (blood type Yt)), GLP1R (glucagon-like peptide 1 receptor), GHR (growth hormone receptor), GSR (glutathione reductase), NQO1 (NAD(P)H dehydrogenase, quinone 1), NR5A1 (nuclear receptor subfamily 5, group A, member 1), GJB2 (gap junction protein, beta 2, 26 kDa), SLC9A1 (solute transporter family 9 (sodium/hydrogen exchanger) member 1), MAOA ( monoamine oxidase A), PCSK9 (subtilisin-kexin type 9 proprotein convertase), FCGR2A (IgG Fc fragment, low affinity IIa, receptor (CD32)), SERPINF1 (serpin peptidase inhibitor, clade F (alpha-2-antiplasmin, pigment epithelium- originating factor), member 1), EDN3 (endothelin 3), DHFR (dihydrofolate reductase), GAS6 (growth arrest specific 6), SMPD1 (sphingomyelin phosphodiesterase 1, acid lysosomal), UCP2 (uncoupling protein 2 (mitochondrial, proton transporter)) , TFAP2A (transcription factor AP-2 alpha (activating enhancer-binding b fir 2 alpha), C4BPA (complement binding component 4 alpha), SERPINF2 (serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium-derived factor), member 2), TYMP (thymidine phosphorylase), ALPP ( alkaline phosphatase, placental (Regan isoenzyme)), CXCR2 (chemokine (C-X-C motif) receptor 2), SLC39A3 (solute transporter family 39 (zinc transporter), member 3), ABCG2 (ATP-binding cassette, subfamily G (WHITE), member 2), ADA (adenosine deaminase), JAK3 (Janus kinase 3), HSPA1A (70 kDa heat shock protein 1A), FASN (fatty acid synthase), FGF1 (fibroblast growth factor 1 (acidic)), F11 (blood clotting factor XI), ATP7A (ATPase, Cu++ transfer, alpha polypeptide), CRI (complement component receptor (3b/4b) 1 (Knops blood type)), GFAP (glial fibrillar acidic protein), ROCK1 (Rho-associated domain containing coiled- coil protein kinase 1), MECP2 (methyl-CpG-binding protein 2 (Rett syndrome)), MYLK (light chain kinase myosin), BCHE (butyrylcholinesterase), LIPE (lipase, hormone-sensitive), PRDX5 (peroxiredoxin 5), ADORA1 (adenosine A1 receptor), WRN (Werner syndrome, RecQ helicase-like), CXCR3 (chemokine (C-X-C motif) receptor 3), CD81 (CD81 molecule), SMAD7 (SMAD 7 family member), LAMC2 (laminin, gamma 2), MAP3K5 (mitogen-activated protein kinase k5), CHGA (chromogranin A (parathyroid secretory protein 1)), IAPP (islet amyloid polypeptide), RHO (rhodopsin), ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), PTHLH (parathyroid hormone-related protein, NRG1 (neuregulin 1), VEGFC (vascular endothelial growth factor C), ENPEP (glutamyl aminopeptidase (aminopeptidase A) ), CEBPB (CCAAT/enhancer binding protein (C/EBP), beta), NAGLU (N-acetylglucosaminidase, alpha), F2RL3 (clotting factor II (thrombin) receptor-like 3), CX3CL1 (chemokine (C-X3 motif -C) 1), BDKRB1 (bradykinin B1 receptor), ADAMTS13 (ADAM metallopeptidase with type 1 thrombospondin motif, 13), EL ANE (neutrophil-expressed elastase), ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase 2), CISH (cytokine-induced SH2-containing protein), GAST (gastrin), MYOC (myocylin, trabecular meshwork-induced glucocorticoid response), ATP1A2 (ATPase, Na + transfer /K+, alpha2 polypeptide), NF1 (neurofibromin 1) GJB1 (gap junction protein, beta 1, 32 kDa), MEF2A (myocyte enhancer factor 2A), VCL (vinculin), BMPR2 (bone morphogenetic protein receptor, type II ( serine/threonine kinase)), TUBB (tubulin, beta) CDC42 (cell cycle protein 42 (GTP-binding protein, 25 kDa)), KRT18 (keratin 18), HSF1 (heat shock protein transcription factor 1), MYB (v-myb myeloblastosis viral oncogene homologue v-myb (avian)), PRKAA2 (protein kinase, AMP-activated, alpha-2 catalytic subunit), ROCK2 (Rho-associated coiled-coil domain-containing protein kinase 2), TFPI (tissue factor pathway inhibitor (lipoprotein -associated coagulation inhibitor)), PRKG1 (protein kin 3a, cGMP-dependent, type I), BMP2 (bone morphogenetic protein 2), CTNND1 (catenin (cadherin-associated protein), delta 1), CTH (cystathionase (cystathionin-gamma-lyase)), CTSS (cathepsin S), VAV2 (guanine nucleotide exchange factor vav 2), NPY2R (neuropeptide Y receptor Y2), IGFBP2 (36 kDa insulin-like growth factor-2 binding protein), CD28 (CD28 molecule), GSTA1 (glutathione-S-transferase alpha 1), PP1A (peptidyl prolyl isomerase A (cyclophilin A)), APOH (apolipoprotein H (beta-2-glycoprotein I)), S100A8 (S100 calcium-binding protein A8), IL11 (interleukin 11), ALOX15 (arachidonate 15-lipoxygenase), FBLN1 (fibulin 1), NR1H3 (nuclear receptor subfamily 1, group H, member 3), SCD (stearoyl-CoA desaturase (delta-9-desaturase)), GIP (gastric inhibitory polypeptide), CHGB (chromogranin B (secretogranin 1) ), PRKCB (protein kinase C, beta), SRD5A1 (steroid 5-alpha-reductase, alpha-polypeptide 1 (3-oxo-5-alpha-steroid-delta-4-dehydrogenase alpha 1)), HSD11B2 (hydroxy steroid-(11-beta)-dehydrogenase 2), CALCRL (calcitonin receptor-like), GALNT2 (UDP-N-acetyl-alpha-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2)), ANGPTL4 ( angiopoietin-like 4), KCNN4 (medium/small calcium-activated potassium channels, subfamily N, member 4), PIK3C2A (phosphoinositide-3-kinase, class 2, alpha polypeptide), HBEGF (heparin-binding epidermal growth factor- similar growth factor), CYP7A1 (cytochrome P450 family 7 subfamily A, polypeptide 1), HLA-DRB5 (major histocompatibility complex class II, DR beta 5), BNIP3 (BCL2/adenovirus E1B 19 kDa -interacting protein 3), GCKR (regulator glucokinase (hexokinase 4)), S100A12 (S100 calcium-binding protein A12), PADI4 (peptidylarginine deiminase type IV), HSPA14 (70 kDa protein thermal shock 14), CXCR1 (chemokine (C-X-C motif) receptor 1), H19 (H19, imprinted transcript expressed in maternal line (non-protein coding)), KRTAPone9-3 (keratin-associated protein 19-3), IDDM2 (insulin-dependent diabetes mellitus 2), RAC2 (ras-associated substrate of botulinum toxin C3 2 (rho family, small GTP-binding protein Rac2)), RYR1 (ryanodine receptor 1 (skeletal)), CLOCK (clock homologue (mouse)), NGFR (nerve growth factor receptor (TNFR superfamily, member 16)), DBH (dopamine beta-hydroxylase (dopamine beta-monooxygenase)), CHRNA4 (cholinergic receptor, nicotinic, alpha 4), CACNA1C (potential-dependent calcium channel, L-type, alpha 1C subunit), PRKAG2 (protein kinase, AMP-activated, non-catalytic gamma-2 subunit), CHAT (choline acetyltransferase), PTGDS (prostaglandin D2 synthase 21 kDa (brain)), NR1H2 (nuclear receptor subfamily 1, group H, member 2), TEK (TEK-tyrosine kinase, endothelial), VEGFB (vascular endothelial growth factor B), MEF2C (myocyte enhancer factor 2C), MAPKAPK2 (mitogen-activated protein kinase-activated protein kinase 2 ), TNFRSF11A (tumor necrosis factor receptor superfamily, member 11a, NFK activator B), HSPA9 (70 kDa heat shock protein 9 (mortalin)), CYSLTR1 (cysteinyl leukotriene receptor 1), MAT1A (methionine adenosyltransferase I, alpha), OPRL1 (opiate receptor-like 1), IMPA1 (inositol (myo) -1 (or 4)-monophosphatase 1), CLCN2 (chloride channel 2), DLD (dihydrolipamide dehydrogenase), PSMA6 (proteasome subunit (prosoma, macropoin), alpha-type, 6), PSMB8 (proteasome subunit (prosoma, macropoin ), beta-type, 8 (large multifunctional peptidase 7)), CHI3L1 (chitinase 3-like protein 1 (cartilage glycoprotein-39)), ALDH1B1 (aldehyde dehydrogenase family 1, member B1), PARP2 (poly (ADP-ribose) polymerase 2), STAR (steroidogenic acute regulatory protein) LBP (lipopolysaccharide-binding protein), ABCC6 (ATP-binding cassette, subgroup C (CFTR/MRP), member 6), RGS2 (G protein signaling regulator 2, 24 kDa), EFNB2 (ephrin-B2), GJB6 (gap junction protein, beta 6, 30 kDa), APOA2 (apolipoprotein A-II), AMPD1 (adenosine monophosphate deaminase 1), DYSF (dysferlin, muscle dist. limb girdles 2B (autosomal recessive)), FDFT1 (farnesyl diphosphate farnesyl transferase 1), EDN2 (endothelin 2), CCR6 (chemokine (C-C motif) receptor 6), GJB3 (gap junction protein, beta 3, 31 kDa), IL1RL1 (interleukin 1 receptor-like protein 1), ENTPD1 (ectonucleoside triphosphate diphosphohydrolase 1), BBS4 (Bardet-Biedl syndrome 4), CELSR2 (cadherin, EGF LAG seven-pass G-type receptor 2 (flamingo, Drosophila homologue)), F11R (F11 receptor), RAPGEF3 (Guanine Nucleotide Exchange Factor (GEF) 3), HYAL1 (Hyaluron Glucosaminidase 1), ZNF259 (Zinc Finger Protein 259), ATOX1 (ATX1 Homolog of Antioxidant Protein 1 (Yeast)), ATF6 (Transcription Activating Factor 6) KHK (ketohexokinase (fructokinase)), SAT1 (spermidine/spermine N1-acetyltransferase 1), GGH (gamma-glutamyl hydrolase (conjugation, folylpolygammaglutamyl hydrolase)), TIMP4 (TIMP 4 metallopeptidase inhibitor), SLC4A4 (solute transporter family 4, sodium, bicarbonate cotransporter member 4), PDE2A (pho cGMP stimulated sphodiesterase 2A), PDE3B (cGMP inhibited phosphodiesterase 3B), FADS1 (fatty acid desaturase 1), FADS2 (fatty acid desaturase 2), TMSB4X (thymosin beta 4, X-linked), TXNIP (thioredoxin-interacting protein) , LIMS1 (LIM and antigen-like domains of senescent cells 1), RHOB (ras homology gene family member B), LY96 (lymphocyte antigen 96), FOXO1 (forkhead box O1 protein), PNPLA2 (patatin-like phospholipase domain containing protein 2), TRH (thyrotropin-releasing hormone), GJC1 (gap junction protein, gamma 1 45 kDa), SLC17A5 (solute transporter family 17 (anion/sugar transporter), member 5), FTO (associated with fat mass and obesity ), GJD2 (gap junction protein, delta 2, 36 kDa), PSRC1 (proline/serine-rich domain coiled-coil 1), CASP12 (caspase 12 (gene/pseudogene)), GPBAR1 (bile acid receptor coupled to G proteins 1), PXK (PX domain containing serine/threonine kinase), IL33 (interleukin 33), TRIB1 (tribbles 1 homolog (Drosophila)), PBX4 (leukemia pre-B cell homeobox 4), NUPRI (nuclear protein, transcription regulator, 1), 15-Sep (15 kDa selenoprotein), CILP2 (cartilage intermediate layer protein 2), TERC (telomerase component RNA), GGT2 (gamma-glutamyltransferase 2), MT-CO1 (mitochondrially encoded cytochrome oxidase with I) and UOX (urate oxidase, pseudogene). Any of these sequences can be targeted by the CRISPR-Cas system, for example to eliminate a mutation.

[001232] In yet another embodiment, the chromosomal sequence may further be selected from Pon1 (paraoxonase 1), LDLR (LDL receptor), ApoE (apolipoprotein E), Apo B-100 (apolipoprotein B-100), ApoA (apolipoprotein (a) ), ApoA1 (apolipoprotein A1), CBS (cystation B-synthase), glycoprotein IIb/IIb, MTHRF (5,10-methylenetetrahydrofolate reductase (NADPH) and their combinations. In one iteration, chromosomal sequences and proteins encoded by chromosomal sequences involved in the development of cardio - vascular diseases can be selected from CacnalC, Sodl, Pten, Ppar (alpha), Apo E, leptin and combinations thereof as a target or targets for the CRISPR-Cas system.

Treatment of liver and kidney diseases

[001233] The present invention also provides for delivery of the CRISPR-Cas system described herein, eg, C2c1 or C2c3 effector protein systems, to the liver and/or kidney. Delivery strategies to induce cellular uptake of the therapeutic nucleic acid include physical force or vector systems such as virus, lipid or complex based delivery or nanocarriers. From initial applications of lesser clinical relevance, where nucleic acids were delivered to kidney cells by systemic high-pressure injection (needleless injection), a wide range of gene therapeutic viral and non-viral carriers are already being used to target post-transcriptional events in different models of kidney disease on animals in vivo (Csaba Revesz and Péter Hamar (2011). Delivery Methods to Target RNAs in the Kidney, Gene Therapy Applications, Prof. Chunsheng Kang (Ed,), ISBN:978-953-307-541-9, InTech, Available from: http://www.intechopen.com/books/gene-therapy-a.pplications/delivery-methods-to-target-rnas-inthe-kidney) Delivery methods to the kidney may include those described in Yuan et al. (Am J Physiol Renal Physiol 295: F605-F617, 2008) who investigated whether in vivo delivery of small interfering RNAs (siRNAs) targeting the 12/15-lipoxygenase (12/15-LO) pathway of arachidonic acid metabolism, improve kidney injury and diabetic nephropathy (DN) in a mice model of type I diabetes mellitus injected with streptozocin. To achieve greater in vivo access and siRNA expression in the kidney, Yuan et al. cholesterol-conjugated double-stranded oligonucleotide 12/15-LO RNAs were used. Approximately 400 µg of siRNA was injected subcutaneously into mice. The methods of Yuang et al. can be applied to the CRISPR Cas system of the present invention, a 1-2 g subcutaneous injection of cholesterol-conjugated CRISPR Cas is provided for human delivery to the kidneys.

[001234] Molitoris et al. ( J AmSocNephrol 20: 1754-1764, 2009) used proximal tubular cells (PTC) as an oligonucleotide reabsorption site in the kidney to test the effectiveness of siRNA targeting p53, a key protein in the apoptotic pathway, to prevent kidney damage. Naked synthetic siRNA to p53 is administered intravenously 4 hours after ischemic injury, and most successfully protects both PTC and kidney function. Data from Molitoris et al. show that rapid siRNA delivery to proximal tubule cells follows intravenous administration. To obtain a dose-response curve, rats were administered the following doses of siP53, 0.33; 1, 3, or 5 mg/kg, at the indicated time intervals, resulting in cumulative doses of 1.32; 4, 12 and 20 mg/kg, respectively. All doses of siRNA tested had an SCr-lowering effect on the first day, with higher doses being effective for about five days compared to ischemic control rats treated with phosphate buffered saline (PBS). Cumulative doses of 12 and 20 mg/kg provided the best protective effect. The methods of Molitoris et al. can be applied to the nucleic acid targeting system according to the present invention, providing a total dose of 12 and 20 mg/kg in humans for delivery to the kidneys.

[001235] Thompson et al. ( Nucleic Acid Therapeutics , Volume 22, Number 4, 2012) disclose the toxicological and pharmacokinetic properties of synthetic small interfering RNA 15NP after intravenous administration in rodents and non-human primates. 15NP is designed to target the RNA interference (RNAi) pathway to temporarily inhibit the expression of the pro-apoptotic p53 protein and is being developed to protect cells from acute ischemic/reperfusion injury, such as acute kidney injury, that can occur during underlying cardiac surgery and delayed function. graft, which can occur after a kidney transplant. Doses of 800 mg/kg 15NP in rodents and 1000 mg/kg 15NP in non-human primates were expected to induce side effects, which in monkeys resulted in various types of effects on the blood: subclinical complement activation and slightly prolonged clotting time. In rats, no side effects were observed with the administration of the 15NP rat analogue, indicating that these effects are likely those of a class of synthetic RNA duplexes and not toxicity associated with the putative pharmacological activity of 15NP. Taken together, these data support clinical trials for intravenous 15NP to preserve renal function after acute ischemia/reperfusion injury. The no observed side effect dose (NOAEL) in monkeys was 500 mg/kg. No effects on cardiovascular, respiratory and neurological parameters were observed in monkeys after intravenous administration at doses up to 25 mg/kg. Thus, a similar dose may be contemplated for intravenous administration of CRISPR Cas to human kidneys.

[001236] Shimizu et al. ( JAmSocNephrol 21: 622-633, 2010) developed a system to target small interfering RNA (siRNA) delivery to glomeruli using poly(ethylene-glycol)-poly(L-lysine). The siRNA/nanocarrier complex was approximately 10 to 20 nm in diameter, which would allow it to travel through the fenestrated endothelium to access the mesangium after intraperitoneal injection of the fluorescent siRNA/nanocarrier complexes. Shimizu et al. observed miRNA circulation in the bloodstream for a long time. Repeated intraperitoneal administration of mitogen-activated protein kinase 1 (MAPK1) siRNA/nanocarrier complex suppressed MAPK1 glomerular mRNA and protein expression in a mouse model of glomerulonephritis. To study the accumulation of siRNAs labeled with Cy5 siRNAs in complex with PIC nanocarriers (0.5 ml, 5 nmol siRNA content), naked Cy5-labeled siRNAs (0.5 ml, 5 nmol) or Cy5-labeled siRNAs encapsulated in HVJ- E (0.5 ml, 5 nmol siRNA content) was administered to BALBc mice. The method of Shimizu et al. can be applied to the nucleic acid targeting system according to the present invention, providing a dose of about 10-20 micromoles of CRISPRCas in complex with nanocarriers in an amount of about 1-2 liters for intraperitoneal administration and delivery to the human kidneys.

[001237] Methods of delivery to the kidneys are summarized in the following table:

Delivery method carrier target RNA Disease Model functional analysis Author Hydrodynamic/lipid TransIT in vivo gene delivery system p85a Acute renal failure Ischemia-reperfusion Absorption, biodistribution Larson et al., Surgery, (Aug 2007), Voi. 142, no. 2.pp. (262-269) Hydrodynamic/lipid Lipofectamine 2000 Fas Acute renal failure Ischemia-reperfusion Blood urea nitrogen, Fas immunohistochemistry, histological count Harnar et al., Proc Natl Acad Sci. (Oct 2004), Voi. 101, no. 41,
pp. (14883-14888) hydrodynamic N/A Elements of the apoptotic cascade Acute renal failure Ischemia-reperfusion N/A Zheng et al., Am.
J Pathol, (Oct 2008), Voi. 173, no. 4, pp. (973-980) hydrodynamic N/A Nuclear factor kappa-B Acute renal failure Ischemia-reperfusion N/A Feng et al., Transplantation, (May 2009), Voi. 87, no. 9, pp. (1283-1289) Hydrodynamic / Viral Lipofectamine 2000 Apoptosis transcription factor antagonist Acute renal failure Ischemia-reperfusion Apoptosis, oxidative stress, caspase activation, membrane lipid peroxidation Xie & Guo, Am.
Soc Nephrol, (Dec 2006), Voi. 17, no. 12, pp. (3336-3346) hydrodynamic Hydrodynamic delivery system pBAsi mU6 Neo/ TransIT-EE Gremlin diabetic nephropathy Streptozocin-induced diabetes Proteinuria, serum creatinine, glomerular and tubular diameter, type IV collagen expression, BMP7 Q. Zhang et al., PloS ONE, (Jul 2010), Voi. 5, no. 7, el 1709, PP-(1-13) Viral/lipid Vector pSUPER/lipofectamine Type II TGF-β receptors Interstitial renal fibrosis Unilateral ureteral obstruction a-SMA expression, collagen content, blood pressure, serum albumin, serum urea nitrogen, serum creatinine, kidney weight, urinary sodium uptake Kushibikia et al.,
1 Controlled Release, (Jul
2005), Voi. 105, no. 3, pp. (318-331) Viral Adeno-associated virus-2 Mineralcorticoid receptors Kidney damage caused by hypertension Cold-induced hypertension Wang et al., Gene Therapy. (Jul 2006), Voi. 13. No. 14, pp. (1097-1103) Hydrodynamic / Viral pU6 vector Luciferase N/A N/A Kobayashi et al., Journal of Pharmacology
and Experimental Therapeutics, (Feb 2004), Vol. 308, no. 2, pp. (688-693) lipid Lipoproteins, albumin apoBl, apoM N/A N/A Absorption, attachment affinity for lipoproteins and albumin wolfram et al.
Nature
biotechnology,
(Sep 2007), Vol. 25, no. 10, pp. (1149-1157) lipid Lipofectamine 2000 p53 Acute renal failure Ischemic and cispatin-induced acute failure Histological scoring, apoptosis Molitoris et al., J Am Soc Nephrol, (Aug 2009), Vol.
20, no. 8, pp. (1754-1764) lipid DOTAP/DOPE,
DOTAP/DO
PE/DOPE-
PEG2000 COX-2 breast adenocarcinoma MDA-MB-231 mouse carrying breast cancer xenograft Cell viability, uptake Mikhaylova et al., Cancer Gene Therapy, (Mar
2011), Vol. 16, no.
3, pp. (217-226)) lipid Cholesterol 12/15-lipoxygenase diabetic nephropathy Streptozotocin-induced diabetes Albuminuria, urinary creatinine, histology, type I and IV collagen, TGF-β, fibronectin, plasminogen activator inhibitor 1 Yuan et al. Am J Physiol Renal Physiol, (Jun
2008), Vol. 295, pp. (F605-F617) lipid Lipofectamine 2000 Membrane 1 mitochondria 44 (TIM44) diabetic nephropathy Streptozotocin-induced diabetes Cell division and apoptosis, histology, ROS, mitochondrial import of Mn-SOD and glutathione peroxidase, cellular membrane polarization Y. Zhang et al., J Am Soc Nephrol, (Apr 2006), Vol. 17, no. 4, pp. (1090-1101) Hydrodynamic/lipid Proteoliposomes RLIP76 renal carcinoma Caki-2 mouse carrying a kidney cancer xenograft Absorption Singhai et al.,
Cancer Res, (May 2009), Vol. 69, no. 10, pp.
(4244-4251) Polymeric PEGylated PEI Luciferase pGL3 N/A N/A Absorption, biodistribution, erythrocyte aggregation Malek et al., Toxicology and Applied
pharmacy,
(Apr 2009), Vol. 236, no. 1, pp. (97-108) Polymeric PEGylated poly-L-lysine MAPK1 Undulating glomerulonephritis Glomerulonephritis Proteinuria, glomerulosclerosis, TGF-β, fibronectin, plasminogen activator inhibitor 1 Shimizu et al.,.1 Am Soc
Nephrology, (Apr 2010), Vol. 21, no. 4, pp. (622-633) polymeric/using nanoparticles Hyaluronic Acid/Quantum Dot/PEI VEGF kidney cancer, melanoma B16F1 mouse carrying a melanoma tumor Biodistribution, tumor volume, endocytosis Jiang et ah. Molecular Pharmaceutics,
(May-Jun 2009), Vol. 6, no. 3, pp. (727-737) polymeric/using nanoparticles pegylated polycaprolactone nanofiber GAPDH N/A N/A Cell viability, uptake Cao et al, J Controlled Release, (Jun 2010), Vol. 144, no. 2, pp. (203-212) Aptameric Spiegelmer
mNOX-E36 Chemokine ligand 2 CC Glomerosclerosis Uninephrectomized mouse Urinary albumin, urinary creatinine, histopathology, glomerular filtration rate, macrophage count, serum Ccl2, Mac-2+, Ki~67+ Ninichuk et al., Am J Pathol, (Mar 2008), Vol. 17.2, no. 3, pp. (628-637) Aptameric Aptamer NOX-F37 Vasopressin Chronic heart failure N/A Attachment affinity to D-AVP, inhibition of AVP signaling, urinary osmolability and sodium concentration Purschke et al., Proc Natl Acad Sci, (Mar 2006), Vol. 103, no. 13, pp. (3173-5178)

[001238] Targeting to liver cells is contemplated. This may be in vitro or in vivo. Hepatocytes are preferred. Delivery of a CRISPR protein such as C2c1 or C2c3 here can be done via viral vectors, especially AAV vectors (and in particular AAV2/6). They can be administered intravenously.

[001239] A preferred target for the liver, whether in vitro or in vivo , is the albumin gene. This is a so-called "safe harbor" because albumin is expressed at very high levels and therefore some reduction in albumin synthesis is tolerated after successful gene editing. It is also preferred that the high levels of expression seen with the albumin promoter/enhancer allow suitable levels of normal or transgenic synthesis (from an inserted donor template) even if only a small fraction of the hepatocytes are edited.

[001240] Wechsler et al. was shown (post from the 57th Annual Meeting and Exposition of the American Society of Hematology - abstract available online at https://ash.confex.com./ash/2015/webprogram/Paper86495.html and submitted December 6, 2015) that albumin intron 1 is a suitable target site. In their work, zinc fingers were used to cut DNA at a given target site, and suitable guide sequences could be generated to guide cleavage at the same site using the CRISPR protein.

[001241] The use of targets in highly expressed genes (genes with highly active enhancers/promoters), such as albumin, may also allow the use of a promoterless donor matrix, as shown by Wechsler et al., and this is also widely applicable to organs other than the liver . Other examples of highly expressed genes are also known.

Liver-associated blood disorders, especially hemophilia and in particular hemophilia B

[ 000019] Successful gene editing of hepatocytes in mice (both in vitro and in vivo ) and in non-human primates ( in vivo ) has shown that treatment of blood diseases through gene editing/genetic engineering in hepatocytes is possible. In particular, the expression of the human F9 (hF9) gene in hepatocytes of non-human primates has been shown, which also indicates the possibility of treating hemophilia B in humans.

[000020] Wechsler et al. reported at the 57th Annual Meeting and Exposition of the American Society of Hematology (abstract submitted December 6, 2015 and available online at https://ash.confex.com/ash/2015/webprogrammpaper86495.html) that they successfully expressed human F9 (hF9) from hepatocytes in non-human primates by in vivo gene editing. This was achieved using 1) two zinc finger nucleases targeting intron 1 of the albumin locus and 2) a human F9 donor template construct. Zinc fingers and a donor matrix were encoded in selected IV adeno-associated virus hepatotropic serotypes s2/6 (AAV2/6), which resulted in the targeted insertion of a corrected copy of the hF9 gene into the albumin locus in a portion of hepatic hepatocytes.

[000021] The albumin locus was chosen as a "safe harbor" because the production of this most abundant plasma protein exceeds 10 g/day, and a moderate decrease in this level is well tolerated. Genome-edited hepatocytes produced normal hFIX (hF9) in therapeutic amounts, in contrast to albumin, which is driven by a highly active albumin enhancer/promoter. Targeted integration of the hF9 transgene in the albumin locus and splicing of this gene into the albumin transcript were shown.

[000022] Mouse studies: C57BL/6 mice injected with delivery vehicle (n=20) or AAV2/6 vectors (n=25) encoding mouse surrogate reagents at 1.0 x 10 ¹³ vector genomes (vg)/kg by injection into the tail vein. Plasma hFIX ELISA analysis in treated mice showed peak levels of 50-1053 ng/ml which were maintained throughout the 6 month study. FIX activity analysis based on mouse plasma confirmed biological activity commensurate with expression levels.

[000023] Studies in non-human primates: a single intravenous co-infusion of AAV2/6 vectors encoding target albumin specific zinc finger nucleases targeting NHP and donor human F9 at 1×10 ¹³ vg/kg (n=5/group) resulted in k > 50 ng/mL (> 1% of normal) in this large animal model. The use of higher doses of AAV2/6 (up to 1.5×10 ¹⁴ vg/kg) resulted in plasma hFIX levels up to 1000 ng/mL (or 20% of normal) in several animals and up to 2000 ng/mL (or 50% from normal) in one animal throughout the study (3 months).

[000024] Treatment was well tolerated in mice and non-human primates without significant toxicological outcomes associated with AAV2/6 ZFN + donor treatment in both species at therapeutic doses. Sangamo (California, USA) has since approached the FDA and received approval to conduct the world's first human clinical trial for in vivo genome editing applications. This is based on EMEA approval for Glybera gene therapy in the treatment of lipoprotein lipase deficiency.

[000025] Accordingly, in some embodiments, it is preferable to use any or all of the following:

* AAV (especially AAV2/6) vectors, preferably administered intravenously;

* Albumin as a target for gene editing/transgene/template insertion - especially in intron 1 of albumin;

* human F9 donor matrix; and/or "promoterless donor matrix".

Hemophilia B.

[000026] Accordingly, in some embodiments, it is preferred that the present invention be used to treat hemophilia B. As such, it is preferred that the template be provided and that it be the human F9 gene. It is understood that the hF9 template contains the correct or "correct" version of hF9 so that the treatment is effective.

[000027] In an alternative embodiment, the F9 version of hemophilia B can be delivered so as to create a model organism, cell or cell line (e.g., a cell, cell line or model organism based on a mouse or non-human primate), model organism, cell or cell lineage having or carrying the hemophilia B phenotype, i.e. inability to produce wild-type F9.

Hemophilia A

[00 0028] In some embodiments, the F9 (factor IX) gene can be replaced with the F8 (factor VIII) gene described above, resulting in the treatment of hemophilia A (by providing the correct F8 gene) and/or the creation of a model organism, cell, or cell hemophilia A lines (by providing the wrong version of the hemophilia A F8 gene).

Hemophilia C

[001242] In some embodiments, the F9 gene (factor IX) can be replaced by the F11 gene (factor XI) described above, resulting in the treatment of hemophilia C (by providing the correct F11 gene) and/or the creation of a model organism, cell or cell line with hemophilia C (by providing an incorrect version of the hemophilia C F11 gene).

Treatment of diseases of the epithelium and lungs

[001243] The present invention provides for delivery of the CRISPR-Cas systems described herein, eg, C2c1 or C2c3 effector protein systems, to one or both lungs.

[001244] Although AAV-2 based vectors were originally proposed to deliver CFTR to the CF airways, other serotypes such as AAV-1, AAV-5, AAV-6 and AAV-9 show improved gene transfer efficiency in a variety of models. lung epithelium (see, for example, Li et al, Molecular Therapy, vol. 17 no. 12, 2067-2077 Dec 2009). AAV-1 has been demonstrated to be 100 times more efficient than AAV-2 and AAV-5 at transducing human airway epithelial cells in vitro , although AAV-1 has transduced mouse tracheal airway epithelium in vivo with an efficiency equal to that of AAV-5. Other studies have shown that AAV-5 is 50 times more efficient than AAV-2 at delivering genes to human airway epithelium (HAE) in vitro and significantly more efficient in mouse airway epithelium in vivo . AAV-6 has also been shown to be more effective than AAV-2 in human airway epithelial cells in vitro and mouse airway cells in vivo . The more recent AAV-9 isolate showed greater gene transfer efficiency than AAV-5 in mouse nasal and alveolar epithelium in vivo , with gene expression detected after more than 9 months, suggesting that AAV can mediate long-term gene expression in vivo . This is the desirable property for a CFTR gene delivery vector. In addition, it has been demonstrated that AAV-9 can be introduced into the mouse lung without loss of CFTR expression and minimal immune consequences. CF and non-CF HAE cultures can be inoculated on the apical surface with 100 ml of AAV vectors for several hours (see e.g. Liet al., Molecular Therapy, vol. 17 no, 12, 2067-2077 Dec 2009) . The MOI can vary from 1×10 ³ to 4×10 ⁵ vector genomes/cell, depending on the virus concentration and the goals of the experiments. The above vectors are intended for the delivery and/or administration of the present invention.

[001245] Zamora et al. ( Am JRespirCritCareMed Vol. 183. pp. 531-538, 2011) described an example of the use of RNA interference therapy for the treatment of a human infectious disease, as well as a randomized trial of an antiviral drug in respiratory syncytial virus (RSV) infected lung transplant recipients. Zamora et al. conducted a randomized, double-blind, placebo-controlled study in LTX recipients with RSV respiratory tract infection. Patients were allowed to receive standard care for RSV. Aerosol ALN-RSV01 (0.6 mg/kg) or placebo was administered daily for 3 days. This study demonstrates that RSV-targeted RNAi-based treatments can be safely administered to LTX recipients with RSV infection. Three daily doses of ALN-RSV01 did not result in exacerbation of respiratory tract symptoms, deterioration in lung function, and systemic inflammatory effects such as cytokine or CRP induction. The pharmacokinetics showed only a low transient systemic exposure after inhalation, which is consistent with preclinical animal data showing that ALN-RSV01 administered intravenously or by inhalation is rapidly cleared from the circulation via exonuclease treatment and renal excretion. The methods of Zamora et al. may be applied to the nucleic acid targeting system of the present invention, and aerosolized CRISPR Cas, for example at a dosage of 0.6 mg/kg, may be considered for the present invention.

[001246] Individuals receiving therapy for lung diseases may, for example, receive a pharmaceutically effective amount of an AAV vector aerosol system per lung delivered endobronchially on spontaneous breathing. Thus, aerosol delivery is preferred for AAV delivery in general. For delivery, an adenovirus or an AAV particle can be used. Suitable gene constructs, each operably linked to one or more regulatory sequences, can be cloned into a delivery vector. In this case, the following constructs are given as examples: Cbh or EF1a promoter for Cas (C2c1 or C2c3), U6 or H1 promoter for guide RNA): Preferably use of CFTR delta508 guide sequence, deltaF508 mutation recovery template and codon-optimized C2c1 or C2c3 enzyme , optionally with one or more Nuclear Localization Signals or Sequence (NLS), e.g., two (2) NLS. Non-NLS designs are also contemplated.

Treatment of diseases of the muscular system

[001247] The present invention involves the introduction of the CRISPR-Cas system, for example, systems of C2c1 or C2c3 effector proteins into muscle.

[001248] Bortolanza et al. ( MolecularTherapyvol. 19 no. 11, 2055-2064 Nov. 2011) showed that systemic delivery of expression cassettes for RNA interference in mouse FRG1 after the onset of fasciomuscular dystrophy (FSHD) resulted in a dose-dependent long-term knockdown of FRG1 without evidence of toxicity. Bortolanza et al. found that a single 5×10 ¹² intravenous injection of rAAV6-sh1FRG1 preserves muscle histopathology and muscle function in FRG1 mice. Similarly, 200 ml containing 2×10 ¹² or 5×10 ¹² vg vector in saline was injected into the tail vein using a Terumo-25 syringe. The method of Bortolanza et al. can be applied to an AAV expressing CRISPR Cas and a dose of approximately 2×10 ¹⁵ or 2×10 ¹⁶ vg of the vector can be administered to a human.

[001249] Dumonceaux et al. ( Molecular Therapy vol. 18 no. 5, 881-887 May 2010) inhibited the myostatin pathway using an RNA interference method directed against ostatin receptor mRNA (sh-AcvRIIb) AcvRIIb. Recovery of quasi-dystrophin was mediated by the vectorized U7 skipping technique (U7-DYS). Adeno-associated vectors carrying either the sh-AcvrIIb construct alone, or the U7-DYS construct alone, or a combination of both constructs, were injected into the tibialis anterior muscle of dystrophy mdx mice. Injections were performed using 10 ¹¹ AAV viral genomes. The method of Dumonceaux et al. may be applied to an AAV expressing CRISPR Cas and administration to a human may be at a dose of, for example, from about 10 ¹⁴ to about 10 ¹⁵ vg of the vector.

[001250] Kinouchi et al. ( GeneTherapy (2008) 15, 1126-1130) report the efficacy of in vivo delivery of small interfering RNA into skeletal muscle tissue of normal or diseased mice via formation of atelocollagen (ATCOL) chemically unmodified siRNA nanoparticles. ATCOL-mediated topical application of siRNA targeting myostatin, a negative skeletal muscle growth regulator, in murine skeletal muscle or intravenously caused a marked increase in muscle mass within weeks of application. These results imply that ATCOL-mediated siRNA application is a powerful tool for future therapeutic use in diseases such as muscle atrophy. Msts-miRNA (final concentration, 10 mM) was mixed with ATCOL (final concentration for local application, 0.5%) (AteloGene, Kohken, Tokyo, Japan) according to the manufacturer's instructions. After anesthetizing mice (20-week-old male C57BL/6) with Nembutal (25 mg/kg, ip), the Mst-siRNA/ATCOL complex was injected into the masseter and biceps femoris. The method of Kinouchi et al. can be applied to CRISPR Cas by injecting into the human body, for example, doses of 500 to 1000 ml of a 40 μM solution into a muscle. Hagstrom et al. (MolecularTherapy, Vol. 10, No. 2, August 2004) describe an intravascular, non-viral methodology that provides efficient and repeatable delivery of nucleic acids to muscle cells (myofibrils) in all mammalian limb muscles. The procedure involves the injection of naked plasmid DNA or siRNA into a distal vein of an extremity that is temporarily isolated with a tourniquet or blood pressure cuff. Delivery of the nucleic acid to the myofibrils is facilitated by rapid injection in sufficient volume to allow extravasation of the nucleic acid solution into the muscle tissue. High levels of transgene expression in skeletal muscles were achieved in both small and large animals with minimal toxicity. Evidence was also obtained for miRNA delivery to limb muscles. For intravenous injection of plasmid DNA in rhesus monkeys, a three-piece clamp was connected to two syringe pumps (model PHD 2000, Harvard Instruments), each with a single syringe. Five minutes after the injection of papaverine, pDNA was injected (from 15.5 to 25.7 mg in 40-100 ml of isotonic solution) at a rate of 1.7 or 2.0 ml/s. The dose can be increased for plasmid DNA expressing the CRISPR Cas of the present invention by injection of 300 to 500 mg in 800-2000 ml of human saline. For adenoviral vector injections in the rat, 2×10 ⁹ infectious particles were injected in 3 ml of normal saline (NSS). The dose can be increased for an adenoviral vector expressing CRISPR Cas according to the present invention, with an injection of about 1×10 ¹³ infectious particles injected in 10 liters of human NSS. For siRNA, the rat was injected into the great saphenous vein with 12.5 µg of siRNA, and the primates were injected with 750 µg of siRNA into the great saphenous vein. The dose may be increased for the CRISPR Cas of the present invention, for example by injecting from about 15 to 50 mg into the human great saphenous vein.

[001251] See also, for example, WO2013163628 A2, "Genetic Correction of Mutated Genes", a Duke University published application describing attempts to correct, for example, frameshift mutations that cause premature stop codons and a truncated gene product that can be corrected by nuclease-mediated non-homologous end joining, such as those responsible for Duchenne muscular dystrophy ("DMD"), a recessive, fatal, X-linked disease that results in muscle degeneration due to mutations in the gene dystrophin. Most dystrophin mutations that cause DMD are exon deletions that disrupt the reading frame and cause premature termination of translation in the dystrophin gene. Dystrophin is a cytoplasmic protein that provides structural stability to the cell membrane dystroglycan complex, which is responsible for regulating the integrity and function of muscle cells. The dystrophin gene or "DMD gene", used interchangeably herein, is 2.2 million bp. at the Xp21 locus. The primary transcription is about 2400 kb and the mature mRNA is about 14 kb. 79 exons code for a protein that contains more than 3500 amino acids. Exon 51 is often adjacent to frameshifting deletions in DMD patients and is the target of clinical trials for oligonucleotide-based exon exclusion. A clinical trial of an exon 51-exclusive compound recently reported significant functional benefit at 48 weeks, with an average of 47% more dystrophin-positive fibers from baseline. Mutations in exon 51 are ideal for permanent correction by non-homologous end joining (NHEJ) genome editing.

[001252] The methods described in US Patent Publication No. 20130145487 assigned to Cellectis relate to variants of using a meganuclease to cleave a target sequence from the human dystrophin (DMD) gene, and can also be modified for the nucleic acid targeting system of the present invention.

[001253] The present invention also provides for the delivery of the CRISPR-Cas system described herein, for example, effector protein systems such as C2c1 or C2c3, to the skin.

[001254] Hickerson et al. ( MolecularTherapy-NucleicAcids (2013) 2, el29) described a motorized microneedle device for delivering self-delivered (sd) siRNA to human or mouse skin. The main problem of transferring miRNA skin therapy to the clinic is the development of effective delivery systems. Significant efforts have been invested in various skin delivery technologies, but have shown limited success. In a clinical study in which skin was treated with siRNA, pain associated with the use of tiny hypodermic needles prevented additional patients from being enrolled for the study, highlighting the need for improved delivery methods that are patient-centered and cause little or no pain. were painless for the patient. Microneedles are an efficient way to deliver large charged compounds, including siRNAs, across the primary barrier, the stratum corneum, and are generally considered less painful than conventional hypodermic needles. Motorized punch-type devices, including the motorized microneedle (MMNA) device used by Hickerson et al, have been shown to be safe. Use of such devices has been shown in hairless mice to cause little or no pain, as evidenced by (i) widespread use in the cosmetics industry and (ii) limited testing in which almost all volunteers found that device use was much less painful. than the flu shot, suggesting that siRNA delivery using this device would result in much less pain than in a previous clinical study using hypodermic needles. The MMNA device (sold as Triple-M or Tri-M by Bomtech Electronic Co, Seoul, South Korea) has been adapted to deliver siRNAs to mouse and human skin. A solution of self-delivering siRNAs (up to 300 μl of 0.1 mg/ml RNA) was introduced into the chamber of a disposable Tri-M cassette (Bomtech), which was set to a depth of 0.1 mm. For human skin treatment, de-identified skin: (obtained immediately after surgical procedures) was manually stretched and attached to a cork platform prior to processing. All intradermal injections were performed using an insulin syringe with a 0.5 inch 28 gauge needle. The MMNA device and method of Hickerson et al. can be used and/or adapted to deliver CRISPR Cas according to the present invention, for example, at a dose of up to 300 μl of 0.1 mg/ml CRISPR Cas to the skin.

[001255] Leachman et al. ( MolecularTherapy , vol. 18 no. 2, 442-446 Feb. 2010) refers to a phase Ib clinical trial for the treatment of the rare skin disorder pachyonychia congenita (PV), an autosomal dominant syndrome involving disabling palmoplantar keratoderma , using small interfering RNA (siRNA). This siRNA, called TD101, specifically and efficiently targets the N171K mutant keratin 6a (K6a) mRNA without affecting the wild-type K6a mRNA.

[001256] Zheng et al. (PNAS, July 24, 2012, vol. 109, no. 30, 11975-11980) show that spherical nucleic acid nanoparticle conjugates (SNA-NC), gold cores, surrounded by a dense shell of highly oriented covalently immobilized siRNA, freely penetrate in almost 100% of keratinocytes in vitro , in mouse skin and human epidermis within hours of application. Zheng et al. demonstrated that a single application of 25 nM SNA-NC for epidermal growth factor receptors (EGFR) for 60 hours demonstrates effective disruption of this gene in human skin. A similar dose can be provided for CRISPR Cas immobilized in SNA-NC for injection into the skin.

malignant tumor

[001257] In some embodiments, the invention provides for the treatment, prevention, or diagnosis of cancer. The target is preferably one or more of the FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, TRAC or TRBC genes. The cancer may be one or more of lymphomas, chronic lymphocytic leukemia (CLL), B-cell lymphoblastic leukemia (B-ALL), acute lymphoblastic leukemia, acute myeloid leukemia, non-Hodgkin's lymphoma (NHL), diffuse large cell lymphoma (DLCL), multiple myeloma, renal cell carcinoma (RCC), neuroblastoma, colorectal cancer, breast cancer, ovarian cancer, melanoma, sarcoma, prostate, lung, esophageal, hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma, head and neck cancer, and medulloblastoma . This can be accomplished with an engineered chimeric T cell receptor (CAR) antigen. This is described in WO2015161276, the contents of which are incorporated herein by reference and described herein below.

[001258] Target genes suitable for the treatment or prevention of cancer may include, in some embodiments, those described in WO 02015048577, the contents of which are incorporated herein by reference.

Usher syndrome or retinitis pigmentosa-39

[001259] In some embodiments, the treatment, prevention, or diagnosis of Usher's syndrome or retinitis pigmentosa-39 ( retinitis pigmentosa -39) is contemplated. The target is preferably the USH2A gene. In some embodiments, the G deletion at position 2299 (2299delG) is corrected. This is described in WO2015134812A1, the contents of which are incorporated herein by reference.

Cystic fibrosis (CF)

[001260] In some embodiments, the treatment, prevention, or diagnosis of cystic fibrosis is provided. The target is preferably SCNN1A or the CFTR gene. This is described in W02015157070, the contents of which are incorporated herein by reference.

[001261] Schwank et al. (CellS:.emCell, 13: 653-58, 2013) used CRISPR-Cas9 to correct a defect associated with cystic fibrosis in human stem cells. The group's target was the ion channel gene, cystic fibrosis transmembrane conductor receptor (CFTR). A deletion in CFTR leads to protein misfolding in patients with cystic fibrosis. Using cultured intestinal stem cells derived from cell samples from two children with cystic fibrosis, Schwank et al. were able to repair the defect using CRISPR together with a donor plasmid containing the insertion repair sequence. The researchers then grew the cells into intestinal "organoids" or miniature intestines and showed that they functioned normally. In this case, approximately half of the clonal organelles underwent proper genetic correction.

HIV and AIDS

[001262] In some embodiments, the treatment, prevention, or diagnosis of HIV and AIDS is provided. The HIV target is preferably the CCR5 gene. This is described in WO2015148670A1, the contents of which are incorporated herein by reference.

Beta Thalassemia

[001263] In some embodiments, the treatment, prevention, or diagnosis of beta thalassemia is contemplated. The target is preferably the BCL11A gene. This is described in WO2015148860, the description of which is incorporated herein by reference.

sickle cell anemia

[001264] In some embodiments, the treatment, prevention, or diagnosis of sickle cell anemia (SCD) is contemplated. The target is preferably the HBB or BCL11A gene. This is described in WO2015148863, the description of which is incorporated herein by reference.

Herpes simplex virus 1 and 2

[001265] In some embodiments, treatment, prevention, or diagnosis of HSV-1 (herpes simplex virus 1) is provided. The target is preferably the UL19, UL30, UL48 or UL50 gene in HSV-1. This is described in WO2015153789, the description of which is incorporated herein by reference.

[001266] In other embodiments, treatment, prevention, or diagnosis of HSV-2 (herpes simplex virus 2) is provided. The target is preferably the UL19, UL30, UL48 or UL50 gene in HSV-2. This is described in WO2015153791, the contents of which are incorporated herein by reference.

[001267] In some embodiments, the treatment, prevention, or diagnosis of primary open angle glaucoma (POAG) is contemplated. The target is preferably the MYOC gene. This is described in WO2015153780, the contents of which are incorporated herein by reference.

Adoptive cell therapy

[001268] The present invention also contemplates the use of the CRISPR-Cas system described herein, for example. C2c1 or C2c3 to modify cells for adoptive therapy. Accordingly, aspects of the present invention include the adoptive transmission of cells of the immune system, such as T cells, specific for selected antigens, such as tumor-associated antigens (see Maus et al., 2014, Adoptive Immunotherapy for Cancer or Viruses, Annual Review of Immunology , Vol. 32: 189-225; Rosenberg and Restifo, 2015, Adoptive cell transfer as personalized immunotherapy for human cancel, Science Vol, 348 no. 6230 pp. 62-68; and, Restifo et al., 2015, Adoptive immunotherapy for cancer: harnessing the T cell response. Nat. Rev. Immunol. 12(4): 269-281; and Jenson and Riddell, 2014, Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells. Immunol Rev. 257( 1): 127-144). For example, various strategies can be used to genetically modify T cells by changing the specificity of the T cell receptor (TCR), for example, by introducing new TCRα and β chains with a selected peptide specificity (see US Pat. No. 8,697,854; PCT Patent Application Publications: W02003020763, W02004033685, WQ2004044004, W02005114215, W02006000830, W02008038002, W02008074322, W02005113595, WO2006125962, WO2013166321, WO2013039889, WO2014018863, WO2014083173, патент США 8088379).

[001269] As an alternative to, or in addition to, TCR modification, chimeric antigen receptors (CARs) can be used to generate immunopositive cells, such as T cells, specific for selected targets, such as cancer cells, with a wide range of receptor chimeric constructs as described (see 5843728, 5851828, 5912170, 6004811, 6284240, 6392013, 6410014, 6753162, 8211422 and PCT Publication WO9215322). Alternative CAR designs can be characterized as belonging to later generations. First generation CARs typically consist of a single chain variable fragment of an antibody specific for an antigen, e.g. containing a VL linked to a VH of a specific antibody linked by a flexible linker, e.g. via a CD8α loop domain and a CD8α transmembrane domain, with transmembrane and intracellular signaling domains, such as CD3cξ and FcRγ (scFν-CD3ξ or scFv-FcRγ, see US Pat. No. 7,741,465, US Pat. No. 5,912,172, US Pat. No. 5,906,936). Second generation CARs include intracellular domains of one or more co-stimulatory molecules such as CD28, OX40 (CD134), or 4-1BB (CD137) in the endodomain (e.g., scFv-CD28/OX4O/4-lBB-CD3Q, see U.S. Patent Nos. 8911993, 8916381, 8975071, 9017584, 9102760, 9102761), third-generation CARs include a combination of costimulatory endodomains such as the CD3ξ chain, CD97, CD11a-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, or CD28 (e.g., scFv-CD28-4-1BB-CD3ξ or scFv-CD28-OX40-CD3ξ, see U.S. Patent 8,906,682, U.S. Patent No. 8,399,645, U.S. Patent No. 5,686,281; PCT Publication No. WO2014134165; PCT Publication No. W02012079000). Alternatively, costimulation can be orchestrated by expression of CARs in antigen-specific T cells chosen to be activated and expanded upon interaction of their native αβTCR with, for example, antigen on professional antigen presenting cells, with concomitant costimulation. In addition, additional modified receptors on immunoresponsive cells may be provided, for example, to improve targeting of T cell attack and/or minimize side effects.

[001270] Alternative methods can be used to transform immunopositive target cells, such as protoplast fusion, lipofection, transfection, or electroporation. A wide variety of vectors can be used, such as retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated viral vectors, plasmids, or transposons such as the Sleeping Beauty transposon (see US Pat. Nos. 6,489,458; 7,148,203; 7,166,682; CARs, for example, using 2nd generation antigen-specific CARs signaling via CD3ξ and either CD28 or CD137. Viral vectors may, for example, include vectors based on HIV, SV40, EBV, HSV or BPV.

[001271] Cells that are targets for subsequent transformation may, for example, include T cells, natural killer (NK), cytotoxic T lymphocytes (CTL), regulatory T cells, human embryonic stem cells, tumor infiltrating lymphocytes (TIL ) or a pluripotent stem cell from which lymphoid cells can be differentiated. T cells expressing the desired CAR can be selected, for example, by co-cultivation with γ-irradiated activated and proliferating cells (AaPC) that co-express cancer antigen and co-stimulatory molecules. Can be carried out increase in the number of engineered CART cells, for example, by co-culturing on AaPC in the presence of soluble factors such as IL-2 and IL-21. Such an increase in number can be performed, for example, in such a way as to provide a memory of CAR + T cells (which can, for example, be analyzed using a non-enzymatic digital array and/or multi-panel flow cytometry). Thus, CART cells can be provided that have specific cytotoxic activity against antigen-binding tumors (optionally in combination with the production of desired chemokines such as interferon-γ). CAR T cells of this type can, for example, be used in animal models, for example, in order to destroy tumor xenografts.

[001272] The following approaches can be adapted to provide methods of treating and/or increasing the survival of an individual having a disease, such as a neoplasm, for example, by administering an effective amount of an immunopositive cell containing an antigen-recognizing receptor that binds a selected antigen, where the binding activates an immunoreactive cell, thereby curing or preventing a disease (such as a neoplasm, a pathogenic infection, an autoimmune disorder, or an allogeneic transplant reaction). Dosages in T cell therapy may, for example, include administration of 106 to 109 cells/kg, with or without a course of lymphodepletion, for example with cyclophosphamide.

[001273] In one embodiment, treatment can be given to patients undergoing immunosuppressive treatment. A cell or population of cells may be resistant to at least one immunosuppressant due to inactivation of a gene encoding a receptor for that immunosuppressant. Not limited only by theory, immunosuppressive treatment should promote the selection and expansion of T cells responsible for the immune response in accordance with the invention within the patient himself.

[001274] The introduction of cells or populations of cells in accordance with the present invention can be carried out by any convenient method, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The cells or population of cells can be administered to a patient subcutaneously, intradermally, intratumorally, intranasally, intramedullary, intramuscularly, by intravenous or intralymphatic injection, or intraperitoneally. In one embodiment, the cell compositions of the present invention. Preferably administered by intravenous injection.

[001275] Administration of cells or cell population may consist of administration of 10 ⁴ -10 ⁹ cells per kg of body weight, preferably 10 ⁵ to 10 ⁶ cells/kg of body weight, including all integer cell numbers in these ranges. Dosing in T-cell CAR therapy may, for example, include the administration of 10 ⁶ to 10 ⁹ cells/kg, with or without a course of cell depletion, for example, with cyclophosphamide. The cells or population of cells can be administered in one or more doses. In another embodiment, an effective amount of cells is administered as a single dose. In another embodiment, an effective amount of cells is administered in more than one dose over a period of time. The timing of administration is within the competence of the attending physician and depends on the clinical condition of the patient. The cells or population of cells may be obtained from any source such as a blood bank or donor. While individual needs vary, determining the optimal ranges for the effective amount of a given cell type for a particular disease or condition is within the skill of the art. An effective amount means an amount that provides a therapeutic or prophylactic benefit. The dosage administered will depend upon the age, health and weight of the recipient, the type of treatment being concomitantly received, if any, the frequency of treatment, and the nature of the effect desired.

[001276] In another embodiment, an effective amount of cells or a composition containing these cells is administered parenterally. The introduction may be intravenous. The introduction can be directly made by injection into the tumor.

[001277] To protect against possible adverse reactions, artificial immunopositive cells can be equipped with a transgenic safety switch in the form of a transgene that makes the cells vulnerable to being affected by a particular signal. For example, the herpes simplex virus (TK) thymidine kinase gene can be used, for example, by introducing into allogeneic T lymphocytes used as donor lymphocyte infusions after stem cell transplantation (Greco et al., Improving the safety of cell therapy with the TK-suicide gene Front Pharmacol 2015: 6: 95). In such cells, administration of a nucleoside prodrug such as ganciclovir or acyclovir causes cell death. Alternative designs of the safety switch include an inducible caspase, for example initiated by the introduction of a small molecule dimerizer that combines two non-functional icasp9 molecules to form an active enzyme. A wide range of alternative approaches to control cell proliferation have been described (see U.S. Patent Publication No. 20130071414, Patent Application Publication PCTWO2011146862, Patent Application Publication PCTWO2014011987, Patent Application Publication PCTWO2013040371, Zhou et al. BLOOD, 2014, 903-25:35: ; Di Stasi et al, The New England Journal of Medicine 2011; 365: 1673-1683; Sadelain M, New England Journal of Medicine, 2011; 365: 1735-173; Ramos et al., Stem Cells 28(6): 1107-15 (2010)).

[001278] A further improvement in adoptive therapy may be genome editing using the CRISPR-Cas system. As described herein, to adapt immunopositive cells to alternative implementations, such as providing edited CART cells (see Poirot et al., 2015, Multiplex genome edited T-cell manufacturing platform. for "off-the-shelf" adoptive T-cell immunotherapies, CancerRes 75 (18); 3853). For example, immunopositive cells can be removed to remove the expression of any or all of a class of HLA type II and/or type I molecules, or to knock out selected genes that can inhibit a desired immune response, such as the PD1 gene.

[001279] Cells can be modified using any of the CRISPR system and method of using it described in this application. CRISPR systems can be delivered to an immune cell by any of the methods described herein. In preferred embodiments, cells are edited ex vivo and then delivered to an individual in need thereof. Immune system cells, CART cells, or any cells used to transfer cells for this type of therapy can be edited. Editing can be done to eliminate potential alloreactive T cell receptors (TCRs), destroy the target of the chemotherapeutic agent, block the immune checkpoint, activate T cells, and/or enhance the differentiation and/or proliferation of functionally depleted or dysfunctional CD8+ T cells (see PCT patent publications: WO2013176915, WO2014059173, WO2014172606, WO2014184744 and WO2014191128). Editing can lead to gene inactivation.

[001280] When a gene is inactivated, it is assumed that the gene of interest is not expressed in a functional protein form. In a specific embodiment, the CRISPR system specifically catalyzes cleavage in one target gene, thereby inactivating said gene. Induced nucleic acid strand breaks are typically repaired through various homologous recombination or non-homologous end joining (NHEJ) mechanisms. However, NHEJ is an imperfect repair process that often results in DNA sequence changes at the cleavage site. Non-homologous end joining (NHEJ) repair often results in small insertions or deletions (Indel) and can be used to create specific gene knockouts. Cells that have undergone cleavage-induced mutagenesis can be identified and/or selected by methods known in the art.

[001281] T cell receptors (TCRs) are cell surface receptors that are involved in the activation of T cells in response to the appearance of an antigen. The TCR usually consists of two chains, α and β, which assemble to form a heterodimer and associate with CD3 transducing subunits to form a T cell receptor complex present on the cell surface. Each α and β chain of the TCR consists of an immunoglobulin-like N-terminal variable (V) and constant (C) region, a hydrophobic transmembrane domain, and a short cytoplasmic region. With respect to immunoglobulin molecules, the variable region of the α and β chains is generated by V(D)J, creating a wide variety of antigen-specific features in the T cell population. However, unlike immunoglobulins that recognize intact antigen, T cells are activated by treated peptide fragments in combination with an MHC molecule, introducing an additional change in T cell antigen recognition known as MHC restriction. Recognition of MHC differences between donor and recipient through the T cell receptor leads to T cell proliferation and the potential development of graft versus host disease (GVHD). TCR inactivation can lead to elimination of TCRs from the surface of T cells, preventing alloantigen recognition and thus development of rejection (GVHD). However, disruption of the TCR usually results in the elimination of the CD3 signaling component and alters the pathway for further expansion of T cells.

[001282] Allogeneic cells have been shown to be rapidly rejected by the host's immune system. It has been demonstrated that allogeneic leukocytes present in non-irradiated blood will persist for no more than 5-6 days (Boni, Muranski et al, 2008 Blood 1; H2 (12): 4746-54). Thus, in order to prevent rejection of allogeneic cells, the host's immune system usually must be suppressed to some extent. However, despite the ease of adoptive cell transfer, the use of immunosuppressants also has a detrimental effect on administered therapeutic T cells. Therefore, in order to effectively use the adoptive immunotherapy approach under these conditions, the injected cells must be resistant to immunosuppressive treatment. Thus, in a specific embodiment, the present invention further comprises the step of modifying T cells to render them resistant to an immunosuppressant, preferably by inactivating at least one gene encoding the target of the immunosuppressant. An immunosuppressive agent is an agent that suppresses immune function by one of several mechanisms of action. The immunosuppressive agent may be, but is not limited to, a calcineurin inhibitor, a rapamycin target, an interleukin-2 receptor α-chain blocker, an inosine monophosphate dehydrogenase inhibitor, a dihydrofolic acid reductase inhibitor, a corticosteroid, or an immunosuppressive antimetabolite. The present invention makes it possible to confer immunosuppressive resistance on T cells for immunotherapy by inactivating the target of the immunosuppressant in T cells. As non-limiting examples of targets for an immunosuppressive agent, receptors for an immunosuppressive agent such as: CD52, glucocorticoid receptor (GR), a member of the FKBP family of families, and a member of the cyclophilin family of families can be used.

[001283] Immune checkpoints are inhibitory pathways that slow or stop immune responses and prevent excessive tissue damage from uncontrolled immune cell activity. In certain embodiments, the target of the immune checkpoint is the programmed death gene 1 (PD-l or CD279) (PDCD1). In other embodiments, the immune control point is a cytotoxic T-lymphocyte antigen (CTLA-4). In further embodiments, the target immune control point is another member of the CD28 and CTLA4 Ig superfamily, such as BTLA, LAGS, ICOS, PDL1, or KIR. In further embodiments, the target immune control point is a member of the TNFR superfamily, such as CD40, OX40, CD137, GITR, CD27, or TIM-3.

[001284] Additional immune checkpoints include tyrosine phosphatase 1 (SHP-1) containing Src homology domain 2 (Watson HA, et al., SHP-1: the next checkpoint target for cancer immunotherapy? Biochem Soc Trans. 2016 Apr 15;44 (2):356-62). SHP-1 is a widely expressed inhibitory protein tyrosine phosphatase (PTP). In T cells, it is a negative regulator of antigen-dependent activation and proliferation. It is a cytosolic protein and thus is not amenable to antibody-mediated modalities, but its role in activation and proliferation makes it an attractive target for genetic manipulation in adaptive transfer strategies such as chimeric antigen receptor (CAR) T cells. Immune checkpoints may also include T-cell immunoreceptor with Ig domains and ITEM (TIGIT/Vstm3/WUCAM/VSIG9) and VISTA (LeMercierI, et al., (2015) Beyond CTLA-4 and PD-1, the generation of negative checkpoint regulators Front Immunol 6:418).

[001285] WO2014172606 relates to the use of MT1 and/or MT1 inhibitors to increase the proliferation and/or activity of depleted CD8+ T cells and to decrease the production of CD8+ T cells (eg, decrease functionally depleted or refractory CD8+ immune cells). In some embodiments, metallothioneins target gene editing in adoptively transferred T cells.

[001286] In some embodiments, gene editing targets may be at least one target locus involved in the expression of an immune control protein. Such targets may include, but are not limited to: CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, ICOS (CD278), PDL1, KIR, LAG3, HAVCR2, BTLA, CD 160, TIGIT, CD96, CRTAM, LAIRI, SIGLEC7, SIGLEC9 CD244 (2B4) , IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, VISTA, GUCY1A2, GUCY1A3, GUCY1B2, GUCYIB3, MT1, MT2, CD40, OX40, CD137, GITR, CD27, SHP-l, or TIM-3.B in preferred embodiments, the target is a gene locus involved in the expression of the PD-1 or CTLA-4 genes. In other preferred embodiments, the following gene combinations are targeted, but limited to: PD-1 and TIGIT.

[001287] In other embodiments, at least two genes are edited. Gene pairs may include, but are not limited to: PD1 and TCRα, PD1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Tim3 and TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRα, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIRI and TCRα, LAIRI and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ.

[001288] Regardless of whether before or after genetic modification of T cells, T cells can be activated and expanded, usually using the methods described, for example, in US patents 6352694; 6534055; 6905680; 5858358; 6887466; 6905681; 7144575; 7232566; 7175843; 5883223; 6905874; 6797514; 6867041; and 7,572,631. T cell expansion can be carried out both in vitro and in vivo .

[001289] The practice of the present invention uses, unless otherwise indicated, conventional methods of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of those skilled in the art. See Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F, M. Ausubel, et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Flames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)).

[001290] The practice of the present invention uses, unless otherwise indicated, conventional methods for creating a generation of genetically modified mice. Sec Marten H. Hofker and Jan van Deursen, TRANSGENIC MOUSE METHODS AND PROTOCOLS, 2nd edition (2011).

Trigger factors of genes

[001291] The present invention also contemplates the use of the CRISPR-Cas system described herein, e.g., C2c1 or C2c3 effector protein, a system for providing RNA-driven gene triggers, e.g., in systems analogous to gene expression triggers described, e.g. described in PCT patent publication WO 2015/105928. Systems of this type may, for example, provide methods for modifying germline eukaryotic cells by introducing into the cell a nucleic acid sequence encoding an RNA-targeted DNA nuclease and one or more guide RNAs. Guide RNAs can be designed to be complementary to one or more target sites on the genomic DNA of the germline cell. A nucleic acid sequence encoding an RNA-targeted DNA nuclease and a nucleic acid sequence encoding guide RNAs can be provided on constructs between flanking sequences with promoters positioned such that a germline cell can express an RNA-targeting DNA nuclease, and guide RNAs along with any desired product coding sequences that are also located between the flanking sequences. The flanking sequences typically include a sequence that is identical to the corresponding sequence on the selected target chromosome, so that the flanking sequences work with the components encoded by the construct to facilitate the introduction of foreign nucleic acid construct sequences into the genomic DNA to cut the site of interest with such mechanisms like homologous recombination to make the germ cell homozygous for a foreign nucleic acid. Thus, gene trigger systems are able to introgress desired cargo genes throughout the breeding population (Gantz et al., 2015, Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles PNAS 2015, published ahead of print 23 November 2015 , doi: 10.1073/pnas.1521077112; Esvelt et al., 2014). In some embodiments, target sequences can be selected that have few potential off-target sites in the genome. Targeting multiple sites at a target locus using multiple guide RNAs can increase the frequency of cleavage and hinder the evolution of trigger-resistant alleles. Shortened guide RNAs can reduce off-target cutting. Instead of a single nuclease, paired nickases can be used to further increase specificity. Gene trigger constructs may include cargo sequences encoding transcriptional regulators, for example, to activate homologous recombination genes and/or suppress non-homologous end junctions. Target regions can be selected within an important gene so that nonhomologous end joining events can cause lethality rather than create a trigger-resistant allele. Gene trigger constructs can be designed to operate over a range of temperatures Cho et al. 2013, Rapid and Tunable Control of Protein Stability in Caenorhabditis elegans Using a Small Molecule, PLoS ONE 8(8): e72393. doi:10.1371/journal.pone.0072393).

Xenotransplantation

[001292] The present invention also contemplates the use of the CRISPR-Cas system described herein, eg C2c1 or C2c3, to provide RNA-directed DNA nucleases adapted for use in modified tissues for transplantation. For example, RNA-based DNA nucleases can be used to knock out, knock down, or disrupt selected genes in an animal such as a transgenic pig (e.g., a strain of pigs transgenic for the human heme oxygenase-1 gene), for example, disrupting the expression of genes that encode epitopes recognized by the human immune system, ie. xenoantigen genes. Pig candidate genes for the disorder may, for example, include genes for α(1,3)-galactosyltransferase and cytidine-monophosphate--N-acetylneuraminic acid hydroxylase (see PCT publication WO 2014/066505). In addition, genes encoding endogenous retroviruses, such as genes encoding all porcine endogenous retroviruses, can be disrupted (see Yang et al., 2015, Genome-wide inactivation of porcine endogenous retroviruses (PERVs), Science 27 November 2015: Vol. 350 no. 6264 pp. 1101-1104). In addition, RNA-directed DNA nucleases can be used to target the site for integration of additional xenograft donor animal genes, such as the human CD55 gene, to improve protection against particularly acute rejection.

General Gene Therapy Issues

[001293] Examples of disease-associated genes and polynucleotides, as well as disease-specific information, are available from the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimor, Md.) and the National Center for Biotechnology Information, National Library of Medicine , (Bethesda Md.)), which are available online.

[001294] Mutations in these genes and pathways can lead to the production of incorrect proteins or proteins in inappropriate amounts that affect function. Other examples of disease genes are provided in US provisional application 61/736527, filed December 12, 2012, incorporated by reference. Such genes, proteins and pathways can be targeted by a polynucleotide of the CRISPR complex of the present invention. Examples of disease-related genes and polynucleotides are listed in Tables 1 and 2. Examples of genes and polynucleotides associated with biochemical signaling cascades are listed in Table 3.

Table 1

Diseases/disorders Genes neoplasm PTEN; ATM; ATR; EGFR; ERBB2; ERBB3; ERBB4; notch1; notch2; notch3; notch4; ACT; AKT2; AK13; HIF; HIFla; HIF3a; met; H.R.G.; Bcl2; PPARα; PPARγ; WT1 (Wilms tumor); members of the FGF receptor family (5 members: 1, 2, 3, 4, 5); CDKN2a; APC; RB (retinoblastoma); MEN1, VHL; BRCA1; BRCA2, AR (androgen receptor); TSG10I; IGF, IGF receptor; Igf1 (4 variants); Igf2 (3 variants); Igf1 receptor; Igf 2 receptor; Bax, Bcl2; caspase family (9 members: 1, 2, 3, 4, 6, 7, 8, 9, 12); Kras; Ape age-related macular degeneration Abcr; Ccl2; Cc2; cp (ceruloplasmin); Timp3; cathepsin D; vldlr; Ccr2 Schizophrenia Neuregulin 1 (Nrg1); Erb4 (neuregulin receptor); complexin 1 (Cplxl); tryptophan hydroxylase 1 Tph1; tryptophan hydroxylase 2 Tph2; neurexin 1; GSK3; GSK3a; GSK3b, 5-HTT (Slc6a4); COMT; DRD (Drd1a); SLC6A3; DAOA; DTNBP1; Dao (Dao1) Diseases due to trinucleotide repeats HTT (Huntington's disease); SBMA/SMAX1/AR (Kennedy's disease); FXN/X25 (Friedrich's ataxia); ATX3 (Machado-Joseph disease); ATXN1 and ATXN2 (spinal ataxia); DMPK (myotonic dystrophy); utrophin-1 and Atn1 (DRPLA disease); SVR (Creb-BP - general instability); VLDLR (Alzheimer's disease), Atxn7; Atxn10 Fragile X Syndrome FMR2; FXR1; FXR2; mGLURS Diseases associated with secretase APH-1 (alpha and beta); presenilin (Psen1); nicotrin (Ncstn); PEN-2 Other NosI; Parpl; Natl; Nat2 Prion related diseases prp amyotrophic lateral sclerosis SOD1, ALS2; STEX; FUS, TARDBP, VEGF (VEGF-a; VEGF-b; VEGF-c) Drug addict Prkce (alcohol); Drd2; Drd4, ABAT (alcohol); GRIA2; Grm5; Grinl; Htlb; Grin2a; Drd3; Pdyn; Grial (alcohol) Autism mecp2; BZRAP1; MDGA2; SemaS.A; neurexin 1, fragile X chromosome (FMR2 (AFF2); FXR1; FXR2; MglurS) Alzheimer's disease E1; CHIP; UGH; UBB; Tau; LRP; PICALAI; clusterin; PSI; SORL1; CR1; vldlr; Ubal; Uba3; CHIP28 (Aqp1, aquaporin 1); Uchll; Uchl3; APP Inflammation IL-10; IL-1 (IL-1a; IL-lb); IL-13; IL-17 (IL-17a (CTLA8); IL-17b; H.-i7c; IL-17d; IL-17f, IL-23; Cx3cr1; ptpn22; TNFa, NOD2/CARD15 for IBD; IL-6, IL- 12(II,-12a; IL-12b), CTLA4; Cx3cl1 Parkinson's disease x-synuclein; DJ-1; LRRK2; parkin; P1NK1

table 2

Diseases and disorders of the blood and its clotting Anemia (CDAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH1, PSN1, RHAG, RH50A, NRAMP2, SPTB, ALAS2, ANH1, ASB, ABCB7, ABC7, ASAT); naked lymphocyte syndrome (TAPBP, TPSN, TAP2, ABCB3, PSF2, RING1L, MHC2TA, C2TA, RFX5, RFXAP, RFX5), bleeding disorders (TBXA2R, P2RX1, P2X1); factor H and factor H-like 1 (HF1, CFH, HUS); factor V and factor VIII (MCFD2); factor VII deficiency (F7); factor X deficiency (F10); factor XI deficiency (FI 1); factor XII deficiency (FI2, HAF); factor XIIIA deficiency (F13A1, F13A); factor XIIIB (F13B) deficiency; Fanconi Anemia , BACH!, FANCJ, PHF9, FANCL, FANCM, KIAA1596); diseases of the class of hemophagocytic lymphohistocytosis (PRF1, HPLH2. UNC13D^ MUNCI3-4, HPLIUy HLH3, FHL3); hemophilia A (F8, F8C, HEMA); hemophilia B (F9, HEMB), hemorrhagic diseases (PI, ATT, F5); leukocyte deficiencies and diseases (ITGB2, CD 18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4); sickle cell anemia (HBB); thalassemia (HBA2, HBB, HBD, LCRB, HBA1). Diseases and disorders associated with cell dysregulation and oncology B-cell non-Hodgkin's lymphoma (BCL7A, BCL7); leukemia (TALI, TCL5, SCL, TAL2, FLT3, NBS1, NBS, ZNFN1A1, IK1, LYF1, HOXD4, HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AF10, ARHGEF12, LARG, KIAA0382, CALM, CLTH, SEVR, SEVR, CHIC2, BTL, FLT3, KIT, PBT, LPP, NPM1, NUP214, D9S46E, CAN, CAIN, RUNX1, CBFA2, AML1, WHSC1L1, NSD3, FLT3, AF1Q, NPM1, NUMAI, ZNF145, PLZF, PML, MYL, STAT5B, AF10, CALM, CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF 1, VRNF, WSS, NFNS, PTPN11, PTP2C, SHP2, NS1, BCL2 , CCND1, PRAD1, BCL1, TCRA, GATA1, GF1, ERYF1, NFE1, ABL1, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN, CAIN). Inflammatory and immune-related diseases and disorders AIDS (KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1, IFNG, CXCL12, SDF1); autoimmune lymphoproliferative syndrome (TNFRSF6, APT1, FAS, CD95, ALPS1A); combined immunodeficiency (IL2RG, SCIDX 1, SCIDX, IMD4); HIV-1 (CCL5, SCYA5, D17S136E, TCP228), HIV exposure or HIV infection (IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5)); immunodeficiencies (CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGMI, IGM, FOXP3, IPEX, AIID, HRSh, RGOX, TNFRSF14B, TACI); inflammation (IL-10, IL-1 (IL-la, IL-lb), IL-13, EL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-17f) , 11-23, Cx3cr 1, ptpn22, TNFa, NOD2/CARD15 for JBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cll); severe combined immunodeficiencies (SCID) (JAK3, JAKL, DCLRE1C, ARTEMIS, SCIDA, RAG 1, RAG2, ADA, PTPRC, Metabolic, hepatic, renal and protein diseases and disorders Amyloid neuropathy (TTR, PALB); amyloidosis (APOA1, APP, AAA, CVAP, ADI, GSN, FGA, LYZ, TTR, PALB); cirrhosis (KRTI8, KRT8, CIRH1A, NAIC, TEX292, KIAA1988); cystic fibrosis (CFTR, ABCC7, CF, MRP7); glycogen storage diseases (SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM); hepatic adenoma, 142330 (TCF1, HNF1A, MODYS), liver failure, early onset, and neurological disorders (SCOD1, SCO1), hepatic lipase deficiency (LIPC), hepatoblastoma, cancer and carcinomas (CTNNB1, PDGFRL, PDGRL, PRLTS, AXIN1, AXEN, CTNNB1, TP53, P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5; medullary cystic kidney disease (UMOD, IINFJ, FJIIN, MCKD2, ADMCKD2); phenylketonuria (PAH, PKUI, QDPR, DHPR, PTS); polycystic renal and hepatic diseases (FCYT, PKHD1, ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63). Muscular/skeletal diseases and disorders Becker muscular dystrophy (DMD, BMD, MYF6), Duchenne muscular dystrophy (DMD, BMD); Emery-Dreyfuss muscular dystrophy (LMNA, LMN1, EMD2, FPLD, CMD1AJHGPS, LGMD1B, LMNA, LMN1, EMD2, FPLD, CMD1A); facial-shoulder-shoulder muscular dystrophy (FSHMD1A, FSHD1A); muscular dystrophy (FKRP, MDC 1C, LGMD2I, LAMA2, LAMM, LARGE, KIA.A.0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B, SGCG, LGMD2C, DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E, SGCD, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRA132, HT2A, LGMD2H, FKRP, MDC1C, LGMD2L TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C, SEPN1 , SEEN, RSMD1, PLEC1, PLTN, EBS1); osteopetrosis (LRP5, BMND1, LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTM1, GL, TCIRGE TIRC7, OS 116, OPTB1); muscular atrophy (VAPB, VAPC, ALS8, SMN1, SMA1, SMA.2, SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2, CATF1, SMARD1). Neurological and neuronal diseases and disorders amyotrophic lateral sclerosis (SOD1, ALS2, STEX, FUS, TARDBP, VEGF (VEGF-a, VEGF-b, VEGF-c); Alzheimer's disease (APP, AAA, CVAP, ADI, APOE, AD2, PSEN2, AD4, STM2, APBB2, FE65L1, NOS3, PLAU, URK, ACE, DCP1, ACE1, MPO, PACIP'i, PAXIPIL, PTIP, A2M, BLMH, BMH, PSEN1, AD3); autism (Mecp2, BZRAP1, MDGA2, SemaSA, neurexin 1, GLO1, MECP2, RTF PPMX, MRX16,' MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2); fragile X syndrome (FMR2, FXR1, FXR2, mGLUR5); Huntington's disease and related diseases (HD, IT15, PRNP, PRJP, JPH3, JP3, HDL2, TBP, SCA17), Parkinson's disease (NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJI, PARK?, LRRK2, PARKS, P1NK1, PARK6 , UCHL 1, PARKS, SNCA, NACP, PARK 1, PARK4, PRKN, PARK2, PDJ, DBH, NDUFV2); Rett syndrome (MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9, MECP2, RTT, PPMX, MRX16 , MRX79, x-synuclein, DJ-1); schizophrenia (neuregulin 1 (Nrgl), Erb4 (neuregulin receptor), complexin 1 (Cplx1), Tph1 tryptophan hydroxylase 1, Tph2, hydro tryptophan xylase 2, neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (SicbaJ) COMT, DRD (Drdla), SLC6A3, DAOA, DTNBP1, Dao (Daol)); secretase related diseases (APH-1 (alpha and beta), presenilin (Psen1), nikastrin, (Ncstn), PEN-2, Nosl, Parpl, Nat1, Nat2); trinucleotide repeat diseases (HIT (Huntinton's disease), SBMA/SMAX1/AR (Kennedy's disease), FXN/X25 (Friedrich's ataxia), ATX3 (Machado-Joseph's disease), ATXN1 and ATXN2 (spinal ataxia), DMPK (myotonic dystrophy), utrophin-1 and Atnl (DRPLA disease), CBP (Creb-BP - general instability), ALDER (Alzheimer's disease), Atxn7, Atxn10). Eye diseases and disorders Age-related macular degeneration (Abcr, Ccl2, Cc2, cp (ceruloplasmin), Timp3, cathepsin D, Vldlr, Ccr2); cataract (CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, CRYA1, PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP 19, CRYGD, CRYG4, BFSP2, CP49 , CP47, HSF4, CTM, HSF4, CTM, MFP, AQPO, CRYAB, CRYA2, CTPP2, CRYBBL CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA, CRYA1, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAEZ, CCM1, CAM, KRJT1); corneal opacity and dystrophy (APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3, CDG2, TACSTD2, TROP2, Ml SI, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, GFD); congenital flat cornea (KERA, CNA2); glaucoma (MYOC, TIGR, GLC1A, JOAG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1B1, GLC3A, OPA1, NTG, NPG, CYP1B1, GLC3A); Leber's congenital amaurosis (CRB1, RP12, CRX, CORD2, CRD, RPGRIP1, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4, GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3); macular degeneration (ELOVL4, ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, VMD2).

Table 3

Cellular function Genes PI3K/AKT signaling pathway PRKCE; ITGAM; ITGA5; IRAKI; PRKAA2; EIF2AK2; PTEN; E1F4E; PRKCZ; GRK6; MAPK1; TSC1; PLK1; AKT2; IKBKB; PIK3CA; CDK8; CDKN1B; NFKB2; BCL2; PIK3CB; PPP2R1A; MAPK8; BCL2L1; MAPK3; TSC2; ITGA1; KRAS; EIF4EBP1; RELA; PRKCD; NOS3; PRKAA1; MAPK9; CDK2; PPP2CA; PIM1; ITGB7; YWHAZ; ILK; TP53; RAF1; IKBKG; RELB; DYRK1A; CDKN1A; ITGBI; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; CHUK; PDPK1; PPP2R5C; CTNNB1, MAP2K1; NFKB1; PAK3,1TGB3, CCND1; GSK3A; FRAP1; SFN, ITGA2; TTC; CSNK1A1; BRAF; GSK3B; AKT3; FOXO1; SGK; HSP90AA1; RPS6KBI ERK/MAPK signaling pathway PRKCE; ITGAM; ITGA5; HSPB1; IRAK 1; PRKAA2; EIF2AK2; RAC1; RAP1A; TLN1; EIF4E; ELKI; GRK6; MAPK1; RAC2, PLK1; AKT2; PIK3CA; CDK8; CREB1; PRKCI; PTK2; FOS; RPS6KA4, PIK3CB; PPP2R1A; PIK3C3, MAPK8; MAPK3; ITGA1; ETSI; KRAS; MYCN; EIF4EBP1; PPARG; PRKCD; PRKAA1; MAPK9; SRC; CDK2; PPP2CA; PIM1; PIK3C2A; ITGB7, YWHAZ; PPP1CC; KSR1; PXN; RAF1; FYN; DYRKIA, ITGBI; MAP2K2; PAK4; PIK3R.1; STAT3; PPP2R5C; MAP2K1; PAK3; ITGB3; ESR1; ITGA2; MYC; TTC; CSNK1A1; CRKL; BRAF; ATF4; PRKCA; SRF; STAT1; SGK Signaling pathway of glucocorticoid receptors RAC1; TAF4B; EP300; SMAD2; TRAF6; PCAF; ELKI; MAPK1; SMAD3; AKT2; IKBKB; NCOR2; UBE2I; PIK3CA; CREB1 FOS; HSPA5; NFKB2; BCL2, MAP3K14; STAT5B; PIK3CB; PIK3C3; MAPK8; BCL2L1, MAPK3; TSC22D3; STAMPS); NRIP1; KRAS; MAPK13; RELA; STAT5A; MAPK9; NOS2A; PBX1; NR3C1; PIK3C2A; CDKN1C; TRAF2; SERPINEl; NCOA3; MARK14; TNF; RAF1; IKBKG; MAP3K7; CREBBP; CDKN1A; MAP2K2; JAK1; P.8; NCOA2; ACT1, JAK2, PIK3R1; CHUK; STAT3; MAP2KI, NFKBI, TGFBR1; ESR1; SMAD4; CEBPB; JUN; AR; AKT3; CCL2; MMP1; STAT1; IL6; HSP90AA1 Axon direction signaling pathway PRKCE; ITGAM; ROCK 1; ITGA5; CXCR4; ADAM12; IGF1; RAC1; RAP1A; EIF4E; PRKCZ; NRP1; NTRK2; ARHGEF7; SMO; ROCK2; MARK 1; PGF; RAC2; PTPN11; GNAS; AKT2; PIK3CA; ERBB2; PRKCI; PTK2; CFL1; GNAQ; PIC3CB; CXCLI2; PIC3C3; WNT11; PRKD1; GNB2L1; AND YOU; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PIK3C2A; ITGB7; GLI2; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; ADAM17; ACT 1; PIK3R1; GUI WNT5A; ADAM 10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; CRKL; RND1; GSK3B; AKT3; PRKCA ephrin receptor signaling pathway PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; IRAKI; PRKAA2; EIF2AK2; RAC 1; RAP1A; GRK6; ROCK2, MARK1; PGF; RAC2; PTPN11; GNAS; REC1, AKT2; DDK 1; CDK8; CREB1, PTK2; CFL1, GNAQ; MAP3KI4; CXCL12; MARKS; GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PRKAA1; MARK9; SRC; CDK2; PIM1; ITGB7; PXN; RAF1; FYN; DYRK1A; ITGB1; MAP2K2; PAK4; AKT1; JAK2; STAT3; ADAM10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; TTC; CSNK1A1; CRKL, BRAF; PTPN13; ATF4; AKT3, SGK Actin cytoskeleton signaling pathway ACTN4; PRKCE, ITGAM; ROCK1; ITGA5; IRAKI; PRKAA2; EIF2AK2; RACE INS; ARHGEF7; GRK6, ROCK2, MARK K RAC2; PLK1; AKT2, PIK3CA; CDK8, PTK2; CFL1; PIK3CB; MYH9; DIAPH1; PIK3C3; MAPK8; F2R; MAPK3; SLC9A1; ITGA1; KRAS; RHOA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; ITGB7; PPP1CC; PXN; VIL2; RAF1; GSN; DYRK1A; ITGBl; MAP2K2; PAK4; PIP5K1A; PIK3R.1, MAP2K1; PAK3; ITGB3; CDC42, ARC; ITGA2; TTC; CSNK1A1, CRKL; BRAF; VAV3; SGK Huntington's disease signaling pathway PRKCE, IGF1; EP300; RCOR1, PRKCZ; HDAC4; TGM2; MARK 1; CAPNS1; AKT2; EGFR; NCOR2; SP1; CAPN2, PIK3CA; HDAC5; CREB1; PRKCI; HSPA5; REST; GNAQ; PIK3CB; PIK3C3; MAPK8; IGF1R; PRKD1; GNB2L1; BCL2L1; CAPN1; MAPK3; CASP8; HDAC2; HDAC7A; PRKCD; HDAC11; MAPK9; HDAC9; PIK3C2A; HDAC3; TP53; CASP9; CREBBP; AKT1; PIK3R1; PDPK1; CASP1; APAF1; FRAP1; CASP2; JUN; BAX; ATF4; ACTH; PRKCA; CLTC; SGK; HDAC6; CASP3 Apoptosis signaling pathway PRKCE; ROCK1; BID; IRAK1; PRKAA2; E1F2AK2; VAK1; BIRC4; GRK6; MARK1; CAPNS1; REC 1; ACT2; IKBKB; CAPN2; CDK8; FAS; NFKB2; BCL2; MAP3K14; MARK8; BCL2L1; CAPNI; MARK3; CASP8; KRAS; RELA; PRKCD; PRKAA1; MARK9; CDK2; PIM1; TP53; TNF; RAF1; IKBKG; RELB; CASP9; DYRK1A; MAP2K2; CHUK; APAF1; MAP2K1; ARCV1; RAK3; LMNA; CASP2; BIRC2; TTK; CSNKIA1; BRAF; VAC; PRKCA; SGK; CASP3; BIRC3; PARP1 B-cell receptor signaling pathway RACE PTEN; LYN; ELK ; MARK RAC2; PTPN11; ACT2; IKBKB; PIK3CA; CREB1; SYK; NFKB2; SAMK2A, MAR3K14; REC3CB; PIK3C3; MARK8; BCL2L1; ABL1; MARK3; ETS1; KRAS; MARK13; RELA; PTPN6; MARK9; EGRI; PIK3C2A; VTK; MARK14; RAF1; IKBKG; RELB; MARK 7; MAP2K2;.ACT E PIK3R1; CHUK; MAP2K1; NFKB1; CDC42; GSK3A; FRAP1; BCL6; ALL; JUN; GSK3B; ATF4; ACTH; VAV3; RPS6KB1 Signaling pathway of leukocyte diapedesis ACTN4; CD44; PRKCE; ITGAM; ROCK1; CXCR4; CYBA; RACE RAP1A; PRKCZ; ROCK2; RAC2; PTRA1E; MMP14; PIK3CA; PRKCI; RTK2; REC3CB; CXCLI2; PIK3C3; MARK8; PRKD1; ABL1; MARK 10; CYBB; MARKE3; RHOA; PRKCD; MARK9; SRC; Р1КЗС2А; VTK; MARK 14; NOX1; PXN; VIL2; VASP; ITGB1; MAP2K2; CTNND1; PIK3R1; CTNNB1; CLDN1; CDC42; F11R; ITK; CRKL; VAV3; CTTN; PRKCA; MMP1; MMP9 Integrin signaling pathway ACTN4; ITGAM; ROCK1; ITGA5; RACE PTEN; RAP1A; TLN1; ARHGEF7; MAPK1; RAC2; CAPNS1; AKT2; CAPN2; PIK3CA; PTK2; PIC3CB; PDC3C3; MAPK8; CAV1; CAPN1; ABL1; MARKZ; ITGA1; KRAS; RHOA; SRC; PIK3C2A; ITGB7; PPP1CC; ILK; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; ACTE PDK3R.1; TNK2; MAP2K1; RAK3; ITGB3; CDC42; RND3; ITGN2; CRKL; BRAF; GSK3B; ACT3 Acute phase response signaling pathway IRAK1; SOD2; MYD88; TRAF6; ELK1; MARK1; PTPN11; AKT2; IKBKB; PIK3CA; FOS; NFKB2; MNP3K14; PIC3CB; MNPK8; RIPK1; MARKZ; IL6ST; KRAS; MARK13; IL6R; RELA; SOCS1; MAPK9; FTL; NR3C1; TRAF2; SERPINE1; MARK 14; TNF; RAF1; PDK1; IKBKG; RELB; MNP3K7; MAP2K2; ACT JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; FRAP1; CEBPB; JUN; ACTH; IL1R1; IL6 PTEN signaling pathway ITGAM; 1TGA.5; RAC1; PTEN; PRKCZ; BCL2LII, MARK 1; RAC2, AKT2; EGFR; IKBKB; CBL; PIK3CA; CDKN1B; PTK2; NFKB2; BCL2; PIK3CB; BCL2L1; MAPK3; ITGA1; KRAS; ITGB7; ILK; PDGFRB; INSR; RAFT; IKBKG; CASP9; CDKN1A; ITGB1; MAP2K2; ACT PIK3R1; CHUK; PDGFRA; PDPK1; MAP2K1; NFKBI; ITGB3; CDC42; CCND1; GSK3A; ITGA2; GSK3B; AKT3; FOXO1; CASP3; RPS6KB1 p53 signaling pathway PTEN, EP300, BBC3, PCAF; FASN; BRCA1; GADD45A; BIRC5; AKT2; PIK3CA, SNEKE TP53INP1; BCL2; PIK3CB, PIK3C3; MAPK8; THBS1; ATR; BCL2L1, E2FI; PMAIP1; CHEK2; TNFRSF10B; TP73; RBI; HDAC9; CDK2; PHK3C2A; MAPK14; TP53; LRDD; CDKN1A; HIPK2; AKT1; PIK3R1; RRM2B; APAF1; CTNNB1; SIRT1; CCND1; PRKDC; ATM; SFN; CDKN2A; JUN; SNAI2; GSK3B; BAX; AKT3 Aryl hydrocarbon receptor signaling pathway HSPB1; EP300; FASN; TGM2; RXRA; MAPK1; NQO1; NCOR2; SP1; ARNT; CDKN1B; FOS; SNEK1; SMARCA4; NFKB2; MAPK8; ALDH1A1; ATR; E2F1; MAPK3; NRIP1; CHEK2; RELA; TP73; GSTPI; RBI; SRC; CDK2; AHR; NFE2L2; NCOA3; TP53; TNF; CDKN1A; NCOA2, APAF1; NFKBI; CCND1; ATM; ESR1; CDKN2A; MYC; JUN; ESR2; BAX, IL6; CYP1B1; HSP90AA1 Signaling pathway of xenobiotic metabolism PRKCE, EP300; PRKCZ; RXRA, MARK NQO1; NCOR2, PIK3CA, ARNT; PRKCI, NFKB2, CAMK2A; PIK3CB; PPP2R1A; RPKSPZ; MAPK8; PRKD1; ALDHEA1; MAPK3; NRIP1; KRAS; MARK 13; PRKCD; GSTPI; MAPK9; NOS2A; ABCB1; AHR; PPP2CA; FTL; NFE2L2; PIK3C2A; PPARGC1A; MARK14; TNF; RAF1; CREBBP; MAP2K2; PIK3R1; PPP2R5C; MAP2K1; NFKBI; REAPL; PRKCA; EIF2AK3; IL6; CYP1B1; HSP90AA1 SAPK/JNK signaling pathway PRKCE; IRAKI; PRKAA2; EIF2AK2; RAC1; ELKI, GRK6; MARK GADD45A; RAC2; PLK1; AKT2; PIK3CA; FADD; CDK8; PIK3CB; PIK3C3; MAPK8; RIPK1; GNB2L1; IRS1; MAPK3; MARK 10; DAXX; KRAS; PRKCD; PRKAA1; MAPK9; CDK2; PIMT; PIK3C2A; TRAF2; TP53; LCK; MAP3K7; DYRK1A; MAP2K2; PIK3R1; MAP2K1; PAK3; CDC42; JUN; TTC; CSNK1A1; CRKL; BRAE; SGK PPAr/RXR signaling pathway PRKAA2; EP300; INS, SMAD2; TRAF6, PPARA; FASN; RXRA; MAPK1; SMAD3; GNAS; IKBKB; NCOR2, ABCA1, GNAQ; NFKB2; MAP3K14, STAT5B; MAPK8; IRS1; MAPK3; KRAS; RELA; PRKAA1; PPARGC1A; NCOA3; STAMPS; INSR; RAF 1, IKBKG, RELB; MAP3K7; CREBBP; MAP2K2; JAK2; CHUK; MAP2K1; NFKB1, TGFBR1; SMAD4; JUN; IL1R1; PRKCA; IL6; HSP90AA1; ADIPQQ NF-KB signaling pathway IRAKI; EIF2AK2; EP300; INS; MYD88; PRKCZ; T1LAF6; TBK1; AKT2; EGFR; IKBKB; PIK3CA; BTRC; NFKB2; MAP3K14; PIK3CB; PIK3C3; MAPK8; RIPK1; HDAC2; KRAS; RELA; PIK3C2A, TRAF2; TLR4; PDGFRB; TNF; INSR; LCK; IKBKG; RELB; MAP3K7; CREBBP; AKT1; PIK3R1; CHUK; PDGFRA; NFKBI; TLR2; ALL; GSK3B; ACTH, TNFAIP3; IL1R1 Neuregulin signaling pathway ERBB4; PRKCE; ITGAM; ITGA5; PTEN; PRKCZ; ELK1; MAPKI; PTPNI1; AKT2; EGFR; ERBB2; PRKCI; CDKN1B; STAT5B; PRKD1; MAPK3; ITGA1; KILLS; PRKCD; STATSA; SRC; ITGB7; RAF1; ITGB1; MAP2K2; ADAM17; ACT PIK3R1; PDPK1; MAP2K1; ITGB3; LRLG; FRAP1; PSENI; ITGA2; MYC; NRG1; CRKL; AKT3; PRKCA; HSP90AA1; RPS6KB1 Wnt and beta-catenin signaling pathway CD44; EP300; LRP6; DVL3; CSNK1E; GJA1; SMO; AKT2; PINT; CDH1; BTRC; GNAQ; MARK2; PPP2R1A; WNT11; SRC; DKKI; PPP2CA; SOX6; SFRP2; ILK; LEF1; SOX9; TP53; MAP3K7; CREBBP; TCF7L2; ACT PPP2R5C; WNT5A; LRP5; CTNNB1; TGFBR1; CCND1; GSK3A; DVL1; APC; CDKN2A; MYC; CSNK1A1; GSK3B; AKT3; SOX2 Insulin receptor signaling pathway PTEN; INS; EIF4E; PTPNI; PRKCZ; MARKE TSCI; PTPN11; AKT2; CBL; PIK3CA; PRKCI; PIK3CB; PIK3C3; MAPK8; IRS1; MAPK3; TSC2; KILLS; EIF4EBP1; SLC2A4; PIK3C2A; PPP1CC; INSR; RAF1; FYN; MAP2K2; JAKE AKT1; JAK2; PIK3RI; PDPK1; MAP2KI; GSK3A; FRAP1; CRKL; GSK3B; AKT3; FOXO1; SGK; RPS6KB1 IL-6 signaling pathway HSPB1; TRAF6; MAPKAPK2; ELK1; MAPKI; PTPN11; IKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK3; MARK10; IL6ST; KRAS; STAMPS; IL6IL RELA; SOCS1; MAPK9; ABCB1; TRAF'2; MARK14; TNF; RLF1; IKBKG; RELB; MAP3K7; MAP2K2; ILS; JAK2; CHUK; STAT3; MAP2K1; NFKBI; CEBPB; JUN; IL1R1; SRF; IL6 Hepatic cholestasis PRKCE; IRAKI; INS; MYD88; PRKCZ; TRAF6; PPARA; RXRA; IKBKB; PRKCI; NFKB2; MAP3K14; MAPK8; PRKD1; MARK 10; RELA; PRKCD; BRAND ABCB1; TRAF2; TLR4; TNF; INSR; IKBKG; RELB; MAP3K7; IL8; CHUK; NR1H2; TJP2; NFKBI; ESRI, SREBF1; FGFR4; JUN; IL1R1; PRKCA; IL6 IGF-1 signaling pathway IGF1; PRKCZ; ELK1; MARK 1, PTPN11; NEDD4; AKT2; PIK3CA; PRKCI; PTK2; FOS; PIK3CB; PIK3C3; MARKS; IGFIR; IRS1; MAPK3; IGFBP7; KRAS; PIK3C2A; YWHAZ; PXN; RAFI; CASP9; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; IGFBP2; SFN; JUN; CYR61; AKT3; FOXOI, S.R.F.; CTGF; RPS6KBI NRF2-mediated response to oxidative stress PRKCE; EP300; SOD2; PRKCZ; MAPK1; SQSTM1; NQO1; PIK3CA; PRKCI; FOS; PIK3CB; PIK3C3; MARKS; PRKDI, MAPK3; KRAS; PRKCD; GSTP1; MAPK9; FTL; NFE2:E2; PIK3C2A; MARK 14; RAF; MAP3K7; CREBBP; MAP2K2; ACT1; PIK3R1; MAP2K1; PPIB; JUN; KEAPi; GSK3B; ATF4; PRKCA; EIF2AK3; HSP90AA1 Hepatic fibrous/hepatic stellate cell activation EDN1, IGF1; KDR; FLT1; SMAO2; FGFR1, MET; PGF; SMAD3; EGFR; FAS; CSFI; NFKB2; BCL2; MYH9; IGFIR; IL6R; RELA; TLR4; PDGFRB; TNF; RELB; ILS, PDGFRA, NFKBI, TGFBRI; SMAD4; VEGFA; BAX; IL1R1; CCL2; HGF; MMP1; STAT1; IL6; CTGF; MMP9 PPAR signaling pathway EP300; INS; TRAF6; PPARA; RXRA; MAPK1; IKBKB; NCOR2; FOS; NFKB2; MAP3K14; STAT5B; MAPK3; NRIP1; KRAS; PPARG; RELA; STATSA; TRAF2; PPARGC1A; PDGFRB, TNF, INSR; RAFI, IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; CHUK; PDGFRA; MAP2K1; NFKBI; JUN; UR1, HSP90AA1 Signaling pathway Fc ε RI PRKCE, RAC1; PRKCZ; LYN; MAPK1; RAC2; PTPN11; AKT2; PIK3CA; SYK; PRKCI, PIK3CB; PIK3C3; MARKS; PRKDI, MAPK3; MAPK10; KRAS; MAPK13; PRKCD; MAPK9; PIK3C2A; VTK; MAPK14; TNF; RAFI; FYN; MAP2K2; automatic transmission, PIK3R1; PDPK1; MAPRK1; ACTH; VAV3; PRKCA G protein-coupled receptor signaling pathway PRKCE; RAP1A; RGS16; MAPK1; GNAS; AKT2; IKBKB; PIK3CA; CREB1; GNAQ; NFKB2, CAMK2A; PIK3CB; PIK3C3, MAPK3; KRAS; RELA, SRC, PIK3C2A; RAFI, IKBKG; RELB, FYN; MAP2K2; AKT1; PFK3P1; CHUK; PDPK1; STATS; MAP2K1; NFKBI; BRAF; ATIN, AKT3; PRKCA Inositol phosphate metabolism PRKCE; IRAKI; PRKAA2; EIF2AK2; PTEN; GRK6; MAPK1; PLK1; AKT2; PIK3CA; CDK8; PIK3CB; PIK3C3; MAPK8; MAPK3; PRKCD: PRKAA1; MAPK9; CDK2; PIMI; RPSZS2A; DYRK1A; MAP2K2; PIP5K1A; PIK3RI; MAP2K1; PAK3; ATM; TTC; CSNK1A1; BRAE; SGK PDGF signaling pathway EIF2AK2, ELK 1; ABL2; MARKT, PIK3CA; FOS, PIK3CB; PIK3C3; MAPK8; CAV1; ABL1; MAPK3; KRAS, SRC, PIK3C2A; PDGFRB, RAFI; MAP2K2, J.AKI, JAK2, PIK3R1; PDGFRA; STAT3; SPHK1; MAP2K1; MYC; JUN; CRKL; PRKCA; SRF; STAT1; SPHK2 VEGF signaling pathway ACTN4; ROCK1; KDR; FLT1; ROCK2; MAPK1; PGF; AKT2; PIK3CA; ARNT; PTK2; BCL2; PIK3CB; PIK3C3; BCL2L1; MARKZ; KIRAS; H1F1A; NOS3; PIK3C2A; PXN; RAF1; MAP2K2; EEAVE1; ACT PIK3R1; MAP2K1; SFN; VEGFA; AKT3; FOXO1; PRKCA natural killer signaling pathway PRKCE; RAC1; PRKCZ; MARK 1; RAC2; PTPN11; KJR2DL3; AKT2; PIK3CA; SYK; PRKC1; PIK3CB; RPSSSZ; PRKD1; MARKZ; KRAS; PRKCD; PTPN6; RPSZS2A; LCK; RAF1; FYN; MAP2K2; PAK4; AKT1; PIK3R1; MAP2K1; PAK3; AKT3; VAV3; PRKCA Cell cycle: G1/S checkpoint regulation HDAC4; SMAD3; SUV39H1; HDAC5; CDKN1B; BTRC; ATR; ABL1;; E2F1; HDAC2; HDAC7A; RB1; HDAC11; HDAC9; CDK2; E2F2; HDAC3; TP53; CDKN1A; CCND1; E2F4; ATM; RBL2; SMAD4; CDKN2A; ICC; NRG1; GSK3B; RBL1; HDAC6 T-cell receptor signaling pathway RAC1; ELK1; MAPK1; IKBKB; CBL; RPSASA; FOS; NFKB2; PIK3CB; Р1КЗСЗ; MAPK8; MARKZ; KRAS; RELA; PIK3C2A; VTK; LCK, RAF1; IKBKG; RELB; FYN; MAP2K2; PIK3R1; CHUK; MAP2K1; NFKB1; 1TK; BCL10; JUN; VAV3 Death receptor signaling pathway CRADD; HSPB1; BID; BIRC4; TBK1; IKBKB; FADD; FAS; NFKB2; BCL2; MAP3K14; MAPK8; R1PK1; CASP8; DAXX; TNFRSF10B; RELA; TRAF2; TNF; IKBKG; RELB; CASP9; CHUK; APAF1; NFKB1; CASP2; BIRC2; CASP3; BIRC3 FGF signaling pathway RAC1; FGFR1; MET, MAPKAPK2; MARKS PTPN11; AKT2; PIC3CA; CREB1; PIK3CB; PIK3C3; MAPK8; MARK3; MAPK13; PTPN6; PIK3C2A; MAPK14; RAF1; AKT1; PIK3R1; STATS; MAP2K1; FGFR4; CRKL; ATF4; AKT3; PRKCA; HGF GM-CSF signaling pathway LYN; ELK1; MAPK1; PTPN11; AKT2; RPSASA; CAMI2A; STAT5B; PIC3CB; PIK3C3; GNB2L1; BCL2L1; MAPK3; ETS1; KRAS; RUNX1; P1M1; PIK3C2A; RAF1; MAP2K2; AKT1; JAK2; PIK3R1; STAT3; MAP2K1; CCND1; AKT3; STAT1 Amyotrophic lateral sclerosis signaling pathway BID; GHL RAC1; BIRC4; PGF; CAPNS1; CAPN2; P1KZSA; BCL2; R1KZSV; PIK3C3; BCL2L1; CAN1; PIK3C2A; TP53; CASP9; PIK3R1; RAB5A; CASP1; APAF1; VEGFA; BIRC2; BAX; AKT3; CASP3; BIRC3 JAK/Stat Signaling Path PTPN1; MAPK1; PTPN11; AKT2; RPSASA; STAT5B; PIC3CB; RPSSSZ; MARKZ; KRAS; SOCS1; STAT5A; PTPN6; PBC3C2A; RAF1; CDKN1A; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; STAT3; MAP2K1; FRAP1; AKT3; STATI Nicotinate and nicotinamide metabolism PRKCE; IRAKI; PRKAA2; EIF2AK2; GRK6; MAC1; PLK1; AKT2; CDK8; MAPK8; MAPK3; PRKCD; PRKAA1; PBEF1; BRAND CDK2; PIMI, DYRK1A; MAP2K2; MAP2K1; PAK3; NT5E; TTC; CSNK1A1; BRAF; SGK Chemokine signaling pathway CXCR4; ROCK2; MAPK1; PTK2; FOS; CFL1; GNAQ; CAMK2A; CXCL12; MAPK8; MAPK3; KRAS; MARK 13; RHOA; CCR3; SRC; PPPICC; MAPK14; NOX1; RAF1; MAP2K2; MAP2K1; JUN; CCL2; PRKCA IL-2 signaling pathway ELK1; MAPK1; PTPN11; AKT2; PIK3CA; SYK; FOS; STAT5B; PIK3CB; PIK3C3, MAPK8; MAPK3; KRAS; SOCS1; STAT5A; PIK3C2A; LCK; RAF1; MAP2K2; JAKI, AKT1; PIK3RI, MAP2K1; JUN; AKT3 synaptic long-term depression PRKCE; IGF1; PRKCZ; PRDX6, LYN, MAPK1; GNAS, PRKCI; GNAQ; PPP2R1A; IGF1R; PRKD1; MAPK3; KRAS; GRN; PRKCD; NOS3; NOS2A; PPP2CA; YWHAZ; RAFT; MAP2K2; PPP2R5C; MAP2K1; PRKCA Estrogen receptor signaling pathway TAF4B; EP300; CARMI; PCAF; MARK I; NCOR2; SMARCA4; MAPK3; NRIPI; KRAS; SRC; NR3C1; HDAC3; PPARGC1A; RBM9; NCOA3; RAF1; CREBBP; MAP2K2; NCOA2; MAP2K1; PRKDC; ESR1; ESR2 Protein ubiquitination pathway TRAF6; SMURF I; BIRC4; BRCA1; UCHL1; NEDD4; CBL; UBE2I; BTRC; HSPA5; USP7; USP10; FBXW7; USP9X; STLB1, USP22; B2M; BIRC2; PARK2; USP8; USP1; VHL; HSP90AA1; BIRC3 IL-10 signaling pathway TRAIL; CCR1; ELK1; IKBKB; SP1; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; MAPK14; TNF; IKBKG; RELB; MAP3K7; JAKI; CHUK; STAT3; NFKBI; JUN; IL1RI; IL6 VDR/RXR activation PRKCE; EP300; PRKCZ; RXRA; GADD45A; HES1; NCOR2; SP1; PRKCI; CDKN1B; PRKD1; PRKCD; RUNX2; KLF4; YY1; NCOA3; CDKN1A; NCOA2; SPP1; LRP5; CEBPB; FOXO1; PRKCA TGF-β signaling pathway EP300; SMAD2; SMURF 1; MARK 11; SMAD3; SMAD1; FOS; MAPK8; MAPK3; KRAS; BRAND RUNX2; SERPINE1; RAFI; MAP3K7; CREBBP; MAP2K2; MAP2K1; TGFBR1; SMAD4; JUN; SMAD5 Signaling pathway of Toll-like receptors IRAKI; EIF2AK2; MYD88; TRAF6; PPARA; ELK1; IKBKB; FOS; NFKB2; MAP3K14; MARK8; MARK 13; RELA; TLR4; MAPK14; IKBKG; RUE MAP3K7; CHUK; NFKBI; TLR2; JUV p38 MARK signaling pathway HSPB1; IRAKI; TRAF6; MAPKAPK2; ELKI; FADD; FAS; CREBI; DDIT3; RPS6KA4; DAXX; MARK 13; TRAF2; STAMPS; TNF; MAP3K7, TGFBR1; MYC; ATF4; 1L1R1; SRF; STAT1 Neurotrophin/TRK signaling pathway NTRK2; MARK1; PTPN11; PIK3CA; CREBI; FOS; PIK3CB; PFK3C3; MAPK8; MARKZ; KRAS; PIK3C2A; RAFI; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; CDC42; JUN; ATF4 FXR/RXR activation INS; PPARA; FASN; RXRA; AKT2; SDC 1; MAPK8; AROV; MAPK10; PPARG; MTTP MAPK9; PPARGC1A; TNF; CREBBP; ACTE SREBF1; FGFR4; AKT3; FOXO1 synaptic long-term potentiation PRKCE; RAP1A; EP300; PRKCZ; MAPK1; CREBI; PRKCI; GNAQ; CAMK2A; PRKD1; MARKZ; KRAS; PRKCD; PPP1CC; RAF1; CREBBP; MAP2K2; MAP2K1; ATF4; PRKCA calcium signaling pathway RAP1A; EP300; HDAC4; MAPK1; HDACS; CREBI; CAMK2A; MYH9; MARKZ; FIDAC2; HDAC7A; FIDAC11; HDAC9; HDAC3; CREBBP; CALR; CAMKK2; ATF4; HDAC6 EGF signaling pathway ELK1; MAPK1; EGFR; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; MARKZ; PIC3C2A; RAF1; JAK1; PIK3R1; STAT3; MAP2K1; JUN; PRKCA; SRF; STAT1 Hypoxia signaling pathway in the cardiovascular system EDN1; PTEN; EP300; NQO1; UBE2I; CREBI; ARNT; HIF1A; SLC2A4; NOS3; TP53; LDHA; AKT1; ATM; VEGFA; JUN; ATF4; VHL; HSP90AA1 LPS/IL-1-mediated inhibition of RXR function IRAKI; MYD88; TRAIL; PPARA; RXRA; ABCA1; MAPK8; ALDFHA1; GSTP1; MAPK9; ABCB1; TRAF2; TLR4; TNF; MAP3K7; NR1H2; SREBF1; JUN, IL1R1 LXR/RXR activation FASN, RXRA; NCOR2; ABCA1; NFKB2; IRF3; RELA, NOS2A; TLR4; TNF; RELB; LDER; NR1H2; NFKB1; SREBF1; IL1R1; CCL2; IL6; MMP9 Amyloid processing PRKCE; CSNK1E; MAPK1; CAPNS1; AKT2; CIAN2; CAPN1; MARKZ; MAPK13; MARCH; MARK14; AKT1; PSEN1; CSNK1A1; GSK3B; AKT3; APP IL-4 signaling pathway AKT2; PIK3CA; PIK3CB; PIK3C3; 1RS1; KRAS; SOCS1; PTPN6; NR3C1; PIK3C2A; JAK1; ACT1; JAK2; PIK3R1; FRAP1; AKT3; RPS6KBI Cell cycle: DNA damage checkpoint regulation in G2/M EP300; PCAF; BRCA1; GADD45A; PLK1; BTRC; CHEK1; ATR; CHEK2; YWHAZ; TP53; CDKN1A; PRKDC; ATM; SFN; CDKN2A Nitric oxide signaling pathway in the cardiovascular system KDR; FETEPGF; AKT2; PIK3CA; PIK3CB; PIK3C3; CAV1; PRKCD; NOS3; PIK3C2A; AKT1; PIK3R.1; VEGFA; AKT3; HSP90AA1 Purine metabolism NME2; SMARCA4; MYH9; RRM2; ADAR; E112AK4; PCM2; ENTPD1; RAD51; RRM2B; TJP2; RADS1C; NT5E; POLD1; NME1 cAMP-mediated signaling pathway RAP-LA; BRANDS GNAS; CREB1; CAMK2A; MAPK3; SRC; RAF1; MAP2K2; STAT3; MAP2K1; BRAF; ATF4 Mitochondrial dysfunction SOD2; MAPK8; CASP8; MARK 10; MAPK9; CASP9; PARK7; PSEN1; PARK2; APP; CASP3 Notch signaling pathway HES1; JAG1; NUMB; NOTCH4; AD AM 17; NO TCH2; PSEN1; NOTCH3; NOTCH1; DLL4 ER stress signaling pathway HSPA5; MARK8; XBP1; TRAF2; ATF6; CASP9; ATF4; EIF2AK3; CASP3 Pyrimidine metabolism NME2; AICDA; RRM2; EIF2AK4; ENTPD1; RRM2B; NT5E; POLD1; NME1 Parkinson's disease signaling pathway UCHL1; MAPK8; MAPK13; MAPK14; CASP9; PARK7; PARK2; CASP3 Cardiac and beta-adrenergic signaling pathway GNAS; GNAQ; PPP2R1A; GNB2L1; PPP2CA; PPP1CC; PPP2R5C Glycolysis/gluconeogenesis HK2; GCK; GPI; ALDH1A1; PCM2; LDHA; HK1 Interferon signaling pathway IRF1; SOCS1; JAK1; JAK2; IFITM1; STATE IFIT3 Sonic hedgehog gene signaling pathway ARRB2; SMO; GLI2; DYRK1A; GLII; GSK3B; DYRK1B Glycerophospholipid metabolism PLD1; GRN; GPAM; YWHAZ; SPHK1; SPHK2 Degradation of phospholipids PRDX6; PLD1; GRN; YWHAZ; SPHK1; SPHK2 Tryptophan metabolism SIAH2; PRMT5; NEDD4; ALDH1AI; CYP1B1; SIAH1 Lysine degradation SUV39H1; EHMT2; NSD1; SETD7; PPP2R5C Nucleotide excision repair pathway ERCC5; ERCC4; XPA; XPC; ERCC1 Metabolism of starch and sucrose UCHL1; HK2; GCK; GPI; HK1 Amino sugar metabolism NQO1; HK2; GCK; HK1 Metabolism of arachidonic acid PRDX6; GRN; YWHAZ; CYP1B1 Signaling pathway of circadian rhythms CSNK1E; CREB1; AT14; NR1D1. blood clotting system BDKRB1; F2R; SERPINE1; F3 Dopamine receptor signaling pathway PPP2R1A; PPP2CA; PPP1CC; PPP2R5C Metabolism of glutathione DDH2; GSTP1; ANPEP; IDH1 Glycerolipid metabolism ALDH1A1; GPAM; SPHK1; SPHK2 Linoleic acid metabolism PRDX6; GRN; YWHAZ; CYP1B1 Methionine metabolism DNMT1, DNMT3B, AHCY; DNMT3A pyruvate metabolism GLO1, ALDH1A1; PCM2; LDHA Metabolism of arginine and proline ALDH1A1; NOS3; NOS2A Eicosanoid signaling pathway PRDX6; GRN; YWHAZ Metabolism of fructose and mannose HK2; GCK; HK1 Metabolism of galactose HK2; GCK; HKI Biosynthesis of stilbene, coumarin and lignin PRDX6; PRDX1; TYR Pathway of antigen presentation CALR; B2M Biosynthesis of steroids NQOIs; DHCR7 Butanoate metabolism ALDHLA1; NLGN1 Citric acid cycle IDH2; IDH1 Fatty acid metabolism ALDHfA1; CYP1B1 Glycerophospholipid metabolism PRDX6; CHKA Histidine metabolism PRMT5; ALDH1A1 Metabolism of inositol ERGIL; APEX1 Metabolism of xenobiotics by cytochrome p450 GSTP1; CYP1B1 Methane metabolism PRDX6; PRDX1 Phenylalanine metabolism PRDX6; PRDX1 Propanoate metabolism ALDHLAl; LDHA Metabolism of selenoamino acids PRMT5, AHCY sphingolipid metabolism SPHK1; SPHK2 Aminophosphonate metabolism PRMT5 Androgen and estrogen metabolism PRMT5 Ascorbate and aldarate metabolism ALDH1A1 Biosynthesis of bile acids ALDH1A1 Cysteine metabolism LDHA Biosynthesis of fatty acids FASN Glutamate receptor signaling pathway GNB2L1 NRF2-mediated response to oxidative stress PRDX1 Pentose phosphate pathway GPI Interconversions of pentose and glucuronate UCHL1 Retinol metabolism ALDH1A1 Metabolism of riboflavin TYR Tyrosine metabolism PRMT5, TYR Biosynthesis of ubiquinone PRMT5 Degradation of valine, leucine and isoleucine ALDH1A1 Metabolism of glycine, serine and threonine CHKA Lysine degradation ALDH1A1 Pain/taste TRPM5; TRPAI Pain TRPM7; TRPC5; TRPC6; TRPCi; cnrl; cm2; Grk2; Trpal; pome; cgrp; crf; pka; era; Nr2b; TRPM5; Prkaca; Prkacb; Prkar1a; Prkar2a Mitochondrial function AIF; CytC; SMAC (Diablo); Aiftn-1; Aifm-2 Developmental neuroscience BMP-4; chordin (Chrd); noggin (Nog); WNT (Wnt2; Wnt2b; Wnt3a, Wnt4; Wnt5a; Wnt6; Wnt7b; Wnt8b; Wnt9a; Wnt9b; Wnt10a; Wnt10b, Wnt16); beta-catenin; Dkk-1; Frizzled-related proteins, Otx-2; Gbx2, FGF-8; Relin; Dab1; unc-86 (Pou4f1 or Brn3a); Numb; Rein

[001295] Embodiments of the invention also relate to methods and constructs related to gene knockout, gene amplification, and repair of specific mutations associated with DNA repeat instability and neurological diseases (Robert D. Wells, Tetsuo Ashizawa, Genetic Instabilities and Neurological Diseases, Second Edition , Academic Press, Oct 13, 2011-Medical). Specific aspects of tandem repeat sequences have been found to be responsible for more than twenty human diseases (New insights into repeat instability: role of RNA-DNA hybrids. Mclvor El, Polak U, Napierala M. RNA. Biol. 2010 Sep-Oct; 7( 5):551-8). These effector protein systems can be used to correct these defects in genomic instability.

[001296] Some further aspects of the invention relate to the correction of defects associated with a wide range of genetic diseases, which are further described on the website of the National Institutes of Health under the topic "Genetic diseases". Genetic brain disorders may include (but are not limited to) adrenoleukodystrophy, corpus callosum agenesis, Aicardi syndrome, Alpers' disease, Alzheimer's disease, Barth's syndrome, Batten's disease, CADASIL, cerebellar degeneration, Fabry disease, Gerstmann-Straussler-Scheinker disease, Huntington's disease and other triplet repeat diseases, Leigh disease, Lesch-Nychen syndrome, Menkes disease, mitochondrial myopathies, NINDS colpocephaly. These diseases are described further on the website of the National Institutes of Health in the subsection "Genetic diseases".

Development and application of Cas9

[001297] The present invention may be further illustrated and expanded by describing the development and use of CRISPR-Cas9, and in particular in terms of the delivery of the CRISPR protein complex and the use of RNA-guided endonuclease in cells and organisms, as set forth in the following articles. incorporated by reference:

> Multiplex genome engineering using CRISPR/Cas systems. Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., & Zhang, F. Science Feb 15;339(6121):819-23 (2013);

> RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Jiang W., Bikard D., Cox D., Zhang F, Marraffini L.A. Nat Biotechnol Mar;31(3):233-9 (2013);

> One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Wang H., Yang H., Shivalila CS., Dawlaty MM., Cheng AW., Zhang F., Jaenisch R. Cell May 9;153(4):910-8 (2013);

> Optical control of mammalian endogenous transcription and epigenetic states. Konermann S, Brigham MD, Trevino AE, Hsu PD, Heidenreich M, Cong L, Platt RT Scott DA, Church GM, Zhang F. Nature. Aug 22;500(7463):472-6. doi: 10.1G38/Naturel2466. Epub 2013 Aug 23 (2013);

> Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity, Ran, FA., Hsu, PD., Lin, CY., Gootenberg, JS., Konermann, S., Trevino, AE., Scott, DA., Inoue, A., Matoba, S., Zhang, Y., & Zhang, F. Cell Aug 28. pii: S0092-8674(13)01015-5 (2013-A);

> DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P., Scott, D., Weinstein, J., Ran, FA., Konermann, S., Agarwala, V., Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, TJ., Marraffini, LA., Bao, G., & Zhang, F. Nat Biotechnold doi: 10.1038/nbt.2647 (2013);

> Genome engineering using the CRISPR-Cas9 system. Ran, FA., Hsu, PD., Wright, J., Agarwala, V., Scott, DA., Zhang, F. Nature Protocols Nov;8(l l):2281-308(2013-B);

> Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem, O., Sanjana, NE., Hartenian, E., Shi, X., Scott, DA., Mikkelson, T., Heckl, D., Ebert, BL., Root, DE., Doench, JG., Zhang, F. Science Dec 12. (2013). [Epub ahead of print];

- Crystal staicture of cas9 in complex with guide RNA and target DNA. Nishimasu, H., Ran, FA., Hsu, PD., Konermann, S., Shehata, SI, Dohmae, N., Ishitani, R., Zhang, F., Nureki, (), Cell Feb 27, 156( 5):935-49 (2014);

- Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells. Wu X., Scott DA., Kriz AJ., Chiu AC., Hsu PD., Dadon DB, Cheng AW., Trevino AE, Konermann S., Chen S., Jaenisch R., Zhang F., Sharp PA. Nat Biotehnol. Apr 20. doi: 10. lQ38/nbt.2889 (2014);

- CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling. Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, Dahlman JE, Parnas G, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, Heidenreich M, Xavier RJ, Danger R, Anderson DG, Hacohen N , Regev A, Feng G, Sharp PA, Zhang F. Cell 159(2): 440-455 DOI: 10.1016/j. cell.2014.09.014(2014);

- Development and Applications of CRISPR-Cas9 for Genome Engineering, Hsu PD, Lander ES, Zhang F., Cell. Jun 5,157(6): 1262-78 (2014).

- Genetic screens in human cells using the CRISPR/Cas9 system, Wang T, Wei JJ, Sabatini DM, Lander ES., Science. January 3; 343(6166): 80-84. doi: 10.1126/science. 1246981 (2014);

- Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation, Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, Sullender M, Ebert BL, Xavier RJ, Root DE., (published online September 3, 2014) Nat Biotehnol. Dec;32(12): 1262-7 (2014);

- In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9, Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J, Sur M, Zhang F., (published online 19 October 2014) Nat Bioteehnol , Jan, 33(1): 102-6 (2015);

- Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex, Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F. , Nature. Jan 29;517(7536):583-8 (2015).

- A split-Cas9 architecture for inducible genome editing and transcription modulation, Zetsche B, Volz SE, Zhang F., (published online 02 February 2015) Nat Bioteehnol. Feb;33(2): 139-42 (2015);

- Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and Metastasis, Chen S, Sanjana NE, Zheng K, Shalem O, Lee K, Shi X, Scott DA, Song J, Pan JQ, Weissleder R, Lee H, Zhang F, Sharp P.A. Cell 160, 1246-1260, March 12, 2015 (multiplex screen in mouse), and

- In vivo genome editing using Staphylococcus aureus Cas9, Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem Q, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F., (published online 01 April 2015), Nature. Apr 9;520(7546): 186-91 (2015).

- Shalem et. al, "High-throughput functional genomics using CRISPR-Cas9," Nature Reviews Genetics 16, 299-311 (May 2015).

- Xu et al., "Sequence determinants of improved CRISPR sgRNA design," Genome Research 25, 1147-1157 (August 2015).

- Parnas et al., "A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks," Cell 162, 675-686 (July 30, 2015).

- Ramanan et al., CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus," Scientific Reports 5:10833, doi: 10.1038/srepl0833 (June 2, 2015),

- Nishimasu et al., Crystal Structure of Staphylococcus aureus Cas9," Cell 162, 1113-1126 (Aug, 27, 2015),

- BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis, Canver et al., Nature 527(7577): 192-7 (Nov. 12, 2015) doi: 10.1038/naturel5521. Epub 2015 Sep 16.

- Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System, Zetsche et al..Cell 163, 759-71 (Sep 25, 2015),

- Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems. Shmakov et al, Molecular Cell, 60(3), 385-397 doi: 10.1016/j.molcel.2015,10.008 Epub October 22, 2015.

- Rationally engineered Cash nucleases with improved specificity, Slaymaker et al., Science 2015 Dec 1. pii: aad5227. [Epub ahead of print],

each of which is incorporated herein by reference may be considered in the practice of the present invention and is briefly discussed below:

- Cong et al. developed type II CRISPR-Cas systems for use in eukaryotic cells based on both Streptococcus thermophilus Cas9 and Streptococcus pyogenes Cas9 and showed that Cas9 nucleases can be targeted by short RNA to induce precise DNA cleavage in human and mouse cells. Their study also showed that Cas9, converted into a nicking enzyme, can be used to facilitate homologous repair in eukaryotic cells with minimal mutagenic activity. In addition, their study showed that multiple guide sequences can be encoded into a single CRISPR sequence to enable simultaneous editing of multiple sites at endogenous genomic loci in the mammalian genome, demonstrating the ease of programming and broad applicability of RNA-based nuclease technology. This ability to use RNA to program sequence-specific DNA cleavage in the cell has defined a new class of tools for genomic engineering. These studies have also shown that other CRISPR loci could possibly be transplanted into mammalian cells to mediate cleavage at target sites in the mammalian genome. It is important to note that some aspects of the CRISPR-Cas system may be further improved in the future to increase its efficiency and versatility.

- Jiang et al. used regularly clustered short palindromic repeats (CRISPR) coupled to the endonuclease Cas9, combined with double RNAs, to introduce precise mutations into the genomes of Streptococcus pneumoniae and Escherichia coli . This approach is based on a dual RNA: Cas9-directed cleavage at a target genomic site eliminates unmutated cells and bypasses the need for marker selection or negative selection systems. The study reports reprogramming the specificity of the double RNA complex: Cas9 by altering the sequence of a short CRISPRRNA (cr-RNA) to introduce single- and multi-nucleotide changes in editing matrices. The study showed that the simultaneous use of two crRNAs provided multiplex mutagenesis. In addition, when the approach was used in combination with recombination, in S. pneumoniae , almost 100% of the cells that were recovered using the described approach contained the desired mutation, and in E. coli , 65% of the cells that were recovered contained the mutation.

- Wang et al., 2013 used the CRISPR/Cas system to one-step generate mice carrying mutations in multiple genes that were traditionally generated in multiple steps by sequential recombination in embryonic stem cells and/or laborious breeding of mice with a single mutation. The CRISPR/Cas system will significantly accelerate the in vivo study of functionally redundant genes and epistatic gene interactions.

- Konermann et al. (2013) reviewed the need for versatile and robust technologies for optical and chemical modulation of DNA-binding domains based on the CRISPR Cas9 enzyme, as well as effector-like transcription activators (TALEs).

- Ran et al. (2013-A) described an approach that combined a Cas9 nicase mutant with paired guide RNAs to introduce targeted double-strand breaks. This concerns the Cas9 nuclease from the microbial CRISPR-Cas system, which targets specific genomic loci with a guide sequence that can tolerate certain mismatches with the target DNA and thereby promote unwanted off-target mutagenesis. Since single strand breaks in the genome are repaired with high fidelity, simultaneous insertion of single strand breaks with appropriately biased guide RNAs is required for double strand breaks and increases the number of specific bases recognized for targeted cleavage. The authors demonstrated that the use of paired nicks can reduce off-target activity by 50-100-fold in cell lines and facilitate gene knockout in mouse zygotes without compromising target cleavage efficiency. This versatile strategy allows for a wide range of genome editing applications that require high specificity.

- Hsu et al. (2013) characterized the specificity of targeting carried out by SpCas9 in human cells to provide data on the choice of target sites and avoid off-targeting. The study evaluated >700 guide RNA variants and SpCas9-induced insertion/deletion (indel mutation) rates at >100 predicted genomic non-target loci in 293T and 293FT cells. The authors showed that SpCas9 tolerate mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position, and distribution of mismatches. The authors also showed that SpAas9-mediated cleavage is not affected by DNA methylation and that the dose of SpCas9 and single-stranded guide RNA (sgRNA) can be titrated to minimize off-target modification. In addition, to facilitate applications for mammalian genome engineering, the authors have developed software for selecting and validating target sequences, as well as off-target analysis.

- Ran et al. (2013-B) described a toolkit for Cas9-mediated genome editing by non-homologous end joining (NHEJ) or homologous repair (HDR) in mammalian cells, as well as the creation of modified cell lines for subsequent functional studies. To minimize off-target cleavage, the authors described a strategy for introducing double stranded breaks using a Cas9 nicase mutant with paired guide RNAs. The protocol provided by the authors experimentally provided guidelines for selecting target sites, evaluating cleavage efficiency, and analyzing off-target activity. Studies have shown that, starting from target development, gene modification can be achieved in as little as 1-2 weeks, and modified clonal cell lines can be obtained within 2-3 weeks.

- Shalem et al. described a new way to study gene function on a genome-wide scale. Their research showed that delivery of a genome-wide CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enabled both negative and positive selection screening in human cells. First, the authors showed the use of the GeCKO library to identify genes required for cell viability in cancer and pluripotent stem cells. Further, in a model of melanoma, the authors screened for genes whose loss is associated with resistance to vemurafenib, a therapeutic drug that inhibits the mutant BRAF protein kinase. Their research showed that top-ranking candidates included the previously tested NF1 and MED12 genes, as well as the new ones: NF2, CUL3, TADA2B, and TADA1. The authors observed a high level of agreement between independent guide RNAs targeting the same gene and a high confirmation rate of the result obtained, and thus demonstrated the possibility of screening the whole genome using Cas9.

- Nishimasu et al. examined the crystal structure of Streptococcus pyogenes Cas9 in complex with single-stranded guide RNA (sg-RNA) and its target DNA at a resolution of 2.5 A°. A "bilobed" architecture of the structure was found, consisting of a subunit for target recognition and a nuclease subunit accommodating the sg-RNA:DNA heteroduplex in a positively charged groove on their surface. While the subunit ("lobe") for recognition is required for binding sg-RNA and DNA, the nuclease subunit ("lobe") contains HNH and RuvC nuclease domains that are properly positioned to cleave complementary and non-complementary strands of the target DNA respectively. The nuclease subunit also contains a carboxyl terminal domain responsible for interaction with the sequence adjacent to the protospacer (PAM). This high-resolution structure and accompanying functional analyzes revealed the molecular mechanism of Cas9's RNA-directed DNA targeting, thus paving the way for the rational design of new, versatile genome-editing technologies.

- Wu et al. mapped genome-wide binding sites for catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single-stranded guide RNA (sgRNA) in mouse embryonic stem cells (mESC). The authors showed that each of the four sgRNAs tested dCas9 targets between tens and thousands of genomic regions, often characterized by a 5-nucleotide primer region in the sgRNA and an NGG adjacent protospacer motif (PAM). The inaccessibility of chromatin reduces the binding of dCas9 to other sites with the appropriate "seed"sequences; thus, 70% of non-target sites are associated with genes. The authors showed that targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 revealed only one site mutated above background levels. The authors proposed a two-state model for Cas9 binding and cleavage in which a "seed" sequence match causes binding, but cleavage requires extensive base pairing with the target DNA.

- Platt et al. created a Cre-dependent mouse with the Cas9 knockin. The authors demonstrated both in vivo and ex vivo genome editing using adeno-associated virus (AAV), lentivirus, or particle-mediated guide RNA delivery to neurons, immune cells, and endothelial cells.

- Hsu et al. (2014) - Review article discussing the history of CRISPR-Cas9 from yogurt to genome editing, including cell genetic screening.

- Wang et al. (2014) refers to a loss-of-function pooled genetic screening method suitable for both positive and negative selection that uses a genome-wide lentiviral single-stranded guide RNA (sgRNA) library.

- Doench et al. created a pool of single-stranded guide RNAs (sgRNAs) by alternating all possible target sites of a group of six endogenous mouse and three endogenous human genes and quantified their ability to produce null alleles of their target gene by antibody staining and flow cytometry. The authors showed that PAM optimization improved activity and also provided an interactive tool for the design of single-stranded sg-RNA guides.

- Swiech et al. demonstrated that AAV-mediated genome editing with SpCas9 could allow for retrospective genetic studies of gene function in the brain.

- Konermann et al. (2015) discuss the possibility of attaching multiple effector domains, such as transcription activators, functional and epigenomic regulators, at appropriate positions on the guide molecule, such as a hairpin or tetraloop with and without linkers.

- Zetsche et al. demonstrate that the Cas9 enzyme can be halved and therefore the assembly of Cas9 can be controlled for activation.

- Chen et al. using CRISPR-Cas9 multiplex genome-wide screening in vivo , genes regulating lung metastases were found in mice.

- Ran et al. (2015) investigated SaCas9 and its ability to edit genomes and demonstrated that biochemical analyzes cannot be extrapolated. Shalem et al. (2015) describe methods for using catalytically inactive Cas9 fusions (dCas9) for synthetic repression (CRISPRi) or activation (CRISPRa) of expression and demonstrate the benefits of using Cas9 for genome-wide screening, including ordered and pooled screens, knockout approaches that inactivate genomic loci, and strategies that modulate transcription activity.

Final changes

- Shalem et al. (2015) described methods for using catalytically inactive Cas9 fusions (dCas9) for synthetic repression (CRISPRi) or activation (CRISPRa) of expression and demonstrated the benefits of using Cas9 for genome-wide screening, including sequenced and pooled screens, knockout approaches that inactivate genomic loci, and strategies modulating transcriptional activity.

- Xu et al. (2015) evaluated DNA sequence characteristics that contribute to the effectiveness of single-stranded guide RNA (sg-RNA) in CRISPR-based screening. The authors investigated the efficiency of CRISPR/Cas9 knockout and the preference for nucleotides at the cleavage site. The authors also found that the preferred sequences for CRISPR interference/CRISPR activation (CRISPRi/a) differ significantly from those for CRISPR/Cas9 knockout.

- Paraas et al. (2015) introduced genome-wide pooled CRISPR-Cas9 libraries into dendritic cells to identify genes that control tumor necrosis factor (Tnf) induction by bacterial lipopolysaccharide (LPS). Known regulators of Tlr4 signaling and previously unknown candidates have been identified and classified into three functional modules with clear influence on canonical responses to LPS.

- Ramananetal (2015) demonstrated the cleavage of viral episomal DNA (cccDNA) in infected cells. The HBV genome exists in the nuclei of infected hepatocytes as a 3.2 kb double-stranded episomal DNA termed covalently closed circular DNA (cccDNA), which is a key component of the HBV life cycle whose replication is not inhibited by current therapy. The authors showed that single-stranded guide RNA molecules (sg-RNA) specifically targeting highly conserved regions of HBV effectively suppress viral replication and depleted cccDNA.

- Nishimasu et al. (2015) reported the crystal structures of SaCas9 in complex with a single guide RNA (sgRNA) and its double-stranded DNA targets containing a 5'-TTGAAT-3' PAM sequence and a 5'-TTGGGT-3' PAM sequence. Structural comparison of SaCas9 with SpCas9 revealed both structural conservatism and divergence, explaining their distinct PAM specificity and orthologous recognition of sgRNA.

- Canver et al. (2015) demonstrated a functional study of non-coding genomic elements based on CRISPR-Cas9. The authors developed CRISPR-Cas9 pooled guide RNA libraries for in situ saturation mutagenesis of human and mouse BCL11A enhancers, which revealed important features of these enhancers.

- Zetsche et al. (2015) reported the characterization of Cpf1, a CRISPR class 2 nuclease from Francisellanovicida U112, which has distinct features from Cas9. Cpf1 is the only RNA-directed endonuclease lacking tracr-RNA, using a T-rich motif adjacent to a protospacer, and cleaving DNA by stepwise double-strand break.

- Shmakov et al. (2015) reported three different classes of CRISPR-Cas systems. Two enzymes of the CRISPR system (C2c1 and C2c3) contain RuvC-like endonuclease domains that are distantly related to Cpf1. Unlike Cpf1, C2c1 depends on both cr-RNA and tracr-RNA for DNA cleavage. The third enzyme (C2c2) contains two predicted HEPN RNase domains and is independent of tracr-RNA.

- Slaymaker et al. (2016) reported the use of structural protein engineering to improve the specificity of Streptococcus pyogenes (SpCas9) Cas9. The authors developed "extended specificity" variants of SpCas9 (eSpCas9) that maintained robust target cleavage with reduced off-target effects.

[001298] Also, "Dimeric CRISPR RNA-guided Fok1 nucleases for highly specific genome editing", Shengdar Q. Tsai, Nicolas Wyvekens, CydKhayter, Jennifer A. Foden, Vishal Thapar, Deepak Reyon, Mathew J, Goodwin, Martin J, Aryee, J. Keith Joung Nature Biotechnology 32(6): 569-77 (2014), refers to dimeric RNA-directed Fok1 nucleases that recognize extended sequences and can edit endogenous genes with high efficiency in human cells.

[001299] With regard to general information about CRISPR-Cas systems, their components and the delivery of such components, including methods, materials, delivery vehicles, vectors, particles, AAV, as well as their manufacture and use, including with regard to quantities and formulas 8999641, 8993233, 8945839, 8932814, 8906616, 8895308, 8889418, 8889356, 8871445, 8865406, 8795965; US Patent Application Publication US 2014-0310830 (US APP. Ser. No. 14/105031), US 2014-0287938 A1 (US App. Ser. No. 14/213991), US 2014-0273234 A1 (U.S. App, Ser . No. 14/293674), US2014-0273232 A1 (U.S. App. Ser. No. 14/290575), US 2014-0273231 (U.S. App. Ser. No. 14/259420), US 2014-0256046 A1 (U.S. App . Ser. No. 14/226274), US 2014-0248702 A1 (U.S. App. Ser. No. 14/258458), US 2014-0242700 A1 (U.S. App. Ser. No. 14/222930), US 2014-0242699 A1 (U.S. App. Ser. No. 14/183512), US 2014-0242664 A1 (U.S. App. Ser. No. 14/104990), US 2014-0234972 A1 (U.S. App. Ser. No. 14/183471) , US 2014-0227787 A1 (U.S. App. Ser. No. 14/256912), US 2014-0189896 A1 (U.S. App. Ser. No. 14/105035), US 2014-0186958 (U.S. App. Ser. No. 14 /105017), US 2014-0186919 Al (U.S. App. Ser. No. 14/104977), US 2014-0186843 A1 (U.S. App. Ser. No. 14/104900), US 2014-0179770 A1 (US. App. Ser. No. 14/104837) and US 2014-0179006 A1 (US. App. Ser. No. 14/183,486), US 2014-0170753 (US App. Ser. No. 14/183429), US 2015-0184139 ( U S. app. Ser. no. 14/324960), 14/054414; European patents: EP 2 764 103 (EP13824232.6), EP 2 784 162 (EP14170383.5) and EP 2 771 468 (EP13818570.7); and PCT patent application publications: WO 2014/093661 (PCT/US2013/074743), WO 2014/093694 (PCT/US2013/074790), WO 2014/093595 (PCT/US2013/074611), WO 2014/093718 (PCTAJS2013/074825 ), WO 2014/093709 (PCT/US2013/074812), WO 2014/093622 (PCT/US2013/074667), WO 2014/093635 (PCT/US2013/074691), WO 2014/093655 (PCT/US2013/074736) , WO 2014/093712 (PCT/US2013/074819), WO 2014/093701 (PCT/US2013/074800), WO 2014/018423 (PCT/US2013/051418), WO 2014/204723 (PCT/US2014/041790), WO 2014/204724 (PCT/US2014/041800) PCT/US2014/041808) US2014/069925), WO 2015/089427 (PCT7US2014/070068), WO 2015/089462 (PCT/US2014/070127), WO 2015/089419 (PCT/US2014/070057), WO 2015/089465 (PCT/07014) , WO 2015/089486 (PCT/US2014/070175), PCT/US2015/051691, PCT/US20 15/051830. Also cited is US Provisional Application 61/758468; 61/802174; 61/806375; 61/814263; 61/819803 and 61/828130, filed January 30, 2013; March 15, 2013; March 28, 2013; April 20, 2013; May 6, 2013 and May 28, 2013, respectively. Also cited is U.S. Provisional Application 61/836123, filed June 17, 2013. See also U.S. Supplemental Applications 61/835931, 61/835936, 61/836127, 61/836101, 61/836080, and 61/835973, filed June 17, 2013. Further reference is made to U.S. Provisional Applications 61/862468 and 61/862355 filed August 5, 2013, 61/871301 filed August 28, 2013, 61/960777 filed September 25, 2013 and 61/961980 filed October 28. year 2013. Further reference is made to: PCX/US2014/041803, PCT/US2014/041809, PCT/US2014/041804 and PCT/US2014/041806, each filed June 10, 2014; PCT/US2014/041808 filed June 11, 2014; and PCT/US2014/62558, filed October 28, 2014, and US Patent Nos: 61/915148, 61/915150, 61/915153, 61/915203, 61/915251, 61/915301, 61/915267, 61/915260 and 61/915397 filed December 12, 2013; 61/757972 and 61/768959 filed January 29, 2013 and February 25, 2013; 61/835936, 61/836127, 61/836101, 61/836080, 61/835973 and 61/835931, filed June 17, 2013; 62/010888 and 62/010879 filed June 11, 2014; 62/010329 and 62/010441 filed June 10, 2014; 61/939228 and 61/939242, each published February 12, 2014, 61/980012, filed April 15, 2010; 62/038358, filed August 17, 2014; 62/054490, 62/055484, 62/055460 and 62/055487, filed September 25, 2014, and 62/069243, filed October 27, 2014. Reference is made to Interim Application US 61/930214 filed January 22, 2014

[001300] Reference is also made to US application 62/180,709, filed June 17, 2015, PROTECTED GUIDE RNAS (PGRNAS); US application 62/091455, filed December 12, 2014, PROTECTED GUIDE RNAS (PGRNAS); US application 62/096708, filed December 24, 2014, PROTECTED GUIDE RNAS (PGRNAS); US application 62/091462, filed December 12, 2014, DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS; U.S. Application 62/096324, filed December 23, 2014, 62/180681, June 17, 2015, and 62/237496, October 5, 2015, DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS; US Application 62/091456, December 12, 2014 and 62/180692, July 17, 2015, ESCORTED AND FUNCTIONALIZED GUIDES FOR CRISPR-CAS SYSTEMS, US Application 62/091461, December 12, 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR -CAS SYSTEMS AND COMPOSITIONS FOR GENOME EDITING AS TO HEMATOPOETIC STEM CELLS (I ISC's); U.S. Application 62/094903, December 19, 2014, UNBIASED IDENTIFICATION OF DOUBLE-STRAND BREAKS AND GENOMIC REARRANGEMENT BY GENOME-WISE INSERT CAPTURE SEQUENCING; U.S. Application 62/096761, December 24, 2014, ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED ENZYME AND GUIDE FUNCTIONAL-CRISPR COMPLEXES; US Application 62/087475, December 4, 2014, FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; US Application 62/055487, September 25, 2014, FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; US Application 62/087546, December 4, 2014, and 62/181687, MULTIFUNCTIONAL CRISPR COMPLEXES AND/OR OPTIMIZED ENZYIFUNCTIONAL-CRISPR COMPLEXES; and US Application 62/098285, December 30, 2014, CRISPR MEDIATED IN VIVO MODELING AND GENETIC SCREENING OF TUMOR GROWTH AND METASTASIS.

[001301] References are made to the following US applications 62/181659, June 18, 2015, and 62/207318, August 19, 2015, ENGINEERING AND OPTIMIZATION OF SYSTEMS, METHODS, ENZYME AND GUIDE SCAFFOLDS OF CAS9 ORTHOLOGS AND VARIANTS FOR SEQUENCE MANIPULATION. Reference is made to US Application 62/181675, June 18, 2015 and Patent Registry Number 46783.01.2128, filed October 22, 2015, NOVEL CRISPR ENZYMES AND SYSTEMS, US Application 62/232067, September 24, 2015, US Application 62/205733, 16 August 2015, US Application 62/201542, August 5, 2015, US Application 62/193507, July 16, 2015, and US Application 62/181739, June 18, 2015, each titled NOVEL CRISPR ENZYMES AND SYSTEMS, and in US application 62/245270, October 22, 2015, NOVEL CRISPR ENZYMES AND SYSTEMS. Also cited are US Application 61/939256, February 12, 2014, and WO 2015/089473 (PCT/US2014/070152), December 12, 2014, each entitled ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED GUIDE COMPOSITIONS WITH NEW ARCHITECTURES FOR SEQUENCE MANIPULATION . Also cited are PCT.AJS2015/045504, August 15, 2015, US Application 62/180699, June 17, 2015, and US Application 62/038358, August 17, 2014, each entitled GENOME EDITING USING CAS9 NICKASES.

[001302] Each of these patents, patent publications and applications and all documents cited therein or in the course of their proceedings ("Attached Documents"), and all documents cited or referred to in documents cited in this appendix, together with any instructions, descriptions, specifications and flow charts of products for any products mentioned in it or in any document and included in this description by reference, are incorporated in this description by reference and can be used in the practice of the invention. All documents (eg, these patents, patent publications and applications, and the documents cited herein) are incorporated herein by reference in the same way as if each individual document were specifically and individually designated for inclusion by reference.

[001303] Also referred to is PCT Application PCT/US14/70057 Patent Registry Number 47627.99.2060 and BI-2013/107 entitled "DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING PARTICLE DELIVERY COMPONENTS" (claiming priority under one or more or all U.S. Provisional Applications: 62/054490 filed September 24, 2014, 62/010441 filed June 10, 2014, and 61/915118, 61/915215 and 61 /915148, each filed December 12, 2013) ("Particle Delivery PCT"), incorporated herein by reference, in relation to a method for producing sg-RNA and,tkrf Cas9, comprising mixing a mixture containing sg-RNA and a Cas9 protein (and optionally an HDR template) with a mixture containing or consisting only of or consisting of a surfactant, a phospholipid, a biodegradable polymer, a lipoprotein and an alcohol; and particles from such a process. For example, when the Cas9 protein and sgRNA are mixed together at a suitable molar ratio, such as 3:1 to 1:3, or 2:1 to 1:2 or 1:1, at a suitable temperature, such as 15-30°C, eg 20-25° C., eg room temperature, for a suitable time, eg 15-45, eg 30 minutes, preferably in sterile, nuclease-free buffer, eg IX PBS. Separately, particle components such as or containing: a surfactant, eg a cationic lipid, eg 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); a phospholipid such as dimyristoylphosphatidylcholine (DMPC); a biodegradable polymer such as a polymer of ethylene glycol or PEG, or a lipoprotein such as low density lipoprotein, eg cholesterol, has been dissolved in an alcohol, preferably a C _1-6 alkyl alcohol such as methanol, ethanol, isopropanol, eg 100% ethanol. Both solutions were mixed together to form particles containing Cas9-sg-RNA complexes. Accordingly, sg-RNA can be pre-combined with the Cas9 protein before the formation of the entire complex in the particle. Formulations can be prepared with other molar ratios of various components known to facilitate the delivery of nucleic acids to cells (e.g., 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-ditretracanoyl-s-glycero- 3-phosphocholine (DMPC), polyethylene glycol (PEG) and cholesterol). For example, volar ratios of DOTAP: DMPC: PEG: cholesterol can be DOTAP 100, DMPC 0, PEG 0, cholesterol 0; or DOTAP 90, DMPC 0, PEG 10, cholesterol 0; or DOTAP 90, DMPC 0, PEG 5, cholesterol 5. DOTAP 100, DMPC 0, PEG 0, cholesterol 0. The present description accordingly covers mixing of sgRNA, Cas9 protein and components that form the particle; as well as particles from such mixing. Embodiments of the present invention may include particles; for example, particles used in a method similar to the PCT particle delivery method, for example, by mixing a mixture containing sg-RNA and/or Cas9, as in the present invention, and components that form a particle, for example, as in for PCT particle delivery, form a particle and particles from such a mixture (or, of course, other particles containing sg-RNA and/or Cas9, as in the present invention).

[001304] The present invention is further illustrated in the following examples, which are provided for illustrative purposes only and are not intended to limit the invention in any way.

Example 1: Origin and Evolution of Adaptive Immunity Systems

[001305] Classification and annotation of CRISPR-Cas systems in archaea and bacteria genomes. CRISPR-Cas loci have more than 50 gene families, while there are no strictly universal genes, rapid evolution occurs, and a significant diversity of loci architecture is observed. Therefore, there is no possible tree and an integrated approach is especially needed. There is now a detailed identification of cas genes within 395 profiles for 93 cas proteins. The classification includes gene expression profiles and loci architecture profiles.

[001306] The latest classification of CRISPR-Cas systems is presented in Fig.1. Class l includes multi-subunit cr-RNA effector complexes (Cascade) and class 2 includes single-subunit cr-RNA effector complexes (Cas-9 type). Figure 2 represents the molecular organization of CRISPR-Cas. Figure 3 shows the structures of type I and III effector complexes: they share a common structure/common nature despite significant differences in sequences. 4 shows CRISPR-Cas as an RNA recognition motif (RRM) based system. Figure 5 shows the phylogeny of cas1, where the recombination of adaptive and cr-RNA effector modules shows a major aspect of the evolution of CRISPR-Cas systems. Figure 6 presents a list of CRISPR-Cas systems, in particular the distribution of types and subtypes of CRISPR-Cas among archaea and bacteria.

[001307] Cas1 protein is not always associated only with CRISPR-Cas systems, so it is possible that there are two branches of separate "solo" Cas 1, which suggests that differences in both function and origin are possible, and detection is also possible. new mobile elements (see Makarova, Krupovic, Koonin, Frontiers Genet 2014). The genomic organization of the three families of casposons may provide possible answers. In addition to cas 1 and PolB, casposons have different genes including nucleases (Krupovic et al. BMC Biology 2014). One family has a protein-primed polymerase, the other family has an RNA-primed polymerase. In addition to members of the phyla Euryarchaeota and Thaumarchaeota, casposons found in several bacteria suggest horizontal gene transfer. Kazposon Cas1 (transposase/integrase) suggests a common basal phylogenetic group in the development of Cas1.

[001308] Bacteria and archaea use the CRISPR system for adaptive immune defense in pro- and eukaryotic cells through genomic manipulation. The cas 1 protein is an already existing method of genomic manipulation. There are similar integration mechanisms in casposons and CRISPR, especially replication-dependent acquisition through copy and paste processes rather than cut and paste (Krupovic et al. BMC Biology 2014). Cas1 is a reference integrase sample (Nunez JK, Lee AS, Engelman A, Doudna JA. Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity. Nature. 2015 Feb 18). There is a certain similarity between terminal casposon reverse repeats and the innate immune locus (Koonin, Kmpovic, Nature Rev Genet, 2015). The evolution of adaptive immunity systems in prokaryotes and animals may have followed parallel paths through the integration of transposons into innate immunity loci (Koonin, Krupovic, Nature Rev Genet, 2015). Transposase RAG1 (the key enzyme of V(D)J recombination of vertebrates) possibly originated from Transib class transposons (Kapitonov VV, Jurka J.. RAG1 core and V(D)J recombination signal sequences were derived from Transib transposons.. PLoS Biol. 2005 Jun;3(6):el81), however, none of the Transibs transposons encode the RAG2 protein. RAG1 and RAG2 encoding transposons are described in Kapitonov, Koonin, Biol Direct 2015 and the phylogeny of the Transib class transposase is presented in Kapitonov, Koonin, Biol Direct 2015. Defensive DNA killing in ciliates evolved from the PiggyMAc transposon, which together with RNA-i form innate immunity (Swart EC, Nowacki M. The eukaryotic way to defend and edit genomes by sRNA-targeted DNA deletion Ann NY Acad Sci 2015).

[001309] The relative stability of the classification means that the most frequently occurring CRISPR-Cas variants are already known. However, the existence of rare and still unclassified variants also suggests that additional types and subtypes are still waiting to be described (Makarova et al, 2015. Evolutionary classification of CRISPR-Cas systems and cas genes).

[001310] Transposons play a key role in the evolution of adaptive immunity and other systems involved in DNA change. Class 1 CRISPR-Cas arose from transposons, but this only concerns the adaptive functional module of the system. Class 2 CRISPR-Cas has both adaptive and effector functions, the modules of which can originate from different transposons.

Example 2: New Predicted Class 2 CRISPR-Cas Systems and Evidence for Their Independent Origin from Transposons

[001311] The CRISPR-Cas systems of adaptive immunity of bacteria and archaea show a huge diversity in the composition of Cas proteins, as well as a diverse structure of genomic loci. However, these systems fall broadly into two classes: Class 1, which has a multi-protein (multi-block) effector complex, and Class 2, where the effector complex consists of a single large protein (single-block complex), such as the cas-9 protein (FIGS. 1A and 1B). ). Applicants have developed a simple bioinformatics technique (FIG. 7) to process ever-growing genomic and metagenomic databases, accompanied by a deepening understanding of CRISPR-cas systems to search for possible candidates for a class 2 CRISPR-Cas system. With the help of our computational methodology, we identified three new variants, each of which is present in different bacteria and contains the cas1 and cas2 genes, as well as a third gene encoding a large protein, which is supposed to function as an effector module. At the first of these loci, a putative effector protein (C2c1p) has a similar science domain to RuvC and resembles the previously described Cpf1 protein, a predicted effector for type V CRISPR-cas systems, thus the new predicted system is classified as a V-B subtype. A comprehensive comparison of protein sequences showed that the effector proteins Cas9 Cpf1 and C2Cip containing the RuvC domain arose from different groups of transposon-encoded TnpB proteins. The second group of new candidates for CRISPR-Cas loci includes a large protein containing two HEPN domains with predicted RNase activity. Given the novelty of the predicted effector protein, these loci were assigned to the new type VI CRISPR-Cas, which is most likely capable of targeting RNA. In general, the results of the analysis show that CRISPR-Cas class 2 systems arose in different, often independent of each other ways, by connecting different Cas1-Cas2 encoding adaptation modules with effector proteins derived from different mobile genetic elements. The path of evolution most likely led to the emergence of various variants of class 2 systems, some of which have not yet been discovered.

[001312] CRISPR-Cas adaptive immunity systems are present in the genomes of 45% of bacteria and 90% of archaea, and they exhibit significant diversity in cas protein composition, sequence, and architecture of genomic loci. According to the architecture of cr-RNA effector complexes, these systems fall into two classes: Class 1 has a multi-subunit effector complex, and Class 2 has a single effector protein (FIGS. 1A and 1B). Class 1 systems are much more common and more diverse than Class 2 systems. In the current classification, Class 1 is represented by 12 distinct subtypes encoded by numerous bacterial and archaeal genomes, while Class 2 includes 3 subtypes, a Type 2 system and a possible Type V , which in general can be found in 10% of sequenced bacterial genomes (together with the only archaeal genome including a possible system of this type). Class 2 systems typically contain only three or four genes in the cas operon, namely the cas1 ~ cas2 gene pair that are involved in adaptation but not interference, one multidomain effector protein that is responsible for interference but also promotes processing and adaptation of preferences. cr-RNA. There is also often a fourth gene with as yet unknown functions that may occur in at least some type II systems. In most cases, the CRISPR cassette and gene for a particular type of RNA, known as tracr-RNA (transactivating small cr-RNA), are found adjacent to class 2 operons (Chylinski, 2014). Tracr-RNA is partially homologous to repeats in the corresponding region of the CRISPR cassette and is essential for the processing of pre-cr-RNA, which is catalyzed by RNAse III, a common bacterial enzyme not associated with CRISPR-cas loci (Deltcheva, 2011) (Chylinski, 20I4; Chylinski , 2013).

[001313] The effector protein of the second type of CRISPR-Cas systems - Cas9 has been studied in great detail in relation to its functions and structure. In various bacteria, Cas9 proteins range from ~950 to over 1600 amino acids and range in size from 950 to 1400 amino acids. It contains two types of nuclease domains, namely the RuvC like domain (RNase H conserved domain) and the HNH (MrcA like conserved domain) domain (Makarova, 2011). The crystal structure of cas9 shows a "bilobed" organization of the protein with a pronounced site of recognition and nuclease activity, while the latter binds to both RuvC and HNH domains (Nishimasu, 2014) (Jinek, 2014). Each of the Cas9 nuclease domains is required for cutting one of the target DNA strands (Jinek, 2012; Sapranauskas, 2011). It has recently been shown that Cas9 is involved in all three stages of the CRISPR immune response, i.e. not only leads to cleavage of the target DNA (interference stage) but also participates in the stages of adaptation and processing of pre-cr-RNA (Jinek, 2012). More specifically, it has been shown that a certain domain in the region with science activity recognizes and binds to the sequence adjacent to the protospacer (PAM) in viral DNA during the adaptation stage (Nishimasu, 2014) (Jinek, 2014) (Heler, 2015; Wei, 2015 ). At the stage of the CRISPR immune response, Cas9 forms a complex with the Cas l and Cas2 proteins, which are directly involved in the incorporation of new spacers in all CRISPR-Cas systems (Heier, 2015; Wei, 2015).

[001314] Cas9 protein coupled to tracr-RNA has recently become a key tool in creating a new generation of genome editing and genetic engineering methods (Gasiunas, 2013; Mali, 2013; Sampson, 2014; Cong, 2015). Such value of Cas9 in genetic engineering is based on the fact that in the second type of CRISPR-Cas, unlike other CRISPR-Cas systems, all operations necessary for recognition of the target DNA and its subsequent cleavage are concentrated within a single, but large, multidomain protein. This feature of type II systems greatly simplifies the development of efficient genome manipulation tools. It is also important that not all variants of cas9 are similar. To date, most of the work on the target protein has been done on cas9 from Streptococcus pyogenes , but other species may offer significant advantages. In this case, recent experiments with cas9, which is 300 amino acids shorter than S. pyogenes, have been packaged into an adeno-associated viral vector, leading to a significant improvement in the ability to use the CRISPR-Cas system for in vivo genome editing (Ran, 2015).

[001315] Type II CRISPR-Cas systems are currently classified into three subtypes ((II-A, II-B and II-C) (Makarova, 2011) (Fonfara, 2014; Chylinski, 2013; Chylinski, 2014). cas1, cas2, and cas9 genes, which are characteristic of all type II loci, subtype II-A is characterized by an additional gene, csn2, which encodes an inactivated ATPase (Nam, 2011, Koo, 2012, Lee, 2012), whose role in the acquisition of new spacers is still poorly studied (Barrangou, 2007; Arslan, 2013) (Heler, 2015) Subtype II-B systems lack csn2, but instead contain the cas4 gene, which, on the contrary, is more typical of type I systems and encodes a 5'-3' exonuclease of the family recB, which promotes the accumulation of spacers by generating recombinogenic DNA ends (Zhang, 2012) (Lemak, 2013, Lemak, 2014) The cas1 and cas2 subtype II-B genes are most closely associated with the corresponding proteins of the CRISPR-Cas type I systems, which implies recombinant the origin of this subtype (Chylinski, 2014).

[001316] Subtype II-C of the CRISPR-Cas systems shows minimal diversity and consists only of the Cas1, cas2 and cas9 genes (Chylinski, 2013; Koonin, 2013; Chylinski, 2014). It is noteworthy, however, that the acquisition of the Campylobacter jejuni spacer by type II-C systems requires the participation of Cas4 encoded by the bacteriophage (Hooton, 2014). Another distinguishing feature of the II-C subtype is the production of some crRNAs by transcription, including transcription from internal alternative promoters, which differs from the processing observed in all other experimentally described CRISPR-Cas systems (Zhang, 2013).

[001317] The existence of type V CRISPR-Cas systems was recently predicted by comparative analysis of bacterial genomes. These putative new CRISPR-Cas systems are present in several bacterial genomes, in particular from the genus Francisella and one archaea, Methanomethylophilus alvus (Vestergaard, 2014). All putative type V loci include Cas1, cas2, a single gene designated as cpf1, and a CRISPR cassette (Schunder, 20I3) (Makarova, 2015). Cpf1 is a large protein (approximately 1300 amino acids) that contains a RuvC-like nuclease domain homologous to the corresponding Cas9 domain, as well as an analog of the typical arginine-rich Cas9 cluster. However, Cpf1 does not have the HNH nuclease domain, which is present in all Cas9 proteins, and the RuvC-like domain is adjacent to the Cpf1 sequence, unlike Cas9, where it contains long insertions including the HNH domain (Chylinski, 2014; Makarova, 2015). These major differences in the domain architectures of Cas9 and Cpf1 suggest that systems containing Cpf1 should be classified as a new type. The composition of the putative type V systems implies that Cpf1 is a single block effector complex and, accordingly, these systems belong to the second class of CRISPR-Cas systems. Some of the putative type V loci encode for Cas4 and thus resemble subtypes of the P-V loci, while others do not have Cas4 and thus are similar to the II-C subtype.

[001318] It has recently been shown that the closest homologues of Cas9 and Cpf1 proteins are TnpB proteins, which are encoded by transposons of the IS605 family and contain a RuvC-like nuclease domain, as well as a zinc finger domain, which has an analogue in Cpf1. In addition, TnpB homologues have been identified that contain an HNH domain inserted into a RuvC-like domain and show high sequence similarity to Cas9. The role of TnpB in transposons remains uncertain, as it has been shown that this protein is not required for transposition.

[001319] Given the homology of Cas9 and Cpf1 and the proteins encoded by transposons, Applicants hypothesized that class 2 CRISPR-Cas systems could arise multiple times by recombination between the transposon and the Cas1-cas2 locus. Accordingly, applicants have developed a simple computational strategy to identify genomic loci that may be candidates for second class variants. In this application, Applicants describe the first application of this approach, which identifies three groups of such candidates, two of which appear to belong to different subtypes of type V, while the third appears to fit type VI. New variants of class 2 CRISPR-Cas systems are of obvious interest as they can be used as tools for editing and regulation of expression.

[001320] Database Search Strategy to Discover New Candidates for Class 2 CRISPR-Cas Locus Applicants used a simple bioinformatics approach to discover new candidates for Class 2 CRISPR-Cas systems (FIG. 7. Schematic). Since the vast majority of CRISPR-Cas loci include the cas1 gene (Makarova, 2011, Makarova, 2015) and the Cas1 sequence is the most conserved for all Cas proteins (Takeuchi, 2012), the Applicants suggested that cas1 is the best possible clue to identify and search for possible new loci using PSI-BLAST. After finding all contigs encoding Cas1 by searching the following WGS (containing the whole genome) and NT (nucleotide) databases in NCBI, protein-coding genes were predicted using GenemarkS in the region of 20 kb. upstream and downstream sequences from the Cas1 gene. These predicted genes were annotated using profiles from the NCBI Conserved Domain Database (CDD) and specific Cas protein profiles (Makarova et al., 2015, Nat Rev Microbiol. 2015, doi: 10.1038/nrmiero3569), and CRISPR cassettes were predicted. using the PILER-CR program. This procedure made it possible to determine the detected CRISPR-Cas loci within already known subtypes. Partial and/or unclassifiable candidate CRISPR-Cas loci containing large (>500 aa) proteins were selected as possible candidates for new class 2 systems based on the characteristic presence of such large single block effector proteins in type II and V systems (Cas9 and Cpf1, respectively). All 63 possible loci identified using these criteria (listed in the table in FIGS. 40A-D) were further analyzed individually using the PSI-BLAST and HHpred programs. Protein sequences encoded at candidate loci were then used as a query to search metagenomic databases for additional homologues, with long contigs found analyzed as above. In this computational pipeline, a total of 53 new loci were generated (some of the originally identified 63 candidate loci were discarded as spurious, while a few incomplete loci missing cas1 were added) with characteristic features of CRISPR-Cas, class 2 systems that , based on the nature of the predicted effector proteins, could be classified into three groups of loci (see Figures 8A and 8B, Figures 9, 14 and 15 and Figures 41A-B, 42A-B and 43A-B). , infecting bacteria that the recently discovered class 2 CRISPR-Cas systems would have are unknown in practice, for each of these systems the inventors have found spacers that correspond to phages or predicted prophages.

[001321] Using the computational strategy described above, Applicants have discovered three new class 2 CRISPR-Cas systems, namely C2e1 and C2c3, which are classified as subtypes of the previously described putative type V and C2c2, which Applicants attribute to the new putative type VI. Applicants present several strategies to prove that these loci encode functional CRISPR-Cas systems. Based on a comparison of genomes, the inventors identified phage-specific spacers for each of the three putative novel systems, and also showed that spacer sets are completely different even in closely related bacterial genomes, indicating active, functioning immunity. Many of these new systems occur in bacterial genomes that do not contain other CRISPR-Cas loci, suggesting that type V and type VI systems can function autonomously. Moreover, even when other CRISPR-Cas systems were identified in the same genomes, the associated repeat structures were clearly distinct from other types in types V and VI, suggesting their independent functionality.

[001322] Assumed VB type system . The first group of possible loci, conventionally designated as C2c1 (class 2, candidate 1), is represented in the bacterial genomes of four major taxa, including Bacilli, Verrucomicrobia, alpha proteobacteria, and delta proteobacteria (Fig. 8A-B "Organization of complete loci of class 2 systems "Fig.41A-B). All C2c1 loci encode a Cas1-Cas4 fusion protein, Cas2, and a large protein referred to by Applicants as C2c1p and are typically found adjacent to the CRISPR cassette (FIG. 9, neighborhood of C2c1, FIGS. 41A-B). In the Cas1 phylogenetic tree, the corresponding protein cluster connects to the IU type system (FIGS. 10A and 10B, FIGS. 10C-W, Cas1 tree) and is the only one in which a Cas1-Cas4 fusion is found. The length of the C2c1p proteins identified here varies from 1100 to 1500 amino acids, for example, may be about 1200 amino acids. HHpred search reveals significant similarity between the C-terminal portion of C2c1p proteins and a subset of TnpB proteins encoded in the IS6G5 family of transposons (FIGS. 13A and 13C). In contrast, no significant similarity was found between C2c1p and Cas9 or Cpf1, which are similar to other groups of TnpB proteins (Chylinski, 2014) (Makarova, 2015; Makarova, 2015). Thus, the domain architecture of C2c1p is similar to that of Cpf1 and differs from that of Cas9 (FIG. 13A), although all three Cas proteins appear to have evolved from the TnpB family (FIG. 11 "Domain Organization of Class 2 Families"; FIG. 13A). The N-terminal region of C2c1p has no significant similarities with other proteins. Secondary structure prediction indicates that this region adopts a predominantly alpha helical conformation. The two TnpB similarity segments encompass three RuvC-like nuclease type catalytic motifs with diagnostic signature D..E..D catalytic amino acid residues (Aravind et al, 2000, Nucleic Acids Res, vol. 28, 3417-3432) (FIG. 12 , "Regions of TnpB Homology in Class 2 Proteins"); the region corresponding to the bridging helix (also known as the arginine-rich cluster), which in the Cas9 protein is involved in cr-RNA binding; and a small region that appears to be analogous to the TnpB zinc finger (however, most C2c1 proteins lack Zn-binding cysteine residues, indicating that such proteins do not bind zinc, moreover, C2c1 contains multiple insertions and deletions in this region, indicating functional divergence (FIG. 13A, FIG. 13D-H, FIG. 131). The retention of catalytic residues (FIG. 13A) clearly indicates that the RuvC homologous domains of all these proteins are active nucleases. N- the terminal regions of C2c1 do not show significant similarity to any known proteins.Secondary structure prediction shows that the N-terminal regions of the C2c1 proteins adopt a mixed α/β conformation (Fig. 13D-H, Fig. 1 131). suggests that C2c1 loci are best classified as a VB subtype, with loci encoding Cpf1 as a VA subtype.

[001323] Although the cas1 genes associated with this system are very similar, the CRISPR repeats in the respective cassettes are highly heterogeneous, although they are all 36-37 bp in length. and can be classified as unstructured (AG folding energy is -0.5-4.5 kcal/mol, while highly palindromic CRISPR have AGs below -7 kcal/mol). According to the CRISPR scheme (Lange, 2013), several V-B subtype repeats have some sequence or structural similarity to type II repeats (FIGS. 41A-T). However, most of the repeats could not be assigned to known families of sequences or structures; they could be assigned to four of the 6 superclasses.

[001324] Considering the possibility that putative V-B subtype CRISPR-Cas are structurally similar to type II systems, Applicants have attempted to identify tracr-RNA at the appropriate genomic loci.

[001325] Comparison of VB-type CRISPR cassette spacers with a non-redundant database of nucleotide sequences resulted in the discovery of several matches with various bacterial genomes. In particular, one of the spacers from Alicyclobacillus acidoterrestris and one of the spacers from Brevibacillus agri corresponded to undefined genes within the predicted prophages integrated into the respective bacterial genomes (FIGS. 41A-L).

[001326] Contemplated Type VI systems. The second group of potential CRISPR-Cas loci, designated C2c2 (class 2, candidate 2), were found in the genomes of 5 major bacterial taxa, including alpha-proteobacteria, bacilli, clostridia, fusobacteria, and bacteroids (Fig. 8A-B. "Organization of complete loci of class 2 systems" in Fig. 42A-B). A number of C2c2 loci include the cas1 and cas2 genes, as well as a large protein (C2c2p) that does not show sequence similarity to C2c1, Cpf1, or Cas9 and the CRISPR cassette; however, unlike C2c1, C2c2p is often encoded next to the CRISPR cassette, but not Cas1-cas2 (FIG. 15, neighborhood of C2c2, FIGS. 42A-B). Although the inventors' computational strategy initially identified C2c2 loci encompassed the cas1 and cas2 genes, subsequent searches have shown that most of these loci can only consist of the c2c2 gene and the CRISPR cassette. Such apparently incomplete loci may either encode defective CRISPR-Cas systems or may function in conjunction with an adaptation module encoded elsewhere in the genome, as has been found, for example, for some type III systems (Majumdar et al., 2015, RNA, vol.21, 1147-1158). In the Cas1 phylogenetic tree, the Cas1 proteins from the C2c2 loci are distributed among two clades. The first clade includes Cas1 from Clostridia and is located in a type II subtree along with a small type III-A branch (FIGS. 10A and 10B, FIGS. 10C-V, Cas1 tree). The second clade consists of Cas1 proteins from Leptotrichia C2c2 loci and is located within a mixed clade that mainly contains Cas1 proteins from CRISPR-Cas type III-A systems. A database search using HHpred and PSI-BLAST found no sequence similarities between C2c2p and other proteins. However, verification of multiple alignments of C2c2p protein sequences led to the identification of two strongly conserved RxxxxH motifs that are characteristic of HEPN domains (Nucleotide Binding Site of Higher Eukaryotic and Prokaryotic Organisms) (Anantharaman et al., 2013, Biol Direct, vol. 8, 15 , Grynberg et al., 2003, Trends in biochemical sciences, vol 28, 224-226) (FIG. 11 and FIG. 13B, FIG. 13J-N). Secondary structure prediction of the protein indicates that these motifs are in structural contexts consistent with the HEPN domain structure, as well as general secondary structure prediction for the corresponding C2c2p portion. HEPN domains are small (-150 a/k) alpha-helical domains with different sequences but highly conserved catalytic motifs that have been shown or predicted to have RNase activity and are often associated with different defense systems ( Anantharaman, 2013) (FIGS. 13B and 16, HEPN RxxxxH motif in the C2c2 family). The HEPN domain sequences are little conserved, except for the RxxxxH catalytic motif. Although the sequences of the two putative C2c2 HEPN domains bear little resemblance to other HEPN domains except for the RxxxxH catalytic motifs, the domain type is strongly supported by secondary structure predictions that indicate that each motif is within compatible structural contexts (Fig. 13B , Fig.13J-N). In addition, the predicted secondary structure of the entire sequence for each putative domain is also consistent with the fold of the HEPN domain (FIGS. 13J-N). Thus, it seems likely that C2c2p contains two active HEPN domains. The HEPN domain is not new to CRISPR-Cas systems, as it is often associated with the CARF (CRISPR-Associated Rossmann Fold) domain in the Csm6 and Csx1 proteins, which are present in many type III CRISPR-Cas systems (Makarova, 2014). These proteins are neither adaptation modules nor effector complexes, but it is hypothesized that they are able to perform some ancillary but not yet characterized functions in related CRISPR systems, namely that they may be components of a related immunity module that is present in most systems. CRISPR-Cas, and works during programmed cell death (apoptosis), and also performs regulatory functions during the CRISPR response (Koonin, 2013; Makarova, 2012; Makarova, 2013). However, C2c2p differs from Csmo and Csx1 in that this larger protein is the only common protein encoded at the C2c2 loci, with the exception of Cas1 and Cas2. Thus, it seems likely that C2c2p is an effector in putative new CRISPR-Cas systems, and HEPN domains are accordingly their catalytic residues. Outside the predicted HEPN domains, the C2c2p sequence showed no similarity to other proteins and was predicted to adopt a mixed alpha/beta secondary structure with no discernible resemblance to any known protein folding methods (FIGS. 13J-N).

[001327] CRISPR cassettes at C2c2 loci are highly heterogeneous, ranging in length from 35 to 39 bp. and unstructured (folding energy from -0.9 to 4.7 kcal/mol). According to CRISPR mapping (Lange, 2013), these CRISPRs do not belong to any of the established structural classes and belong to 3 out of 6 superclasses. Only CRISPR from Listeria seeligeri could be assigned to sequence family number 24, which is commonly associated with type II-C systems (FIGS. 42A-L).

[001328] Analysis of the spacer loci of C2c2 identified one 30 nucleotide region identical to the genomic sequence of Listeria weihenstephanensis and two partial matches with bacteriophage genomes, in particular, the spacer from Listeria weihenstephanensis matched the tail gene of the bacteriophage Listeria (Fig. 42A-L).

[001329] Given the uniqueness of the predicted C2c2 effector complex, these systems can be classified as putative type VI CRISPR-Cas systems. Moreover, since all experimentally characterized and enzymatically active HEPN domains are RNases, type VI systems are likely to act at the level of RNAs, such as mRNAs,

[001330] Contemplated Type V-C systems. The third group of candidate loci includes exclusively metagenomic sequences and, therefore, cannot be assigned to specific taxa. These loci include only two protein-coding genes that encode Cas1 and a large protein designated C2c3 (class 3 candidate 3) (Fig. 8A "Organization of complete class 2 system loci"; Fig. 14, C2c3 neighborhood, Fig. 44A-B ). C2c3 proteins are in the same size range as Cpf1 and C2c1 and similarly contain a TnpB homologous domain at their C-termini, which, in contrast to the corresponding C2c1 domain, showed limited but significant similarity to Cpf1 (Fig. 13A and 13C). The TnpB C2c3 homology regions contain three RuvC-like nuclease-type catalytic motifs, with a diagnostic triad of D..E..D catalytic amino acid residues (Aravind et al., 2000, supra), a region corresponding to the bridging helix (also known as the arginine-rich cluster ), which is involved in cr-RNA binding in Cas9, and a small region that appears to correspond to the "zinc finger" of TnpB (Zn-binding cysteine residues are conserved in C2c3). The retention of catalytic residues clearly indicates that the RuvC homologous domains of all these proteins are active nucleases. The N-terminal regions of C2c1 and C2c3 do not show significant similarity to each other or to any known proteins. Secondary structure prediction shows that the N-terminal regions of C2c3 proteins adopt a mixed cx/j3 conformation. Thus, the general architectures of the C2c1 and C2c3 domains and, in particular, the organization of the RuvC domain are similar to the Cpf1 domains, but differ from the Cas9 architecture. This suggests that the C2c1 and C2c3 loci are more correctly classified as the V-B (see above) and V-C subtypes, respectively, with the loci encoding Cpf1 currently designated as the V-A subtype.

[001331] Among the c2c3 loci, only one contains a CRISPR cassette with uncharacteristically short spacers of 17-18 nucleotides in length. Repeats in cassettes are 25 bp long. and it turns out that they are unstructured and have an energy of 1.6 kcal/mol (FIGS. 43A-F).

[001332] Spacers from a single C2c3 contingent containing a CRISPR cassette are too short to have statistically significant matches. However, several matches were found with sequences from the predicted prophages (FIGS. 43 A-F).

[001333] TnpB protein subtypes with significant similarities to the known (Cas9) and the three putative class 2 effectors described here (Cpf1, C2c1 and C2c3) did not match (FIGS. 13A and 13C). Although the divergence in the sequence of TnpB-like domains is too great to provide a reliable phylogenetic analysis, these data suggest that the four currently known class 2 large effector proteins, Cas9, Cpf1, C2c1, and C2c3, evolved independently of the genes of different transposable genetic elements.

[001334] Although most of the spacers at the new CRISPR-Cas loci described herein were not significantly similar to any available sequences, the existence of spacers that match the phage genome implies that these loci may encode active functional adaptive immune systems. A small proportion of phage-specific spacers is typical of CRISPR-Cas systems and most likely indicates their dynamic evolution and a small proportion of viral diversity, which is represented in modern sequence databases. This interpretation is consistent with the observation that closely related bacterial strains encoding homologous CRISPR-Cas loci typically contain unrelated collections of spacers, exemplified by the C2c2 loci from Listeria weihemtephanensis and Listeria newyorkensis (FIGS. 45A-C).

[001335] Applicants have applied a simple and understandable computational strategy to predict new class 2 CRISPR-cas systems. Previously described class 2 systems, namely type II and putative type V, consisted of the cas1 and cas2 (and in some cases, cas4) genes, containing an adaptation module and one large protein that makes up the effector module. Therefore, Applicants have suggested that any genomic locus containing cas1 and a large protein could be a potential candidate for a new class 2 system and deserve careful investigation. This analysis, using sensitive protein sequence comparison methods, led to the identification of three most likely candidates, two of which are separate subtypes of the previously described putative type V (subtypes VB and VC), while the third can be classified as a new putative type VI, due to the presence new predicted effector protein. Many of these novel systems occur in bacterial genomes that do not contain other CRISPR-Cas loci (FIGS. 44A-E), indicating that type V and type VI systems can function autonomously. The candidate loci discussed herein were tested using functional methods that revealed the expression and processing of the respective mature crRNA producing CRISPR cassettes, identified putative tracr RNA (where possible), demonstrated interference when expressed in E. coli , determined PAM and identified the minimum components required for cleavage of the lysate.

[001336] Type V systems encode predicted effector proteins that resemble Cas9 in general domain architecture, but, unlike Cas9, the RuvC-like domains of Cpf1, C2c1, and C2c3 are adjacent and lack the Cas9-specific inserts, in particular the nuclease domain HNH. The presence of one instead of two nuclease domains indicates that type V effector proteins are mechanically different from Cas9, in which the HNH and RuvC domains are responsible for cleaving complementary and non-complementary target DNA strands, respectively (Chen et al., 2014, The Journal of biological chemistry7, vol.289, 13284-13294, Gasiunas et al., 2012, Proceedings of the National Academy of Sciences of the United States of America, vol.109, E2579-2586; Jinek et al., 2012, Science, vol. 337, 816-821). The predicted type V effector proteins can form dimers in which two RuvC-like domains can cleave opposite strands of the target molecule.

[001337] Putative type VI CRISPR-Cas systems appear to be based on a novel effector protein that contains two predicted HEPN domains that, like previously characterized HEPN domains, may have RNase activity that type VI systems can target and cleave mRNA. mRNA targeting has previously been reported for some type III CRISPR-Cas systems (Hale et al., 2014, Genes Dev, vol. 28, 2432-2443, Hale et al, 2009, Cell, vol. 139, 945-956, Peng et al., 2015, Nucleic acid research, vol.43, 406-417). An alternative possibility is that C2c2 is the first DNase in the HEPN superfamily, possibly with two HEPN domains each cleaving one strand of DNA. Thus, it seems possible to transform C2c1 and C2c2 into genome editing tools with different target classes.

[001338] To test the functionality of these class 2 CRISPR-Cas systems, Applicants have shown that two C2c1 CRISPR cassettes are expressed, processed into mature crRNAs, and capable of interference when expressed in E. coli. These experiments revealed more characteristics of the C2c'l locus, including: (i) processable direct repeats (DR) at the 5' end of cr-RNA, (ii) a 5' PAM, and (iii) the presence of a short RNA with repeat-anti-repeat homology in relation to to the processed 5' DR, i.e. putative tracr-RNA. The opening of processable DRs at the 5' end of crRNA and 5' PAM supports the development that C2c1 is derived from class 1 systems because these systems exhibit processing of the 5' ends (type I and type III) and the 5' end PAM sequences (type I) (Mojica et al., 2009, Microbiology, vol. 155, 733-740; Makarova et al., 2011, Nat Rev Microbioln, vol. 9, 467-477). In this case, an AT-rich PAM sequence was identified for C2c1, in contrast to the GC-rich PAM sequences of well-characterized class II systems. For the C2c1 loci experimentally characterized herein, Applicants have identified crRNAs that are processed to a length that retains the ability to bind and cofold with putative tracr RNAs, indicating that tracr RNAs may be involved and possibly are even necessary for the formation of complexes. The inventors then used C2c1 expression in human cell culture to experimentally verify that tracr-RNA was involved under these conditions and was required for in vitro cleavage of the target DNA by the particular C2c1 nuclease under investigation.

[001339] Applicants have also shown that when the C2c2 locus from L. seeligeri is expressed in E. coli it is processed into a direct repeat (DR) crRNA at the 5' end of 29 nucleotides long, similar results were obtained for the C2c2 locus from L.shahii . In this case, the degenerate repeat is at the beginning of the sequence rather than at the end, as is typical for most CRISPR sequences, and the sequence and cas genes are transcribed co-directionally. Applicants did not detect the putative tracr-RNA in the C2c2 RNA sequencing data. However, the predicted 29 nt long direct repeat (DR) secondary structure shows a stable hairpin that may be potentially important for complex formation with the C2c2 effector protein.

[001340] In combination with the results of previous analyzes, (Chylinski, 2014; Makarova, 2011), the identification of new class 2 CRISPR-Cas systems reveals a major trend in the development of class 2 CRISPR-Cas systems. Effector proteins of two or three types in this class, apparently evolved from a pool of transposable genetic elements that encode TnpB proteins containing a RuvC-like domain. The sequences of the RuvC-like domains of TnpB and the homologous domains of class 2 effector proteins diverged too much for a reliable phylogenetic analysis. However, for Cas9, the effector protein of the Type II system, a specific group of ancestors seems to be easily identified, namely a family of TnpB-like proteins, especially abundant in cyanobacteria, which show relatively high similarity to Cas9 and share an entire domain domain with Cas9. architecture, namely RuvC-like domains, HNH nuclease domains, and arginine-rich bridging helix (Chylinski, 2014) (Figure 11, Figures 13A and 13B, "Class 2 family domain organization"; Figure 12, Figures 13A and 13B, " homology regions of TnpB in class 2 proteins"). Unlike Cas9, it is not possible to assign Cpf1, C2c1, and C2c3 to a specific family, despite retaining all the motifs concentrated in the catalytic residues of RuvC-like nucleases, these proteins show only limited similarity to the generic TnpB profiles. However, given that C2clp shows no detectable sequence similarity to Cpf1, that Cpf1, C2c1, and C2c3 contain distinct insertions between RuvC motifs and apparently unrelated N-terminal regions, it is most likely that Cpf1, C2c1, and C2c3 arose independently from different families. in the TnpB coding element pool.

[001341] Curiously, TnpB proteins appear to be "destined" for use in effector complexes of class 2 CRISPR-Cas systems, so they appear to have been recruited on many different occasions. This utility of TnpB proteins appears to be related to their predicted ability to cut single-stranded DNA when bound to an RNA molecule via an R-rich bridging helix, which has been shown to bind cr-RNA in Cas9 (Jinek, 2014; Nishimasu, 2014 ; Anders et al., 2014, Nature, vol. 513, 569-573). The functions of TnpB in the life cycles of the corresponding transposons are poorly understood. These proteins are not required for transposition, and in one case the TnpB protein was shown to reduce transposition (Pasternak, 2013), but their mechanism of action remains unknown. An experimental study of TnpB is likely to shed light on the mechanical aspects of CRISPR-Cas class 2 systems. It is worth noting that the mechanisms of Cpf1 and C2c1 may be similar to each other, but they differ significantly from Cas9, since the first two proteins lack the HNH domain, which in Cas9 is responsible for breaking one of the target DNA strands (Gasiunas, 2012) (Jinek, 2012) (Chen, 2014). Accordingly, the use of Cpf1 and C2c1 may lead to additional genome editing opportunities.

[001342] From an evolutionary standpoint, it is striking that Class 2 CRISPR-Cas systems appear to have evolved entirely from different transposable genetic elements, given recent evidence that the cas1 genes likely originated from a separate transposon family (Koonin, 2015, Krupovic, 2014) . Moreover, the probable independent origin of effector proteins from different TnpB families, along with different phylogenetic similarities of the corresponding cas1 proteins, strongly suggests that class 2 systems evolved repeatedly through the combination of different adaptation modules and transposon-derived nucleases, which led to the appearance of effector proteins. . This mode of evolution seems to be the final manifestation of the modularity characteristic of the evolution of the CRISPR-Cas system (Makarova, 2015), which implies that additional combinations of adaptive and effector modules may exist in nature.

[001343] Type VI predicted CRISPR-Cas systems contain a predicted effector protein that contains two predicted HEPN domains that are likely to have RNase activity. These HEPN domains are not part of effector complexes in other CRISPR-Cas systems, but are involved in various protective functions, including a predicted supportive role (Anantharaman, 2013) (Makarova, 2015) in various CRISPR Cas systems. The presence of HEPN domains as catalytic units of the predicted effector module implies that type VI systems target and cleave mRNA. mRNA targeting has already been reported for Type III CRISPR-Cas systems (Hale, 2014; Hale, 2009) (Peng, 2015). Despite the fact that HEPN domains have not yet been found in true transposons, they are characterized by high horizontal mobility and are an integral part of mobile elements such as toxin-antitoxin systems (Anantharaman, 2013). Thus, putative type VI systems seem to fit the general paradigm of modular evolution of class 2 CRISPR-Cas from transposable elements, and new variants and new types of systems are expected to be discovered through analysis of genomics and metagenomics data. Considering that the C2c2 protein is not associated with other class 2 effectors (all of which contain RuvC-like domains, even if they are distantly related), the discovery of type VI can be considered as confirmation of the independent origin of other class 2 variants.

[001344] Considering the looming scenario for the evolution of class 2 systems of transposable elements, it seems useful to study the overall evolution of CRISPR-Cas loci and, in particular, the contribution of transposable elements to this process (Fig.53). The precursors of the adaptive immune system most likely arose from the insertion of a casposon (a transposon that codes for Cas) adjacent to a locus that coded for the primitive innate immune system (iKoonin and Krupovic, 2015, Nature reviews Genetics, vol. 16, 184-192; Krupovic et al., 2014, BMC Biology, vols 12, 36). An important result was also the inclusion of a toxin-antitoxin system that passed on the cas2 gene and could appear either in the ancestral caspison or in the changing adaptive immunity locus (FIG. 51).

[001345] Considering the extremely wide distribution of class 1 systems in archaea and bacteria and the distribution of ancient RRM domains (RNA recognition motif, RRM) in them, there is no doubt that the ancestral system belonged to class 1 (Fig.51). Most likely, the ancestral architecture resembled the existing type III and included the enzymatically active Cas10 protein (Makarova et al, 2011, Biol Direct, vol. 6, 38; Makarova et al, 2013, Biochem Soc Trans, vol. 41, 1392-1400). The Cas10 protein is a homologue of B family DNA polymerases and GGDEF family nucleotide cyclases, which shows significant sequence similarity to these enzymes and retains all catalytic amino acid residues (Makarova et al., 2011, Biol Direct, vol. 6, 38, Makarova et al. , 2006, Biol Direct, volume 1, 7). Structural analysis confirmed the presence of a polymerase cyclase-like domain in Cas10 and additionally revealed a second, degenerate and apparently inactive domain of this family (Khachatryan et al., 2015, Phys Rev Lett, vol. 114, 051801; Shao et al., 2013, Structure , vol.21, 376-384, Zhu and Ye, 2012, FEBS Lett, volume 586, 939-945). The exact nature of the catalytic activity of Cas10 remains unclear, but the catalytic residues of the polymerase cyclase-like domain have been shown to be essential for target DNA cleavage (Saniai et al., 2015, Cell, vol. 161, 1164-1174). Cas8 proteins present in Type I CRISPR-Cas systems are similar in size to Cas10 and occupy equivalent positions in effector complexes (Jackson et al., 2014, Science, vol. 345, 1473-1479; Jackson and Wiedenheft, 2015, Mol Cell, vol. 58, 722-728, Staals et al., 2014, Molecular cell, vol. 56, 518-530), suggestive of an evolutionary relationship between the large subunits of Type III and Type I effector complexes. More specifically, Cas8 proteins that diverge greatly in sequence between Type I subtypes may be catalytically inactive derivatives of Cas10 (Makarova et al., 2011, Biol Direct, vol. 6, 38, Makarova et al., 2015). This scenario suggests a plausible trend in evolution, from a type III-like class 1 ancestral system to type I systems. The divergence of type III and type I systems could be accelerated by the production of Cas3 heliase at the time of type I emergence (FIG. 53). Various class 2 types and subtypes then evolved through multiple substitutions of the gene block encoding class 1 effector complexes by introducing transposable elements encoding various nucleases (FIG. 53). This particular direction of evolution stems from the observation that the adaptation modules of the different class 2 variants are derived from the different class 1 types (FIGS. 10A and 10B).

[001346] Class 2 CRISPR-Cas systems appear to be entirely derived from different mobile elements. In particular, there appear to have been at least two (in subtype V-C), but usually three or, in the case of Type II, even four representatives of mobile elements: (i) an ancestral casposon, (ii) a toxin-antitoxin module, which gave rise to Cas 2 (iii) a transposable element, in many cases coding for TnpB, which was the ancestor of a class 2 effector complex, and (iv) in the case of Type II, the HNH nuclease could have been donated to the ancestral transposon by a self-splicing group I or group II intron ( Stoddard, 2005, Q Rev Biophys, vol 38, 49-95) (Fig. 53). The putative V-C loci described herein encode the ultimate minimal CRISPR-Cas system, the only one currently identified that lacks Cas2; Apparently, highly divergent subtypes of V-C Cas1 proteins are able to independently form an adaptation complex without an auxiliary Cas2 subunit. The multiple emergence of class 2 systems from mobile elements is the ultimate manifestation of modularity characteristic of the evolution of CRISPR-Cas (Makarova et al., 2015).

[001347] The demonstration that different varieties of CRISPR-Cas class 2 systems, regardless of whether they evolved from different transposons, implies that additional variants and new types have yet to be identified. Although most, if not all, new CRISPR-Cas systems are expected to be rare, they can exploit new strategies and molecular mechanisms and could be an important resource for new, diverse applications in genome engineering and biotechnology.

[001348] Modular evolution is a key feature of CRISPR-Cas systems. This mode of evolution appears to be most pronounced in class 2 systems, which evolve through the combination of adaptive modules from various other CRISPR-Cas systems with effector proteins that appear to be recruited from transposable elements on several independent occasions. Given the extreme diversity of transposable elements in bacteria, it seems likely that the effector modules of class 2 CRISPR-Cas systems are also very diverse. Here, Applicants used a simple computational method to identify three new variants of CRISPR-Cas systems, but many more are likely to be contained in bacterial genomes that have not yet been sequenced. Although most, if not all, of these new CRISPR-Cas systems are expected to be rare, they can exploit new strategies and molecular mechanisms and will be an important resource for new applications in genome engineering and biotechnology.

[001349] The TBLASTN program with a cutoff value of 0.01 and low complexity filtering disabled was used to search for the Cas1 profile (Makarova et al., 2015) as a query against the NCSI WGS database. Sequences of contigs or whole genomes in which a hit was found (high score result) were retrieved from the same database. The region around the Cas1 gene (region 20 kb from the start of the Cas1 gene and 20 kb from the end of the Cas1 gene) was extracted and translated using GeneMarkS (Besemer et al, 2001, supra). The predicted proteins of each Cas1-coding region were searched using a set of profiles from the CDD database (Marchler-Bauer, 2009) and specific profiles of Cas proteins (Makarova et al., 2015) using the RPS-BLAST program (Marchler-Bauer et al. , 2002, Nucleic Acids Res, Volume 30, 281-283). The previously developed procedure for determining the completeness of CRISPR loci and classifying CRISPR-Cas systems into existing types and subtypes (Makarova et al., 2015) was applied to each locus.

[001350] CRISPRmap (Lange, 2013) was used for reclassification.

[001351] Partial and/or unclassified loci that spanned proteins larger than 500 amino acids were analyzed on an individual basis. Specifically, for each predicted protein encoded by these loci, a search was performed using iterative profile searches with PSI-BLAST (Altschul, 1997), and compositional statistics with low complexity filtering disabled to search for remote similar sequences against a "redundant" database ( NR) of NCBF proteins. For each "non-redundant" protein identified, the WGS database was searched using the TBLAST program (Altschul, 1997). The HHpred program was used with default parameters to identify distant sequence similarity (Soding, 2005), using as queries all the proteins identified in the BLAST search. Multiple sequence alignments were constructed using MUSCLE (Edgar, 2004) and MAFFT (Katoh and Standiey, 2013, Mol Biol Evol, vol, 30, 772-780). Phylogenetic analysis was performed using the FastTree program with the evolutionary WAG model and a discrete gamma model with 20 speed categories (Price et al., 2010, PLoS One, vol. 5, e9490). The secondary structure of the protein was predicted using Jpred 4 (Drozdetskiy, 2015).

[001352] CRISPR repeats were found using PILER-CR (Edgar, 2007, supra) or, for degenerate repeats, CRISPRfinder (Grissa et al., 2007, Nucleic Acids Res, vol. 35, W52-57). The Mfold program (Zukler, 2003, Nucleic Acids Res, vol 31, 3406-3415) was used to find the most stable structures for repeat sequences.

[001353] A search for spacer sequences was performed against the NCBI: NR and WGS databases using MEGABLAST (Morgulis et al., 2008, Bioinformatics, vol. 24, 1757-1764) with default parameters set except that the wordsize parameter was set to 20

[001354] Selected Gene Candidates

[001355] GenID: A; Gene type: C2C1; Organism: 5. bacterium Opitutaceae TAV5; Spacer length - mode (range): 34 (33-37);

DR1:

DR2: no;

tracr-RNA1:

tracr-RNA2: no;

Protein sequence:

[001356] Gene ID: B; Gene type: C2C1; Organism: 7. Bacillus thermoamylovorans strain B4166; Spacer length - mode (range): 37 (35-38);

DR1:

DR2: no; tracr-RNA1;

tracr-RNA2: no;

Protein sequence:

[001357] Gene ID: C; Gene type: C2C1; Organism: 9. Bacillus sp. NSP2.1; Spacer length - mode (range): 36 (35-42);

DR1:

DR2: no; tracr-RNA1:

tracr-RNA2: no;

Protein sequence:

[001358] Gene ID: D; Gene type: C2C2; Organism: 4. Bacterium Lachnospiraceae NK4A144 G619; Spacer length - fashion (range): 35;

DR1:

DR2:

tracr-RNA1: no; tracr-RNA2: no; Protein sequence:

[001359] Gene ID: E; Gene type: C2C2; Organism: 8. Listeria seeligeri serovar 1/2b strain SLCC3954; Spacer length - mode (range): 30;

DR1:

DR2: no; tracr-RNA1:

tracr-RNA2: no; Protein sequence:

[001360] Gene ID: F; Gene type: C2C2;

Organism: 12. Leptotriciawadei F0279;

Spacer length - mode(range): 31;

DR1:

DR2: no;

tracr-RNA1:

tracr-RNA2:

Protein sequence:

[001361] Gene ID: G; Gene type: C2C2;

Organism: l4. Leptotrichiashahii DSM 19757 B031;

Spacer length - mode (range): 30 (30-32);

DR1:

DR2: no;

tracr-RNA1:

tracr-RNA2: no;

Protein sequence:

[001362] Gene ID: H;

Gene type: Cpf1; Organism: Francisellaularensis subsp. novicida U112, Spacer length - mode (range): 31;

DR1:

DR2: no;

tracr-RNA1:

tracr-RNA2: no;

Protein sequence:

[001363] Genes for synthesis

[001364] For genes from A-H, Applicants optimize the genes for human expression and add the following DNA sequence to the end of each gene. Note that this DNA sequence contains a stop codon (underlined), so no stop codon is added to the gene's codon-optimized sequence:

[001365] Avoid the following restriction sites for optimization: BamHI, EcoRI, HindIII, BsmBI, BsaI, BbsI, AgeI, XhoI, Ndel, NotI, Kpnl, BSrGI, SpeI, XbaI, NheI

[001366] These genes have been cloned into a simple mammalian expression vector:

[001367] A

[001368]

[001371] C

[001372]

[001373] D

[001374]

[001377] F

[001378]

[001379] G

[001380]

[001381]H

[001382]

[001383] Loci A to G are cloned and inserted into a low copy number plasmid. A vector that does not contain resistance to Amp is used.

[01384] A-locus

[01385]

Example 3: Further Evaluation of C2c2p and Associated Components

Example 3: Further Evaluation of C2c1 and Related Components

[001398] Applicants have demonstrated in the experiments described that CRISPR-Cas class 2 loci are expressed to form mature crRNA and encode putative tracrRNA and functional RNA-targeting nucleases.

[001399] Applicants analyzed a representative C2c1 locus, ie the C2c1 Alticclobacillis acidoterrestris (AacC2c1) CRISPR locus, and performed RNA sequencing (RNA-seq) and Northern blotting to characterize its transcriptional activity.

[001400] For RNA sequencing , Alicyclobacillus acidoterrestris (ATCC 49025) was cultured using ATCC Medium 1.655 suspension at 50°C and 300 rpm. E. coli containing heterologous constructs were cultured in suspension in Luria medium supplemented with appropriate antibiotics at 37°C and 300 rpm. Both strains were grown under aerobic conditions and harvested in the stationary growth phase.

[001401] To obtain the AacC2c1 locus for heterologous expression, genomic DNA was isolated from Alicyclobacillus acidoterrestris using the Qiagen DNeasy Blood and Tissue kit and the C2c1 locus was amplified by PCR for cloning into pACYC-184.

[001402] RNA was isolated from bacteria in stationary phase, bacteria were first resuspended in TRIzol and then homogenized with zirconium/silicon beads (BioSpec Products) in a BeadBeater (BioSpec Products) for 7 one minute cycles. Total RNA was purified from homogenized samples using the Miniprep Direct-Zol RNA protocol (Zymo), treated with TURBO DNAase (Life Technologies), and 3'-dephosphorylated with T4 polynucleotide kinase (New England Biolabs). rRNA was removed using a Ribo-Zero bacterial rRNA removal kit (Illumina). RNA sequencing libraries were generated from rRNA-poor RNA using a modification of the previously described crRNA sequencing method (Heidrich et al., 2015, Methods Mol Biol, vol. 1311, 1-21). Briefly, transcripts were poly-A-tailed with E. coli poly-(A)-polymerase (New England Biolabs), ligated to 5' RNA adapters using T4 RNA ligase 1 (ssRNA ligase), high concentrations (High Concentration, New England Biolabs) and reverse transcribed with AffmityScript multitemperature reverse transcriptase (Agilent Technologies). cDNA was PCR amplified with barcoded primers using Herculase II polymerase (Agilent Technologies).

[001403] The finished cDNA libraries were sequenced on MiSeq (Alumina). Reads from each sample were identified based on their associated barcodes and aligned to the RefSeq reference genome using BWA (Li and Durbin, 2009, Bioinformatics, vol. 25, 1754-1760). Pairwise alignments were used to isolate entire transcript sequences using the Picard tools (http://broadinstitute.github.io/picard) and these sequences were analyzed using Geneious 8.1.5.

[001404] Whole transcriptome analysis of RNAseq Aac showed that transcription of the CRISPR array occurs in the same orientation as the Cas genes (FIG. 17). The display redundancy of transcripts mapped to the CRISPR array decreased in the 5' to 3' direction (FIG. 46A). This is consistent with the results of Applicants' northern blot. The CRISPR array demonstrated robust processing of a 34 nt crRNA with a 14 nt 5'-direct repeat (DR) and a 20 nt spacer (FIG. 46A).

[001405] Applicants identified a 79 bp redundant small RNA encoded between the cas2 gene and the CRISPR array and transcribed in the same orientation as the CRISPR array (FIGS. 46A and B). The interior of this small RNA contains a sequence that is highly complementary to the CRISPR truncated repeat sequence (anti-repeat), suggesting that this small transcript is a tracr-RNA. When co-foldingin silico processed repeat CRISPR of 14 nucleotides with this putative tracr-RNA formed a stable predicted secondary structure (Fig.46C).

[001406] Applicants also expressed the C2c1 locus of Alicyclobacillus acidoterrestris (ATCC 49025) in E. coli and analyzed its expression using Northern blotting. The procedure was carried out essentially as described in Pougach and Severinov, 2012 (Methods Mol Biol, vol. 905, 73-86). E. coli BL21 AI cells were transformed with plasmid pACYCduet-1 containing the T7 promoter-controlled cas Alicyclobacillus acidoterrestris operon and pCDF-1b plasmid containing a minimal CRISPR cassette with a single spacer. Total RNA was extracted from 5 ml E. coli cells induced with 1 mM arabinose/0.2 mM IPTG and grown to an OD600 of 0.8-1.0. Cells were lysed by a 5 minute treatment with Max Bacterial Enhancement Reagent followed by RNA purification with TRIzol Reagent (Thermo Fisher Scientific). 15 pg of total RNA was separated on a denaturing 8 M urea-12% polyacrylamide gel and electrophoretically transferred to a Hybond-XL membrane (GE Healthcare) using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). The membrane was dried and then cross-linked with ultraviolet radiation. ExpHyb hybridization solution (Clontech) was used to hybridize according to manufacturer's instructions for 1 hour at 40°C with 32P-tagged oligonucleotide probes.

[001407] Based on the observation that the putative tracr RNA in A. acidoterrestris contains a characteristic anti-repeat sequence, Applicants sought to predict potential tracr RNAs for the remainder of the identified C2c1 loci as well as C2c2 and C2c3 by looking for anti-repeat sequences at each locus. In many CRISPR-Cas loci, the repeat located at the far end of the CRISPR sequence from the promoter is degenerate and has a sequence that is clearly different from the rest of the repeats (Biswas et al., 2014, Bioinformatics, vol. 30, 1805-1813). Such degenerate repeats were found in several C2c1 and C2c2 systems (FIGS. 9, 14 and 15), allowing applicants to predict the direction of transcription of the sequence. Integrating this information, putative tracr-RNAs were identified for 4 of 13 C2c1 loci and 4 of 17 C2c2 loci (FIGS. 9, 14 and 15, FIGS. 41A-M, FIGS. 42A-N). In some subtypes II-B and II-C, the CRISPR sequence is transcribed in the opposite direction, starting with a degenerate repeat (Sampson et al., 2013, Nature, vol. 497, 254-257; Zhang et al, 2013, Mol Cell, vol. 50, 488-503). Accordingly, the inventors attempted to predict tracr RNA at different positions relative to the CRISPR sequence, but were unable to identify additional candidate tracr RNA sequences. Of course, tracr-RNA prediction for other loci has been difficult due to a combination of factors such as incomplete repeat complementarity, lack of an associated CRISPR sequence, and/or potential loci incompleteness. In addition, there is a possibility that not all class 2 CRISPR systems require tracr-RNA.

[001408] Given the robust expression of transcripts at the Aac locus and the identification of processed tracr-RNA and cr-RNA, Applicants sought to test the functionality of the AacC2c1 enzyme. To this end, an interference screen was developed to determine if these loci are active and to identify the necessary protospacer adjacent motif (PAM) that determines where the effector protein will cleave in known type II systems (FIG. 47A). A library of plasmids conferring ampicillin resistance and carrying protospacers flanked by 7 random nucleotides of nucleotides was constructed. Although the presence of a DR fragment on the 5' side of the cr-RNA suggests the presence of 5'-PAM, individual libraries with random 7 nucleotide sequences upstream or downstream of the protospacer were analyzed. Over 10 colonies were pooled to represent all 16384 possible PAM sequences. CellsE. colicarrying either the AacC2c1 locus plasmid (FIG. 46D) or the control plasmid (pACYC184) were transformed with 5' or 3' PAM libraries.

[001409] Specifically, randomized PAM plasmid libraries were constructed using synthesized oligonucleotides (IDTs) consisting of 7 randomized nucleotides either upstream or downstream of the spacer 1 target. large Klenow Fragment for the synthesis of the second strand. The dsDNA product was assembled into linearized PUC19 using Gibson cloning. Stabl3E. coli cells were transformed with cloned products and harvested and collected over 10⁷ cells. Plasmid DNA was isolated using the Qiagen maxi kit. 360 ng of the pooled library was transformed into cellsE. coli, transformed with the AacC2c1 locus, and pACYC-184. After transformation, cells were plated on LB-agar plates supplemented with ampicillin and chloramphenicol and grown at 37°C. After 16 hours of growth > 4×10⁶ cells and plasmid DNA were isolated using the Qiagen maxi-prep kit. The PAM target region was amplified and sequenced using Illumina. MiSeq with single ended 150 cycles.

[001410] In this experimental setup, PAM protospacers that are recognized by the AacC2c1 complex will be destroyed, making cells sensitive to ampicillin and expected to show depletion on the screen. After overnight growth of the bacteria, target plasmids were isolated and subjected to deep sequencing. The effectiveness of each PAM was determined by calculating its relative level in the AacC2c1 locus sample compared to the control sample.

[001411] While the 3 nucleotide PAM screen did not show significant PAM depletion, the 5'-PAM library screen identified 364 PAMs that were significantly reduced; all of these PAMs had the sequence NNNNTTN (FIG. 47B). However, bases other than C were slightly preferred at the 5' position immediately adjacent to the protospacer, these results indicate that the 5-TTN motif is recognized by the AacC2c1 complex.

[001412] The proposed PAM was tested using the first spacer of the AacC2c1 locus and all four TTN PAMs (FIG. 47C). Sequences corresponding to both PAM and non-PAM were cloned into digested pUC19 and ligated with T4 ligase (enzymes). Competent E. coli with AacC2c1 locus plasmid or pACYCl84 plasmid was transformed with 20 g of PAM plasmid and plated on LB agar plates supplemented with ampicillin and chloramphenicol. After 18 hours, colonies were counted using OpenCFU (Geissmann 2013, PLoS ONE, vol 8, e54072). The results of this experiment confirm that 5'TTN PAM is required for interference, and this interference is somewhat reduced in the case of 5'TTC PAM (FIG. 47C).

[001413] Applicants then tried to find out whether it is possible to express C2cl nuclease using human cells and at the same time explore its requirements for tracr-RNA. To this end, Applicants generated a codon-optimized human AacC2c1 DNA construct and assembled a protein lysate of protein-expressing HEK293 cells. In particular, a codon-optimized C2c1 protein carrying the N-terminal NLS was developed for human expression. The protein sequence was synthesized and cloned into the pcDNA3.1 Genscript expression plasmid. Using the Lipofectamine 2000 reagent (Life Technologies), 2000 ng of expression plasmids were transfected into HEK293FT cells (6-well plates at 90% confluence). After 48 hours, cells were washed with DPBS (Life Technologies), lysed using lysis buffer (20 mM Hepes pH 7.5, 100 mM KC1, 5 mM MgCL, 1 mM DTT, 5% glycerol, 0.1% Triton X-100 , IX cOmplete Protease Inhibitor Cocktail (Roche)]. splitting analyses.

[001414] Based on the longest crRNA transcript observed in RNA sequencing, Applicants designed a crRNA with a direct repeat (DR) of 20 nucleotides in length and a 22 nucleotide spacer targeting the 639-bp region of the EMX1 locus, which was amplified by PCR from human genomic DNA. Since A. acidoterrestris grows optimally at 50°C (Chang and Kang, 2004, Crit Rev Microbiol, vol. 30, 55-7-4), applicants have suggested that the AacC2c1 protein may work best at this temperature. Therefore, digestion was performed using a mammalian lysate from cells expressing C2c1 protein at 50° C., unless otherwise indicated, in digestion buffer (NEBuffer 3, 5 mM DTT) for 1 hour. In each digestion reaction, 200 μg of target DNA and an equimolar ratio of cr-RNA to tracr-RNA (500 μg of cRNA) were used. RNA was preliminarily annealed by heating at 95°C and slowly cooled to 4°C. The target DNA consisted of either genomic PCR amplicons from the EMX1 gene or the first AacC2c1 protospacer locus cloned into pUC19. The reaction mixture was purified by PCR. (Qiagen) and run on 2% agarose E-gels (Life Technologies).

[001415] At 50°C, Applicants have shown that the longest expressed tracr RNA observed in RNAseq experiments (151 nucleotides) is required for DNA cleavage using C2c1 cell lysate and that cleavage is specific to the complementary region between cRNA and target DNA (FIG. 48A). Since experiments with RNA-seq yielded putative variable length tracr RNA transcripts (FIG. 46A), the present inventors tested a series of 3'-truncated tracr RNA transcripts and found that the shortest tracr RNA cleaving with C2c1 cell lysate , is a sequence of 78 nucleotides (Fig.48B). Using this minimum tracr-RNA, the inventors showed that 50° C. is indeed the optimal cleavage temperature and showed no observed cleavage below 40° C. using C2c1 cell lysate (FIG. 48C).

[001416] To further validate the PAM requirements for C2c1, Applicants designed cRNAs for the PAM protospacer-1 validation constructs used in the above experiments (FIG. 47C) and demonstrated cleavage for TTT, TTA and TTC PAM but not GGA using tracr-RNA of 78 nucleotides (Fig. 4DD).

[001417] Given the discovery of a sequence-specific, redirected DNA cleavage activity for the AacC2c1 nuclease, Applicants hypothesized that, like Cas9, the C2RRRRRTRRANA cr-RNA duplex tracr-RNA could be processed into unidirectional RNA (sg-RNA) by attaching a 3' tracr- RNA of 78 nucleotides to the 5'end of the cr-RNA (Fig. 48E). Target cleavage activity similar to that obtained for the tracr-RNA cr-RNA duplex was observed for sg-RNA with both EMX1 plasmid targets and protospacer 1 (FIG. 48F). These experiments showed that a lysate from human cells expressing C2c1 could cleave the target DNA, identified the temperature optimum of the enzyme, and demonstrated the need for a cr-RNA duplex of tracr-RNA or sg-RNA and 5'-PAM for AacC2c1 nuclease activity.

[001418] In further experiments, applicants study the domain structure and organization, as well as the expression of the CRISPR template in different bacterial strains containing CRISPR loci, designated as subtype V-B, as, for example, shown in figa and 8B. Applicants have optimized methods for obtaining C2c1 loci from other bacterial strains.

[001419] In further experimentation, Applicants are developing pACYC cloning from isolated genomic bacterial strains and cultured bacterial strains to conduct DNA/RNA cleavage experiments in E. coli with effector proteins encoded by CRISPR loci.

[001420] In further experimentation, Applicants are developing bait PAM libraries to evaluate the DNA cutting ability of the C2c1p effector protein described here. Applicants are developing DNA cutting experiments based on cutting the resistance gene transcript.

[001421] In further experiments, Applicants test function in mammalian cells using U6 PCR products: spacer (DR-spacer-DR) (in some aspects, spacers may be referred to as cr-RNA or guide RNA or a similar term as described in this application ) and tracr for the indicated strains indicated as subtype V-B in FIGS. 8A and 8B.

[001422] Example 4: Further evaluation of the C2c1 orthologo locus

Applicants also screened the C2c1 locus from Bacillus thermoamylovorans (Bth): Applicants expressed the B. Thermoamylovorans C2c1 (BthC2c1.) locus in E. coli and collected RNA-Seq data demonstrating transcript generation. The BthC2c1 locus was synthesized using Genscript and cloned into the pET-28 vector. Cells containing plasmids were made competent using a Z-competent kit (Zymo). E. coli containing heterologous constructs were cultured in suspension in Luria medium supplemented with appropriate antibiotics at 37°C and 300 rpm. Bacteria were grown under aerobic conditions and harvested in the stationary growth phase.

[001423] The RNA was isolated and RNA sequencing was performed using the methods outlined in Example 3.

[001424] Full transcriptome sequencing of synthesized BthC2c1. locus cloned at pET-28 in E. coli revealed significant processing of both as spacers, as well as expression of a 91 nt RNA (Fig. 49A) that has a secondary structure and a repeat-anti-repeat-repeat motif similar to Aac tracr RNA (Fig. 49B). The putative tracr sequence would be a ~91 nt long tracr having the following sequence:

CGAGGUUCUGUCUUUUGGUCAGGACAACCGUCUAGCUAUAAGUGCUGCAGGGGU

GUGAGAAAACUCCUAUUGCUGGACGAUGUCUCUUUUAU

[001425] In an embodiment of the invention, a direct repeat (DR) sequence can be processed to a 15 bp polynucleotide having the sequence UACGAGGCAUUAGCAC.

[001426] Applicants have determined a consensus PAM for BthC2c1 from a PAM detection screen. Applicants transformed the PAM library with the appropriate spacer into E. coli cells containing the BthC2c1 locus and compared depletion with pET-28. PAM screening was performed as described for AACC2c1 PAM screening, except that after transformation, cells were plated on LB-agar plates supplemented with ampicillin and kanamycin. This screen showed that BthC2c1 uses 5' T-rich PAM with the ATTN consensus sequence (FIG. 49C).

[001427] Applicants also expressed the C2c1 locus of B. sp. in E. coli and obtained RNAseq data demonstrating transcript generation. The arrow in Fig. 50 indicates the putative tracr RNA associated with the locus, while all reads are shown. The putative tracr sequence may be a 101 nucleotide long tracr having the following sequence:

UCAGUCGCACGCUAUAGGCCAUAAGUCGACUUACAUAUCCGUGCGUGUGCAUUAU

GGGCCCAUCCACAGGUCUAUUCCCACGGAUAAUCACGACUUUCCAC

[001428] In one possible embodiment of the invention, the direct repeat (DR) sequence can be processed up to 20 nucleotides having the sequence GAAAGCUUCGUGGUUAGCAC (FIG. 51). Applicants have also shown co-folding of DR and tracr RNA associated with the C2c1 locus of B. sp. (Fig.52)

Example 5: Adaptation modules of new class 2 systems likely evolved independently from different class 1 units

[001429] Cas1, a specialized DNase and integrase that plays a central role in CRISPR adaptation (Nunez et al., 2014, Nature, Nature structural & molecular biology, vol. 21, 528-534; Nunez et al., 2015, vol. 519, 193-8), is the most conserved Cas protein (Takeuchi et al., 2012, J Bacteriol, vol. 194, 1216-1225) and the only one for which complex phylogenetic analysis is possible (Makarova et al., 2011b, Nat; Rev Microbiol, vol. 9, 467-477, Makarova et al., 2015). In the Cas1 phylogenetic tree, the putative VB subtype (C2c1) is a largely monophyletic group and clusters with type IU (FIGS. 10A and 10B, FIGS. 10C-V). Among all (putative) CRISPR-Cas loci, only types I-U and C2c1 encode the fusion of Cas1 and Cas4 proteins. Together, this common characteristic and the phylogenetic affinity of Cas1 strongly suggest that the adaptation module of the V-B subtype is derived from the adaptive type I-U. Type V-C Cas1 is the most distinct Cas1 sequence variant discovered to date, as shown by the long branch in the phylogenetic tree (FIGS. 10A and 10B). In the Cas1 tree, the type V-C branch is in subtype I-B (FIGS. 10A and 10B), although the positioning of such a rapidly evolving group must be taken with care. Type VI Cas1 proteins are distributed between two clades. The first group includes Cas1 from Leptotrichia and is located in the type II subtree along with a small type III-A branch. The second group includes Cas1 proteins from Clostridia C2c2 loci and refers to a mixed branch that mainly contains Cas1 proteins from Type III-A CRISPR-Cas type systems (FIGS. 10A and 10B).

[001430] Although Cas2 is a small and relatively non-conserved protein for which it is difficult to obtain a reliable phylogeny, all available evidence points to evolutionary coherence of the adaptation module, i.e. co-evolution of the cas1 and cas2 genes (Chylinsky et al., 2014), Nucleic Acids Res. , Vol. 42, 6091-105, Norais et al., 2013, RNA Biol, volume 10, 659-670). Taken together and not limited to any hypothesis, these data show that the adaptation modules of the new Class 2 CRISPR-Cas systems come from different Class 1 variants.

[001431] While preferred embodiments of the present invention have been demonstrated and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications and substitutions can now be developed by those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described in the present description, can be used in the implementation of the invention. It is intended that the following claims define the scope of the invention and that methods and structures falling within the scope of those claims and their equivalents will be so embraced.

--->

SEQUENCE LISTING

<110> THE BROAD INSTITUTE INC.

MASSACHUSETTS INSTITUTE OF TECHNOLOGY

PRESIDENT AND FELLOWS OF HARVARD COLLEGE

RUTGERS, THE STATE UNIVERSITY OF NEW JERSEY

SKOLKOVO INNOVATION OF SCIENCE AND TECHNOLOGY

<120> NOVEL CRISPR ENZYMES AND SYSTEMS

<130> 47627.99.2136

<140> US 15/842,073

<141> 2017-12-14

<150> PCT/US2016/038238

<151> 2016-06-17

<150> 62/245.264

<151> 2015-10-22

<150> 62/181.663

<151> 2015-06-18

<160> 1786

<170>PatentIn version 3.5

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<211> 7

<212> PRT

<213> Simian virus 40

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Pro Lys Lys Lys Arg Lys Val

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<210> 2

<211> 16

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Nucleoplasmin bipartite NLS sequence"

<400> 2

Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

1 5 10 15

<210> 3

<211> 9

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

C-myc NLS sequence"

<400> 3

Pro Ala Ala Lys Arg Val Lys Leu Asp

fifteen

<210> 4

<211> 11

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

C-myc NLS sequence"

<400> 4

Arg Gln Arg Arg Asn Glu Leu Lys Arg Ser Pro

1 5 10

<210> 5

<211> 38

<212> PRT

<213> Homo sapiens

<400> 5

Asn Gln Ser Ser Asn Phe Gly Pro Met Lys Gly Gly Asn Phe Gly Gly

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Arg Ser Ser Gly Pro Tyr Gly Gly Gly Gly Gln Tyr Phe Ala Lys Pro

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Arg Asn Gln Gly Gly Tyr

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<210> 6

<211> 42

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

IBB domain from importin-alpha sequence"

<400> 6

Arg Met Arg Ile Glx Phe Lys Asn Lys Gly Lys Asp Thr Ala Glu Leu

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Arg Arg Arg Arg Val Glu Val Ser Val Glu Leu Arg Lys Ala Lys Lys

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Asp Glu Gln Ile Leu Lys Arg Arg Asn Val

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<210> 7

<211> 8

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Myoma T protein sequence"

<400> 7

Val Ser Arg Lys Arg Pro Arg Pro

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<210> 8

<211> 8

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Myoma T protein sequence"

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Pro Pro Lys Lys Ala Arg Glu Asp

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<210> 9

<211> 8

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<213> Homo sapiens

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<213> Mus musculus

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Arg Lys Leu Lys Lys Lys Ile Lys Lys Leu

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Arg Glu Lys Lys Lys Phe Leu Lys Arg Arg

1 5 10

<210> 15

<211> 20

<212> PRT

<213> Homo sapiens

<400> 15

Lys Arg Lys Gly Asp Glu Val Asp Gly Val Asp Glu Val Ala Lys Lys

1 5 10 15

Lys Ser Lys Lys

twenty

<210> 16

<211> 17

<212> PRT

<213> Homo sapiens

<400> 16

Arg Lys Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys

1 5 10 15

Lys

<210> 17

<211> 4

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 17

Gly Gly Gly Ser

one

<210> 18

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 18

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 19

<211> 30

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 19

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25 30

<210> 20

<211> 45

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 20

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

35 40 45

<210> 21

<211> 60

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 21

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

35 40 45

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

50 55 60

<210> 22

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 22

Gly Gly Gly Gly Ser

fifteen

<210> 23

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 23

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 24

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 24

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

twenty

<210> 25

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 25

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25

<210> 26

<211> 35

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 26

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser

35

<210> 27

<211> 40

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 27

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Gly Gly Gly Gly Ser

35 40

<210> 28

<211> 50

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 28

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

35 40 45

Gly Ser

fifty

<210> 29

<211> 55

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 29

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

20 25 30

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

35 40 45

Gly Ser Gly Gly Gly Gly Ser

50 55

<210> 30

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<220>

<221> MOD_RES

<222> (2)..(2)

<223>Aminohexanoyl

<220>

<221> MOD_RES

<222> (5)..(5)

<223>Aminohexanoyl

<220>

<221> MOD_RES

<222>(8)..(8)

<223>Aminohexanoyl

<220>

<221> MOD_RES

<222> (11)..(11)

<223>Aminohexanoyl

<400> 30

Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg

1 5 10

<210> 31

<211> 20

<212> DNA

<213> Homo sapiens

<400> 31

gagtccgagc agaagaagaa 20

<210> 32

<211> 20

<212> DNA

<213> Homo sapiens

<400> 32

gagtcctagc aggagaagaa 20

<210> 33

<211> 20

<212> DNA

<213> Homo sapiens

<400> 33

gagtctaagc agaagaagaa 20

<210> 34

<211> 12

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 34

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10

<210> 35

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

peptide"

<400> 35

Ala Glu Ala Ala Ala Lys Ala

fifteen

<210> 36

<211> 9

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

'LAGLIDADG' family motif peptide"

<400> 36

Leu Ala Gly Leu Ile Asp Ala Asp Gly

fifteen

<210> 37

<211> 5

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

'GGDEF' family motif peptide"

<400> 37

Gly Gly Asp Glu Phe

fifteen

<210> 38

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 38

gccgcagcga augccguuuc acgaaucguc aggcgg 36

<210> 39

<211> 75

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 39

gcuggagacg uuuuuugaaa cggcgagugc ugcggauagc gaguuucucu uggggaggcg 60

cucgcggcca cuuuu 75

<210> 40

<211> 1388

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 40

Met Ser Leu Asn Arg Ile Tyr Gln Gly Arg Val Ala Ala Val Glu Thr

1 5 10 15

Gly Thr Ala Leu Ala Lys Gly Asn Val Glu Trp Met Pro Ala Ala Gly

20 25 30

Gly Asp Glu Val Leu Trp Gln His His Glu Leu Phe Gln Ala Ala Ile

35 40 45

Asn Tyr Tyr Leu Val Ala Leu Leu Ala Leu Ala Asp Lys Asn Asn Pro

50 55 60

Val Leu Gly Pro Leu Ile Ser Gln Met Asp Asn Pro Gln Ser Pro Tyr

65 70 75 80

His Val Trp Gly Ser Phe Arg Arg Gln Gly Arg Gln Arg Thr Gly Leu

85 90 95

Ser Gln Ala Val Ala Pro Tyr Ile Thr Pro Gly Asn Asn Ala Pro Thr

100 105 110

Leu Asp Glu Val Phe Arg Ser Ile Leu Ala Gly Asn Pro Thr Asp Arg

115 120 125

Ala Thr Leu Asp Ala Ala Leu Met Gln Leu Leu Lys Ala Cys Asp Gly

130 135 140

Ala Gly Ala Ile Gln Gln Glu Gly Arg Ser Tyr Trp Pro Lys Phe Cys

145 150 155 160

Asp Pro Asp Ser Thr Ala Asn Phe Ala Gly Asp Pro Ala Met Leu Arg

165 170 175

Arg Glu Gln His Arg Leu Leu Leu Pro Gln Val Leu His Asp Pro Ala

180 185 190

Ile Thr His Asp Ser Pro Ala Leu Gly Ser Phe Asp Thr Tyr Ser Ile

195 200 205

Ala Thr Pro Asp Thr Arg Thr Pro Gln Leu Thr Gly Pro Lys Ala Arg

210 215 220

Ala Arg Leu Glu Gln Ala Ile Thr Leu Trp Arg Val Arg Leu Pro Glu

225 230 235 240

Ser Ala Ala Asp Phe Asp Arg Leu Ala Ser Ser Leu Lys Lys Ile Pro

245 250 255

Asp Asp Asp Ser Arg Leu Asn Leu Gln Gly Tyr Val Gly Ser Ser Ala

260 265 270

Lys Gly Glu Val Gln Ala Arg Leu Phe Ala Leu Leu Leu Phe Arg His

275 280 285

Leu Glu Arg Ser Ser Phe Thr Leu Gly Leu Leu Arg Ser Ala Thr Pro

290 295 300

Pro Pro Lys Asn Ala Glu Thr Pro Pro Pro Ala Gly Val Pro Leu Pro

305 310 315 320

Ala Ala Ser Ala Ala Asp Pro Val Arg Ile Ala Arg Gly Lys Arg Ser

325 330 335

Phe Val Phe Arg Ala Phe Thr Ser Leu Pro Cys Trp His Gly Gly Asp

340 345 350

Asn Ile His Pro Thr Trp Lys Ser Phe Asp Ile Ala Ala Phe Lys Tyr

355 360 365

Ala Leu Thr Val Ile Asn Gln Ile Glu Glu Lys Thr Lys Glu Arg Gln

370 375 380

Lys Glu Cys Ala Glu Leu Glu Thr Asp Phe Asp Tyr Met His Gly Arg

385 390 395 400

Leu Ala Lys Ile Pro Val Lys Tyr Thr Thr Gly Glu Ala Glu Pro Pro

405 410 415

Pro Ile Leu Ala Asn Asp Leu Arg Ile Pro Leu Leu Arg Glu Leu Leu

420 425 430

Gln Asn Ile Lys Val Asp Thr Ala Leu Thr Asp Gly Glu Ala Val Ser

435 440 445

Tyr Gly Leu Gln Arg Arg Thr Ile Arg Gly Phe Arg Glu Leu Arg Arg

450 455 460

Ile Trp Arg Gly His Ala Pro Ala Gly Thr Val Phe Ser Ser Glu Leu

465 470 475 480

Lys Glu Lys Leu Ala Gly Glu Leu Arg Gln Phe Gln Thr Asp Asn Ser

485 490 495

Thr Thr Ile Gly Ser Val Gln Leu Phe Asn Glu Leu Ile Gln Asn Pro

500 505 510

Lys Tyr Trp Pro Ile Trp Gln Ala Pro Asp Val Glu Thr Ala Arg Gln

515 520 525

Trp Ala Asp Ala Gly Phe Ala Asp Asp Pro Leu Ala Ala Leu Val Gln

530 535 540

Glu Ala Glu Leu Gln Glu Asp Ile Asp Ala Leu Lys Ala Pro Val Lys

545 550 555 560

Leu Thr Pro Ala Asp Pro Glu Tyr Ser Arg Arg Gln Tyr Asp Phe Asn

565 570 575

Ala Val Ser Lys Phe Gly Ala Gly Ser Arg Ser Ala Asn Arg His Glu

580 585 590

Pro Gly Gln Thr Glu Arg Gly His Asn Thr Phe Thr Thr Glu Ile Ala

595 600 605

Ala Arg Asn Ala Ala Asp Gly Asn Arg Trp Arg Ala Thr His Val Arg

610 615 620

Ile His Tyr Ser Ala Pro Arg Leu Leu Arg Asp Gly Leu Arg Arg Pro

625 630 635 640

Asp Thr Asp Gly Asn Glu Ala Leu Glu Ala Val Pro Trp Leu Gln Pro

645 650 655

Met Met Glu Ala Leu Ala Pro Leu Pro Thr Leu Pro Gln Asp Leu Thr

660 665 670

Gly Met Pro Val Phe Leu Met Pro Asp Val Thr Leu Ser Gly Glu Arg

675 680 685

Arg Ile Leu Leu Asn Leu Pro Val Thr Leu Glu Pro Ala Ala Leu Val

690 695 700

Glu Gln Leu Gly Asn Ala Gly Arg Trp Gln Asn Gln Phe Phe Gly Ser

705 710 715 720

Arg Glu Asp Pro Phe Ala Leu Arg Trp Pro Ala Asp Gly Ala Val Lys

725 730 735

Thr Ala Lys Gly Lys Thr His Ile Pro Trp His Gln Asp Arg Asp His

740 745 750

Phe Thr Val Leu Gly Val Asp Leu Gly Thr Arg Asp Ala Gly Ala Leu

755 760 765

Ala Leu Leu Asn Val Thr Ala Gln Lys Pro Ala Lys Pro Val His Arg

770 775 780

Ile Ile Gly Glu Ala Asp Gly Arg Thr Trp Tyr Ala Ser Leu Ala Asp

785 790 795 800

Ala Arg Met Ile Arg Leu Pro Gly Glu Asp Ala Arg Leu Phe Val Arg

805 810 815

Gly Lys Leu Val Gln Glu Pro Tyr Gly Glu Arg Gly Arg Asn Ala Ser

820 825 830

Leu Leu Glu Trp Glu Asp Ala Arg Asn Ile Ile Leu Arg Leu Gly Gln

835 840 845

Asn Pro Asp Glu Leu Leu Gly Ala Asp Pro Arg Arg His Ser Tyr Pro

850 855 860

Glu Ile Asn Asp Lys Leu Leu Val Ala Leu Arg Arg Ala Gln Ala Arg

865 870 875 880

Leu Ala Arg Leu Gln Asn Arg Ser Trp Arg Leu Arg Asp Leu Ala Glu

885 890 895

Ser Asp Lys Ala Leu Asp Glu Ile His Ala Glu Arg Ala Gly Glu Lys

900 905 910

Pro Ser Pro Leu Pro Pro Leu Ala Arg Asp Asp Ala Ile Lys Ser Thr

915 920 925

Asp Glu Ala Leu Leu Ser Gln Arg Asp Ile Ile Arg Arg Ser Phe Val

930 935 940

Gln Ile Ala Asn Leu Ile Leu Pro Leu Arg Gly Arg Arg Trp Glu Trp

945 950 955 960

Arg Pro His Val Glu Val Pro Asp Cys His Ile Leu Ala Gln Ser Asp

965 970 975

Pro Gly Thr Asp Asp Thr Lys Arg Leu Val Ala Gly Gln Arg Gly Ile

980 985 990

Ser His Glu Arg Ile Glu Gln Ile Glu Glu Leu Arg Arg Arg Cys Gln

995 1000 1005

Ser Leu Asn Arg Ala Leu Arg His Lys Pro Gly Glu Arg Pro Val

1010 1015 1020

Leu Gly Arg Pro Ala Lys Gly Glu Glu Ile Ala Asp Pro Cys Pro

1025 1030 1035

Ala Leu Leu Glu Lys Ile Asn Arg Leu Arg Asp Gln Arg Val Asp

1040 1045 1050

Gln Thr Ala His Ala Ile Leu Ala Ala Ala Leu Gly Val Arg Leu

1055 1060 1065

Arg Ala Pro Ser Lys Asp Arg Ala Glu Arg Arg His Arg Asp Ile

1070 1075 1080

His Gly Glu Tyr Glu Arg Phe Arg Ala Pro Ala Asp Phe Val Val

1085 1090 1095

Ile Glu Asn Leu Ser Arg Tyr Leu Ser Ser Gln Asp Arg Ala Arg

1100 1105 1110

Ser Glu Asn Thr Arg Leu Met Gln Trp Cys His Arg Gln Ile Val

1115 1120 1125

Gln Lys Leu Arg Gln Leu Cys Glu Thr Tyr Gly Ile Pro Val Leu

1130 1135 1140

Ala Val Pro Ala Ala Tyr Ser Ser Arg Phe Ser Ser Arg Asp Gly

1145 1150 1155

Ser Ala Gly Phe Arg Ala Val His Leu Thr Pro Asp His Arg His

1160 1165 1170

Arg Met Pro Trp Ser Arg Ile Leu Ala Arg Leu Lys Ala His Glu

1175 1180 1185

Glu Asp Gly Lys Arg Leu Glu Lys Thr Val Leu Asp Glu Ala Arg

1190 1195 1200

Ala Val Arg Gly Leu Phe Asp Arg Leu Asp Arg Phe Asn Ala Gly

1205 1210 1215

His Val Pro Gly Lys Pro Trp Arg Thr Leu Leu Ala Pro Leu Pro

1220 1225 1230

Gly Gly Pro Val Phe Val Pro Leu Gly Asp Ala Thr Pro Met Gln

1235 1240 1245

Ala Asp Leu Asn Ala Ala Ile Asn Ile Ala Leu Arg Gly Ile Ala

1250 1255 1260

Ala Pro Asp Arg His Asp Ile His His Arg Leu Arg Ala Glu Asn

1265 1270 1275

Lys Lys Arg Ile Leu Ser Leu Arg Leu Gly Thr Gln Arg Glu Lys

1280 1285 1290

Ala Arg Trp Pro Gly Gly Ala Pro Ala Val Thr Leu Ser Thr Pro

1295 1300 1305

Asn Asn Gly Ala Ser Pro Glu Asp Ser Asp Ala Leu Pro Glu Arg

1310 1315 1320

Val Ser Asn Leu Phe Val Asp Ile Ala Gly Val Ala Asn Phe Glu

1325 1330 1335

Arg Val Thr Ile Glu Gly Val Ser Gln Lys Phe Ala Thr Gly Arg

1340 1345 1350

Gly Leu Trp Ala Ser Val Lys Gln Arg Ala Trp Asn Arg Val Ala

1355 1360 1365

Arg Leu Asn Glu Thr Val Thr Asp Asn Asn Arg Asn Glu Glu Glu

1370 1375 1380

Asp Asp Ile Pro Met

1385

<210> 41

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 41

guccaagaaa aaagaaauga uacgaggcau uagcac 36

<210> 42

<211> 107

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 42

cuggacgaug ucucuuuuau uucuuuuuuc uuggaucuga guacgagcac ccacauugga

cauuucgcau ggugggugcu cguacuauag guaaaacaaa ccuuuuu 107

<210> 43

<211> 1108

<212> PRT

<213> Bacillus thermoamylovorans

<400> 43

Met Ala Thr Arg Ser Phe Ile Leu Lys Ile Glu Pro Asn Glu Glu Val

1 5 10 15

Lys Lys Gly Leu Trp Lys Thr His Glu Val Leu Asn His Gly Ile Ala

20 25 30

Tyr Tyr Met Asn Ile Leu Lys Leu Ile Arg Gln Glu Ala Ile Tyr Glu

35 40 45

His His Glu Gln Asp Pro Lys Asn Pro Lys Lys Val Ser Lys Ala Glu

50 55 60

Ile Gln Ala Glu Leu Trp Asp Phe Val Leu Lys Met Gln Lys Cys Asn

65 70 75 80

Ser Phe Thr His Glu Val Asp Lys Asp Val Val Phe Asn Ile Leu Arg

85 90 95

Glu Leu Tyr Glu Glu Leu Val Pro Ser Ser Val Glu Lys Lys Gly Glu

100 105 110

Ala Asn Gln Leu Ser Asn Lys Phe Leu Tyr Pro Leu Val Asp Pro Asn

115 120 125

Ser Gln Ser Gly Lys Gly Thr Ala Ser Ser Gly Arg Lys Pro Arg Trp

130 135 140

Tyr Asn Leu Lys Ile Ala Gly Asp Pro Ser Trp Glu Glu Glu Lys Lys

145 150 155 160

Lys Trp Glu Glu Asp Lys Lys Lys Asp Pro Leu Ala Lys Ile Leu Gly

165 170 175

Lys Leu Ala Glu Tyr Gly Leu Ile Pro Leu Phe Ile Pro Phe Thr Asp

180 185 190

Ser Asn Glu Pro Ile Val Lys Glu Ile Lys Trp Met Glu Lys Ser Arg

195 200 205

Asn Gln Ser Val Arg Arg Leu Asp Lys Asp Met Phe Ile Gln Ala Leu

210 215 220

Glu Arg Phe Leu Ser Trp Glu Ser Trp Asn Leu Lys Val Lys Glu Glu

225 230 235 240

Tyr Glu Lys Val Glu Lys Glu His Lys Thr Leu Glu Glu Arg Ile Lys

245 250 255

Glu Asp Ile Gln Ala Phe Lys Ser Leu Glu Gln Tyr Glu Lys Glu Arg

260 265 270

Gln Glu Gln Leu Leu Arg Asp Thr Leu Asn Thr Asn Glu Tyr Arg Leu

275 280 285

Ser Lys Arg Gly Leu Arg Gly Trp Arg Glu Ile Ile Gln Lys Trp Leu

290 295 300

Lys Met Asp Glu Asn Glu Pro Ser Glu Lys Tyr Leu Glu Val Phe Lys

305 310 315 320

Asp Tyr Gln Arg Lys His Pro Arg Glu Ala Gly Asp Tyr Ser Val Tyr

325 330 335

Glu Phe Leu Ser Lys Lys Glu Asn His Phe Ile Trp Arg Asn His Pro

340 345 350

Glu Tyr Pro Tyr Leu Tyr Ala Thr Phe Cys Glu Ile Asp Lys Lys Lys

355 360 365

Lys Asp Ala Lys Gln Gln Ala Thr Phe Thr Leu Ala Asp Pro Ile Asn

370 375 380

His Pro Leu Trp Val Arg Phe Glu Glu Arg Ser Gly Ser Asn Leu Asn

385 390 395 400

Lys Tyr Arg Ile Leu Thr Glu Gln Leu His Thr Glu Lys Leu Lys Lys

405 410 415

Lys Leu Thr Val Gln Leu Asp Arg Leu Ile Tyr Pro Thr Glu Ser Gly

420 425 430

Gly Trp Glu Glu Lys Gly Lys Val Asp Ile Val Leu Leu Pro Ser Arg

435 440 445

Gln Phe Tyr Asn Gln Ile Phe Leu Asp Ile Glu Glu Lys Gly Lys His

450 455 460

Ala Phe Thr Tyr Lys Asp Glu Ser Ile Lys Phe Pro Leu Lys Gly Thr

465 470 475 480

Leu Gly Gly Ala Arg Val Gln Phe Asp Arg Asp His Leu Arg Arg Tyr

485 490 495

Pro His Lys Val Glu Ser Gly Asn Val Gly Arg Ile Tyr Phe Asn Met

500 505 510

Thr Val Asn Ile Glu Pro Thr Glu Ser Pro Val Ser Lys Ser Leu Lys

515 520 525

Ile His Arg Asp Asp Phe Pro Lys Phe Val Asn Phe Lys Pro Lys Glu

530 535 540

Leu Thr Glu Trp Ile Lys Asp Ser Lys Gly Lys Lys Leu Lys Ser Gly

545 550 555 560

Ile Glu Ser Leu Glu Ile Gly Leu Arg Val Met Ser Ile Asp Leu Gly

565 570 575

Gln Arg Gln Ala Ala Ala Ala Ser Ile Phe Glu Val Val Asp Gln Lys

580 585 590

Pro Asp Ile Glu Gly Lys Leu Phe Phe Pro Ile Lys Gly Thr Glu Leu

595 600 605

Tyr Ala Val His Arg Ala Ser Phe Asn Ile Lys Leu Pro Gly Glu Thr

610 615 620

Leu Val Lys Ser Arg Glu Val Leu Arg Lys Ala Arg Glu Asp Asn Leu

625 630 635 640

Lys Leu Met Asn Gln Lys Leu Asn Phe Leu Arg Asn Val Leu His Phe

645 650 655

Gln Gln Phe Glu Asp Ile Thr Glu Arg Glu Lys Arg Val Thr Lys Trp

660 665 670

Ile Ser Arg Gln Glu Asn Ser Asp Val Pro Leu Val Tyr Gln Asp Glu

675 680 685

Leu Ile Gln Ile Arg Glu Leu Met Tyr Lys Pro Tyr Lys Asp Trp Val

690 695 700

Ala Phe Leu Lys Gln Leu His Lys Arg Leu Glu Val Glu Ile Gly Lys

705 710 715 720

Glu Val Lys His Trp Arg Lys Ser Leu Ser Asp Gly Arg Lys Gly Leu

725 730 735

Tyr Gly Ile Ser Leu Lys Asn Ile Asp Glu Ile Asp Arg Thr Arg Lys

740 745 750

Phe Leu Leu Arg Trp Ser Leu Arg Pro Thr Glu Pro Gly Glu Val Arg

755 760 765

Arg Leu Glu Pro Gly Gln Arg Phe Ala Ile Asp Gln Leu Asn His Leu

770 775 780

Asn Ala Leu Lys Glu Asp Arg Leu Lys Lys Met Ala Asn Thr Ile Ile

785 790 795 800

Met His Ala Leu Gly Tyr Cys Tyr Asp Val Arg Lys Lys Lys Trp Gln

805 810 815

Ala Lys Asn Pro Ala Cys Gln Ile Ile Leu Phe Glu Asp Leu Ser Asn

820 825 830

Tyr Asn Pro Tyr Glu Glu Arg Ser Arg Phe Glu Asn Ser Lys Leu Met

835 840 845

Lys Trp Ser Arg Arg Glu Ile Pro Arg Gln Val Ala Leu Gln Gly Glu

850 855 860

Ile Tyr Gly Leu Gln Val Gly Glu Val Gly Ala Gln Phe Ser Ser Arg

865 870 875 880

Phe His Ala Lys Thr Gly Ser Pro Gly Ile Arg Cys Ser Val Val Thr

885 890 895

Lys Glu Lys Leu Gln Asp Asn Arg Phe Phe Lys Asn Leu Gln Arg Glu

900 905 910

Gly Arg Leu Thr Leu Asp Lys Ile Ala Val Leu Lys Glu Gly Asp Leu

915 920 925

Tyr Pro Asp Lys Gly Gly Glu Lys Phe Ile Ser Leu Ser Lys Asp Arg

930 935 940

Lys Leu Val Thr Thr His Ala Asp Ile Asn Ala Ala Gln Asn Leu Gln

945 950 955 960

Lys Arg Phe Trp Thr Arg Thr His Gly Phe Tyr Lys Val Tyr Cys Lys

965 970 975

Ala Tyr Gln Val Asp Gly Gln Thr Val Tyr Ile Pro Glu Ser Lys Asp

980 985 990

Gln Lys Gln Lys Ile Ile Glu Glu Phe Gly Glu Gly Tyr Phe Ile Leu

995 1000 1005

Lys Asp Gly Val Tyr Glu Trp Gly Asn Ala Gly Lys Leu Lys Ile

1010 1015 1020

Lys Lys Gly Ser Ser Lys Gln Ser Ser Ser Glu Leu Val Asp Ser

1025 1030 1035

Asp Ile Leu Lys Asp Ser Phe Asp Leu Ala Ser Glu Leu Lys Gly

1040 1045 1050

Glu Lys Leu Met Leu Tyr Arg Asp Pro Ser Gly Asn Val Phe Pro

1055 1060 1065

Ser Asp Lys Trp Met Ala Ala Gly Val Phe Phe Gly Lys Leu Glu

1070 1075 1080

Arg Ile Leu Ile Ser Lys Leu Thr Asn Gln Tyr Ser Ile Ser Thr

1085 1090 1095

Ile Glu Asp Asp Ser Ser Lys Gln Ser Met

1100 1105

<210> 44

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 44

guucgaaagc uuaguggaaa gcuucguggu uagcac 36

<210> 45

<211> 69

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 45

cacggauaau cacgacuuuc cacuaagcuu ucgaauuuua ugaugcgagc auccucucag 60

69

<210> 46

<211> 1108

<212> PRT

<213> Bacillus sp.

<400> 46

Met Ala Ile Arg Ser Ile Lys Leu Lys Leu Lys Thr His Thr Gly Pro

1 5 10 15

Glu Ala Gln Asn Leu Arg Lys Gly Ile Trp Arg Thr His Arg Leu Leu

20 25 30

Asn Glu Gly Val Ala Tyr Tyr Met Lys Met Leu Leu Leu Phe Arg Gln

35 40 45

Glu Ser Thr Gly Glu Arg Pro Lys Glu Glu Leu Gln Glu Glu Leu Ile

50 55 60

Cys His Ile Arg Glu Gln Gln Gln Arg Asn Gln Ala Asp Lys Asn Thr

65 70 75 80

Gln Ala Leu Pro Leu Asp Lys Ala Leu Glu Ala Leu Arg Gln Leu Tyr

85 90 95

Glu Leu Leu Val Pro Ser Ser Val Gly Gln Ser Gly Asp Ala Gln Ile

100 105 110

Ile Ser Arg Lys Phe Leu Ser Pro Leu Val Asp Pro Asn Ser Glu Gly

115 120 125

Gly Lys Gly Thr Ser Lys Ala Gly Ala Lys Pro Thr Trp Gln Lys Lys

130 135 140

Lys Glu Ala Asn Asp Pro Thr Trp Glu Gln Asp Tyr Glu Lys Trp Lys

145 150 155 160

Lys Arg Arg Glu Glu Asp Pro Thr Ala Ser Val Ile Thr Thr Leu Glu

165 170 175

Glu Tyr Gly Ile Arg Pro Ile Phe Pro Leu Tyr Thr Asn Thr Val Thr

180 185 190

Asp Ile Ala Trp Leu Pro Leu Gln Ser Asn Gln Phe Val Arg Thr Trp

195 200 205

Asp Arg Asp Met Leu Gln Gln Ala Ile Glu Arg Leu Leu Ser Trp Glu

210 215 220

Ser Trp Asn Lys Arg Val Gln Glu Glu Tyr Ala Lys Leu Lys Glu Lys

225 230 235 240

Met Ala Gln Leu Asn Glu Gln Leu Glu Gly Gly Gln Glu Trp Ile Ser

245 250 255

Leu Leu Glu Gln Tyr Glu Glu Asn Arg Glu Arg Glu Leu Arg Glu Asn

260 265 270

Met Thr Ala Ala Asn Asp Lys Tyr Arg Ile Thr Lys Arg Gln Met Lys

275 280 285

Gly Trp Asn Glu Leu Tyr Glu Leu Trp Ser Thr Phe Pro Ala Ser Ala

290 295 300

Ser His Glu Gln Tyr Lys Glu Ala Leu Lys Arg Val Gln Gln Arg Leu

305 310 315 320

Arg Gly Arg Phe Gly Asp Ala His Phe Phe Gln Tyr Leu Met Glu Glu

325 330 335

Lys Asn Arg Leu Ile Trp Lys Gly Asn Pro Gln Arg Ile His Tyr Phe

340 345 350

Val Ala Arg Asn Glu Leu Thr Lys Arg Leu Glu Glu Ala Lys Gln Ser

355 360 365

Ala Thr Met Thr Leu Pro Asn Ala Arg Lys His Pro Leu Trp Val Arg

370 375 380

Phe Asp Ala Arg Gly Gly Asn Leu Gln Asp Tyr Tyr Leu Thr Ala Glu

385 390 395 400

Ala Asp Lys Pro Arg Ser Arg Arg Phe Val Thr Phe Ser Gln Leu Ile

405 410 415

Trp Pro Ser Glu Ser Gly Trp Met Glu Lys Lys Asp Val Glu Val Glu

420 425 430

Leu Ala Leu Ser Arg Gln Phe Tyr Gln Gln Val Lys Leu Leu Lys Asn

435 440 445

Asp Lys Gly Lys Gln Lys Ile Glu Phe Lys Asp Lys Gly Ser Gly Ser

450 455 460

Thr Phe Asn Gly His Leu Gly Gly Ala Lys Leu Gln Leu Glu Arg Gly

465 470 475 480

Asp Leu Glu Lys Glu Glu Lys Asn Phe Glu Asp Gly Glu Ile Gly Ser

485 490 495

Val Tyr Leu Asn Val Val Ile Asp Phe Glu Pro Leu Gln Glu Val Lys

500 505 510

Asn Gly Arg Val Gln Ala Pro Tyr Gly Gln Val Leu Gln Leu Ile Arg

515 520 525

Arg Pro Asn Glu Phe Pro Lys Val Thr Thr Tyr Lys Ser Glu Gln Leu

530 535 540

Val Glu Trp Ile Lys Ala Ser Pro Gln His Ser Ala Gly Val Glu Ser

545 550 555 560

Leu Ala Ser Gly Phe Arg Val Met Ser Ile Asp Leu Gly Leu Arg Ala

565 570 575

Ala Ala Ala Thr Ser Ile Phe Ser Val Glu Glu Ser Ser Asp Lys Asn

580 585 590

Ala Ala Asp Phe Ser Tyr Trp Ile Glu Gly Thr Pro Leu Val Ala Val

595 600 605

His Gln Arg Ser Tyr Met Leu Arg Leu Pro Gly Glu Gln Val Glu Lys

610 615 620

Gln Val Met Glu Lys Arg Asp Glu Arg Phe Gln Leu His Gln Arg Val

625 630 635 640

Lys Phe Gln Ile Arg Val Leu Ala Gln Ile Met Arg Met Ala Asn Lys

645 650 655

Gln Tyr Gly Asp Arg Trp Asp Glu Leu Asp Ser Leu Lys Gln Ala Val

660 665 670

Glu Gln Lys Lys Ser Pro Leu Asp Gln Thr Asp Arg Thr Phe Trp Glu

675 680 685

Gly Ile Val Cys Asp Leu Thr Lys Val Leu Pro Arg Asn Glu Ala Asp

690 695 700

Trp Glu Gln Ala Val Val Gln Ile His Arg Lys Ala Glu Glu Tyr Val

705 710 715 720

Gly Lys Ala Val Gln Ala Trp Arg Lys Arg Phe Ala Ala Asp Glu Arg

725 730 735

Lys Gly Ile Ala Gly Leu Ser Met Trp Asn Ile Glu Glu Leu Glu Gly

740 745 750

Leu Arg Lys Leu Leu Ile Ser Trp Ser Arg Arg Thr Arg Asn Pro Gln

755 760 765

Glu Val Asn Arg Phe Glu Arg Gly His Thr Ser His Gln Arg Leu Leu

770 775 780

Thr His Ile Gln Asn Val Lys Glu Asp Arg Leu Lys Gln Leu Ser His

785 790 795 800

Ala Ile Val Met Thr Ala Leu Gly Tyr Val Tyr Asp Glu Arg Lys Gln

805 810 815

Glu Trp Cys Ala Glu Tyr Pro Ala Cys Gln Val Ile Leu Phe Glu Asn

820 825 830

Leu Ser Gln Tyr Arg Ser Asn Leu Asp Arg Ser Thr Lys Glu Asn Ser

835 840 845

Thr Leu Met Lys Trp Ala His Arg Ser Ile Pro Lys Tyr Val His Met

850 855 860

Gln Ala Glu Pro Tyr Gly Ile Gln Ile Gly Asp Val Arg Ala Glu Tyr

865 870 875 880

Ser Ser Arg Phe Tyr Ala Lys Thr Gly Thr Pro Gly Ile Arg Cys Lys

885 890 895

Lys Val Arg Gly Gln Asp Leu Gln Gly Arg Arg Phe Glu Asn Leu Gln

900 905 910

Lys Arg Leu Val Asn Glu Gln Phe Leu Thr Glu Glu Glu Gln Val Lys Gln

915 920 925

Leu Arg Pro Gly Asp Ile Val Pro Asp Asp Ser Gly Glu Leu Phe Met

930 935 940

Thr Leu Thr Asp Gly Ser Gly Ser Lys Glu Val Val Phe Leu Gln Ala

945 950 955 960

Asp Ile Asn Ala Ala His Asn Leu Gln Lys Arg Phe Trp Gln Arg Tyr

965 970 975

Asn Glu Leu Phe Lys Val Ser Cys Arg Val Ile Val Arg Asp Glu Glu

980 985 990

Glu Tyr Leu Val Pro Lys Thr Lys Ser Val Gln Ala Lys Leu Gly Lys

995 1000 1005

Gly Leu Phe Val Lys Lys Ser Asp Thr Ala Trp Lys Asp Val Tyr

1010 1015 1020

Val Trp Asp Ser Gln Ala Lys Leu Lys Gly Lys Thr Thr Phe Thr

1025 1030 1035

Glu Glu Ser Glu Ser Pro Glu Gln Leu Glu Asp Phe Gln Glu Ile

1040 1045 1050

Ile Glu Glu Ala Glu Glu Ala Lys Gly Thr Tyr Arg Thr Leu Phe

1055 1060 1065

Arg Asp Pro Ser Gly Val Phe Phe Pro Glu Ser Val Trp Tyr Pro

1070 1075 1080

Gln Lys Asp Phe Trp Gly Glu Val Lys Arg Lys Leu Tyr Gly Lys

1085 1090 1095

Leu Arg Glu Arg Phe Leu Thr Lys Ala Arg

1100 1105

<210> 47

<211> 35

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 47

guuuugagaa uagcccgaca uagaggggcaa uagac 35

<210> 48

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 48

guuaugaaaa cagcccgaca uagaggggcaa uagaca 36

<210> 49

<211> 1334

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 49

Met Lys Ile Ser Lys Val Asp His Thr Arg Met Ala Val Ala Lys Gly

1 5 10 15

Asn Gln His Arg Arg Asp Glu Ile Ser Gly Ile Leu Tyr Lys Asp Pro

20 25 30

Thr Lys Thr Gly Ser Ile Asp Phe Asp Glu Arg Phe Lys Lys Leu Asn

35 40 45

Cys Ser Ala Lys Ile Leu Tyr His Val Phe Asn Gly Ile Ala Glu Gly

50 55 60

Ser Asn Lys Tyr Lys Asn Ile Val Asp Lys Val Asn Asn Asn Asn Leu Asp

65 70 75 80

Arg Val Leu Phe Thr Gly Lys Ser Tyr Asp Arg Lys Ser Ile Ile Asp

85 90 95

Ile Asp Thr Val Leu Arg Asn Val Glu Lys Ile Asn Ala Phe Asp Arg

100 105 110

Ile Ser Thr Glu Glu Arg Glu Gln Ile Ile Asp Asp Leu Leu Glu Ile

115 120 125

Gln Leu Arg Lys Gly Leu Arg Lys Gly Lys Ala Gly Leu Arg Glu Val

130 135 140

Leu Leu Ile Gly Ala Gly Val Ile Val Arg Thr Asp Lys Lys Gln Glu

145 150 155 160

Ile Ala Asp Phe Leu Glu Ile Leu Asp Glu Asp Phe Asn Lys Thr Asn

165 170 175

Gln Ala Lys Asn Ile Lys Leu Ser Ile Glu Asn Gln Gly Leu Val Val

180 185 190

Ser Pro Val Ser Arg Gly Glu Glu Arg Ile Phe Asp Val Ser Gly Ala

195 200 205

Gln Lys Gly Lys Ser Ser Lys Lys Ala Gln Glu Lys Glu Ala Leu Ser

210 215 220

Ala Phe Leu Leu Asp Tyr Ala Asp Leu Asp Lys Asn Val Arg Phe Glu

225 230 235 240

Tyr Leu Arg Lys Ile Arg Arg Leu Ile Asn Leu Tyr Phe Tyr Val Lys

245 250 255

Asn Asp Asp Val Met Ser Leu Thr Glu Ile Pro Ala Glu Val Asn Leu

260 265 270

Glu Lys Asp Phe Asp Ile Trp Arg Asp His Glu Gln Arg Lys Glu Glu

275 280 285

Asn Gly Asp Phe Val Gly Cys Pro Asp Ile Leu Leu Ala Asp Arg Asp

290 295 300

Val Lys Lys Ser Asn Ser Lys Gln Val Lys Ile Ala Glu Arg Gln Leu

305 310 315 320

Arg Glu Ser Ile Arg Glu Lys Asn Ile Lys Arg Tyr Arg Phe Ser Ile

325 330 335

Lys Thr Ile Glu Lys Asp Asp Gly Thr Tyr Phe Phe Ala Asn Lys Gln

340 345 350

Ile Ser Val Phe Trp Ile His Arg Ile Glu Asn Ala Val Glu Arg Ile

355 360 365

Leu Gly Ser Ile Asn Asp Lys Lys Leu Tyr Arg Leu Arg Leu Gly Tyr

370 375 380

Leu Gly Glu Lys Val Trp Lys Asp Ile Leu Asn Phe Leu Ser Ile Lys

385 390 395 400

Tyr Ile Ala Val Gly Lys Ala Val Phe Asn Phe Ala Met Asp Asp Leu

405 410 415

Gln Glu Lys Asp Arg Asp Ile Glu Pro Gly Lys Ile Ser Glu Asn Ala

420 425 430

Val Asn Gly Leu Thr Ser Phe Asp Tyr Glu Gln Ile Lys Ala Asp Glu

435 440 445

Met Leu Gln Arg Glu Val Ala Val Asn Val Ala Phe Ala Ala Asn Asn

450 455 460

Leu Ala Arg Val Thr Val Asp Ile Pro Gln Asn Gly Glu Lys Glu Asp

465 470 475 480

Ile Leu Leu Trp Asn Lys Ser Asp Ile Lys Lys Tyr Lys Lys Asn Ser

485 490 495

Lys Lys Gly Ile Leu Lys Ser Ile Leu Gln Phe Phe Gly Gly Ala Ser

500 505 510

Thr Trp Asn Met Lys Met Phe Glu Ile Ala Tyr His Asp Gln Pro Gly

515 520 525

Asp Tyr Glu Glu Asn Tyr Leu Tyr Asp Ile Ile Gln Ile Ile Tyr Ser

530 535 540

Leu Arg Asn Lys Ser Phe His Phe Lys Thr Tyr Asp His Gly Asp Lys

545 550 555 560

Asn Trp Asn Arg Glu Leu Ile Gly Lys Met Ile Glu His Asp Ala Glu

565 570 575

Arg Val Ile Ser Val Glu Arg Glu Lys Phe His Ser Asn Asn Leu Pro

580 585 590

Met Phe Tyr Lys Asp Ala Asp Leu Lys Lys Ile Leu Asp Leu Leu Tyr

595 600 605

Ser Asp Tyr Ala Gly Arg Ala Ser Gln Val Pro Ala Phe Asn Thr Val

610 615 620

Leu Val Arg Lys Asn Phe Pro Glu Phe Leu Arg Lys Asp Met Gly Tyr

625 630 635 640

Lys Val His Phe Asn Asn Pro Glu Val Glu Asn Gln Trp His Ser Ala

645 650 655

Val Tyr Tyr Leu Tyr Lys Glu Ile Tyr Tyr Asn Leu Phe Leu Arg Asp

660 665 670

Lys Glu Val Lys Asn Leu Phe Tyr Thr Ser Leu Lys Asn Ile Arg Ser

675 680 685

Glu Val Ser Asp Lys Lys Gln Lys Leu Ala Ser Asp Asp Phe Ala Ser

690 695 700

Arg Cys Glu Glu Ile Glu Asp Arg Ser Leu Pro Glu Ile Cys Gln Ile

705 710 715 720

Ile Met Thr Glu Tyr Asn Ala Gln Asn Phe Gly Asn Arg Lys Val Lys

725 730 735

Ser Gln Arg Val Ile Glu Lys Asn Lys Asp Ile Phe Arg His Tyr Lys

740 745 750

Met Leu Leu Ile Lys Thr Leu Ala Gly Ala Phe Ser Leu Tyr Leu Lys

755 760 765

Gln Glu Arg Phe Ala Phe Ile Gly Lys Ala Thr Pro Ile Pro Tyr Glu

770 775 780

Thr Thr Asp Val Lys Asn Phe Leu Pro Glu Trp Lys Ser Gly Met Tyr

785 790 795 800

Ala Ser Phe Val Glu Glu Ile Lys Asn Asn Leu Asp Leu Gln Glu Trp

805 810 815

Tyr Ile Val Gly Arg Phe Leu Asn Gly Arg Met Leu Asn Gln Leu Ala

820 825 830

Gly Ser Leu Arg Ser Tyr Ile Gln Tyr Ala Glu Asp Ile Glu Arg Arg

835 840 845

Ala Ala Glu Asn Arg Asn Lys Leu Phe Ser Lys Pro Asp Glu Lys Ile

850 855 860

Glu Ala Cys Lys Lys Ala Val Arg Val Leu Asp Leu Cys Ile Lys Ile

865 870 875 880

Ser Thr Arg Ile Ser Ala Glu Phe Thr Asp Tyr Phe Asp Ser Glu Asp

885 890 895

Asp Tyr Ala Asp Tyr Leu Glu Lys Tyr Leu Lys Tyr Gln Asp Asp Ala

900 905 910

Ile Lys Glu Leu Ser Gly Ser Ser Tyr Ala Ala Leu Asp His Phe Cys

915 920 925

Asn Lys Asp Asp Leu Lys Phe Asp Ile Tyr Val Asn Ala Gly Gln Lys

930 935 940

Pro Ile Leu Gln Arg Asn Ile Val Met Ala Lys Leu Phe Gly Pro Asp

945 950 955 960

Asn Ile Leu Ser Glu Val Met Glu Lys Val Thr Glu Ser Ala Ile Arg

965 970 975

Glu Tyr Tyr Asp Tyr Leu Lys Lys Val Ser Gly Tyr Arg Val Arg Gly

980 985 990

Lys Cys Ser Thr Glu Lys Glu Gln Glu Asp Leu Leu Lys Phe Gln Arg

995 1000 1005

Leu Lys Asn Ala Val Glu Phe Arg Asp Val Thr Glu Tyr Ala Glu

1010 1015 1020

Val Ile Asn Glu Leu Leu Gly Gln Leu Ile Ser Trp Ser Tyr Leu

1025 1030 1035

Arg Glu Arg Asp Leu Leu Tyr Phe Gln Leu Gly Phe His Tyr Met

1040 1045 1050

Cys Leu Lys Asn Lys Ser Phe Lys Pro Ala Glu Tyr Val Asp Ile

1055 1060 1065

Arg Arg Asn Asn Gly Thr Ile Ile His Asn Ala Ile Leu Tyr Gln

1070 1075 1080

Ile Val Ser Met Tyr Ile Asn Gly Leu Asp Phe Tyr Ser Cys Asp

1085 1090 1095

Lys Glu Gly Lys Thr Leu Lys Pro Ile Glu Thr Gly Lys Gly Val

1100 1105 1110

Gly Ser Lys Ile Gly Gln Phe Ile Lys Tyr Ser Gln Tyr Leu Tyr

1115 1120 1125

Asn Asp Pro Ser Tyr Lys Leu Glu Ile Tyr Asn Ala Gly Leu Glu

1130 1135 1140

Val Phe Glu Asn Ile Asp Glu His Asp Asn Ile Thr Asp Leu Arg

1145 1150 1155

Lys Tyr Val Asp His Phe Lys Tyr Tyr Ala Tyr Gly Asn Lys Met

1160 1165 1170

Ser Leu Leu Asp Leu Tyr Ser Glu Phe Phe Asp Arg Phe Phe Thr

1175 1180 1185

Tyr Asp Met Lys Tyr Gln Lys Asn Val Val Asn Val Leu Glu Asn

1190 1195 1200

Ile Leu Leu Arg His Phe Val Ile Phe Tyr Pro Lys Phe Gly Ser

1205 1210 1215

Gly Lys Lys Asp Val Gly Ile Arg Asp Cys Lys Lys Glu Arg Ala

1220 1225 1230

Gln Ile Glu Ile Ser Glu Gln Ser Leu Thr Ser Glu Asp Phe Met

1235 1240 1245

Phe Lys Leu Asp Asp Lys Ala Gly Glu Glu Ala Lys Lys Phe Pro

1250 1255 1260

Ala Arg Asp Glu Arg Tyr Leu Gln Thr Ile Ala Lys Leu Leu Tyr

1265 1270 1275

Tyr Pro Asn Glu Ile Glu Asp Met Asn Arg Phe Met Lys Lys Gly

1280 1285 1290

Glu Thr Ile Asn Lys Lys Val Gln Phe Asn Arg Lys Lys Lys Ile

1295 1300 1305

Thr Arg Lys Gln Lys Asn Asn Ser Ser Asn Glu Val Leu Ser Ser

1310 1315 1320

Thr Met Gly Tyr Leu Phe Lys Asn Ile Lys Leu

1325 1330

<210> 50

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 50

guuuuagucc ucuuucauau agagguaguc ucuuac 36

<210> 51

<211> 99

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 51

augaaaagag gacuaaaacu gaaagaggac uaaaacacca gauguggaua acuauauuag 60

uggcuauuaa aaauucgucg auauuagaga ggaaacuuu 99

<210> 52

<211> 1120

<212> PRT

<213> Listeria seeligeri

<400> 52

Met Trp Ile Ser Ile Lys Thr Leu Ile His His Leu Gly Val Leu Phe

1 5 10 15

Phe Cys Asp Tyr Met Tyr Asn Arg Arg Glu Lys Lys Ile Ile Glu Val

20 25 30

Lys Thr Met Arg Ile Thr Lys Val Glu Val Asp Arg Lys Lys Val Leu

35 40 45

Ile Ser Arg Asp Lys Asn Gly Gly Lys Leu Val Tyr Glu Asn Glu Met

50 55 60

Gln Asp Asn Thr Glu Gln Ile Met His His Lys Lys Ser Ser Phe Tyr

65 70 75 80

Lys Ser Val Val Asn Lys Thr Ile Cys Arg Pro Glu Gln Lys Gln Met

85 90 95

Lys Lys Leu Val His Gly Leu Leu Gln Glu Asn Ser Gln Glu Lys Ile

100 105 110

Lys Val Ser Asp Val Thr Lys Leu Asn Ile Ser Asn Phe Leu Asn His

115 120 125

Arg Phe Lys Lys Ser Leu Tyr Tyr Phe Pro Glu Asn Ser Pro Asp Lys

130 135 140

Ser Glu Glu Tyr Arg Ile Glu Ile Asn Leu Ser Gln Leu Leu Glu Asp

145 150 155 160

Ser Leu Lys Lys Gln Gln Gly Thr Phe Ile Cys Trp Glu Ser Phe Ser

165 170 175

Lys Asp Met Glu Leu Tyr Ile Asn Trp Ala Glu Asn Tyr Ile Ser Ser

180 185 190

Lys Thr Lys Leu Ile Lys Lys Ser Ile Arg Asn Asn Arg Ile Gln Ser

195 200 205

Thr Glu Ser Arg Ser Gly Gln Leu Met Asp Arg Tyr Met Lys Asp Ile

210 215 220

Leu Asn Lys Asn Lys Pro Phe Asp Ile Gln Ser Val Ser Glu Lys Tyr

225 230 235 240

Gln Leu Glu Lys Leu Thr Ser Ala Leu Lys Ala Thr Phe Lys Glu Ala

245 250 255

Lys Lys Asn Asp Lys Glu Ile Asn Tyr Lys Leu Lys Ser Thr Leu Gln

260 265 270

Asn His Glu Arg Gln Ile Ile Glu Glu Leu Lys Glu Asn Ser Glu Leu

275 280 285

Asn Gln Phe Asn Ile Glu Ile Arg Lys His Leu Glu Thr Tyr Phe Pro

290 295 300

Ile Lys Lys Thr Asn Arg Lys Val Gly Asp Ile Arg Asn Leu Glu Ile

305 310 315 320

Gly Glu Ile Gln Lys Ile Val Asn His Arg Leu Lys Asn Lys Ile Val

325 330 335

Gln Arg Ile Leu Gln Glu Gly Lys Leu Ala Ser Tyr Glu Ile Glu Ser

340 345 350

Thr Val Asn Ser Asn Ser Leu Gln Lys Ile Lys Ile Glu Glu Ala Phe

355 360 365

Ala Leu Lys Phe Ile Asn Ala Cys Leu Phe Ala Ser Asn Asn Leu Arg

370 375 380

Asn Met Val Tyr Pro Val Cys Lys Lys Asp Ile Leu Met Ile Gly Glu

385 390 395 400

Phe Lys Asn Ser Phe Lys Glu Ile Lys His Lys Lys Phe Ile Arg Gln

405 410 415

Trp Ser Gln Phe Phe Ser Gln Glu Ile Thr Val Asp Asp Ile Glu Leu

420 425 430

Ala Ser Trp Gly Leu Arg Gly Ala Ile Ala Pro Ile Arg Asn Glu Ile

435 440 445

Ile His Leu Lys Lys His Ser Trp Lys Lys Phe Phe Asn Asn Pro Thr

450 455 460

Phe Lys Val Lys Lys Ser Lys Ile Ile Asn Gly Lys Thr Lys Asp Val

465 470 475 480

Thr Ser Glu Phe Leu Tyr Lys Glu Thr Leu Phe Lys Asp Tyr Phe Tyr

485 490 495

Ser Glu Leu Asp Ser Val Pro Glu Leu Ile Ile Asn Lys Met Glu Ser

500 505 510

Ser Lys Ile Leu Asp Tyr Tyr Ser Ser Asp Gln Leu Asn Gln Val Phe

515 520 525

Thr Ile Pro Asn Phe Glu Leu Ser Leu Leu Thr Ser Ala Val Pro Phe

530 535 540

Ala Pro Ser Phe Lys Arg Val Tyr Leu Lys Gly Phe Asp Tyr Gln Asn

545 550 555 560

Gln Asp Glu Ala Gln Pro Asp Tyr Asn Leu Lys Leu Asn Ile Tyr Asn

565 570 575

Glu Lys Ala Phe Asn Ser Glu Ala Phe Gln Ala Gln Tyr Ser Leu Phe

580 585 590

Lys Met Val Tyr Tyr Gln Val Phe Leu Pro Gln Phe Thr Thr Asn Asn

595 600 605

Asp Leu Phe Lys Ser Ser Val Asp Phe Ile Leu Thr Leu Asn Lys Glu

610 615 620

Arg Lys Gly Tyr Ala Lys Ala Phe Gln Asp Ile Arg Lys Met Asn Lys

625 630 635 640

Asp Glu Lys Pro Ser Glu Tyr Met Ser Tyr Ile Gln Ser Gln Leu Met

645 650 655

Leu Tyr Gln Lys Lys Gln Glu Glu Lys Glu Lys Ile Asn His Phe Glu

660 665 670

Lys Phe Ile Asn Gln Val Phe Ile Lys Gly Phe Asn Ser Phe Ile Glu

675 680 685

Lys Asn Arg Leu Thr Tyr Ile Cys His Pro Thr Lys Asn Thr Val Pro

690 695 700

Glu Asn Asp Asn Ile Glu Ile Pro Phe His Thr Asp Met Asp Asp Ser

705 710 715 720

Asn Ile Ala Phe Trp Leu Met Cys Lys Leu Leu Asp Ala Lys Gln Leu

725 730 735

Ser Glu Leu Arg Asn Glu Met Ile Lys Phe Ser Cys Ser Leu Gln Ser

740 745 750

Thr Glu Glu Ile Ser Thr Phe Thr Lys Ala Arg Glu Val Ile Gly Leu

755 760 765

Ala Leu Leu Asn Gly Glu Lys Gly Cys Asn Asp Trp Lys Glu Leu Phe

770 775 780

Asp Asp Lys Glu Ala Trp Lys Lys Asn Met Ser Leu Tyr Val Ser Glu

785 790 795 800

Glu Leu Leu Gln Ser Leu Pro Tyr Thr Gln Glu Asp Gly Gln Thr Pro

805 810 815

Val Ile Asn Arg Ser Ile Asp Leu Val Lys Lys Tyr Gly Thr Glu Thr

820 825 830

Ile Leu Glu Lys Leu Phe Ser Ser Ser Asp Asp Tyr Lys Val Ser Ala

835 840 845

Lys Asp Ile Ala Lys Leu His Glu Tyr Asp Val Thr Glu Lys Ile Ala

850 855 860

Gln Gln Glu Ser Leu His Lys Gln Trp Ile Glu Lys Pro Gly Leu Ala

865 870 875 880

Arg Asp Ser Ala Trp Thr Lys Lys Tyr Gln Asn Val Ile Asn Asp Ile

885 890 895

Ser Asn Tyr Gln Trp Ala Lys Thr Lys Val Glu Leu Thr Gln Val Arg

900 905 910

His Leu His Gln Leu Thr Ile Asp Leu Leu Ser Arg Leu Ala Gly Tyr

915 920 925

Met Ser Ile Ala Asp Arg Asp Phe Gln Phe Ser Ser Asn Tyr Ile Leu

930 935 940

Glu Arg Glu Asn Ser Glu Tyr Arg Val Thr Ser Trp Ile Leu Leu Ser

945 950 955 960

Glu Asn Lys Asn Lys Asn Lys Tyr Asn Asp Tyr Glu Leu Tyr Asn Leu

965 970 975

Lys Asn Ala Ser Ile Lys Val Ser Ser Lys Asn Asp Pro Gln Leu Lys

980 985 990

Val Asp Leu Lys Gln Leu Arg Leu Thr Leu Glu Tyr Leu Glu Leu Phe

995 1000 1005

Asp Asn Arg Leu Lys Glu Lys Arg Asn Asn Ile Ser His Phe Asn

1010 1015 1020

Tyr Leu Asn Gly Gln Leu Gly Asn Ser Ile Leu Glu Leu Phe Asp

1025 1030 1035

Asp Ala Arg Asp Val Leu Ser Tyr Asp Arg Lys Leu Lys Asn Ala

1040 1045 1050

Val Ser Lys Ser Leu Lys Glu Ile Leu Ser Ser His Gly Met Glu

1055 1060 1065

Val Thr Phe Lys Pro Leu Tyr Gln Thr Asn His His Leu Lys Ile

1070 1075 1080

Asp Lys Leu Gln Pro Lys Lys Ile His His Leu Gly Glu Lys Ser

1085 1090 1095

Thr Val Ser Ser Asn Gln Val Ser Asn Glu Tyr Cys Gln Leu Val

1100 1105 1110

Arg Thr Leu Leu Thr Met Lys

1115 1120

<210> 53

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 53

guuuuagucc ccuucguuuu ugggguaguc uaaauc 36

<210> 54

<211> 113

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 54

gauuuagagc accccaaaag uaaugaaaau uugcaauuaa auaaggaaua uuaaaaaaau60

gugauuuuaa aaaaauugaa gaaauuaaau gaaaaauugu ccaaguaaaa aaa 113

<210> 55

<211> 70

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 55

auuuagauua ccccuuuaau uuauuuuacc auauuuuucu cauaaugcaa acuaauauuc 60

caaaauuuuu 70

<210> 56

<211> 1389

<212> PRT

<213> Leptotrichia wadei

<400> 56

Met Gly Asn Leu Phe Gly His Lys Arg Trp Tyr Glu Val Arg Asp Lys

1 5 10 15

Lys Asp Phe Lys Ile Lys Arg Lys Val Lys Val Lys Arg Asn Tyr Asp

20 25 30

Gly Asn Lys Tyr Ile Leu Asn Ile Asn Glu Asn Asn Asn Lys Glu Lys

35 40 45

Ile Asp Asn Asn Lys Phe Ile Arg Lys Tyr Ile Asn Tyr Lys Lys Asn

50 55 60

Asp Asn Ile Leu Lys Glu Phe Thr Arg Lys Phe His Ala Gly Asn Ile

65 70 75 80

Leu Phe Lys Leu Lys Gly Lys Glu Gly Ile Ile Arg Ile Glu Asn Asn

85 90 95

Asp Asp Phe Leu Glu Thr Glu Glu Val Val Leu Tyr Ile Glu Ala Tyr

100 105 110

Gly Lys Ser Glu Lys Leu Lys Ala Leu Gly Ile Thr Lys Lys Lys Ile

115 120 125

Ile Asp Glu Ala Ile Arg Gln Gly Ile Thr Lys Asp Asp Lys Lys Ile

130 135 140

Glu Ile Lys Arg Gln Glu Asn Glu Glu Glu Ile Glu Ile Asp Ile Arg

145 150 155 160

Asp Glu Tyr Thr Asn Lys Thr Leu Asn Asp Cys Ser Ile Ile Leu Arg

165 170 175

Ile Ile Glu Asn Asp Glu Leu Glu Thr Lys Lys Ser Ile Tyr Glu Ile

180 185 190

Phe Lys Asn Ile Asn Met Ser Leu Tyr Lys Ile Ile Glu Lys Ile Ile

195 200 205

Glu Asn Glu Thr Glu Lys Val Phe Glu Asn Arg Tyr Tyr Glu Glu His

210 215 220

Leu Arg Glu Lys Leu Leu Lys Asp Asp Lys Ile Asp Val Ile Leu Thr

225 230 235 240

Asn Phe Met Glu Ile Arg Glu Lys Ile Lys Ser Asn Leu Glu Ile Leu

245 250 255

Gly Phe Val Lys Phe Tyr Leu Asn Val Gly Gly Asp Lys Lys Lys Ser

260 265 270

Lys Asn Lys Lys Met Leu Val Glu Lys Ile Leu Asn Ile Asn Val Asp

275 280 285

Leu Thr Val Glu Asp Ile Ala Asp Phe Val Ile Lys Glu Leu Glu Phe

290 295 300

Trp Asn Ile Thr Lys Arg Ile Glu Lys Val Lys Lys Val Asn Asn Glu

305 310 315 320

Phe Leu Glu Lys Arg Arg Asn Arg Thr Tyr Ile Lys Ser Tyr Val Leu

325 330 335

Leu Asp Lys His Glu Lys Phe Lys Ile Glu Arg Glu Asn Lys Lys Asp

340 345 350

Lys Ile Val Lys Phe Phe Val Glu Asn Ile Lys Asn Asn Ser Ile Lys

355 360 365

Glu Lys Ile Glu Lys Ile Leu Ala Glu Phe Lys Ile Asp Glu Leu Ile

370 375 380

Lys Lys Leu Glu Lys Glu Leu Lys Lys Gly Asn Cys Asp Thr Glu Ile

385 390 395 400

Phe Gly Ile Phe Lys Lys His Tyr Lys Val Asn Phe Asp Ser Lys Lys

405 410 415

Phe Ser Lys Lys Ser Asp Glu Glu Lys Glu Leu Tyr Lys Ile Ile Tyr

420 425 430

Arg Tyr Leu Lys Gly Arg Ile Glu Lys Ile Leu Val Asn Glu Gln Lys

435 440 445

Val Arg Leu Lys Lys Met Glu Lys Ile Glu Ile Glu Lys Ile Leu Asn

450 455 460

Glu Ser Ile Leu Ser Glu Lys Ile Leu Lys Arg Val Lys Gln Tyr Thr

465 470 475 480

Leu Glu His Ile Met Tyr Leu Gly Lys Leu Arg His Asn Asp Ile Asp

485 490 495

Met Thr Thr Val Asn Thr Asp Asp Phe Ser Arg Leu His Ala Lys Glu

500 505 510

Glu Leu Asp Leu Glu Leu Ile Thr Phe Phe Ala Ser Thr Asn Met Glu

515 520 525

Leu Asn Lys Ile Phe Ser Arg Glu Asn Ile Asn Asn Asp Glu Asn Ile

530 535 540

Asp Phe Phe Gly Gly Asp Arg Glu Lys Asn Tyr Val Leu Asp Lys Lys

545 550 555 560

Ile Leu Asn Ser Lys Ile Lys Ile Ile Arg Asp Leu Asp Phe Ile Asp

565 570 575

Asn Lys Asn Asn Ile Thr Asn Asn Phe Ile Arg Lys Phe Thr Lys Ile

580 585 590

Gly Thr Asn Glu Arg Asn Arg Ile Leu His Ala Ile Ser Lys Glu Arg

595 600 605

Asp Leu Gln Gly Thr Gln Asp Asp Tyr Asn Lys Val Ile Asn Ile Ile

610 615 620

Gln Asn Leu Lys Ile Ser Asp Glu Glu Val Ser Lys Ala Leu Asn Leu

625 630 635 640

Asp Val Val Phe Lys Asp Lys Lys Asn Ile Ile Thr Lys Ile Asn Asp

645 650 655

Ile Lys Ile Ser Glu Glu Asn Asn Asn Asp Ile Lys Tyr Leu Pro Ser

660 665 670

Phe Ser Lys Val Leu Pro Glu Ile Leu Asn Leu Tyr Arg Asn Asn Pro

675 680 685

Lys Asn Glu Pro Phe Asp Thr Ile Glu Thr Glu Lys Ile Val Leu Asn

690 695 700

Ala Leu Ile Tyr Val Asn Lys Glu Leu Tyr Lys Lys Leu Ile Leu Glu

705 710 715 720

Asp Asp Leu Glu Glu Asn Glu Ser Lys Asn Ile Phe Leu Gln Glu Leu

725 730 735

Lys Lys Thr Leu Gly Asn Ile Asp Glu Ile Asp Glu Asn Ile Ile Glu

740 745 750

Asn Tyr Tyr Lys Asn Ala Gln Ile Ser Ala Ser Lys Gly Asn Asn Lys

755 760 765

Ala Ile Lys Lys Tyr Gln Lys Lys Val Ile Glu Cys Tyr Ile Gly Tyr

770 775 780

Leu Arg Lys Asn Tyr Glu Glu Leu Phe Asp Phe Ser Asp Phe Lys Met

785 790 795 800

Asn Ile Gln Glu Ile Lys Lys Gln Ile Lys Asp Ile Asn Asp Asn Lys

805 810 815

Thr Tyr Glu Arg Ile Thr Val Lys Thr Ser Asp Lys Thr Ile Val Ile

820 825 830

Asn Asp Asp Phe Glu Tyr Ile Ile Ser Ile Phe Ala Leu Leu Asn Ser

835 840 845

Asn Ala Val Ile Asn Lys Ile Arg Asn Arg Phe Phe Ala Thr Ser Val

850 855 860

Trp Leu Asn Thr Ser Glu Tyr Gln Asn Ile Ile Asp Ile Leu Asp Glu

865 870 875 880

Ile Met Gln Leu Asn Thr Leu Arg Asn Glu Cys Ile Thr Glu Asn Trp

885 890 895

Asn Leu Asn Leu Glu Glu Phe Ile Gln Lys Met Lys Glu Ile Glu Lys

900 905 910

Asp Phe Asp Asp Phe Lys Ile Gln Thr Lys Lys Glu Ile Phe Asn Asn

915 920 925

Tyr Tyr Glu Asp Ile Lys Asn Asn Ile Leu Thr Glu Phe Lys Asp Asp

930 935 940

Ile Asn Gly Cys Asp Val Leu Glu Lys Lys Leu Glu Lys Ile Val Ile

945 950 955 960

Phe Asp Asp Glu Thr Lys Phe Glu Ile Asp Lys Lys Ser Asn Ile Leu

965 970 975

Gln Asp Glu Gln Arg Lys Leu Ser Asn Ile Asn Lys Lys Asp Leu Lys

980 985 990

Lys Lys Val Asp Gln Tyr Ile Lys Asp Lys Asp Gln Glu Ile Lys Ser

995 1000 1005

Lys Ile Leu Cys Arg Ile Ile Phe Asn Ser Asp Phe Leu Lys Lys

1010 1015 1020

Tyr Lys Lys Glu Ile Asp Asn Leu Ile Glu Asp Met Glu Ser Glu

1025 1030 1035

Asn Glu Asn Lys Phe Gln Glu Ile Tyr Tyr Pro Lys Glu Arg Lys

1040 1045 1050

Asn Glu Leu Tyr Ile Tyr Lys Lys Asn Leu Phe Leu Asn Ile Gly

1055 1060 1065

Asn Pro Asn Phe Asp Lys Ile Tyr Gly Leu Ile Ser Asn Asp Ile

1070 1075 1080

Lys Met Ala Asp Ala Lys Phe Leu Phe Asn Ile Asp Gly Lys Asn

1085 1090 1095

Ile Arg Lys Asn Lys Ile Ser Glu Ile Asp Ala Ile Leu Lys Asn

1100 1105 1110

Leu Asn Asp Lys Leu Asn Gly Tyr Ser Lys Glu Tyr Lys Glu Lys

1115 1120 1125

Tyr Ile Lys Lys Leu Lys Glu Asn Asp Asp Phe Phe Ala Lys Asn

1130 1135 1140

Ile Gln Asn Lys Asn Tyr Lys Ser Phe Glu Lys Asp Tyr Asn Arg

1145 1150 1155

Val Ser Glu Tyr Lys Lys Ile Arg Asp Leu Val Glu Phe Asn Tyr

1160 1165 1170

Leu Asn Lys Ile Glu Ser Tyr Leu Ile Asp Ile Asn Trp Lys Leu

1175 1180 1185

Ala Ile Gln Met Ala Arg Phe Glu Arg Asp Met His Tyr Ile Val

1190 1195 1200

Asn Gly Leu Arg Glu Leu Gly Ile Ile Lys Leu Ser Gly Tyr Asn

1205 1210 1215

Thr Gly Ile Ser Arg Ala Tyr Pro Lys Arg Asn Gly Ser Asp Gly

1220 1225 1230

Phe Tyr Thr Thr Thr Ala Tyr Tyr Lys Phe Phe Asp Glu Glu Ser

1235 1240 1245

Tyr Lys Lys Phe Glu Lys Ile Cys Tyr Gly Phe Gly Ile Asp Leu

1250 1255 1260

Ser Glu Asn Ser Glu Ile Asn Lys Pro Glu Asn Glu Ser Ile Arg

1265 1270 1275

Asn Tyr Ile Ser His Phe Tyr Ile Val Arg Asn Pro Phe Ala Asp

1280 1285 1290

Tyr Ser Ile Ala Glu Gln Ile Asp Arg Val Ser Asn Leu Leu Ser

1295 1300 1305

Tyr Ser Thr Arg Tyr Asn Asn Ser Thr Tyr Ala Ser Val Phe Glu

1310 1315 1320

Val Phe Lys Lys Asp Val Asn Leu Asp Tyr Asp Glu Leu Lys Lys

1325 1330 1335

Lys Phe Lys Leu Ile Gly Asn Asn Asp Ile Leu Glu Arg Leu Met

1340 1345 1350

Lys Pro Lys Lys Val Ser Val Leu Glu Leu Glu Ser Tyr Asn Ser

1355 1360 1365

Asp Tyr Ile Lys Asn Leu Ile Ile Glu Leu Leu Thr Lys Ile Glu

1370 1375 1380

Asn Thr Asn Asp Thr Leu

1385

<210> 57

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 57

guuuuagucc ccuucgauau uggggugguc uauauc 36

<210> 58

<211> 95

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 58

60

uuuaauuuaa aaauauuuua uuagauuaaa guaga 95

<210> 59

<211> 1300

<212> PRT

<213> Leptotrichia shahii

<400> 59

Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr

1 5 10 15

Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys

20 25 30

Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys

35 40 45

Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu

50 55 60

Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser

65 70 75 80

Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys

85 90 95

Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr

100 105 110

Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile

115 120 125

Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln

130 135 140

Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr

145 150 155 160

Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr

165 170 175

Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser

180 185 190

Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu

195 200 205

Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys

210 215 220

Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu

225 230 235 240

Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg

245 250 255

Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr

260 265 270

Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys

275 280 285

Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile

290 295 300

Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys

305 310 315 320

Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser

325 330 335

Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met

340 345 350

Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys

355 360 365

Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln

370 375 380

Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr

385 390 395 400

Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala

405 410 415

Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn

420 425 430

Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala

435 440 445

Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn

450 455 460

Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala

465 470 475 480

Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys

485 490 495

Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys

500 505 510

Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp

515 520 525

Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His

530 535 540

Ile Ser Gln Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His

545 550 555 560

Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val

565 570 575

Pro Leu Tyr Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser

580 585 590

Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly

595 600 605

Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys

610 615 620

Asp Asp Lys Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile

625 630 635 640

Phe Asp Asp Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys

645 650 655

Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val

660 665 670

Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile

675 680 685

Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln

690 695 700

Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe

705 710 715 720

Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp

725 730 735

Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu

740 745 750

Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn

755 760 765

Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr

770 775 780

Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg

785 790 795 800

Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn

805 810 815

Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr

820 825 830

Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala

835 840 845

Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu

850 855 860

Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe

865 870 875 880

His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe

885 890 895

Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His

900 905 910

Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu

915 920 925

Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile

930 935 940

Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile

945 950 955 960

Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn

965 970 975

Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile

980 985 990

Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu

995 1000 1005

Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val

1010 1015 1020

Tyr Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu

1025 1030 1035

Val Phe Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg

1040 1045 1050

Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly

1055 1060 1065

Lys Gln Thr Gly Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser

1070 1075 1080

Lys Ile Cys Pro Val Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys

1085 1090 1095

Tyr Glu Ser Val Ser Lys Ser Gln Glu Phe Phe Ser Lys Phe Asp

1100 1105 1110

Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr Phe Glu Phe Ser Phe

1115 1120 1125

Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr

1130 1135 1140

Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg Asn Ser Asp

1145 1150 1155

Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr Lys Glu

1160 1165 1170

Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His Gly

1175 1180 1185

Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe

1190 1195 1200

Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg

1205 1210 1215

Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val

1220 1225 1230

Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys

1235 1240 1245

Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly

1250 1255 1260

Leu Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu

1265 1270 1275

Gly Lys Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu

1280 1285 1290

Phe Val Gln Asn Arg Asn Asn

1295 1300

<210> 60

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 60

gucuaagaac uuuaaauaau uucuacuguu guagau 36

<210> 61

<211> 71

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 61

aucuacaaaa uuauaaacua aauaaagauu cuuuauaauaa cuuuauauau aaucgaaaug 60

uagagaauuu u 71

<210> 62

<211> 1300

<212> PRT

<213> Francisella ularensis

<400> 62

Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr

1 5 10 15

Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys

20 25 30

Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys

35 40 45

Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu

50 55 60

Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser

65 70 75 80

Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys

85 90 95

Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr

100 105 110

Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile

115 120 125

Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln

130 135 140

Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr

145 150 155 160

Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr

165 170 175

Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser

180 185 190

Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu

195 200 205

Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys

210 215 220

Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu

225 230 235 240

Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg

245 250 255

Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr

260 265 270

Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys

275 280 285

Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile

290 295 300

Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys

305 310 315 320

Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser

325 330 335

Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met

340 345 350

Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys

355 360 365

Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln

370 375 380

Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr

385 390 395 400

Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala

405 410 415

Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn

420 425 430

Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala

435 440 445

Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn

450 455 460

Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala

465 470 475 480

Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys

485 490 495

Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys

500 505 510

Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp

515 520 525

Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His

530 535 540

Ile Ser Gln Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His

545 550 555 560

Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val

565 570 575

Pro Leu Tyr Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser

580 585 590

Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly

595 600 605

Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys

610 615 620

Asp Asp Lys Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile

625 630 635 640

Phe Asp Asp Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys

645 650 655

Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val

660 665 670

Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile

675 680 685

Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln

690 695 700

Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe

705 710 715 720

Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp

725 730 735

Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu

740 745 750

Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn

755 760 765

Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr

770 775 780

Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg

785 790 795 800

Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn

805 810 815

Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr

820 825 830

Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala

835 840 845

Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu

850 855 860

Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe

865 870 875 880

His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe

885 890 895

Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His

900 905 910

Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu

915 920 925

Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile

930 935 940

Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile

945 950 955 960

Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn

965 970 975

Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile

980 985 990

Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu

995 1000 1005

Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val

1010 1015 1020

Tyr Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu

1025 1030 1035

Val Phe Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg

1040 1045 1050

Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly

1055 1060 1065

Lys Gln Thr Gly Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser

1070 1075 1080

Lys Ile Cys Pro Val Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys

1085 1090 1095

Tyr Glu Ser Val Ser Lys Ser Gln Glu Phe Phe Ser Lys Phe Asp

1100 1105 1110

Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr Phe Glu Phe Ser Phe

1115 1120 1125

Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr

1130 1135 1140

Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg Asn Ser Asp

1145 1150 1155

Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr Lys Glu

1160 1165 1170

Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His Gly

1175 1180 1185

Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe

1190 1195 1200

Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg

1205 1210 1215

Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val

1220 1225 1230

Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys

1235 1240 1245

Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly

1250 1255 1260

Leu Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu

1265 1270 1275

Gly Lys Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu

1280 1285 1290

Phe Val Gln Asn Arg Asn Asn

1295 1300

<210> 63

<211> 138

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 63

aaaaggccgg cggccacgaa aaaggccggc caggcaaaaa agaaaaaggg atcctaccca 60

tacgatgttc cagattacgc ttatccctac gacgtgcctg attatgcata cccatatgat 120

gtccccgact atgcctaa 138

<210> 64

<211> 1388

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 64

Met Ser Leu Asn Arg Ile Tyr Gln Gly Arg Val Ala Ala Val Glu Thr

1 5 10 15

Gly Thr Ala Leu Ala Lys Gly Asn Val Glu Trp Met Pro Ala Ala Gly

20 25 30

Gly Asp Glu Val Leu Trp Gln His His Glu Leu Phe Gln Ala Ala Ile

35 40 45

Asn Tyr Tyr Leu Val Ala Leu Leu Ala Leu Ala Asp Lys Asn Asn Pro

50 55 60

Val Leu Gly Pro Leu Ile Ser Gln Met Asp Asn Pro Gln Ser Pro Tyr

65 70 75 80

His Val Trp Gly Ser Phe Arg Arg Gln Gly Arg Gln Arg Thr Gly Leu

85 90 95

Ser Gln Ala Val Ala Pro Tyr Ile Thr Pro Gly Asn Asn Ala Pro Thr

100 105 110

Leu Asp Glu Val Phe Arg Ser Ile Leu Ala Gly Asn Pro Thr Asp Arg

115 120 125

Ala Thr Leu Asp Ala Ala Leu Met Gln Leu Leu Lys Ala Cys Asp Gly

130 135 140

Ala Gly Ala Ile Gln Gln Glu Gly Arg Ser Tyr Trp Pro Lys Phe Cys

145 150 155 160

Asp Pro Asp Ser Thr Ala Asn Phe Ala Gly Asp Pro Ala Met Leu Arg

165 170 175

Arg Glu Gln His Arg Leu Leu Leu Pro Gln Val Leu His Asp Pro Ala

180 185 190

Ile Thr His Asp Ser Pro Ala Leu Gly Ser Phe Asp Thr Tyr Ser Ile

195 200 205

Ala Thr Pro Asp Thr Arg Thr Pro Gln Leu Thr Gly Pro Lys Ala Arg

210 215 220

Ala Arg Leu Glu Gln Ala Ile Thr Leu Trp Arg Val Arg Leu Pro Glu

225 230 235 240

Ser Ala Ala Asp Phe Asp Arg Leu Ala Ser Ser Leu Lys Lys Ile Pro

245 250 255

Asp Asp Asp Ser Arg Leu Asn Leu Gln Gly Tyr Val Gly Ser Ser Ala

260 265 270

Lys Gly Glu Val Gln Ala Arg Leu Phe Ala Leu Leu Leu Phe Arg His

275 280 285

Leu Glu Arg Ser Ser Phe Thr Leu Gly Leu Leu Arg Ser Ala Thr Pro

290 295 300

Pro Pro Lys Asn Ala Glu Thr Pro Pro Pro Ala Gly Val Pro Leu Pro

305 310 315 320

Ala Ala Ser Ala Ala Asp Pro Val Arg Ile Ala Arg Gly Lys Arg Ser

325 330 335

Phe Val Phe Arg Ala Phe Thr Ser Leu Pro Cys Trp His Gly Gly Asp

340 345 350

Asn Ile His Pro Thr Trp Lys Ser Phe Asp Ile Ala Ala Phe Lys Tyr

355 360 365

Ala Leu Thr Val Ile Asn Gln Ile Glu Glu Lys Thr Lys Glu Arg Gln

370 375 380

Lys Glu Cys Ala Glu Leu Glu Thr Asp Phe Asp Tyr Met His Gly Arg

385 390 395 400

Leu Ala Lys Ile Pro Val Lys Tyr Thr Thr Gly Glu Ala Glu Pro Pro

405 410 415

Pro Ile Leu Ala Asn Asp Leu Arg Ile Pro Leu Leu Arg Glu Leu Leu

420 425 430

Gln Asn Ile Lys Val Asp Thr Ala Leu Thr Asp Gly Glu Ala Val Ser

435 440 445

Tyr Gly Leu Gln Arg Arg Thr Ile Arg Gly Phe Arg Glu Leu Arg Arg

450 455 460

Ile Trp Arg Gly His Ala Pro Ala Gly Thr Val Phe Ser Ser Glu Leu

465 470 475 480

Lys Glu Lys Leu Ala Gly Glu Leu Arg Gln Phe Gln Thr Asp Asn Ser

485 490 495

Thr Thr Ile Gly Ser Val Gln Leu Phe Asn Glu Leu Ile Gln Asn Pro

500 505 510

Lys Tyr Trp Pro Ile Trp Gln Ala Pro Asp Val Glu Thr Ala Arg Gln

515 520 525

Trp Ala Asp Ala Gly Phe Ala Asp Asp Pro Leu Ala Ala Leu Val Gln

530 535 540

Glu Ala Glu Leu Gln Glu Asp Ile Asp Ala Leu Lys Ala Pro Val Lys

545 550 555 560

Leu Thr Pro Ala Asp Pro Glu Tyr Ser Arg Arg Gln Tyr Asp Phe Asn

565 570 575

Ala Val Ser Lys Phe Gly Ala Gly Ser Arg Ser Ala Asn Arg His Glu

580 585 590

Pro Gly Gln Thr Glu Arg Gly His Asn Thr Phe Thr Thr Glu Ile Ala

595 600 605

Ala Arg Asn Ala Ala Asp Gly Asn Arg Trp Arg Ala Thr His Val Arg

610 615 620

Ile His Tyr Ser Ala Pro Arg Leu Leu Arg Asp Gly Leu Arg Arg Pro

625 630 635 640

Asp Thr Asp Gly Asn Glu Ala Leu Glu Ala Val Pro Trp Leu Gln Pro

645 650 655

Met Met Glu Ala Leu Ala Pro Leu Pro Thr Leu Pro Gln Asp Leu Thr

660 665 670

Gly Met Pro Val Phe Leu Met Pro Asp Val Thr Leu Ser Gly Glu Arg

675 680 685

Arg Ile Leu Leu Asn Leu Pro Val Thr Leu Glu Pro Ala Ala Leu Val

690 695 700

Glu Gln Leu Gly Asn Ala Gly Arg Trp Gln Asn Gln Phe Phe Gly Ser

705 710 715 720

Arg Glu Asp Pro Phe Ala Leu Arg Trp Pro Ala Asp Gly Ala Val Lys

725 730 735

Thr Ala Lys Gly Lys Thr His Ile Pro Trp His Gln Asp Arg Asp His

740 745 750

Phe Thr Val Leu Gly Val Asp Leu Gly Thr Arg Asp Ala Gly Ala Leu

755 760 765

Ala Leu Leu Asn Val Thr Ala Gln Lys Pro Ala Lys Pro Val His Arg

770 775 780

Ile Ile Gly Glu Ala Asp Gly Arg Thr Trp Tyr Ala Ser Leu Ala Asp

785 790 795 800

Ala Arg Met Ile Arg Leu Pro Gly Glu Asp Ala Arg Leu Phe Val Arg

805 810 815

Gly Lys Leu Val Gln Glu Pro Tyr Gly Glu Arg Gly Arg Asn Ala Ser

820 825 830

Leu Leu Glu Trp Glu Asp Ala Arg Asn Ile Ile Leu Arg Leu Gly Gln

835 840 845

Asn Pro Asp Glu Leu Leu Gly Ala Asp Pro Arg Arg His Ser Tyr Pro

850 855 860

Glu Ile Asn Asp Lys Leu Leu Val Ala Leu Arg Arg Ala Gln Ala Arg

865 870 875 880

Leu Ala Arg Leu Gln Asn Arg Ser Trp Arg Leu Arg Asp Leu Ala Glu

885 890 895

Ser Asp Lys Ala Leu Asp Glu Ile His Ala Glu Arg Ala Gly Glu Lys

900 905 910

Pro Ser Pro Leu Pro Pro Leu Ala Arg Asp Asp Ala Ile Lys Ser Thr

915 920 925

Asp Glu Ala Leu Leu Ser Gln Arg Asp Ile Ile Arg Arg Ser Phe Val

930 935 940

Gln Ile Ala Asn Leu Ile Leu Pro Leu Arg Gly Arg Arg Trp Glu Trp

945 950 955 960

Arg Pro His Val Glu Val Pro Asp Cys His Ile Leu Ala Gln Ser Asp

965 970 975

Pro Gly Thr Asp Asp Thr Lys Arg Leu Val Ala Gly Gln Arg Gly Ile

980 985 990

Ser His Glu Arg Ile Glu Gln Ile Glu Glu Leu Arg Arg Arg Cys Gln

995 1000 1005

Ser Leu Asn Arg Ala Leu Arg His Lys Pro Gly Glu Arg Pro Val

1010 1015 1020

Leu Gly Arg Pro Ala Lys Gly Glu Glu Ile Ala Asp Pro Cys Pro

1025 1030 1035

Ala Leu Leu Glu Lys Ile Asn Arg Leu Arg Asp Gln Arg Val Asp

1040 1045 1050

Gln Thr Ala His Ala Ile Leu Ala Ala Ala Leu Gly Val Arg Leu

1055 1060 1065

Arg Ala Pro Ser Lys Asp Arg Ala Glu Arg Arg His Arg Asp Ile

1070 1075 1080

His Gly Glu Tyr Glu Arg Phe Arg Ala Pro Ala Asp Phe Val Val

1085 1090 1095

Ile Glu Asn Leu Ser Arg Tyr Leu Ser Ser Gln Asp Arg Ala Arg

1100 1105 1110

Ser Glu Asn Thr Arg Leu Met Gln Trp Cys His Arg Gln Ile Val

1115 1120 1125

Gln Lys Leu Arg Gln Leu Cys Glu Thr Tyr Gly Ile Pro Val Leu

1130 1135 1140

Ala Val Pro Ala Ala Tyr Ser Ser Arg Phe Ser Ser Arg Asp Gly

1145 1150 1155

Ser Ala Gly Phe Arg Ala Val His Leu Thr Pro Asp His Arg His

1160 1165 1170

Arg Met Pro Trp Ser Arg Ile Leu Ala Arg Leu Lys Ala His Glu

1175 1180 1185

Glu Asp Gly Lys Arg Leu Glu Lys Thr Val Leu Asp Glu Ala Arg

1190 1195 1200

Ala Val Arg Gly Leu Phe Asp Arg Leu Asp Arg Phe Asn Ala Gly

1205 1210 1215

His Val Pro Gly Lys Pro Trp Arg Thr Leu Leu Ala Pro Leu Pro

1220 1225 1230

Gly Gly Pro Val Phe Val Pro Leu Gly Asp Ala Thr Pro Met Gln

1235 1240 1245

Ala Asp Leu Asn Ala Ala Ile Asn Ile Ala Leu Arg Gly Ile Ala

1250 1255 1260

Ala Pro Asp Arg His Asp Ile His His Arg Leu Arg Ala Glu Asn

1265 1270 1275

Lys Lys Arg Ile Leu Ser Leu Arg Leu Gly Thr Gln Arg Glu Lys

1280 1285 1290

Ala Arg Trp Pro Gly Gly Ala Pro Ala Val Thr Leu Ser Thr Pro

1295 1300 1305

Asn Asn Gly Ala Ser Pro Glu Asp Ser Asp Ala Leu Pro Glu Arg

1310 1315 1320

Val Ser Asn Leu Phe Val Asp Ile Ala Gly Val Ala Asn Phe Glu

1325 1330 1335

Arg Val Thr Ile Glu Gly Val Ser Gln Lys Phe Ala Thr Gly Arg

1340 1345 1350

Gly Leu Trp Ala Ser Val Lys Gln Arg Ala Trp Asn Arg Val Ala

1355 1360 1365

Arg Leu Asn Glu Thr Val Thr Asp Asn Asn Arg Asn Glu Glu Glu

1370 1375 1380

Asp Asp Ile Pro Met

1385

<210> 65

<211> 1108

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 65

Met Ala Thr Arg Ser Phe Ile Leu Lys Ile Glu Pro Asn Glu Glu Val

1 5 10 15

Lys Lys Gly Leu Trp Lys Thr His Glu Val Leu Asn His Gly Ile Ala

20 25 30

Tyr Tyr Met Asn Ile Leu Lys Leu Ile Arg Gln Glu Ala Ile Tyr Glu

35 40 45

His His Glu Gln Asp Pro Lys Asn Pro Lys Lys Val Ser Lys Ala Glu

50 55 60

Ile Gln Ala Glu Leu Trp Asp Phe Val Leu Lys Met Gln Lys Cys Asn

65 70 75 80

Ser Phe Thr His Glu Val Asp Lys Asp Val Val Phe Asn Ile Leu Arg

85 90 95

Glu Leu Tyr Glu Glu Leu Val Pro Ser Ser Val Glu Lys Lys Gly Glu

100 105 110

Ala Asn Gln Leu Ser Asn Lys Phe Leu Tyr Pro Leu Val Asp Pro Asn

115 120 125

Ser Gln Ser Gly Lys Gly Thr Ala Ser Ser Gly Arg Lys Pro Arg Trp

130 135 140

Tyr Asn Leu Lys Ile Ala Gly Asp Pro Ser Trp Glu Glu Glu Lys Lys

145 150 155 160

Lys Trp Glu Glu Asp Lys Lys Lys Asp Pro Leu Ala Lys Ile Leu Gly

165 170 175

Lys Leu Ala Glu Tyr Gly Leu Ile Pro Leu Phe Ile Pro Phe Thr Asp

180 185 190

Ser Asn Glu Pro Ile Val Lys Glu Ile Lys Trp Met Glu Lys Ser Arg

195 200 205

Asn Gln Ser Val Arg Arg Leu Asp Lys Asp Met Phe Ile Gln Ala Leu

210 215 220

Glu Arg Phe Leu Ser Trp Glu Ser Trp Asn Leu Lys Val Lys Glu Glu

225 230 235 240

Tyr Glu Lys Val Glu Lys Glu His Lys Thr Leu Glu Glu Arg Ile Lys

245 250 255

Glu Asp Ile Gln Ala Phe Lys Ser Leu Glu Gln Tyr Glu Lys Glu Arg

260 265 270

Gln Glu Gln Leu Leu Arg Asp Thr Leu Asn Thr Asn Glu Tyr Arg Leu

275 280 285

Ser Lys Arg Gly Leu Arg Gly Trp Arg Glu Ile Ile Gln Lys Trp Leu

290 295 300

Lys Met Asp Glu Asn Glu Pro Ser Glu Lys Tyr Leu Glu Val Phe Lys

305 310 315 320

Asp Tyr Gln Arg Lys His Pro Arg Glu Ala Gly Asp Tyr Ser Val Tyr

325 330 335

Glu Phe Leu Ser Lys Lys Glu Asn His Phe Ile Trp Arg Asn His Pro

340 345 350

Glu Tyr Pro Tyr Leu Tyr Ala Thr Phe Cys Glu Ile Asp Lys Lys Lys

355 360 365

Lys Asp Ala Lys Gln Gln Ala Thr Phe Thr Leu Ala Asp Pro Ile Asn

370 375 380

His Pro Leu Trp Val Arg Phe Glu Glu Arg Ser Gly Ser Asn Leu Asn

385 390 395 400

Lys Tyr Arg Ile Leu Thr Glu Gln Leu His Thr Glu Lys Leu Lys Lys

405 410 415

Lys Leu Thr Val Gln Leu Asp Arg Leu Ile Tyr Pro Thr Glu Ser Gly

420 425 430

Gly Trp Glu Glu Lys Gly Lys Val Asp Ile Val Leu Leu Pro Ser Arg

435 440 445

Gln Phe Tyr Asn Gln Ile Phe Leu Asp Ile Glu Glu Lys Gly Lys His

450 455 460

Ala Phe Thr Tyr Lys Asp Glu Ser Ile Lys Phe Pro Leu Lys Gly Thr

465 470 475 480

Leu Gly Gly Ala Arg Val Gln Phe Asp Arg Asp His Leu Arg Arg Tyr

485 490 495

Pro His Lys Val Glu Ser Gly Asn Val Gly Arg Ile Tyr Phe Asn Met

500 505 510

Thr Val Asn Ile Glu Pro Thr Glu Ser Pro Val Ser Lys Ser Leu Lys

515 520 525

Ile His Arg Asp Asp Phe Pro Lys Phe Val Asn Phe Lys Pro Lys Glu

530 535 540

Leu Thr Glu Trp Ile Lys Asp Ser Lys Gly Lys Lys Leu Lys Ser Gly

545 550 555 560

Ile Glu Ser Leu Glu Ile Gly Leu Arg Val Met Ser Ile Asp Leu Gly

565 570 575

Gln Arg Gln Ala Ala Ala Ala Ser Ile Phe Glu Val Val Asp Gln Lys

580 585 590

Pro Asp Ile Glu Gly Lys Leu Phe Phe Pro Ile Lys Gly Thr Glu Leu

595 600 605

Tyr Ala Val His Arg Ala Ser Phe Asn Ile Lys Leu Pro Gly Glu Thr

610 615 620

Leu Val Lys Ser Arg Glu Val Leu Arg Lys Ala Arg Glu Asp Asn Leu

625 630 635 640

Lys Leu Met Asn Gln Lys Leu Asn Phe Leu Arg Asn Val Leu His Phe

645 650 655

Gln Gln Phe Glu Asp Ile Thr Glu Arg Glu Lys Arg Val Thr Lys Trp

660 665 670

Ile Ser Arg Gln Glu Asn Ser Asp Val Pro Leu Val Tyr Gln Asp Glu

675 680 685

Leu Ile Gln Ile Arg Glu Leu Met Tyr Lys Pro Tyr Lys Asp Trp Val

690 695 700

Ala Phe Leu Lys Gln Leu His Lys Arg Leu Glu Val Glu Ile Gly Lys

705 710 715 720

Glu Val Lys His Trp Arg Lys Ser Leu Ser Asp Gly Arg Lys Gly Leu

725 730 735

Tyr Gly Ile Ser Leu Lys Asn Ile Asp Glu Ile Asp Arg Thr Arg Lys

740 745 750

Phe Leu Leu Arg Trp Ser Leu Arg Pro Thr Glu Pro Gly Glu Val Arg

755 760 765

Arg Leu Glu Pro Gly Gln Arg Phe Ala Ile Asp Gln Leu Asn His Leu

770 775 780

Asn Ala Leu Lys Glu Asp Arg Leu Lys Lys Met Ala Asn Thr Ile Ile

785 790 795 800

Met His Ala Leu Gly Tyr Cys Tyr Asp Val Arg Lys Lys Lys Trp Gln

805 810 815

Ala Lys Asn Pro Ala Cys Gln Ile Ile Leu Phe Glu Asp Leu Ser Asn

820 825 830

Tyr Asn Pro Tyr Glu Glu Arg Ser Arg Phe Glu Asn Ser Lys Leu Met

835 840 845

Lys Trp Ser Arg Arg Glu Ile Pro Arg Gln Val Ala Leu Gln Gly Glu

850 855 860

Ile Tyr Gly Leu Gln Val Gly Glu Val Gly Ala Gln Phe Ser Ser Arg

865 870 875 880

Phe His Ala Lys Thr Gly Ser Pro Gly Ile Arg Cys Ser Val Val Thr

885 890 895

Lys Glu Lys Leu Gln Asp Asn Arg Phe Phe Lys Asn Leu Gln Arg Glu

900 905 910

Gly Arg Leu Thr Leu Asp Lys Ile Ala Val Leu Lys Glu Gly Asp Leu

915 920 925

Tyr Pro Asp Lys Gly Gly Glu Lys Phe Ile Ser Leu Ser Lys Asp Arg

930 935 940

Lys Leu Val Thr Thr His Ala Asp Ile Asn Ala Ala Gln Asn Leu Gln

945 950 955 960

Lys Arg Phe Trp Thr Arg Thr His Gly Phe Tyr Lys Val Tyr Cys Lys

965 970 975

Ala Tyr Gln Val Asp Gly Gln Thr Val Tyr Ile Pro Glu Ser Lys Asp

980 985 990

Gln Lys Gln Lys Ile Ile Glu Glu Phe Gly Glu Gly Tyr Phe Ile Leu

995 1000 1005

Lys Asp Gly Val Tyr Glu Trp Gly Asn Ala Gly Lys Leu Lys Ile

1010 1015 1020

Lys Lys Gly Ser Ser Lys Gln Ser Ser Ser Glu Leu Val Asp Ser

1025 1030 1035

Asp Ile Leu Lys Asp Ser Phe Asp Leu Ala Ser Glu Leu Lys Gly

1040 1045 1050

Glu Lys Leu Met Leu Tyr Arg Asp Pro Ser Gly Asn Val Phe Pro

1055 1060 1065

Ser Asp Lys Trp Met Ala Ala Gly Val Phe Phe Gly Lys Leu Glu

1070 1075 1080

Arg Ile Leu Ile Ser Lys Leu Thr Asn Gln Tyr Ser Ile Ser Thr

1085 1090 1095

Ile Glu Asp Asp Ser Ser Lys Gln Ser Met

1100 1105

<210> 66

<211> 1108

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 66

Met Ala Ile Arg Ser Ile Lys Leu Lys Leu Lys Thr His Thr Gly Pro

1 5 10 15

Glu Ala Gln Asn Leu Arg Lys Gly Ile Trp Arg Thr His Arg Leu Leu

20 25 30

Asn Glu Gly Val Ala Tyr Tyr Met Lys Met Leu Leu Leu Phe Arg Gln

35 40 45

Glu Ser Thr Gly Glu Arg Pro Lys Glu Glu Leu Gln Glu Glu Leu Ile

50 55 60

Cys His Ile Arg Glu Gln Gln Gln Arg Asn Gln Ala Asp Lys Asn Thr

65 70 75 80

Gln Ala Leu Pro Leu Asp Lys Ala Leu Glu Ala Leu Arg Gln Leu Tyr

85 90 95

Glu Leu Leu Val Pro Ser Ser Val Gly Gln Ser Gly Asp Ala Gln Ile

100 105 110

Ile Ser Arg Lys Phe Leu Ser Pro Leu Val Asp Pro Asn Ser Glu Gly

115 120 125

Gly Lys Gly Thr Ser Lys Ala Gly Ala Lys Pro Thr Trp Gln Lys Lys

130 135 140

Lys Glu Ala Asn Asp Pro Thr Trp Glu Gln Asp Tyr Glu Lys Trp Lys

145 150 155 160

Lys Arg Arg Glu Glu Asp Pro Thr Ala Ser Val Ile Thr Thr Leu Glu

165 170 175

Glu Tyr Gly Ile Arg Pro Ile Phe Pro Leu Tyr Thr Asn Thr Val Thr

180 185 190

Asp Ile Ala Trp Leu Pro Leu Gln Ser Asn Gln Phe Val Arg Thr Trp

195 200 205

Asp Arg Asp Met Leu Gln Gln Ala Ile Glu Arg Leu Leu Ser Trp Glu

210 215 220

Ser Trp Asn Lys Arg Val Gln Glu Glu Tyr Ala Lys Leu Lys Glu Lys

225 230 235 240

Met Ala Gln Leu Asn Glu Gln Leu Glu Gly Gly Gln Glu Trp Ile Ser

245 250 255

Leu Leu Glu Gln Tyr Glu Glu Asn Arg Glu Arg Glu Leu Arg Glu Asn

260 265 270

Met Thr Ala Ala Asn Asp Lys Tyr Arg Ile Thr Lys Arg Gln Met Lys

275 280 285

Gly Trp Asn Glu Leu Tyr Glu Leu Trp Ser Thr Phe Pro Ala Ser Ala

290 295 300

Ser His Glu Gln Tyr Lys Glu Ala Leu Lys Arg Val Gln Gln Arg Leu

305 310 315 320

Arg Gly Arg Phe Gly Asp Ala His Phe Phe Gln Tyr Leu Met Glu Glu

325 330 335

Lys Asn Arg Leu Ile Trp Lys Gly Asn Pro Gln Arg Ile His Tyr Phe

340 345 350

Val Ala Arg Asn Glu Leu Thr Lys Arg Leu Glu Glu Ala Lys Gln Ser

355 360 365

Ala Thr Met Thr Leu Pro Asn Ala Arg Lys His Pro Leu Trp Val Arg

370 375 380

Phe Asp Ala Arg Gly Gly Asn Leu Gln Asp Tyr Tyr Leu Thr Ala Glu

385 390 395 400

Ala Asp Lys Pro Arg Ser Arg Arg Phe Val Thr Phe Ser Gln Leu Ile

405 410 415

Trp Pro Ser Glu Ser Gly Trp Met Glu Lys Lys Asp Val Glu Val Glu

420 425 430

Leu Ala Leu Ser Arg Gln Phe Tyr Gln Gln Val Lys Leu Leu Lys Asn

435 440 445

Asp Lys Gly Lys Gln Lys Ile Glu Phe Lys Asp Lys Gly Ser Gly Ser

450 455 460

Thr Phe Asn Gly His Leu Gly Gly Ala Lys Leu Gln Leu Glu Arg Gly

465 470 475 480

Asp Leu Glu Lys Glu Glu Lys Asn Phe Glu Asp Gly Glu Ile Gly Ser

485 490 495

Val Tyr Leu Asn Val Val Ile Asp Phe Glu Pro Leu Gln Glu Val Lys

500 505 510

Asn Gly Arg Val Gln Ala Pro Tyr Gly Gln Val Leu Gln Leu Ile Arg

515 520 525

Arg Pro Asn Glu Phe Pro Lys Val Thr Thr Tyr Lys Ser Glu Gln Leu

530 535 540

Val Glu Trp Ile Lys Ala Ser Pro Gln His Ser Ala Gly Val Glu Ser

545 550 555 560

Leu Ala Ser Gly Phe Arg Val Met Ser Ile Asp Leu Gly Leu Arg Ala

565 570 575

Ala Ala Ala Thr Ser Ile Phe Ser Val Glu Glu Ser Ser Asp Lys Asn

580 585 590

Ala Ala Asp Phe Ser Tyr Trp Ile Glu Gly Thr Pro Leu Val Ala Val

595 600 605

His Gln Arg Ser Tyr Met Leu Arg Leu Pro Gly Glu Gln Val Glu Lys

610 615 620

Gln Val Met Glu Lys Arg Asp Glu Arg Phe Gln Leu His Gln Arg Val

625 630 635 640

Lys Phe Gln Ile Arg Val Leu Ala Gln Ile Met Arg Met Ala Asn Lys

645 650 655

Gln Tyr Gly Asp Arg Trp Asp Glu Leu Asp Ser Leu Lys Gln Ala Val

660 665 670

Glu Gln Lys Lys Ser Pro Leu Asp Gln Thr Asp Arg Thr Phe Trp Glu

675 680 685

Gly Ile Val Cys Asp Leu Thr Lys Val Leu Pro Arg Asn Glu Ala Asp

690 695 700

Trp Glu Gln Ala Val Val Gln Ile His Arg Lys Ala Glu Glu Tyr Val

705 710 715 720

Gly Lys Ala Val Gln Ala Trp Arg Lys Arg Phe Ala Ala Asp Glu Arg

725 730 735

Lys Gly Ile Ala Gly Leu Ser Met Trp Asn Ile Glu Glu Leu Glu Gly

740 745 750

Leu Arg Lys Leu Leu Ile Ser Trp Ser Arg Arg Thr Arg Asn Pro Gln

755 760 765

Glu Val Asn Arg Phe Glu Arg Gly His Thr Ser His Gln Arg Leu Leu

770 775 780

Thr His Ile Gln Asn Val Lys Glu Asp Arg Leu Lys Gln Leu Ser His

785 790 795 800

Ala Ile Val Met Thr Ala Leu Gly Tyr Val Tyr Asp Glu Arg Lys Gln

805 810 815

Glu Trp Cys Ala Glu Tyr Pro Ala Cys Gln Val Ile Leu Phe Glu Asn

820 825 830

Leu Ser Gln Tyr Arg Ser Asn Leu Asp Arg Ser Thr Lys Glu Asn Ser

835 840 845

Thr Leu Met Lys Trp Ala His Arg Ser Ile Pro Lys Tyr Val His Met

850 855 860

Gln Ala Glu Pro Tyr Gly Ile Gln Ile Gly Asp Val Arg Ala Glu Tyr

865 870 875 880

Ser Ser Arg Phe Tyr Ala Lys Thr Gly Thr Pro Gly Ile Arg Cys Lys

885 890 895

Lys Val Arg Gly Gln Asp Leu Gln Gly Arg Arg Phe Glu Asn Leu Gln

900 905 910

Lys Arg Leu Val Asn Glu Gln Phe Leu Thr Glu Glu Glu Gln Val Lys Gln

915 920 925

Leu Arg Pro Gly Asp Ile Val Pro Asp Asp Ser Gly Glu Leu Phe Met

930 935 940

Thr Leu Thr Asp Gly Ser Gly Ser Lys Glu Val Val Phe Leu Gln Ala

945 950 955 960

Asp Ile Asn Ala Ala His Asn Leu Gln Lys Arg Phe Trp Gln Arg Tyr

965 970 975

Asn Glu Leu Phe Lys Val Ser Cys Arg Val Ile Val Arg Asp Glu Glu

980 985 990

Glu Tyr Leu Val Pro Lys Thr Lys Ser Val Gln Ala Lys Leu Gly Lys

995 1000 1005

Gly Leu Phe Val Lys Lys Ser Asp Thr Ala Trp Lys Asp Val Tyr

1010 1015 1020

Val Trp Asp Ser Gln Ala Lys Leu Lys Gly Lys Thr Thr Phe Thr

1025 1030 1035

Glu Glu Ser Glu Ser Pro Glu Gln Leu Glu Asp Phe Gln Glu Ile

1040 1045 1050

Ile Glu Glu Ala Glu Glu Ala Lys Gly Thr Tyr Arg Thr Leu Phe

1055 1060 1065

Arg Asp Pro Ser Gly Val Phe Phe Pro Glu Ser Val Trp Tyr Pro

1070 1075 1080

Gln Lys Asp Phe Trp Gly Glu Val Lys Arg Lys Leu Tyr Gly Lys

1085 1090 1095

Leu Arg Glu Arg Phe Leu Thr Lys Ala Arg

1100 1105

<210> 67

<211> 1334

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 67

Met Lys Ile Ser Lys Val Asp His Thr Arg Met Ala Val Ala Lys Gly

1 5 10 15

Asn Gln His Arg Arg Asp Glu Ile Ser Gly Ile Leu Tyr Lys Asp Pro

20 25 30

Thr Lys Thr Gly Ser Ile Asp Phe Asp Glu Arg Phe Lys Lys Leu Asn

35 40 45

Cys Ser Ala Lys Ile Leu Tyr His Val Phe Asn Gly Ile Ala Glu Gly

50 55 60

Ser Asn Lys Tyr Lys Asn Ile Val Asp Lys Val Asn Asn Asn Asn Leu Asp

65 70 75 80

Arg Val Leu Phe Thr Gly Lys Ser Tyr Asp Arg Lys Ser Ile Ile Asp

85 90 95

Ile Asp Thr Val Leu Arg Asn Val Glu Lys Ile Asn Ala Phe Asp Arg

100 105 110

Ile Ser Thr Glu Glu Arg Glu Gln Ile Ile Asp Asp Leu Leu Glu Ile

115 120 125

Gln Leu Arg Lys Gly Leu Arg Lys Gly Lys Ala Gly Leu Arg Glu Val

130 135 140

Leu Leu Ile Gly Ala Gly Val Ile Val Arg Thr Asp Lys Lys Gln Glu

145 150 155 160

Ile Ala Asp Phe Leu Glu Ile Leu Asp Glu Asp Phe Asn Lys Thr Asn

165 170 175

Gln Ala Lys Asn Ile Lys Leu Ser Ile Glu Asn Gln Gly Leu Val Val

180 185 190

Ser Pro Val Ser Arg Gly Glu Glu Arg Ile Phe Asp Val Ser Gly Ala

195 200 205

Gln Lys Gly Lys Ser Ser Lys Lys Ala Gln Glu Lys Glu Ala Leu Ser

210 215 220

Ala Phe Leu Leu Asp Tyr Ala Asp Leu Asp Lys Asn Val Arg Phe Glu

225 230 235 240

Tyr Leu Arg Lys Ile Arg Arg Leu Ile Asn Leu Tyr Phe Tyr Val Lys

245 250 255

Asn Asp Asp Val Met Ser Leu Thr Glu Ile Pro Ala Glu Val Asn Leu

260 265 270

Glu Lys Asp Phe Asp Ile Trp Arg Asp His Glu Gln Arg Lys Glu Glu

275 280 285

Asn Gly Asp Phe Val Gly Cys Pro Asp Ile Leu Leu Ala Asp Arg Asp

290 295 300

Val Lys Lys Ser Asn Ser Lys Gln Val Lys Ile Ala Glu Arg Gln Leu

305 310 315 320

Arg Glu Ser Ile Arg Glu Lys Asn Ile Lys Arg Tyr Arg Phe Ser Ile

325 330 335

Lys Thr Ile Glu Lys Asp Asp Gly Thr Tyr Phe Phe Ala Asn Lys Gln

340 345 350

Ile Ser Val Phe Trp Ile His Arg Ile Glu Asn Ala Val Glu Arg Ile

355 360 365

Leu Gly Ser Ile Asn Asp Lys Lys Leu Tyr Arg Leu Arg Leu Gly Tyr

370 375 380

Leu Gly Glu Lys Val Trp Lys Asp Ile Leu Asn Phe Leu Ser Ile Lys

385 390 395 400

Tyr Ile Ala Val Gly Lys Ala Val Phe Asn Phe Ala Met Asp Asp Leu

405 410 415

Gln Glu Lys Asp Arg Asp Ile Glu Pro Gly Lys Ile Ser Glu Asn Ala

420 425 430

Val Asn Gly Leu Thr Ser Phe Asp Tyr Glu Gln Ile Lys Ala Asp Glu

435 440 445

Met Leu Gln Arg Glu Val Ala Val Asn Val Ala Phe Ala Ala Asn Asn

450 455 460

Leu Ala Arg Val Thr Val Asp Ile Pro Gln Asn Gly Glu Lys Glu Asp

465 470 475 480

Ile Leu Leu Trp Asn Lys Ser Asp Ile Lys Lys Tyr Lys Lys Asn Ser

485 490 495

Lys Lys Gly Ile Leu Lys Ser Ile Leu Gln Phe Phe Gly Gly Ala Ser

500 505 510

Thr Trp Asn Met Lys Met Phe Glu Ile Ala Tyr His Asp Gln Pro Gly

515 520 525

Asp Tyr Glu Glu Asn Tyr Leu Tyr Asp Ile Ile Gln Ile Ile Tyr Ser

530 535 540

Leu Arg Asn Lys Ser Phe His Phe Lys Thr Tyr Asp His Gly Asp Lys

545 550 555 560

Asn Trp Asn Arg Glu Leu Ile Gly Lys Met Ile Glu His Asp Ala Glu

565 570 575

Arg Val Ile Ser Val Glu Arg Glu Lys Phe His Ser Asn Asn Leu Pro

580 585 590

Met Phe Tyr Lys Asp Ala Asp Leu Lys Lys Ile Leu Asp Leu Leu Tyr

595 600 605

Ser Asp Tyr Ala Gly Arg Ala Ser Gln Val Pro Ala Phe Asn Thr Val

610 615 620

Leu Val Arg Lys Asn Phe Pro Glu Phe Leu Arg Lys Asp Met Gly Tyr

625 630 635 640

Lys Val His Phe Asn Asn Pro Glu Val Glu Asn Gln Trp His Ser Ala

645 650 655

Val Tyr Tyr Leu Tyr Lys Glu Ile Tyr Tyr Asn Leu Phe Leu Arg Asp

660 665 670

Lys Glu Val Lys Asn Leu Phe Tyr Thr Ser Leu Lys Asn Ile Arg Ser

675 680 685

Glu Val Ser Asp Lys Lys Gln Lys Leu Ala Ser Asp Asp Phe Ala Ser

690 695 700

Arg Cys Glu Glu Ile Glu Asp Arg Ser Leu Pro Glu Ile Cys Gln Ile

705 710 715 720

Ile Met Thr Glu Tyr Asn Ala Gln Asn Phe Gly Asn Arg Lys Val Lys

725 730 735

Ser Gln Arg Val Ile Glu Lys Asn Lys Asp Ile Phe Arg His Tyr Lys

740 745 750

Met Leu Leu Ile Lys Thr Leu Ala Gly Ala Phe Ser Leu Tyr Leu Lys

755 760 765

Gln Glu Arg Phe Ala Phe Ile Gly Lys Ala Thr Pro Ile Pro Tyr Glu

770 775 780

Thr Thr Asp Val Lys Asn Phe Leu Pro Glu Trp Lys Ser Gly Met Tyr

785 790 795 800

Ala Ser Phe Val Glu Glu Ile Lys Asn Asn Leu Asp Leu Gln Glu Trp

805 810 815

Tyr Ile Val Gly Arg Phe Leu Asn Gly Arg Met Leu Asn Gln Leu Ala

820 825 830

Gly Ser Leu Arg Ser Tyr Ile Gln Tyr Ala Glu Asp Ile Glu Arg Arg

835 840 845

Ala Ala Glu Asn Arg Asn Lys Leu Phe Ser Lys Pro Asp Glu Lys Ile

850 855 860

Glu Ala Cys Lys Lys Ala Val Arg Val Leu Asp Leu Cys Ile Lys Ile

865 870 875 880

Ser Thr Arg Ile Ser Ala Glu Phe Thr Asp Tyr Phe Asp Ser Glu Asp

885 890 895

Asp Tyr Ala Asp Tyr Leu Glu Lys Tyr Leu Lys Tyr Gln Asp Asp Ala

900 905 910

Ile Lys Glu Leu Ser Gly Ser Ser Tyr Ala Ala Leu Asp His Phe Cys

915 920 925

Asn Lys Asp Asp Leu Lys Phe Asp Ile Tyr Val Asn Ala Gly Gln Lys

930 935 940

Pro Ile Leu Gln Arg Asn Ile Val Met Ala Lys Leu Phe Gly Pro Asp

945 950 955 960

Asn Ile Leu Ser Glu Val Met Glu Lys Val Thr Glu Ser Ala Ile Arg

965 970 975

Glu Tyr Tyr Asp Tyr Leu Lys Lys Val Ser Gly Tyr Arg Val Arg Gly

980 985 990

Lys Cys Ser Thr Glu Lys Glu Gln Glu Asp Leu Leu Lys Phe Gln Arg

995 1000 1005

Leu Lys Asn Ala Val Glu Phe Arg Asp Val Thr Glu Tyr Ala Glu

1010 1015 1020

Val Ile Asn Glu Leu Leu Gly Gln Leu Ile Ser Trp Ser Tyr Leu

1025 1030 1035

Arg Glu Arg Asp Leu Leu Tyr Phe Gln Leu Gly Phe His Tyr Met

1040 1045 1050

Cys Leu Lys Asn Lys Ser Phe Lys Pro Ala Glu Tyr Val Asp Ile

1055 1060 1065

Arg Arg Asn Asn Gly Thr Ile Ile His Asn Ala Ile Leu Tyr Gln

1070 1075 1080

Ile Val Ser Met Tyr Ile Asn Gly Leu Asp Phe Tyr Ser Cys Asp

1085 1090 1095

Lys Glu Gly Lys Thr Leu Lys Pro Ile Glu Thr Gly Lys Gly Val

1100 1105 1110

Gly Ser Lys Ile Gly Gln Phe Ile Lys Tyr Ser Gln Tyr Leu Tyr

1115 1120 1125

Asn Asp Pro Ser Tyr Lys Leu Glu Ile Tyr Asn Ala Gly Leu Glu

1130 1135 1140

Val Phe Glu Asn Ile Asp Glu His Asp Asn Ile Thr Asp Leu Arg

1145 1150 1155

Lys Tyr Val Asp His Phe Lys Tyr Tyr Ala Tyr Gly Asn Lys Met

1160 1165 1170

Ser Leu Leu Asp Leu Tyr Ser Glu Phe Phe Asp Arg Phe Phe Thr

1175 1180 1185

Tyr Asp Met Lys Tyr Gln Lys Asn Val Val Asn Val Leu Glu Asn

1190 1195 1200

Ile Leu Leu Arg His Phe Val Ile Phe Tyr Pro Lys Phe Gly Ser

1205 1210 1215

Gly Lys Lys Asp Val Gly Ile Arg Asp Cys Lys Lys Glu Arg Ala

1220 1225 1230

Gln Ile Glu Ile Ser Glu Gln Ser Leu Thr Ser Glu Asp Phe Met

1235 1240 1245

Phe Lys Leu Asp Asp Lys Ala Gly Glu Glu Ala Lys Lys Phe Pro

1250 1255 1260

Ala Arg Asp Glu Arg Tyr Leu Gln Thr Ile Ala Lys Leu Leu Tyr

1265 1270 1275

Tyr Pro Asn Glu Ile Glu Asp Met Asn Arg Phe Met Lys Lys Gly

1280 1285 1290

Glu Thr Ile Asn Lys Lys Val Gln Phe Asn Arg Lys Lys Lys Ile

1295 1300 1305

Thr Arg Lys Gln Lys Asn Asn Ser Ser Asn Glu Val Leu Ser Ser

1310 1315 1320

Thr Met Gly Tyr Leu Phe Lys Asn Ile Lys Leu

1325 1330

<210> 68

<211> 1120

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 68

Met Trp Ile Ser Ile Lys Thr Leu Ile His His Leu Gly Val Leu Phe

1 5 10 15

Phe Cys Asp Tyr Met Tyr Asn Arg Arg Glu Lys Lys Ile Ile Glu Val

20 25 30

Lys Thr Met Arg Ile Thr Lys Val Glu Val Asp Arg Lys Lys Val Leu

35 40 45

Ile Ser Arg Asp Lys Asn Gly Gly Lys Leu Val Tyr Glu Asn Glu Met

50 55 60

Gln Asp Asn Thr Glu Gln Ile Met His His Lys Lys Ser Ser Phe Tyr

65 70 75 80

Lys Ser Val Val Asn Lys Thr Ile Cys Arg Pro Glu Gln Lys Gln Met

85 90 95

Lys Lys Leu Val His Gly Leu Leu Gln Glu Asn Ser Gln Glu Lys Ile

100 105 110

Lys Val Ser Asp Val Thr Lys Leu Asn Ile Ser Asn Phe Leu Asn His

115 120 125

Arg Phe Lys Lys Ser Leu Tyr Tyr Phe Pro Glu Asn Ser Pro Asp Lys

130 135 140

Ser Glu Glu Tyr Arg Ile Glu Ile Asn Leu Ser Gln Leu Leu Glu Asp

145 150 155 160

Ser Leu Lys Lys Gln Gln Gly Thr Phe Ile Cys Trp Glu Ser Phe Ser

165 170 175

Lys Asp Met Glu Leu Tyr Ile Asn Trp Ala Glu Asn Tyr Ile Ser Ser

180 185 190

Lys Thr Lys Leu Ile Lys Lys Ser Ile Arg Asn Asn Arg Ile Gln Ser

195 200 205

Thr Glu Ser Arg Ser Gly Gln Leu Met Asp Arg Tyr Met Lys Asp Ile

210 215 220

Leu Asn Lys Asn Lys Pro Phe Asp Ile Gln Ser Val Ser Glu Lys Tyr

225 230 235 240

Gln Leu Glu Lys Leu Thr Ser Ala Leu Lys Ala Thr Phe Lys Glu Ala

245 250 255

Lys Lys Asn Asp Lys Glu Ile Asn Tyr Lys Leu Lys Ser Thr Leu Gln

260 265 270

Asn His Glu Arg Gln Ile Ile Glu Glu Leu Lys Glu Asn Ser Glu Leu

275 280 285

Asn Gln Phe Asn Ile Glu Ile Arg Lys His Leu Glu Thr Tyr Phe Pro

290 295 300

Ile Lys Lys Thr Asn Arg Lys Val Gly Asp Ile Arg Asn Leu Glu Ile

305 310 315 320

Gly Glu Ile Gln Lys Ile Val Asn His Arg Leu Lys Asn Lys Ile Val

325 330 335

Gln Arg Ile Leu Gln Glu Gly Lys Leu Ala Ser Tyr Glu Ile Glu Ser

340 345 350

Thr Val Asn Ser Asn Ser Leu Gln Lys Ile Lys Ile Glu Glu Ala Phe

355 360 365

Ala Leu Lys Phe Ile Asn Ala Cys Leu Phe Ala Ser Asn Asn Leu Arg

370 375 380

Asn Met Val Tyr Pro Val Cys Lys Lys Asp Ile Leu Met Ile Gly Glu

385 390 395 400

Phe Lys Asn Ser Phe Lys Glu Ile Lys His Lys Lys Phe Ile Arg Gln

405 410 415

Trp Ser Gln Phe Phe Ser Gln Glu Ile Thr Val Asp Asp Ile Glu Leu

420 425 430

Ala Ser Trp Gly Leu Arg Gly Ala Ile Ala Pro Ile Arg Asn Glu Ile

435 440 445

Ile His Leu Lys Lys His Ser Trp Lys Lys Phe Phe Asn Asn Pro Thr

450 455 460

Phe Lys Val Lys Lys Ser Lys Ile Ile Asn Gly Lys Thr Lys Asp Val

465 470 475 480

Thr Ser Glu Phe Leu Tyr Lys Glu Thr Leu Phe Lys Asp Tyr Phe Tyr

485 490 495

Ser Glu Leu Asp Ser Val Pro Glu Leu Ile Ile Asn Lys Met Glu Ser

500 505 510

Ser Lys Ile Leu Asp Tyr Tyr Ser Ser Asp Gln Leu Asn Gln Val Phe

515 520 525

Thr Ile Pro Asn Phe Glu Leu Ser Leu Leu Thr Ser Ala Val Pro Phe

530 535 540

Ala Pro Ser Phe Lys Arg Val Tyr Leu Lys Gly Phe Asp Tyr Gln Asn

545 550 555 560

Gln Asp Glu Ala Gln Pro Asp Tyr Asn Leu Lys Leu Asn Ile Tyr Asn

565 570 575

Glu Lys Ala Phe Asn Ser Glu Ala Phe Gln Ala Gln Tyr Ser Leu Phe

580 585 590

Lys Met Val Tyr Tyr Gln Val Phe Leu Pro Gln Phe Thr Thr Asn Asn

595 600 605

Asp Leu Phe Lys Ser Ser Val Asp Phe Ile Leu Thr Leu Asn Lys Glu

610 615 620

Arg Lys Gly Tyr Ala Lys Ala Phe Gln Asp Ile Arg Lys Met Asn Lys

625 630 635 640

Asp Glu Lys Pro Ser Glu Tyr Met Ser Tyr Ile Gln Ser Gln Leu Met

645 650 655

Leu Tyr Gln Lys Lys Gln Glu Glu Lys Glu Lys Ile Asn His Phe Glu

660 665 670

Lys Phe Ile Asn Gln Val Phe Ile Lys Gly Phe Asn Ser Phe Ile Glu

675 680 685

Lys Asn Arg Leu Thr Tyr Ile Cys His Pro Thr Lys Asn Thr Val Pro

690 695 700

Glu Asn Asp Asn Ile Glu Ile Pro Phe His Thr Asp Met Asp Asp Ser

705 710 715 720

Asn Ile Ala Phe Trp Leu Met Cys Lys Leu Leu Asp Ala Lys Gln Leu

725 730 735

Ser Glu Leu Arg Asn Glu Met Ile Lys Phe Ser Cys Ser Leu Gln Ser

740 745 750

Thr Glu Glu Ile Ser Thr Phe Thr Lys Ala Arg Glu Val Ile Gly Leu

755 760 765

Ala Leu Leu Asn Gly Glu Lys Gly Cys Asn Asp Trp Lys Glu Leu Phe

770 775 780

Asp Asp Lys Glu Ala Trp Lys Lys Asn Met Ser Leu Tyr Val Ser Glu

785 790 795 800

Glu Leu Leu Gln Ser Leu Pro Tyr Thr Gln Glu Asp Gly Gln Thr Pro

805 810 815

Val Ile Asn Arg Ser Ile Asp Leu Val Lys Lys Tyr Gly Thr Glu Thr

820 825 830

Ile Leu Glu Lys Leu Phe Ser Ser Ser Asp Asp Tyr Lys Val Ser Ala

835 840 845

Lys Asp Ile Ala Lys Leu His Glu Tyr Asp Val Thr Glu Lys Ile Ala

850 855 860

Gln Gln Glu Ser Leu His Lys Gln Trp Ile Glu Lys Pro Gly Leu Ala

865 870 875 880

Arg Asp Ser Ala Trp Thr Lys Lys Tyr Gln Asn Val Ile Asn Asp Ile

885 890 895

Ser Asn Tyr Gln Trp Ala Lys Thr Lys Val Glu Leu Thr Gln Val Arg

900 905 910

His Leu His Gln Leu Thr Ile Asp Leu Leu Ser Arg Leu Ala Gly Tyr

915 920 925

Met Ser Ile Ala Asp Arg Asp Phe Gln Phe Ser Ser Asn Tyr Ile Leu

930 935 940

Glu Arg Glu Asn Ser Glu Tyr Arg Val Thr Ser Trp Ile Leu Leu Ser

945 950 955 960

Glu Asn Lys Asn Lys Asn Lys Tyr Asn Asp Tyr Glu Leu Tyr Asn Leu

965 970 975

Lys Asn Ala Ser Ile Lys Val Ser Ser Lys Asn Asp Pro Gln Leu Lys

980 985 990

Val Asp Leu Lys Gln Leu Arg Leu Thr Leu Glu Tyr Leu Glu Leu Phe

995 1000 1005

Asp Asn Arg Leu Lys Glu Lys Arg Asn Asn Ile Ser His Phe Asn

1010 1015 1020

Tyr Leu Asn Gly Gln Leu Gly Asn Ser Ile Leu Glu Leu Phe Asp

1025 1030 1035

Asp Ala Arg Asp Val Leu Ser Tyr Asp Arg Lys Leu Lys Asn Ala

1040 1045 1050

Val Ser Lys Ser Leu Lys Glu Ile Leu Ser Ser His Gly Met Glu

1055 1060 1065

Val Thr Phe Lys Pro Leu Tyr Gln Thr Asn His His Leu Lys Ile

1070 1075 1080

Asp Lys Leu Gln Pro Lys Lys Ile His His Leu Gly Glu Lys Ser

1085 1090 1095

Thr Val Ser Ser Asn Gln Val Ser Asn Glu Tyr Cys Gln Leu Val

1100 1105 1110

Arg Thr Leu Leu Thr Met Lys

1115 1120

<210> 69

<211> 1152

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 69

Met Lys Val Thr Lys Val Asp Gly Ile Ser His Lys Lys Tyr Ile Glu

1 5 10 15

Glu Gly Lys Leu Val Lys Ser Thr Ser Glu Glu Asn Arg Thr Ser Glu

20 25 30

Arg Leu Ser Glu Leu Leu Ser Ile Arg Leu Asp Ile Tyr Ile Lys Asn

35 40 45

Pro Asp Asn Ala Ser Glu Glu Glu Asn Arg Ile Arg Arg Glu Asn Leu

50 55 60

Lys Lys Phe Phe Ser Asn Lys Val Leu His Leu Lys Asp Ser Val Leu

65 70 75 80

Tyr Leu Lys Asn Arg Lys Glu Lys Asn Ala Val Gln Asp Lys Asn Tyr

85 90 95

Ser Glu Glu Asp Ile Ser Glu Tyr Asp Leu Lys Asn Lys Asn Ser Phe

100 105 110

Ser Val Leu Lys Lys Ile Leu Leu Asn Glu Asp Val Asn Ser Glu Glu

115 120 125

Leu Glu Ile Phe Arg Lys Asp Val Glu Ala Lys Leu Asn Lys Ile Asn

130 135 140

Ser Leu Lys Tyr Ser Phe Glu Glu Asn Lys Ala Asn Tyr Gln Lys Ile

145 150 155 160

Asn Glu Asn Asn Val Glu Lys Val Gly Gly Lys Ser Lys Arg Asn Ile

165 170 175

Ile Tyr Asp Tyr Tyr Arg Glu Ser Ala Lys Arg Asn Asp Tyr Ile Asn

180 185 190

Asn Val Gln Glu Ala Phe Asp Lys Leu Tyr Lys Lys Glu Asp Ile Glu

195 200 205

Lys Leu Phe Phe Leu Ile Glu Asn Ser Lys Lys His Glu Lys Tyr Lys

210 215 220

Ile Arg Glu Tyr Tyr His Lys Ile Ile Gly Arg Lys Asn Asp Lys Glu

225 230 235 240

Asn Phe Ala Lys Ile Ile Tyr Glu Glu Ile Gln Asn Val Asn Asn Ile

245 250 255

Lys Glu Leu Ile Glu Lys Ile Pro Asp Met Ser Glu Leu Lys Lys Ser

260 265 270

Gln Val Phe Tyr Lys Tyr Tyr Leu Asp Lys Glu Glu Leu Asn Asp Lys

275 280 285

Asn Ile Lys Tyr Ala Phe Cys His Phe Val Glu Ile Glu Met Ser Gln

290 295 300

Leu Leu Lys Asn Tyr Val Tyr Lys Arg Leu Ser Asn Ile Ser Asn Asp

305 310 315 320

Lys Ile Lys Arg Ile Phe Glu Tyr Gln Asn Leu Lys Lys Leu Ile Glu

325 330 335

Asn Lys Leu Leu Asn Lys Leu Asp Thr Tyr Val Arg Asn Cys Gly Lys

340 345 350

Tyr Asn Tyr Tyr Leu Gln Val Gly Glu Ile Ala Thr Ser Asp Phe Ile

355 360 365

Ala Arg Asn Arg Gln Asn Glu Ala Phe Leu Arg Asn Ile Ile Gly Val

370 375 380

Ser Ser Val Ala Tyr Phe Ser Leu Arg Asn Ile Leu Glu Thr Glu Asn

385 390 395 400

Glu Asn Asp Ile Thr Gly Arg Met Arg Gly Lys Thr Val Lys Asn Asn

405 410 415

Lys Gly Glu Glu Lys Tyr Val Ser Gly Glu Val Asp Lys Ile Tyr Asn

420 425 430

Glu Asn Lys Gln Asn Glu Val Lys Glu Asn Leu Lys Met Phe Tyr Ser

435 440 445

Tyr Asp Phe Asn Met Asp Asn Lys Asn Glu Ile Glu Asp Phe Phe Ala

450 455 460

Asn Ile Asp Glu Ala Ile Ser Ser Ile Arg His Gly Ile Val His Phe

465 470 475 480

Asn Leu Glu Leu Glu Gly Lys Asp Ile Phe Ala Phe Lys Asn Ile Ala

485 490 495

Pro Ser Glu Ile Ser Lys Lys Met Phe Gln Asn Glu Ile Asn Glu Lys

500 505 510

Lys Leu Lys Leu Lys Ile Phe Lys Gln Leu Asn Ser Ala Asn Val Phe

515 520 525

Asn Tyr Tyr Glu Lys Asp Val Ile Ile Lys Tyr Leu Lys Asn Thr Lys

530 535 540

Phe Asn Phe Val Asn Lys Asn Ile Pro Phe Val Pro Ser Phe Thr Lys

545 550 555 560

Leu Tyr Asn Lys Ile Glu Asp Leu Arg Asn Thr Leu Lys Phe Phe Trp

565 570 575

Ser Val Pro Lys Asp Lys Glu Glu Lys Asp Ala Gln Ile Tyr Leu Leu

580 585 590

Lys Asn Ile Tyr Tyr Gly Glu Phe Leu Asn Lys Phe Val Lys Asn Ser

595 600 605

Lys Val Phe Phe Lys Ile Thr Asn Glu Val Ile Lys Ile Asn Lys Gln

610 615 620

Arg Asn Gln Lys Thr Gly His Tyr Lys Tyr Gln Lys Phe Glu Asn Ile

625 630 635 640

Glu Lys Thr Val Pro Val Glu Tyr Leu Ala Ile Ile Gln Ser Arg Glu

645 650 655

Met Ile Asn Asn Gln Asp Lys Glu Glu Lys Asn Thr Tyr Ile Asp Phe

660 665 670

Ile Gln Gln Ile Phe Leu Lys Gly Phe Ile Asp Tyr Leu Asn Lys Asn

675 680 685

Asn Leu Lys Tyr Ile Glu Ser Asn Asn Asn Asn Asp Asn Asn Asp Ile

690 695 700

Phe Ser Lys Ile Lys Ile Lys Lys Asp Asn Lys Glu Lys Tyr Asp Lys

705 710 715 720

Ile Leu Lys Asn Tyr Glu Lys His Asn Arg Asn Lys Glu Ile Pro His

725 730 735

Glu Ile Asn Glu Phe Val Arg Glu Ile Lys Leu Gly Lys Ile Leu Lys

740 745 750

Tyr Thr Glu Asn Leu Asn Met Phe Tyr Leu Ile Leu Lys Leu Leu Asn

755 760 765

His Lys Glu Leu Thr Asn Leu Lys Gly Ser Leu Glu Lys Tyr Gln Ser

770 775 780

Ala Asn Lys Glu Glu Thr Phe Ser Asp Glu Leu Glu Leu Ile Asn Leu

785 790 795 800

Leu Asn Leu Asp Asn Asn Arg Val Thr Glu Asp Phe Glu Leu Glu Ala

805 810 815

Asn Glu Ile Gly Lys Phe Leu Asp Phe Asn Glu Asn Lys Ile Lys Asp

820 825 830

Arg Lys Glu Leu Lys Lys Phe Asp Thr Asn Lys Ile Tyr Phe Asp Gly

835 840 845

Glu Asn Ile Ile Lys His Arg Ala Phe Tyr Asn Ile Lys Lys Tyr Gly

850 855 860

Met Leu Asn Leu Leu Glu Lys Ile Ala Asp Lys Ala Lys Tyr Lys Ile

865 870 875 880

Ser Leu Lys Glu Leu Lys Glu Tyr Ser Asn Lys Lys Asn Glu Ile Glu

885 890 895

Lys Asn Tyr Thr Met Gln Gln Asn Leu His Arg Lys Tyr Ala Arg Pro

900 905 910

Lys Lys Asp Glu Lys Phe Asn Asp Glu Asp Tyr Lys Glu Tyr Glu Lys

915 920 925

Ala Ile Gly Asn Ile Gln Lys Tyr Thr His Leu Lys Asn Lys Val Glu

930 935 940

Phe Asn Glu Leu Asn Leu Leu Gln Gly Leu Leu Leu Lys Ile Leu His

945 950 955 960

Arg Leu Val Gly Tyr Thr Ser Ile Trp Glu Arg Asp Leu Arg Phe Arg

965 970 975

Leu Lys Gly Glu Phe Pro Glu Asn His Tyr Ile Glu Glu Ile Phe Asn

980 985 990

Phe Asp Asn Ser Lys Asn Val Lys Tyr Lys Ser Gly Gln Ile Val Glu

995 1000 1005

Lys Tyr Ile Asn Phe Tyr Lys Glu Leu Tyr Lys Asp Asn Val Glu

1010 1015 1020

Lys Arg Ser Ile Tyr Ser Asp Lys Lys Val Lys Lys Leu Lys Gln

1025 1030 1035

Glu Lys Lys Asp Leu Tyr Ile Arg Asn Tyr Ile Ala His Phe Asn

1040 1045 1050

Tyr Ile Pro His Ala Glu Ile Ser Leu Leu Glu Val Leu Glu Asn

1055 1060 1065

Leu Arg Lys Leu Leu Ser Tyr Asp Arg Lys Leu Lys Asn Ala Ile

1070 1075 1080

Met Lys Ser Ile Val Asp Ile Leu Lys Glu Tyr Gly Phe Val Ala

1085 1090 1095

Thr Phe Lys Ile Gly Ala Asp Lys Lys Ile Glu Ile Gln Thr Leu

1100 1105 1110

Glu Ser Glu Lys Ile Val His Leu Lys Asn Leu Lys Lys Lys Lys

1115 1120 1125

Leu Met Thr Asp Arg Asn Ser Glu Glu Leu Cys Glu Leu Val Lys

1130 1135 1140

Val Met Phe Glu Tyr Lys Ala Leu Glu

1145 1150

<210> 70

<211> 1389

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 70

Met Gly Asn Leu Phe Gly His Lys Arg Trp Tyr Glu Val Arg Asp Lys

1 5 10 15

Lys Asp Phe Lys Ile Lys Arg Lys Val Lys Val Lys Arg Asn Tyr Asp

20 25 30

Gly Asn Lys Tyr Ile Leu Asn Ile Asn Glu Asn Asn Asn Lys Glu Lys

35 40 45

Ile Asp Asn Asn Lys Phe Ile Arg Lys Tyr Ile Asn Tyr Lys Lys Asn

50 55 60

Asp Asn Ile Leu Lys Glu Phe Thr Arg Lys Phe His Ala Gly Asn Ile

65 70 75 80

Leu Phe Lys Leu Lys Gly Lys Glu Gly Ile Ile Arg Ile Glu Asn Asn

85 90 95

Asp Asp Phe Leu Glu Thr Glu Glu Val Val Leu Tyr Ile Glu Ala Tyr

100 105 110

Gly Lys Ser Glu Lys Leu Lys Ala Leu Gly Ile Thr Lys Lys Lys Ile

115 120 125

Ile Asp Glu Ala Ile Arg Gln Gly Ile Thr Lys Asp Asp Lys Lys Ile

130 135 140

Glu Ile Lys Arg Gln Glu Asn Glu Glu Glu Ile Glu Ile Asp Ile Arg

145 150 155 160

Asp Glu Tyr Thr Asn Lys Thr Leu Asn Asp Cys Ser Ile Ile Leu Arg

165 170 175

Ile Ile Glu Asn Asp Glu Leu Glu Thr Lys Lys Ser Ile Tyr Glu Ile

180 185 190

Phe Lys Asn Ile Asn Met Ser Leu Tyr Lys Ile Ile Glu Lys Ile Ile

195 200 205

Glu Asn Glu Thr Glu Lys Val Phe Glu Asn Arg Tyr Tyr Glu Glu His

210 215 220

Leu Arg Glu Lys Leu Leu Lys Asp Asp Lys Ile Asp Val Ile Leu Thr

225 230 235 240

Asn Phe Met Glu Ile Arg Glu Lys Ile Lys Ser Asn Leu Glu Ile Leu

245 250 255

Gly Phe Val Lys Phe Tyr Leu Asn Val Gly Gly Asp Lys Lys Lys Ser

260 265 270

Lys Asn Lys Lys Met Leu Val Glu Lys Ile Leu Asn Ile Asn Val Asp

275 280 285

Leu Thr Val Glu Asp Ile Ala Asp Phe Val Ile Lys Glu Leu Glu Phe

290 295 300

Trp Asn Ile Thr Lys Arg Ile Glu Lys Val Lys Lys Val Asn Asn Glu

305 310 315 320

Phe Leu Glu Lys Arg Arg Asn Arg Thr Tyr Ile Lys Ser Tyr Val Leu

325 330 335

Leu Asp Lys His Glu Lys Phe Lys Ile Glu Arg Glu Asn Lys Lys Asp

340 345 350

Lys Ile Val Lys Phe Phe Val Glu Asn Ile Lys Asn Asn Ser Ile Lys

355 360 365

Glu Lys Ile Glu Lys Ile Leu Ala Glu Phe Lys Ile Asp Glu Leu Ile

370 375 380

Lys Lys Leu Glu Lys Glu Leu Lys Lys Gly Asn Cys Asp Thr Glu Ile

385 390 395 400

Phe Gly Ile Phe Lys Lys His Tyr Lys Val Asn Phe Asp Ser Lys Lys

405 410 415

Phe Ser Lys Lys Ser Asp Glu Glu Lys Glu Leu Tyr Lys Ile Ile Tyr

420 425 430

Arg Tyr Leu Lys Gly Arg Ile Glu Lys Ile Leu Val Asn Glu Gln Lys

435 440 445

Val Arg Leu Lys Lys Met Glu Lys Ile Glu Ile Glu Lys Ile Leu Asn

450 455 460

Glu Ser Ile Leu Ser Glu Lys Ile Leu Lys Arg Val Lys Gln Tyr Thr

465 470 475 480

Leu Glu His Ile Met Tyr Leu Gly Lys Leu Arg His Asn Asp Ile Asp

485 490 495

Met Thr Thr Val Asn Thr Asp Asp Phe Ser Arg Leu His Ala Lys Glu

500 505 510

Glu Leu Asp Leu Glu Leu Ile Thr Phe Phe Ala Ser Thr Asn Met Glu

515 520 525

Leu Asn Lys Ile Phe Ser Arg Glu Asn Ile Asn Asn Asp Glu Asn Ile

530 535 540

Asp Phe Phe Gly Gly Asp Arg Glu Lys Asn Tyr Val Leu Asp Lys Lys

545 550 555 560

Ile Leu Asn Ser Lys Ile Lys Ile Ile Arg Asp Leu Asp Phe Ile Asp

565 570 575

Asn Lys Asn Asn Ile Thr Asn Asn Phe Ile Arg Lys Phe Thr Lys Ile

580 585 590

Gly Thr Asn Glu Arg Asn Arg Ile Leu His Ala Ile Ser Lys Glu Arg

595 600 605

Asp Leu Gln Gly Thr Gln Asp Asp Tyr Asn Lys Val Ile Asn Ile Ile

610 615 620

Gln Asn Leu Lys Ile Ser Asp Glu Glu Val Ser Lys Ala Leu Asn Leu

625 630 635 640

Asp Val Val Phe Lys Asp Lys Lys Asn Ile Ile Thr Lys Ile Asn Asp

645 650 655

Ile Lys Ile Ser Glu Glu Asn Asn Asn Asp Ile Lys Tyr Leu Pro Ser

660 665 670

Phe Ser Lys Val Leu Pro Glu Ile Leu Asn Leu Tyr Arg Asn Asn Pro

675 680 685

Lys Asn Glu Pro Phe Asp Thr Ile Glu Thr Glu Lys Ile Val Leu Asn

690 695 700

Ala Leu Ile Tyr Val Asn Lys Glu Leu Tyr Lys Lys Leu Ile Leu Glu

705 710 715 720

Asp Asp Leu Glu Glu Asn Glu Ser Lys Asn Ile Phe Leu Gln Glu Leu

725 730 735

Lys Lys Thr Leu Gly Asn Ile Asp Glu Ile Asp Glu Asn Ile Ile Glu

740 745 750

Asn Tyr Tyr Lys Asn Ala Gln Ile Ser Ala Ser Lys Gly Asn Asn Lys

755 760 765

Ala Ile Lys Lys Tyr Gln Lys Lys Val Ile Glu Cys Tyr Ile Gly Tyr

770 775 780

Leu Arg Lys Asn Tyr Glu Glu Leu Phe Asp Phe Ser Asp Phe Lys Met

785 790 795 800

Asn Ile Gln Glu Ile Lys Lys Gln Ile Lys Asp Ile Asn Asp Asn Lys

805 810 815

Thr Tyr Glu Arg Ile Thr Val Lys Thr Ser Asp Lys Thr Ile Val Ile

820 825 830

Asn Asp Asp Phe Glu Tyr Ile Ile Ser Ile Phe Ala Leu Leu Asn Ser

835 840 845

Asn Ala Val Ile Asn Lys Ile Arg Asn Arg Phe Phe Ala Thr Ser Val

850 855 860

Trp Leu Asn Thr Ser Glu Tyr Gln Asn Ile Ile Asp Ile Leu Asp Glu

865 870 875 880

Ile Met Gln Leu Asn Thr Leu Arg Asn Glu Cys Ile Thr Glu Asn Trp

885 890 895

Asn Leu Asn Leu Glu Glu Phe Ile Gln Lys Met Lys Glu Ile Glu Lys

900 905 910

Asp Phe Asp Asp Phe Lys Ile Gln Thr Lys Lys Glu Ile Phe Asn Asn

915 920 925

Tyr Tyr Glu Asp Ile Lys Asn Asn Ile Leu Thr Glu Phe Lys Asp Asp

930 935 940

Ile Asn Gly Cys Asp Val Leu Glu Lys Lys Leu Glu Lys Ile Val Ile

945 950 955 960

Phe Asp Asp Glu Thr Lys Phe Glu Ile Asp Lys Lys Ser Asn Ile Leu

965 970 975

Gln Asp Glu Gln Arg Lys Leu Ser Asn Ile Asn Lys Lys Asp Leu Lys

980 985 990

Lys Lys Val Asp Gln Tyr Ile Lys Asp Lys Asp Gln Glu Ile Lys Ser

995 1000 1005

Lys Ile Leu Cys Arg Ile Ile Phe Asn Ser Asp Phe Leu Lys Lys

1010 1015 1020

Tyr Lys Lys Glu Ile Asp Asn Leu Ile Glu Asp Met Glu Ser Glu

1025 1030 1035

Asn Glu Asn Lys Phe Gln Glu Ile Tyr Tyr Pro Lys Glu Arg Lys

1040 1045 1050

Asn Glu Leu Tyr Ile Tyr Lys Lys Asn Leu Phe Leu Asn Ile Gly

1055 1060 1065

Asn Pro Asn Phe Asp Lys Ile Tyr Gly Leu Ile Ser Asn Asp Ile

1070 1075 1080

Lys Met Ala Asp Ala Lys Phe Leu Phe Asn Ile Asp Gly Lys Asn

1085 1090 1095

Ile Arg Lys Asn Lys Ile Ser Glu Ile Asp Ala Ile Leu Lys Asn

1100 1105 1110

Leu Asn Asp Lys Leu Asn Gly Tyr Ser Lys Glu Tyr Lys Glu Lys

1115 1120 1125

Tyr Ile Lys Lys Leu Lys Glu Asn Asp Asp Phe Phe Ala Lys Asn

1130 1135 1140

Ile Gln Asn Lys Asn Tyr Lys Ser Phe Glu Lys Asp Tyr Asn Arg

1145 1150 1155

Val Ser Glu Tyr Lys Lys Ile Arg Asp Leu Val Glu Phe Asn Tyr

1160 1165 1170

Leu Asn Lys Ile Glu Ser Tyr Leu Ile Asp Ile Asn Trp Lys Leu

1175 1180 1185

Ala Ile Gln Met Ala Arg Phe Glu Arg Asp Met His Tyr Ile Val

1190 1195 1200

Asn Gly Leu Arg Glu Leu Gly Ile Ile Lys Leu Ser Gly Tyr Asn

1205 1210 1215

Thr Gly Ile Ser Arg Ala Tyr Pro Lys Arg Asn Gly Ser Asp Gly

1220 1225 1230

Phe Tyr Thr Thr Thr Ala Tyr Tyr Lys Phe Phe Asp Glu Glu Ser

1235 1240 1245

Tyr Lys Lys Phe Glu Lys Ile Cys Tyr Gly Phe Gly Ile Asp Leu

1250 1255 1260

Ser Glu Asn Ser Glu Ile Asn Lys Pro Glu Asn Glu Ser Ile Arg

1265 1270 1275

Asn Tyr Ile Ser His Phe Tyr Ile Val Arg Asn Pro Phe Ala Asp

1280 1285 1290

Tyr Ser Ile Ala Glu Gln Ile Asp Arg Val Ser Asn Leu Leu Ser

1295 1300 1305

Tyr Ser Thr Arg Tyr Asn Asn Ser Thr Tyr Ala Ser Val Phe Glu

1310 1315 1320

Val Phe Lys Lys Asp Val Asn Leu Asp Tyr Asp Glu Leu Lys Lys

1325 1330 1335

Lys Phe Lys Leu Ile Gly Asn Asn Asp Ile Leu Glu Arg Leu Met

1340 1345 1350

Lys Pro Lys Lys Val Ser Val Leu Glu Leu Glu Ser Tyr Asn Ser

1355 1360 1365

Asp Tyr Ile Lys Asn Leu Ile Ile Glu Leu Leu Thr Lys Ile Glu

1370 1375 1380

Asn Thr Asn Asp Thr Leu

1385

<210> 71

<211> 1300

<212> PRT

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polypeptide"

<400> 71

Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr

1 5 10 15

Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys

20 25 30

Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys

35 40 45

Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu

50 55 60

Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser

65 70 75 80

Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys

85 90 95

Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr

100 105 110

Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile

115 120 125

Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln

130 135 140

Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr

145 150 155 160

Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr

165 170 175

Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser

180 185 190

Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu

195 200 205

Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys

210 215 220

Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu

225 230 235 240

Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg

245 250 255

Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr

260 265 270

Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys

275 280 285

Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile

290 295 300

Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys

305 310 315 320

Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser

325 330 335

Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met

340 345 350

Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys

355 360 365

Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln

370 375 380

Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr

385 390 395 400

Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala

405 410 415

Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn

420 425 430

Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala

435 440 445

Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn

450 455 460

Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala

465 470 475 480

Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys

485 490 495

Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys

500 505 510

Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp

515 520 525

Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His

530 535 540

Ile Ser Gln Ser Glu Asp Lys Ala Asn Ile Leu Asp Lys Asp Glu His

545 550 555 560

Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val

565 570 575

Pro Leu Tyr Asn Lys Ile Arg Asn Tyr Ile Thr Gln Lys Pro Tyr Ser

580 585 590

Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly

595 600 605

Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys

610 615 620

Asp Asp Lys Tyr Tyr Leu Gly Val Met Asn Lys Lys Asn Asn Lys Ile

625 630 635 640

Phe Asp Asp Lys Ala Ile Lys Glu Asn Lys Gly Glu Gly Tyr Lys Lys

645 650 655

Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val

660 665 670

Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile

675 680 685

Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln

690 695 700

Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe

705 710 715 720

Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp

725 730 735

Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu

740 745 750

Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn

755 760 765

Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr

770 775 780

Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg

785 790 795 800

Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn

805 810 815

Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr

820 825 830

Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala

835 840 845

Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu

850 855 860

Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe

865 870 875 880

His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe

885 890 895

Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His

900 905 910

Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu

915 920 925

Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile

930 935 940

Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile

945 950 955 960

Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn

965 970 975

Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile

980 985 990

Ala Lys Leu Val Ile Glu Tyr Asn Ala Ile Val Val Phe Glu Asp Leu

995 1000 1005

Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val

1010 1015 1020

Tyr Gln Lys Leu Glu Lys Met Leu Ile Glu Lys Leu Asn Tyr Leu

1025 1030 1035

Val Phe Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg

1040 1045 1050

Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly

1055 1060 1065

Lys Gln Thr Gly Ile Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser

1070 1075 1080

Lys Ile Cys Pro Val Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys

1085 1090 1095

Tyr Glu Ser Val Ser Lys Ser Gln Glu Phe Phe Ser Lys Phe Asp

1100 1105 1110

Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr Phe Glu Phe Ser Phe

1115 1120 1125

Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr

1130 1135 1140

Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg Asn Ser Asp

1145 1150 1155

Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr Lys Glu

1160 1165 1170

Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His Gly

1175 1180 1185

Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe

1190 1195 1200

Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg

1205 1210 1215

Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val

1220 1225 1230

Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys

1235 1240 1245

Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly

1250 1255 1260

Leu Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu

1265 1270 1275

Gly Lys Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu

1280 1285 1290

Phe Val Gln Asn Arg Asn Asn

1295 1300

<210> 72

<211> 7403

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 72

tatccggtcg aatcgagaat gacgaccgct acgtcttgga ctacgaagcc gtggcccttg 60

ccgatgctct cggtgtggat gttgccgacc tgttccgcaa gatcgattgc cccaagaacc 120

tgctgcgcag gcgggcaggg taggggagcg gtttccggcg gagattttcg gaggcgccgg 180

taacgttatg tcggggaatt tgctatacat cgacgataat tagttttgtt gattcaggat 240

cgaaatgcgc tcaaacaaag aacgttccgc gtttccctca tgcgctacta cgcccacacc 300

gccatctttc ggcacgcaaa caaagcagat gggttgcctg tcaatggggtg atcattgcct 360

gaagttacca tccatcaata atataaatca tccttactcc gaatgtccct caatcgcatc 420

tatcaaggcc gcgtggcggc cgtcgaaaca ggaacggcct tagcgaaagg taatgtcgaa 480

tggatgcctg ccgcaggagg cgacgaagtt ctctggcagc accacgaact tttccaagct 540

gccatcaact actatctcgt cgccctgctc gcactcgccg acaaaaacaa tcccgtactt 600

ggcccgctga tcagccagat ggataatccc caaagccctt accatgtctg gggaagtttc 660

cgccgccaag gacgtcagcg cacaggtctc agtcaagccg ttgcacctta tatcacgccg 720

ggcaataacg ctcccaccct tgacgaagtt ttccgctcca ttcttgcggg caacccaacc 780

gaccgcgcaa ctttggacgc tgcactcatg caattgctca aggcttgtga cggcgcgggc 840

gctatccagc aggaaggtcg ttcctactgg cccaaattct gcgatcctga ctccactgcc 900

aacttcgcgg gagatccggc catgctccgg cgtgaacaac accgcctcct ccttccgcaa 960

gttctccacg atccggcgat tactcacgac agtcctgccc ttggctcgtt cgacacttat 1020

tcgattgcta cccccgacac cagaactcct caactcaccg gccccaaggc acgcgcccgt 1080

cttgagcagg cgatcaccct ctggcgcgtc cgtcttcccg aatcggctgc tgacttcgat 1140

cgccttgcca gttccctcaa aaaaattccg gacgacgatt ctcgccttaa ccttcaggggc 1200

tacgtcggca gcagtgcgaa aggcgaagtt caggcccgtc ttttcgccct tctgctattc 1260

cgtcacctgg agcgttcctc ctttacgctt ggccttctcc gttccgccac cccgccgccc 1320

aagaacgctg aaacacctcc tcccgccggc gttcctttac ctgcggcgtc cgcagccgat 1380

ccggtgcgga tagcccgtgg caaacgcagt tttgtttttc gcgcattcac cagtctcccc 1440

tgctggcatg gcggtgataa catccatccc acctggaagt cattcgacat cgcagcgttc 1500

1560

tgtgcggaac ttgaaactga tttcgactac atgcacggac ggctcgccaa gattccggta 1620

aaatacacga ccggcgaagc cgaaccgccc cccattctcg caaacgatct ccgcatcccc 1680

ctcctccgcg aacttctcca gaatatcaag gtcgacaccg cactcaccga tggcgaagcc 1740

gtctcctatg gtctccaacg ccgcaccatt cgcggtttcc gcgagctgcg ccgcatctgg 1800

cgcggccatg cccccgctgg cacggtcttt tccagcgagt tgaaagaaaa actagccggc 1860

gaactccgcc agttccagac cgacaactcc accaccatcg gcagcgtcca actcttcaac 1920

gaactcatcc aaaacccgaa atactggccc atctggcagg ctcctgacgt cgaaaccgcc 1980

cgccaatggg ccgatgccgg ttttgccgac gatccgctcg ccgcccttgt gcaagaagcc 2040

gaactccagg aagacatcga cgccctcaag gctccagtca aactcactcc ggccgatcct 2100

gagtattcaa gaaggcaata cgatttcaat gccgtcagca aattcggggc cggctcccgc 2160

tccgccaatc gccacgaacc cgggcagacg gagcgcggcc acaacacctt taccaccgaa 2220

atcgccgccc gtaacgcggc ggacgggaac cgctggcggg caacccgt ccgcatccat 2280

tactccgctc cccgccttct tcgtgacgga ctccgccgac ctgacaccga cggcaacgaa 2340

gccctggaag ccgtcccttg gctccagccc atgatggaag ccctcgcccc tctcccgacg 2400

cttccgcaag acctcacagg catgccggtc ttcctcatgc ccgacgtcac cctttccggt 2460

gagcgtcgca tcctcctcaa tcttcctgtc accctcgaac cagccgctct tgtcgaacaa 2520

ctgggcaacg ccggtcgctg gcaaaaccag ttcttcggct cccgcgaaga tccattcgct 2580

ctccgatggc ccgccgacgg tgctgtaaaa accgccaagg ggaaaaccca cataccttgg 2640

caccaggacc gcgatcactt caccgtactc ggcgtggatc tcggcacgcg cgatgccggg 2700

gcgctcgctc ttctcaacgt cactgcgcaa aaaccggcca agccggtcca ccgcatcatt 2760

ggtgaggccg acggacgcac ctggtatgcc agccttgccg acgctcgcat gatccgcctg 2820

ccgggggagg atgcccggct ctttgtccgg ggaaaactcg ttcaggaacc ctatggtgaa 2880

cgcgggcgaa acgcgtctct tctcgaatgg gaagacgccc gcaatatcat ccttcgcctt 2940

ggccaaaatc ccgacgaact cctcggcgcc gatccccggc gccattcgta tccggaaata 3000

aacgataaac ttctcgtcgc ccttcgccgc gctcaggccc gtcttgcccg tctccagaac 3060

cggagctggc ggttgcgcga ccttgcagaa tcggacaagg cccttgatga aatccatgcc 3120

gagcgtgccg gggagaagcc ttctccgctt ccgcccttgg ctcgcgacga tgccatcaaa 3180

agcaccgacg aagccctcct ttcccagcgt gacatcatcc ggcgatcctt cgttcagatc 3240

gccaacttga tccttcccct tcgcggacgc cgatgggaat ggcggcccca tgtcgaggtc 3300

ccggattgcc acatccttgc gcagagcgat cccggtacgg atgacaccaa gcgtcttgtc 3360

gccggacaac gcggcatctc tcacgagcgt atcgagcaaa tcgaagaact ccgtcgtcgc 3420

tgccaatccc tcaaccgtgc cctgcgtcac aaacccggag agcgtcccgt gctcggacgc 3480

cccgccaagg gcgaggaaat cgccgatccc tgtcccgcgc tcctcgaaaa gatcaaccgt 3540

ctccgggacc agcgcgttga ccaaaccgcg catgccatcc tcgccgccgc tctcggtgtt 3600

cgactccgcg ccccctcaaa agaccgcgcc gaacgccgcc atcgcgacat ccatggcgaa 3660

tacgaacgct ttcgtgcgcc cgctgatttt gtcgtcatcg aaaacctctc ccgttatctc 3720

agctcgcagg atcgtgctcg tagtgaaaac acccgtctca tgcagtggtg ccatcgccag 3780

atcgtgcaaa aactccgtca gctctgcgag acctacggca tccccgtcct cgccgtcccg 3840

gcggcctact catcgcgttt ttcttcccgg gacggctcgg ccggattccg ggccgtccat 3900

3960

4020

ggactctttg accggctcga ccggttcaac gccgggcatg tcccgggaaa accttggcgc 4080

acgctcctcg cgccgctccc cggcggccct gtgtttgtcc ccctcgggga cgccacaccc 4140

atgcaggccg atctgaacgc cgccatcaac atcgccctcc ggggcatcgc ggctcccgac 4200

cgccacgaca tccatcaccg gctccgtgcc gaaaacaaaa aacgcatcct gagcttgcgt 4260

ctcggcactc agcgcgagaa agcccgctgg cctggaggag ctccggcggt gacactctcc 4320

actccgaaca acggcgcctc tcccgaagat tccgatgcgt tgcccgaacg ggtatccaac 4380

ctgtttgtgg acatcgccgg tgtcgccaac ttcgagcgag tcacgatcga aggagtctcg 4440

caaaaattcg ccaccggggcg tggcctttgg gcctccgtca agcaacgtgc atggaaccgc 4500

4560

ccgatgtaac cattgcttca ttacatctga gtctcccctc aatccctctg ccccatgcgt 4620

gatataacct ccacctcatg tcccggatcg gcgccggcaa cctgtagttc ccttccatcc 4680

tccaacactc ccgcagatcg cgatccgctg ccgccgatgc cggtgcgccg ccttcacaac 4740

tatctctact gtccgcggct tttttatctc cagtgggtcg agaatctctt tgaggaaaat 4800

gccgacacca ttgccggcag cgccgtgcat cgtcacgccg acaaacctac gcgttacgat 4860

gatgaaaaag ccgaggcact tcgcactggt ctccctgaag gcgcgcacat acgcagcctt 4920

cgcctggaaa acgcccaact cggtctcgtt ggcgtggtgg atatcgtgga gggaggcccc 4980

gacggactcg aactcgtcga ctacaaaaaa ggttccgcct tccgcctcga cgacggcacg 5040

ctcgctccca aggaaaacga caccgtgcaa cttgccgcct acgctcttct cctggctgcc 5100

gatggtgcgc gcgttgcgcc catggcgacg gtctattacg ctgccgatcg ccggcgtgtc 5160

accttcccgc tcgatgacgc cctctacgcc cgcacccgtt ccgccctcga agaggcccgc 5220

gccgttgcaa cctcggggcg catacctccg ccgctcgtct ctgacgtccg ctgcctccat 5280

tgttcctcct atgcgctttg ccttccccgc gagtccgcct ggtggtgccg ccatcgcagc 5340

acgccgcggg gagccggcca cacccccatg ttgccggggct ttgaggatga cgccgccgcc 5400

attcaccaaa tctccgaacc tgacaccgag ccaccacccg atcttgccag ccagcctccc 5460

cgtcccccgc ggctcgatgg agaattgttg gttgtccaga ctccgggagc gatgatcgga 5520

caaagcggcg gtgagtttac cgtgtccgtc aagggtgagg ttttgcgcaa gcttccggtt 5580

catcaactcc gggccattta cgtttacgga gccgtgcaac tcacggcgca tgctgtgcag 5640

accgcccttg aggaggatat cgacgtctcc tattttgcgc ccagcggccg ctttcttggc 5700

ctcctccgcg gcctgcccgc atccggcgtg gatgcgcgtc tcgggcaata caccctgttt 5760

cgcgaaccct ttggccgtct ccgtctcgcc tgcgaggcga ttcgggccaa gatccataac 5820

cagcgcgtcc tcctcatgcg taacggcgag cccggggagg gcgtcttgcg cgaactcgcc 5880

cgtctgcgcg acgccaccag tgaggcgact tcgctcgacg aactcctcgg catcgagggc 5940

atcgccgcgc atttctattt ccagtatttt cccaccatgc tgaaagaacg ggcggcctgg 6000

6060

tcgttcggtt acagcgtgtt gtccaaggaa cttgccggcg tctgccacgc tgttggccta 6120

gacccgtttt tcggcttcat gcaccagccg cgttacgggc gccccgcact cgctctcgat 6180

ctgatggagg agtttcgcc tctcatcgcc gacagtgttg ccctgaatct catcaaccgt 6240

ggcgaactcg acgaagggga ctttatccgg tcggccaatg gcaccgcgct caatgatcgg 6300

ggccgccggc gtttttggga ggcatggttc cggcgtctcg acagcgaagt cagccatcct 6360

gaatttggtt acaagatgag ctatcgacgg atgcttgaag tgcaggcgcg ccagctatgg 6420

cgctatgtgc gcggtgacgc cttccgctac cacggattca ccacccgttg attccgatgt 6480

cagatccccg ccgccgttat cttgtgtgtt acgacatcgc caatccgaag cgattgcgcc 6540

aagtggccaa gctgctggag agctatggca cgcgtctgca atactcggtt ttcgaatgtc 6600

6660

accaagacca ggtgttattt gtttcgcttg gccccgaagc caacgatgcc acgttgatca 6720

tcgccacgct tgggctccct tataccgtgc gctcgcgagt gacgattatc tgacccataa 6780

6840

ccgaactcag cagaccttta tgcggctaag gccaatgatc atccatccta ccgccattgg 6900

6960

ctcgcggcca cttttacaga ggagatgttc gggcgaactg gccgacctaa caaggcgtac 7020

ccggctcaaa atcgaggcac gctcgcacgg gatgatgtaa ttcgttgttt ttcagcatac 7080

cgtgcgagca cgggccgcag cgaatgccgt ttcacgaatc gtcaggcggc ggggagaagt 7140

catttaataa ggccactgtt aaaagccgca gcgaatgccg tttcacgaat cgtcaggcgg 7200

gcagtggatg tttttccatg aggcgaagaa tttcatcgcc gcagtgaatg ccgtttcacc 7260

attgatgaag aatgcgaggt gaaaacagag aaattgggtc aactctatca ctcttattca 7320

gccatcgttt caagaaagga tacctcgtat tggatacaac acagctcgtt cgttctctct 7380

acctccctcg acaatctcaa gga 7403

<210> 73

<211> 6789

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 73

taataaaatt gaaatatcac tatggattat tgtaatatta ccataaagat aggtgacgtt 60

tttttgaaaa ttgtaaacct aatttgaaga aaaccaatta aaaatcgctt cggctttttt 120

ttaagtgcca ggtagcattg atgctaaccc atgtgtaata aaggtttgtt ttccttcggg 180

gcacgaacac attataaggg aaacctaaag attccctttc ttgtttaata ttataaccag 240

tgaaaataag aataatgcac ctaaaactaa tatacagaaa ataagaatta aaagtactaa 300

tatatacatc ataattgatg ttagtattag 360

ttttatttta atttctaaac ataagaattt gaaaaggatg tgtttattat ggcgacacgc 420

agttttattt taaaaattga accaaatgaa gaagttaaaa agggattatg gaagacgcat 480

gaggtattga atcatggaat tgcctactac atgaatattc tgaaactaat tagacaggaa 540

gctatttatg aacatcatga acaagatcct aaaaatccga aaaaagtttc aaaagcagaa 600

atacaagccg agttatggga ttttgtttta aaaatgcaaa aatgtaatag ttttacacat 660

gaagttgaca aagatgttgt tttttaacatc ctgcgtgaac tatatgaaga gttggtccct 720

agttcagtcg agaaaaaggg tgaagccaat caattatcga ataagtttct gtacccgcta 780

gttgatccga acagtcaaag tgggaaaggg acggcatcat ccggacgtaa acctcggtgg 840

tataatttaa aaatagcagg cgacccatcg tgggaggaag aaaagaaaaa atgggaagag 900

gataaaaaga aagatcccct tgctaaaatc ttaggtaagt tagcagaata tgggcttatt 960

ccgctattta ttccatttac tgacagcaac gaaccaattg taaaagaaat taaatggatg 1020

gaaaaaagtc gtaatcaaag tgtccggcga cttgataagg atatgtttat ccaagcatta 1080

1140

gaaaaggaac acaaaacact agaggaaagg ataaaagagg acattcaagc atttaaatcc 1200

cttgaacaat atgaaaaaga acggcaggag caacttctta gagatacatt gaatacaaat 1260

gaataccgat taagcaaaag aggattacgt ggttggcgtg aaattatcca aaaatggcta 1320

1380

aaacatccac gagaagccgg ggactattct gtctatgaat ttttaagcaa gaaagaaaat 1440

cattttattt ggcgaaatca tcctgaatat ccttatttgt atgctacatt ttgtgaaatt 1500

gacaaaaaaa agaaagacgc taagcaacag gcaactttta ctttggctga cccgattaac 1560

catccgttat gggtacgatt tgaagaaaga agcggttcga acttaaacaa atatcgaatt 1620

ttaacagagc aattacacac tgaaaagtta aaaaagaaat taacagttca acttgatcgt 1680

ttaatttatc caactgaatc cggcggttgg gaggaaaaag gtaaagtaga tatcgttttg 1740

ttgccgtcaa gacaatttta taatcaaatc ttccttgata tagaagaaaa ggggaaacat 1800

gcttttactt ataaggatga aagtattaaa ttccccctta aaggtacact tggtggtgca 1860

agagtgcagt ttgaccgtga ccatttgcgg agatatccgc ataaagtaga atcaggaaat 1920

1980

aagtctttga aaatacatag ggacgatttc cccaagttcg ttaattttaa accgaaagag 2040

ctcaccgaat ggataaaaga tagtaaaggg aaaaaattaa aaagtggtat agaatccctt 2100

gaaattggtc tacgggtgat gagtatcgac ttaggtcaac gtcaagcggc tgctgcatcg 2160

2220

ggaactgagc tttatgctgt tcaccgggca agttttaaca ttaaattacc gggtgaaaca 2280

ttagtaaaat cacgggaagt attgcggaaa gctcgggagg acaacttaaa attaatgaat 2340

2400

agagagaagc gtgtaactaa atggatttct agacaagaaa atagtgatgt tcctcttgta 2460

2520

2580

tggcgaaaat cattaagtga cgggagaaaa ggtctttacg gaatctccct aaaaaatatt 2640

gatgaaattg atcgaacaag gaaattcctt ttaagatgga gcttacgtcc aacagaacct 2700

ggggaagtaa gacgcttgga accaggacag cgttttgcga ttgatcaatt aaaccaccta 2760

aatgcattaa aagaagatcg attaaaaaag atggcaaata cgattatcat gcatgcctta 2820

ggttactgtt atgatgtaag aaagaaaaag tggcaggcaa aaaatccagc atgtcaaatt 2880

aaaggtcccg ttttgaaaac 2940

tcaaaactga tgaagtggtc acggagagaa attccacgac aagtcgcctt acaaggtgaa 3000

3060

accgggtcgc cgggaattcg ttgcagtgtt gtaacgaaag aaaaattgca ggataatcgc 3120

3180

gaaggagact tatatccaga taaaggtgga gaaaagttta tttctttatc aaaggatcga 3240

3300

3360

gtttatattc cggagagcaa ggaccaaaaa caaaaaataa ttgaagaatt tggggaaggc 3420

tattttattt taaaagatgg tgtatatgaa tggggtaatg cggggaaact aaaaattaaa 3480

aaaggttcct ctaaacaatc atcgagtgaa ttagtagatt cggacatact gaaagattca 3540

3600

3660

atattgattt ctaagttaac aaatcaatac tcaatatcaa caatagaaga tgattcttca 3720

3780

3840

aggtgaggaa tggtaagaaa ggaaaattta tatgaaccaa ccgattccta ttcgaatgtt 3900

aaatgaaata catgtccaaa agctatttga 3960

tgagaatgca gatacagttg aaggaagtgc acagcatgag cgggcagaaa gaagcaaaag 4020

accaagtaaa atgggaccaa aggaattatg gggtgaggcg ccaagaagtc ttaagcttgg 4080

tgatgagctg ttaaatatta ccggtgttct tgatgccata agtcatgaag agaacagttg 4140

gatcccggtt gaatcaaaac acagttccgc accggatgga ttgaaccctt ttaaagtaga 4200

tggctttcta cttgacgggt ctgcatggcc aaacgatcaa attcaacttt gtgcacaagg 4260

cttgctcttg aatgccaatg gatacccgtg tgattatggg tatttatttt atcgtggtaa 4320

taagaaaaag gtgaaaattt attttactga agatttaatc gctgccacaa agtactatat 4380

taaaaaagca cacgagatac tagtattatc tggtgatgaa tcagctattc ctaagccttt 4440

aattgattct aataagtgtt ttcgctgttc tttaaactat atctgtcttc cggatgaaac 4500

gaactatcta ttagggggcaa gttcaacaat tcgtaaaatt gtgccttcaa ggacagatgg 4560

tggcgtttta tatgtatcag agtctggtac aaaattagga aaatcgggtg aggagttaat 4620

cattcagtat aaagatggcc aaaagcaggg tgttcctata aaagatatta ttcaagtttc 4680

gttaattgga aatgttcaat gctcaacgca attacttcat tttttaatgc aatcaaatat 4740

tcctgtaagt tatttatcat cccacggtcg tttgattggt gtcagttcat ctttagttac 4800

aaaaaatgtt ttaacaaggc agcaacagtt cattaaattt acaaatcctg agtttggact 4860

aaatctagca aaacaaattg tttatgccaa gattcgaaat caacgaactt tacttagaag 4920

aaatgggggg agtgaggtaa aggagatttt aacagattta aaatctttaa gtgacagtgc 4980

actgaacgca atatcaatag aacaattacg gggtattgaa gggatttctg caaaacatta 5040

tttcgcagga tttccgttta tgttgaaaaa tgaattacgt gaattgaatt taatgaaagg 5100

gcgtaatagg agaccgccaa aagatcctgt aaatgtactt ctttctcttg gttatacttt 5160

5220

ttaccatcgt ccagaagcag gtcgaccggc tctagtatta gatgttatgg aaacatttcg 5280

accacttatt gtagacagta ttgtcatccg agctttgaat acgggtgaaa tctcattaaa 5340

atagttttat ataggaaaag atagttgtca attattaaaa catggccgcg attccttttt 5400

tgccatttat gaaagaagaa tgcatgaaac tattaccgat ccaattttcg gctataagat 5460

tagctatcgc cgtatgctcg atttgcacat tcgaatgctt gcaaggttta ttgaagggga 5520

actgccggaa tataaaccat taatgacccg gtgagtttgt ttattaggtt aaaagaaggt 5580

gaagacatgc agcaatacgt ccttgtttct tatgatattt cggaccaaaa aagatggaga 5640

aaagtattta aactgatgaa aggatacgga gaacatgttc aatattccgt attcatatgc 5700

cagttaactg aattacagaa ggcaaaatta caagcctctt tagaagacat tatccatcat 5760

aagaatgacc aagtaatgtt tgttcacatc gggccagtga aagatggtca actatctaaa 5820

aaaatctcaa caattgggaa agaatttgtt ccattggatt taaagcggct tatattttga 5880

aaagatatag caaagaaatc ttatgaaaaa aatacaaaaa tatattgtta aaaaataggg 5940

aatattatat aatggactta cgaggttctg tcttttggtc aggacaaccg tctagctata 6000

6060

cttggatctg agtacgagca cccacattgg acatttcgca tggtgggtgc tcgtactata 6120

ggtaaaacaa acctttttaa gaagaataca aaaataacca caatattttt taaaaggaat 6180

acccaagccc acccatagccc 6240

aaaaccccct gcggtccaag aaaaaagaaa tgatacgagg cattagcacc ggggagaagt 6300

catttaataa ggccactgtt aaaagtccaa gaaaaaagaa atgatacgag gcattagcac 6360

aacaatataa acgactactt taccgtgttc aagaaaaaag aaatgatatg aggcattagc 6420

6480

atccggtatc atttcttcac ttctttctgc acataaaaaa gcacctaact atttggataa 6540

gttaagtgct tttatttccg tttgaagttg tctattgctt ttttcttcat atcttcaaat 6600

tttttctgtt tctcagagtc aactttacca actgtaatcc cttttctttt tggcattggg 6660

gtatctttcc accttagtgt gttcataagg cttatattta tcactcattg tattcctcca 6720

acacaattat aatttttccg tcatcctcaa tccaaccgtc aactgtgaca aaagacgaat 6780

ctctcttat 6789

<210> 74

<211> 6214

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 74

gtttcatttg gaaagggaga gcattggctt ttctctttgt aaataaagtg caagctttgt 60

aataagcttc tagtggagaa gtgattgttt gaatcaccca atgcacacgc actaaagtta 120

gacgaaccta taattcgtat tagtaagtat agtacatgaa gaaaaatgca acaagcattt 180

actctctttt aaataaagaa ttgatagctg ttaatattga tagtatatta taccttatag 240

atgttcgatt ttttttgaaa ttcaaaaatc atacttagta aagaaaggaa ataacgtcat 300

ggacaagcga aagcgtagaa gttacgagtt taggtgggaa gcgggaggca ccagtcatgg 360

caatccgtag cataaaacta aaactaaaaa cccacacagg cccggaagcg caaaacctcc 420

gaaaaggaat atggcggacg catcggttgt taaatgaagg cgtcgcctat tacatgaaaa 480

tgctcctgct ctttcgtcag gaaagcactg gtgaacggcc aaaagaagaa ctacaggaag 540

aactgatttg tcacatacgc gaacagcaac aacgaaatca ggcagataaa aatacgcaag 600

cgcttccgct agataaggca ctggaagctt tgcgccaact atatgaactg cttgtcccct 660

cctcggtcgg acaaagtggc gacgcccaga tcatcagccg aaagtttctc agcccgctcg 720

tcgatccgaa cagcgaaggc ggcaaaggta cttcgaaggc aggggcaaaa cccacttggc 780

agaagaaaaa agaagcgaac gacccaacct gggaacagga ttacgaaaaa tggaaaaaaa 840

gacgcgagga agacccaacc gcttctgtga ttactacttt ggaggaatac ggcattagac 900

cgatctttcc cctgtacacg aacaccgtaa cagatatcgc gtggttgcca cttcaatcca 960

atcagtttgt gcgaacctgg gacagagaca tgcttcaaca agcgattgaa agactgctca 1020

1080

ctcaactgaa cgagcaactc gaaggcggtc aggaatggat cagcttgcta gagcagtacg 1140

aagaaaaccg agagcgagag cttagggaaa acatgaccgc tgccaatgac aagtatcgga 1200

ttaccaagcg gcaaatgaaa ggctggaacg agctgtacga gctatggtca acctttcccg 1260

ccagtgccag tcacgagcaa tacaaagagg cgctcaagcg tgtgcagcag cgactgagag 1320

ggcggtttgg ggatgctcat ttcttccagt atctgatgga agagaagaac cgcctgatct 1380

ggaaggggaa tccgcagcgt atccattatt ttgtcgcgcg caacgaactg acgaaacggc 1440

tggaggaagc caagcaaagc gccacgatga cgttgcccaa tgccaggaag catccattgt 1500

gggtgcgctt cgatgcacgg ggaggaaatt tgcaagacta ctacttgacg gctgaagcgg 1560

acaaaccgag aagcagacgt tttgtaacgt ttagtcagtt gatatggcca agcgaatcgg 1620

gatggatgga aaagaaagac gtcgaggtcg agctagcttt gtccaggcag ttttaccagc 1680

1740

cgggctcgac gtttaacgga cacttggggg gagcaaagct acaactggag cggggcgatt 1800

tggagaagga agaaaaaaac ttcgaggacg gggaaatcgg cagcgtttac cttaacgttg 1860

tcattgattt cgaacctttg caagaagtga aaaatggccg cgtgcaggcg ccgtatggac 1920

aagtactgca actcattcgt cgccccaacg agtttcccaa ggtcactacc tataagtcgg 1980

agcaacttgt tgaatggata aaagcttcgc cacaacactc ggctggggtg gagtcgctgg 2040

catccggttt tcgtgtaatg agcatagacc ttgggctgcg cgcggctgca gcgacttcta 2100

ttttttctgt agaagagagt agcgataaaa atgcggctga ttttttcctac tggattgaag 2160

gaacgccgct ggtcgctgtc catcagcgga gctatatgct caggttgcct ggtgaacagg 2220

2280

ttcaaatcag agtgctcgcc caaatcatgc gtatggcaaa taagcagtat ggagatcgct 2340

gggatgaact cgacagcctg aaacaagcgg ttgagcagaa aaagtcgccg ctcgatcaaa 2400

cagaccggac attttgggag gggattgtct gcgacttaac aaaggttttg cctcgaaacg 2460

2520

aagccgttca ggcatggcgc aagcgctttg ctgctgacga gcgaaaaggc atcgcaggtc 2580

tgagcatgtg gaacatagaa gaattggagg gcttgcgcaa gctgttgatt tcctggagcc 2640

gcaggacgag gaatccgcag gaggttaatc gctttgagcg aggccatacc agccaccagc 2700

gtctgttgac ccatatccaa aacgtcaaag aggatcgcct gaagcagtta agtcacgcca 2760

ttgtcatgac tgccttgggg tatgtttacg acgagcggaa acaagagtgg tgcgccgaat 2820

acccggcttg ccaggtcatt ctgtttgaaa atctgagcca gtaccgttct aacctggatc 2880

gctcgaccaa agaaaactcc accttgatga agtgggcgca tcgcagcatt ccgaaatacg 2940

tccacatgca ggcggagcca tacgggattc agattggcga tgtccggggcg gaatattcct 3000

ctcgttttta cgccaagaca ggaacgccag gcattcgttg taaaaaggtg agaggccaag 3060

acctgcaggg cagacggttt gagaacttgc agaagaggtt agtcaacgag caatttttga 3120

3180

tgttcatgac cttgacagac ggaagcggaa gcaaggaggt cgtgtttctc caggccgata 3240

ttaacgcggc gcacaatctg caaaaacgtt tttggcagcg atacaatgaa ctgttcaagg 3300

3360

tgcaggcaaa gctgggcaaa gggctttttg tgaaaaaatc ggatacagcc tggaaagatg 3420

3480

agtcgccga acaactggaa gactttcagg agatcatcga ggaagcagaa gaggcgaaag 3540

gaacataccg tacactgttc cgcgatccta gcggagtctt ttttcccgaa tccgtatggt 3600

3660

ggtttttgac aaaggctcgg taagggtgtg caagggagt gaatggcttg tcctggatac 3720

ctgtccgcat gctaaatgaa attcagtatt gtgagcgact gtaccatatt atgcatgtgc 3780

aggggctgtt tgaggaaagc gcagacacgg tcgaaggagc agcacaacac aagcgtgcag 3840

agacacatct gcgcaaaagc aaggcagcgc cggaagagat gtggggggac gctccgttta 3900

gcttgcagct cggcgaccct gtgcttggca ttacgggaaa gctggatgcc gtctgtctgg 3960

aagaaggtaa gcagtggatt ccggtagaag gaaagcattc ggcgtcgcca gaaggcgggc 4020

agatgttcac tgtaggcgtg tattcgctgg acggttctgc ctggcccaac gaccaaatcc 4080

aattgtgtgc gcaaggcttg ctgcttcgcg cgaatggata tgaatccgat tatggctact 4140

tatactaccg tggcaataaa aagaaggttc gcattccttt ttcgcaggaa ctcatagcgg 4200

ctactcacgc ctgcattcaa aaagctcatc agcttcggga agccgaaatt ccccctccgt 4260

tgcaggagtc gaaaaagtgc tttcgatgct cgttaaatta cgtatgcatg cctgacgaga 4320

cgaattacat gttggggttg agcgcaaaca tcagaaagat tgtgcccagt cgtccagatg 4380

gcggggtact gtatgttaca gagcaggggg caaaactggg cagaagcgga gaaagcttga 4440

ccatcacctg ccggggcgaa aagatagacg aaatcccgat caaagacttg attcacgtga 4500

gcttgatggg gcatgtgcaa tgctctacgc agcttctgca caccttgatg aactgtggcg 4560

tccacgtcag ctacttgact acgcatggca cattgacagg aataatgact ccccctttat 4620

cgaaaaacat tcgaacaaga gccaagcagt ttatcaaatt tcagcacgcg gagatcgccc 4680

ttggaatcgc gagaagggtc gtgtatgcga aaatttccaa tcagcgcacg atgctgcgcc 4740

gcaatggctc accagataaa gcagttttaa aagagttaaa agagcttaga gatcgcgcgt 4800

gggaggcgcc atcactggaa atagtgagag gtatcgaggg acgtgcagca cagttgtaca 4860

tgcagtttt ccctaccatg ttaaagcacc cagtagtaga cggtatggcg atcatgaacg 4920

gtcgcaaccg tcgcccgccc aaagatccgg tcaatgcgct gctctccctc ggctatacgc 4980

ttctttcacg ggatgtttac tccgcatgtg ccaatgtcgg actcgatcca ctgttcggct 5040

ttttccatac gatggagccg ggcagaccag ctttggcact cgatctgatg gaaccgttcc 5100

gcgccttgat tgccgatagc gtagcgatac gtaccttgaa tacggaggaa ctcaccctcg 5160

gggactttta ttgggaaaa gacagttgtt atttgaaaaa ggcaggaaga caaacgtatt 5220

tcgctgccta tgaaagacgg atgaacgaga cgctgacgca tccgcaattt gggtataagc 5280

tcagctatcg ccgtatgctg gagctggaag caaggttttt ggcccggtat ctggatggag 5340

agctggtgga atatacgccg ctcatgacaa ggtaggaaat gaccatgcga caatttgttc 5400

tggtaagcta tgatattgcc gatcaaaaac gttggagaaa agtattcaag ctgatgaagg 5460

ggcaaggcga gcacgtccag tactcggtgt ttctgtgcca actcaccgag attcagcaag 5520

ccaagctaaa ggtaagcctg gcggagctgg ttcaccatgg agagaccag gtcatgtttg 5580

taaaaatcgg cccagtgacg agagatcaac tggacaagcg gatatctact gttggcaggg 5640

agtttctgcc tcgcgatttg accaaattta tctattaagg aatgaagaaa gctagttgta 5700

acaaaagtgg aaaaagagta aaataaaggt gtcagtcgca cgctataggc cataagtcga 5760

cttacatatc cgtgcgtgtg cattatgggc ccatccacag gtctattccc acggataatc 5820

acgactttcc actaagcttt cgaattttat gatgcgagca tcctctcagg tcaaaaaagc 5880

cgggggatgc tcgaactctt tgtgggcgta ggctttccag agttttttag gggaagaggc 5940

agccgatgga taagaggaat ggcgattgaa ttttggcttg ctcgaaaaac gggtctgtaa 6000

ggcttgcggc tgtagggggtt gagtgggaag gagttcgaaa gcttagtgga aagcttcgtg 6060

gttagcaccg gggagaagtc atttaataag gccactgtta aaagttcgaa agcttagtgg 6120

aaagcttcgt ggttagcacg ctaaagtccg tctaaactac tgagatctta aatcggcgct 6180

caaataaaaa acctcgctaa tgcgaggttt cagc 6214

<210> 75

<211> 12338

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 75

gaagttatgt tgataaaatg gtttatgaaa acgtgagtct gtggtagtat tataaacaat 60

gatggaataa agtgtttttt gcgccgcacg gcatgaattc aggggttagc ttggttttgt 120

gtataaataa atgttctaca tatttatttt gttttttgcg ccgcaaaatg caactgaaag 180

ccgcatctag agcaccctgt agaagacagg gttttgagaa tagcccgaca tagagggcaa 240

tagacacggg gagaagtcat ttaataaggc cactgttaaa agttttgaga atagcccgac 300

atagagggca atagactttt gcttcgtcac ggatggactt cacaatggca acaacgtttt 360

gagaatagcc cgacatagtt atagagatgt ataaatataa ccgataaaca ttgactaatt 420

tgttgaagtc agtgtttatc ggttttttgt gtaaatatag gagttgttag aatgatactt 480

540

aggttaaaga gcatggcagg aatagtgacc tgtgatgaag atgatggtag aattaaaagt 600

aaaaacaata ttggataagg aaaataattc aatagataaa aaatttaggg 660

ggaaaaatga aaatatcaaa agtcgatcat accagaatgg cggttgctaa aggtaatcaa 720

cacaggagag atgagattag tgggattctc tataaggatc cgacaaagac aggaagtata 780

gattttgatg aacgattcaa aaaactgaat tgttcggcga agatacttta tcatgtattc 840

aatggaattg ctgagggaag caataaatac aaaaatattg ttgataaagt aaataacaat 900

ttagataggg tcttatttac aggtaagagc tatgatcgaa aatctatcat agacatagat 960

actgttctta gaaatgttga gaaaattaat gcatttgatc gaatttcaac agaggaaaga 1020

gaacaaataa ttgacgattt gttagaaata caattgagga aggggttaag gaaaggaaaa 1080

gctggattaa gagaggtatt actaattggt gctggtgtaa tagttagaac cgataagaag 1140

caggaaatag ctgattttct ggagatttta gatgaagatt tcaataagac gaatcaggct 1200

aagaacataa aattgtctat tgagaatcag gggttggtgg tctcgcctgt atcaagggga 1260

1320

gagaaagagg cactatctgc atttctgtta gattatgctg atcttgataa gaatgtcagg 1380

1440

gatgttatgt ctttaactga aattccggca gaagtgaatc tggaaaaaga ttttgatatc 1500

tggagagatc acgaacaaag aaaggaagag aatggagatt ttgttggatg tccggacata 1560

cttttggcag atcgtgatgt gaagaaaagt aacagtaagc aggtaaaaat tgcagagagg 1620

1680

attgaaaagg atgatggaac atactttttt gcaaataagc agataagtgt attttggatt 1740

catcgcattg aaaatgctgt agaacgtata ttaggatcta ttaatgataa aaaactgtat 1800

agattacgtt taggatatct aggagaaaaa gtatggaagg acatactcaa ttttctcagc 1860

ataaaataca ttgcagtagg caaggcagta ttcaattttg caatggatga tctgcaggag 1920

aaggatagag atatagaacc cggcaagata tcagaaaatg cagtaaatgg attgacttcg 1980

tttgattatg agcaaataaa ggcagatgag atgctgcaga gagaagttgc tgttaatgta 2040

gcattcgcag caaataatct tgctagagta actgtagata ttccgcaaaa tggagaaaaa 2100

2160

ggtattctga aatctatact tcagtttttt ggtggtgctt caacttggaa tatgaaaatg 2220

tttgagattg catatcatga tcagccaggt gattacgaag aaaactacct atatgacatt 2280

attcagatca tttactcgct cagaaataag agctttcatt tcaagacata tgatcatggg 2340

gataagaatt ggaatagaga actgatagga aagatgattg agcatgatgc tgaaagagtc 2400

2460

gatctaaaga aaatattgga tctcttgtat agcgattatg caggacgtgc atctcaggtt 2520

ccggcattta acactgtctt ggttcgaaag aactttccgg aatttcttag gaaagatatg 2580

ggctacaagg ttcattttaa caatcctgaa gtagagaatc agtggcacag tgcggtgtat 2640

tacctatata aagagattta ttacaatcta tttttgagag ataaagaggt aaagaatctt 2700

ttttatactt cattaaaaaa tataagaagt gaagtttcgg acaaaaaaca aaagttagct 2760

tcagatgatt ttgcatccag gtgtgaagaa atagaggata gaagtcttcc ggaaatttgt 2820

cagataataa tgacagaata caatgcgcag aactttggta atagaaaagt taaatctcag 2880

2940

ttagcaggtg ctttttctct ttatttgaag caggaaagat ttgcatttat tggtaaggca 3000

aaccctatac catacgaaac aaccgatgtt aagaattttt tgcctgaatg gaaatccgga 3060

atgtatgcat cgtttgtaga ggagataaag aataatcttg atcttcaaga atggtatatc 3120

gtcggacgat tccttaatgg gaggatgctc aatcaattgg caggaagcct gcggtcatac 3180

atacagtatg cggaagatat agaacgtcgt gctgcagaaa ataggaataa gcttttctcc 3240

aagcctgatg aaaagattga agcatgtaaa aaagcggtca gagtgcttga tttgtgtata 3300

3360

gcagattatc ttgaaaaata tctcaagtat caggatgatg ccattaagga attgtcagga 3420

3480

gtaaatgccg gacagaagcc tatcttacag agaaatatcg tgatggcaaa gctttttgga 3540

ccagataaca ttttgtctga agttatggaa aaggtaacag aaagtgccat acgagaatac 3600

3660

gaacaggaag atctgctaaa gttccaaaga ttgaaaaacg cagtagaatt cggggatgtt 3720

actgaatatg ctgaggttat taatgagctt ttaggacagt tgataagttg gtcatatctt 3780

agggagggg atctattata tttccagctg ggattccatt acatgtgtct gaaaaacaaa 3840

tctttcaaac cggcagaata tgtggatatt cgtagaaata atggtacgat tatacataat 3900

gcgatacttt accagattgt ttcgatgtat attaatggac tggatttcta tagttgtgat 3960

aaagaaggga aaacgctcaa accaattgaa acaggaaagg gcgtaggaag taagatagga 4020

caatttataa agtattccca gtatttatac aatgatccgt catataagct tgagatctat 4080

aatgcaggat tagaagtttt tgaaaacatt gatgaacatg ataatattac agatcttaga 4140

aagtatgtgg atcattttaa gtattatgca tatggtaata aaatgagcct gcttgatctg 4200

4260

aatgtgttgg agaatatcct tttaaggcat tttgtaattt tctatccgaa gtttggatca 4320

4380

gagcagagcc tcacatcgga agacttcatg tttaagcttg acgacaaagc aggagaagaa 4440

gcaaagaagt ttccggcaag ggatgaacgt tatctccaga caatagccaa gttgctctat 4500

aaattgagga tatgaacaga ttcatgaaga aaggagaaac gataaataaa 4560

aaagttcagt ttaatagaaa aaagaagata accaggaaac aaaagaataa ttcatcaaac 4620

4680

gttgtagata attgataggt aaaagctgac cggagccttt ggctccggac agttgtatat 4740

aagaggatat taatgactga aaatgatttt tgttggaagt cagttttttc tgtggaaagc 4800

gaaatcgaat atgatgagta tgcatatggc agaagagctg tagaaggcga gaatacatat 4860

gattacatta ctaaggaaga aagaccggaa cttaatgacg aatatgtagc gagacgttgc 4920

attttcggta aaaaagcagg aaaaatatcc aggtcggatt ttagtaggat aagatctgcg 4980

ttggatcatg cgatgataaa taatacacat acagcatttg ccagatttat cactgaaaat 5040

ctgacgagac tcaatcacaa agaacatttt ctgaatgtga cacgtgcata ttctaaacct 5100

gattctgaaa aattgataca accgagatac tggcagtcgc ctgtagttcc aaaggataaa 5160

caaatatatt atagcaagaa tgcgattaaa aaatggtgtg gttacgaaga tgatattccg 5220

cctcgttctg tgatagttca gatgtgtcta ttgtggggga ctgatcatga agaggcagat 5280

catatccttc gcagttcagg atacgcggcg cttagtcctg ttgtacttcg agatcttatc 5340

tatatgtatt atctggatca tcaggatttg caaaaaaatg agttgatatg ggaagtaaaa 5400

aagcagttgg atcacttcga tttgacaaat agaaattatg atacaaatcc ttttgatgta 5460

gggggcagcg taaatgatca tatctgtgaa ctgagcgagc atatagcgaa ggctcattat 5520

atttatgaga gggctaagga aggaccattg caaaatgtaa ttcgggatat tttgggagat 5580

acacctgccc tttattctga aatggcattt cctcagctag catctataaa caggtgtgct 5640

tgcaattcgc tttcttcata tcaaaaaaat atttttgata ctgacatagc tatatatgca 5700

gatgaaaagg acacaagagg taaatcagac cgtatccttg ttgagggcgc atcttcgaaa 5760

tggtatgaat tgaagaaacg cgatgctaat aatgtcaaaa tttctgaaaa gctgagtata 5820

ctcaatacta ttcttaaatt taatagtgtt ttttgggaag aatgttacct tgatggaaat 5880

ataaaacaat cgagcggaaa gcgatctgag gcaggaaaaa ttctttatgg tcgcgacaac 5940

ggaaaagaaa atgtcggagt ttcaaaattg gaattggtgc ggtatatgat agctgcaggt 6000

6060

6120

gactatattc tgatatatgc atggggattt agggaaaata tcattaaaaa gaacagtaat 6180

gtgaattctt tggatgaaaa gactagaaaa gtgcagtttc cgtttataaa gttactcatg 6240

gcaattgcaa gagatatcca gatacttata tgttcagcac atgaaaaaac agtcgatgag 6300

tcatctcgaa atgcagcaaa gaagatagat atattgggaa attatattcc ttttcagatt 6360

catcttcaga gaactaaaaa agatggtgga agagtggtaa tggatacatt gtgtgctgat 6420

tggattgcgg attatgaatg gtacattgat cttgagaaag gaacacttgg atgagcagtg 6480

6540

ttttagaaga aaagaaatgt tcggataaac taactgcact gcttgggaat tactgcatac 6600

6660

6720

tacgtttgct tctcataaag agaattctgg aactcgtgtg tgcttttcac gaaaaaaaat 6780

ggtattgtct cagtatttca ccgggaatgc tcatggttga agattttgat ataccgatgg 6840

gaaatgtcgg aaaagtattg atatatgatt tcagaaatcc tgttccgttc gagtcagtaa 6900

atgaaagaca taattttaac gtttcaaata aatacacttc accggagctg ctcatccatt 6960

aaaaatcaga tttgtattct gttgcaaaaa 7020

ttgcggaaac aataatagga gattttaaca gtattattgc aaatggaaat ttgatactac 7080

7140

aatcgtcgga aaatatgctt tcagtatttg aaaatttgat caaagaaaat tgtttttttg 7200

aaaaaaacga ttatacatct atgtttcatc aggcgtatga caattttttt gaatggcagg 7260

aatgtttgat atcaccggat cacttggata aaaatatgtt cgaggcagct ttatcaaatc 7320

ttgaggatca gctgcttagg gttgatattg ataagtatag agcagagtac ttctataagc 7380

ttctccgaga gttgtctaat aaatataaaa atacaattac tgatgaacaa aaggtaaggt 7440

tggcaatact tggaatcaga gcgaaaaata atctgggaaa aagttttgat gcattggaaa 7500

tatatgagtc agtacgtgat ttagaaacta tgttggagga gatggcagag cttagtcctg 7560

tcattgcttc gacatatatg gattgctacc gatatgcaga tgcgcagaaa gtggcggaag 7620

aaaacattat caggcttcat aatagtaata ttcgtatgga gaaaaaaaga atactgcttg 7680

gaaggtcata tagttcaaaa gggtgcagca tggggtttca gcatattctt ggtgcggatg 7740

agtcatttga acaggcttta tatttcttta acgaaaagga caatttttgg aaagaaatat 7800

ttgagagcag aaatttagag gacagcgata gacttataaa gtctttacga agcaatacgc 7860

atattacgct gtttcattac atgcaatatg catgtgaaac aaggagaaag gaattatatg 7920

gagcactttc agacaaatat tttataggta aagaatggac agaaagactc aaagcatata 7980

taagcaacaa ggatatatgg aaaaactatt atgagatata tattctgcta aagggtattt 8040

attgcttcta tccagaagtc atgtgttcgt ctgcgtttta tgatgaaatc caaaaaatgt 8100

acgatcttga atttgaaaag gaaaaaatgt tttacccatt gagtctgata gaactgtatc 8160

ttgctctgat agagataaaa gttaatggga gtctgacgga gaatgccgag aagttgttta 8220

aacaggcatt gacacatgac aatgaagtca aaaaaggaaa tatgaatatt cagaccgcca 8280

tttggtatcg aatatatgca ctgtataacg atgtaaaaga tgaaactgat aagaataaaa 8340

ggcttttaaa acggcttatg attctttgcc gacgatttgg ttgggcggat atgtatagtg 8400

ctttggagaa ggatgggaag ttaattgatt ttttgagatt tgaggtatgt taaatgataa 8460

8520

cgataatgat tgcggggata atctatgatg acaaggggaa agagtatgat gctgagaatg 8580

aacgctacag gatatccagt tatctgcgag cagtatgtga cagtttgggt gcgaaatacc 8640

ctcaggatct acattcaaat agtaatggaa ataaggcgac tgttgggaaa gtaaaatgta 8700

aaattggtga aacactaaag gaattcttga gagaaggaac ctatgaaaaa aaggaattgc 8760

cgacaaagaa cggttattta aataagagat ctggaaaata tgtaatgttt gcagaactca 8820

ggagtagtca gggagttaaa aagcgtgtta gtggttggaa tgacaatgat ctgactcagg 8880

atgaaaaggt cagcaatctg taccttcata tggcagaaaa tgccgttgtc agaatgctct 8940

tccataatcc tatatatgaa gatgtaacag atgtaaatct ctattttccc acgcgaaaag 9000

ttgttctgaa agatagagat agagaatacg ataaacaaga tttcaaaata tatggtgata 9060

aggacaagtg cgaagcagaa agcgggagat tggtgcatta tgatatcgtg tcatcggatt 9120

tttaccgtac gataatggag aacgaatgta caagaattaa taaaaagcaa ttaaatgttc 9180

attatatgaa cacaagccca atttcgtact gggagaaaaa tgaaaaatat aatacatttt 9240

tatatttggc tgacatagtt tgttctatgc tggattatta caaaaagggt tcgagtccgg 9300

9360

tcttatttgg gtatgatgat atagatgaca aatacatgga ggctgtagat gcagtaggac 9420

agggagagta ttttcatgcg ctggatatta tatatgatgc ggaatgtagt ggaagtgaat 9480

ttgagaagca ctacaaagat tattggtttc caaagcttat aaaaaagata cgaataacag 9540

9600

atcttgatca gcagaaactt ttgtggattt ttgaggaaat caaagctatc gtcgataagg 9660

gagattttgg aaagaaatat catacagatc aggttatgtt tgatatgtgt aatgccggta 9720

ttgctgtgta caatcatatc ggagattttg ggactgcaaa ggaatactat gatgagtgca 9780

tgaaacacac tggggatgtg gatctggtaa agatacttcg tgcatcaaat aaaatggtgg 9840

tctttcttga cgatgctttt aggtatggtg acgcgacaga acgtgccagg aagaatgttg 9900

aataccaaaa agctttgcac gatataaaga gtgagatttg tccggaaaag aaagatgaag 9960

acttgaacta tgccatatcg ctcagtcaat ttggacaggc gcttgcgtgt gaaaaaaatt 10020

ctgatgcaga gagtgttttc ctagagtcgt tgcggcatat gaggaaaggg actgccaatt 10080

10140

atcgagaaaa aacaaaggac tattttggaa gtgaaaaacc aaaggaacag ctgaaagaat 10200

tgctgaagtt atcgggaaag gatgatagta tagttacttt caaatttgca atgtatgtct 10260

10320

aggacatacg tgagactctt gtaaagaaga aaatgagtga acatatggtt ggacatccgt 10380

gggagttgat ttataaatat ctggcatttc ttttttatcg tgatggaaat tgtgaagctg 10440

ctgaaaaata tattcataaa agtgaagagt gcttggaaac acaaggactg actatagatg 10500

cgattattca taatggtaag tatgaatatg cagaattgtc aggtgacgag gagatgatgg 10560

caagagagaa agcgtacttt gatgaaaaag ggatagatag aaaaaatgtt tgtactttta 10620

10680

ggtattacct gtcatttttt gatgaaataa gctacttttt gcctaaaaaa cgaaactgtt 10740

ggtgttttat gatgattgtg tcaacaaaag agagcaaaag aagaggagaa aagtaatgtc 10800

10860

ttgtggatat gtgttagtcg aagaagctaa agtagtgtgc acagaatgtg gaactgaggt 10920

agagagtggc gctgctgtat gtccgaagtg cggctgtcct gtaaatgata gtgagacgcc 10980

tcagaaagtt gaagtgacta gggtaaatgt atcttccgta atcagcaaaa aagtcgttgt 11040

aagcatactg atcgcagtga ttacaattgc aggttttttc tatggagtga agtattcgca 11100

11160

gcttgcttcg ctaatgatgc ttcaaggagc ttcggatgca gaaacttgtg ggaatttggt 11220

taggaaagtg tggagcaact gcatttataa ggagagggat gaagaaaccg acaagtatac 11280

gtgtgatagc aggggtgcag gatggtttta tgatgatttt aatgatgcat taatggctct 11340

ttacagtgac agcagttttg gcaagaagat aaatgaaatc aaaaacggtc aggaaaccgt 11400

11460

11520

11580

tagccggatg aagttgtatt tagattaaac tattgaggaa aaaatggagg tgctttaatg 11640

cgggggagaa actgtggagg gtcatcaggc gacggactgc tggtacttct cgtactgctt 11700

11760

gaacgtaaag atctgggtat gggtatgatt attgtcggga tagttctata tgtattatta 11820

gaggtttttt aatgtgagtt tctgtggtaa actataaaag tacaagcttt tgcgccgcac 11880

cgcataaata gcggatttat gaccattatt tggtgaaaaa aatggtgtac acctgtgttt 11940

ttttgttttg cgccgcaaaa tgcgccacgg aaccgcatgc agagcaccct gcaagagaca 12000

gggttatgaa aacagcccga cataggggc aatagacacg gggagaagtc atttaataag 12060

gccactgtta aaagttatga aaacagcccg acatagaggg caatagacat aaagaccaaa 12120

aacagggtcat ctgcatactg tgttatgaaa acagcccgat atagaggggtg tgagagatat 12180

12240

atggaaaggc gtatttaatg aaatgctgat ctgttgattt gaattaacaa aaaaaggtcg 12300

ccccacggat gacaaaaaca tccggggggcg accctttt 12338

<210> 76

<211> 6098

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 76

tactgtgtgc ataagtcttc cttagatcca taggtacagc agttttattt attagcctta 60

120

cgtgcaaact ttaaatatat tgattatatc ctttacattg gttgttttaa ttactattat 180

taagtaggaa tacgatatac ctctaaatga aagaggacta aaacccgcca aaagtatcag 240

aaaatgttat tgcagtaaga gactacctct atatgaaaga ggactaaaac ttttaacagt 300

ggccttatta aatgacttct gtaagagact acctctatat gaaagaggac taaaacgtct 360

aatgtggata agtataaaaa cgcttatcca tcatttaggt gttttatttt tttgtgatta 420

tatgtacaat agaagagaga aaaaaatcat tgaggtgaaa actatgagaa ttactaaagt 480

agaggttgat agaaaaaaag tactaatttc tagggataaa aacggggggca agttagttta 540

tgaaaatgaa atgcaagata atacagaaca aatcatgcat cacaaaaaaa gttcttttta 600

660

tcatggatta ttacaagaaa atagtcaaga aaaaataaaa gtttcagatg tcactaaact 720

taatatctca aatttcttaa atcatcgttt caaaaaaagt ttatattatt ttcctgaaaa 780

tagtcctgac aaaagcgaag aatacagaat agaaataaat ctctcccaat tgttagaaga 840

tagcttaaaa aaacagcaag ggacatttat atgttgggaa tcttttagca aagacatgga 900

attatacatt aattgggcgg aaaattatat ttcatcaaaa acgaagctaa taaaaaaatc 960

cattcgaaac aatagaattc aatctactga atcaagaagt ggacaactaa tggatagata 1020

1080

ccaacttgaa aaattgacta gtgctttaaa agctactttt aaagaagcga agaaaaacga 1140

caaagagatt aactataagc ttaagtccac tctccaaaac catgaaagac aaataataga 1200

agaattgaag gaaaattccg aactgaacca atttaatata gaaataagaa aacatcttga 1260

1320

aggagaaatc caaaaaatag taaatcatcg gttgaaaaat aaaatagttc aacgcattct 1380

ccaagaaggg aaattagctt cttatgagat tgaatcaaca gttaactcta attccttaca 1440

aaaaattaaa attgaagaag catttgcctt aaagtttatc aatgcttgtt tatttgcttc 1500

1560

1620

cttctctcaa gaaataactg ttgatgacat tgaattagct tcatgggggc tgagaggagc 1680

cattgcacca ataagaaatg aaataattca tttaaagaag catagctgga aaaaattttt 1740

taataaccct actttcaaag tgaaaaaaag taaaataata aatgggaaaa cgaaagatgt 1800

tacatctgaa ttcctttata aagaaacttt atttaaggat tatttctata gtgagttaga 1860

ttctgttcca gaattgatta ttaataaaat ggaaagtagc aaaattttag attattattc 1920

cagtgaccag cttaaccaag tttttacaat tccgaatttc gaattatctt tactgacttc 1980

ggccgttccc tttgcaccta gctttaaacg agtttatttg aaaggctttg attatcagaa 2040

tcaagatgaa gcacaaccgg attataatct taaattaaat atctataacg aaaaagcctt 2100

taattcggag gcatttcagg cgcaatattc attatttaaa atggtttatt atcaagtctt 2160

2220

attaaacaaa gaacggaaag gttacgccaa agcatttcaa gatattcgaa agatgaataa 2280

agatgaaaag ccctcagaat atatgagtta cattcagagt caattaatgc tctatcaaaa 2340

aaagcaagaa gaaaaagaga aaattaatca ttttgaaaaa tttataaatc aagtgtttat 2400

taaaggtttc aattctttta tagaaaagaa tagattaacc tatatttgcc atccaaccaa 2460

aaacacagtg ccagaaaatg ataatataga aatacctttc cacacggata tggatgattc 2520

caatattgca ttttggctta tgtgtaaatt attagatgct aaacaactta gcgaattacg 2580

taatgaaatg ataaaattca gttgttcctt acaatcaact gaagaaataa gcacatttac 2640

caaggcgcga gaagtgattg gtttagctct tttaaatggc gaaaaaggat gtaatgattg 2700

2760

ggaattgctt caatcattgc cgtacacaca agaagatggt caaacacctg taattaatcg 2820

aagtatcgat ttagtaaaaa aatacggtac agaaacaata ctagagaaat tattttcctc 2880

2940

ggagaaaata gcacagcaag agagtctaca taagcaatgg atagaaaagc ccggtttagc 3000

ccgtgactca gcatggacaa aaaaatacca aaatgtgatt aatgatatta gtaattacca 3060

atgggctaag acaaaggtcg aattaacaca agtaaggcat cttcatcaat taactattga 3120

3180

taattatatt ttagaaagag agaactctga gtatagagtt acaagttgga tattattaag 3240

tgaaaataaa aataaaaata aatataacga ctacgaattg tataatctaa aaaatgcctc 3300

tataaaagta tcatcaaaaa atgatcccca gttaaaagtt gatcttaagc aattacgatt 3360

aaccttagag tacttagaac tttttgataa ccgattgaaa gaaaaacgaa ataacatttc 3420

3480

3540

aattttaagc tctcatggaa tggaagtgac atttaaacca ctatatcaaa ccaatcatca 3600

3660

3720

gtaattcttt taaagcacat taattacctc taaatgaaaa gaggactaaa actgaaagag 3780

3840

agaggaaact ttagatgaag atgaaatgga aattaaaaga aaatgacgtt cgcaaagggg 3900

tggtggtcat tgagtaaaat tgacatcgga gaagtaaccc actttttaca aggtctaaag 3960

aaaagtaacg aaaacgcccg aaaaatgata gaagacattc aatcggctgt caaagcctac 4020

gctgatgata caactttaaa aggaaaagca gtggattctt cacaaagata ctttgatgaa 4080

acgtatactg ttatttgtaa aagtatcata gaagcattag atgaaagcga agagagatta 4140

caacaatata ttcatgattt tggagatcaa gtggattctt cacctaacgc acgaattgat 4200

gcggaattac tacaagaagc aatgagtagg ttagctgaca taaagcggaa gcaagaagca 4260

cttatgcaat ccttatcttc ttctacagca acgctttacg aaggcaagca acaagcgtta 4320

cacactcaat tcacggatgc gctggagcaa gaaaaaatat tggaacgcta tattactttt 4380

gaacaaactc acgggaattt ttttgactca tttggagaac ttgtctatcg aacgggacaa 4440

gcagtgcgtg aattagctaa taacgtcaca ttcgagagcc aaacaggaag ctatcatttt 4500

gataaaatag atgcttctag attccaaact ttgcaagaaa tgttgccaaa ggcaaagaaa 4560

aaagcattta attttaatga ctaccaaata acatggaatg gcaccacgca ccttttatgg 4620

aaaaatggta aagtggatgc agaagcaacc aaagcttata acgaggcgaa actgaatgga 4680

aagctaccaa aggaaggtaa tgtagcaaca caagatgcag aactattaaa aggcattttg 4740

gcttcactga aaaacaagaa agatcctatc actggagcag atataagcag tgtgcatgta 4800

ttatctatcc ttagcgggct cgcattctcc tatacagctg ggaattataa gggaagaaaa 4860

cttactgttc caaaaagttt cttagacaaa ttaaagaaaa accgaaaatc taaagtacct 4920

aaactatcta gtttatcaga aaaacaacaa ctaaaactcg caaataaata caagaaaaaa 4980

tcacctattc caattccaga tgatgctaaa atcaaagctc agacgaaaaa ggctggttat 5040

gaacaaatat cttataaatg gaaagagaat gggataacct ttgaagttag atggcatact 5100

aggacaccag gtgcaccaaa ggaacaagga aatacgtttg ttatagaaag aaaaattcag 5160

ggtacagcag aagggaaaac aaaagttcaa caaatattgg ttggagataa taagtgggtg 5220

agtaaaagtg agtggcaaaa ggctataact gataagaaaa atggtgtaag tacctcggag 5280

5340

attattataa aggttttgag ggatatccag agatagattt ttatacgtat atagatgata 5400

tgaaattggg tatagcaatg tgggaaggat actttgacaa cattatgaaa gaaattaatc 5460

caagtaacgg aagatggact tcattagcgt attattatca tttagatgag gggtggtatg 5520

atgaaagtcc ttgggaaata ccaagtaata cagaagcatt agaattattg gaaacaatcc 5580

atatatctaa tctagatact atcacacaag agatattact taaattaata aatttattaa 5640

agaagaatat aaatagacaa gtttatattg aatactcata aaaaagatga ttatgatata 5700

ttatagaaca aacgaacaag ccccaaatac gaggtttgtt cgtttgtttt caatataatt 5760

atttgccacc aagtgagata ttacggtttt aaatagctta tttgacgata ccaaaccctg 5820

ataagagaaa gaagaaagag aaagctggtg tagttgtttt aagtgaacta gataaaaaat 5880

taatagcaaa acttgaaaaa gatggtgtga aaatatcaaa agaagatgtt ataggaataa 5940

aataattgcc agatgatgag aaatcgtttg gctggaaaaa ggaaatccat ccgctggatt 6000

tgagcatatt cttattgaac atggtgaaca atttgctaaa tagggaattt caaaagctga 6060

gttacctgat tttttgatga ctgctttaga aaaggaaa 6098

<210> 77

<211> 6222

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 77

attctttaaa aatatctaat aatttattta ctatatactc taatacatct tttaacctat 60

ctaaaacatc atcacctaca acatcccaaa aatcatctaa aaagttaaaa aaatccatct 120

ttatcaactc ctatatctat tttttattgt gtaattcctg agttacaaaa ccattataac 180

acgtattaca cacgtagtca atacttcaaa aaaattttttt gtatattttt ttgaataagt 240

aaataaaaag agctgtgtag ctctttatta aaatcaatat tttttattttg ttaacaaact 300

tagacaacat taaatttaga aacctatata tatttcagta cttttcattt ttaggtagtc 360

taaatcagaa atggttttgt ctaaatgatg tatgtaagtt ttagtcccct tcgtttttag 420

ggtagtctaa atcagaagtc atttaataag gccactgtta aaagttttag tccccttcgt 480

ttttagggta gtctaaatcc catccaaatt atgggataat atgttacttt ttattttaat 540

600

tttttctcat aatgcaaact aatattccaa aatttttgtt tcttttctta tgatcttttc 660

tccgatagtt atttctccag ataagatttt catttttttg aattgatctt ctgttagaat 720

taatgttctt actgatgaat ttttctggaac tatcattgac aactgatttt cataggaaat 780

tattttttct tttgtgctag aacttacaat gtatactgat ttttgtacct gataatatcc 840

ttttcttata atttcttttc taaattttgc atattctttt ttttcttttc ctgtttgcat 900

tggaaaatca tacattagaa tccctacata attagtactc ataatcctct atccttaact 960

caggaatttc tacttctgac atttctcctg taaaataatt tctaatatta tctaaaaaat 1020

1080

1140

atacgatata atctaccatt ggacgaaata tttcaataat atcatctgca aaattataat 1200

tattaaattg tgaactgtga tgtattccca aacttggatg aaatccttta gccacaattt 1260

ttgaagagat taagcttctc aaaaccatat acccataatt taatgccgaa tttgtcccgt 1320

cttcaccaaa tctcttaaat tttttcccaa aaagttcacc aaaatacatt cttgcagcaa 1380

ttgcttcctg atgttccgct tcttttcctt ttaatctaat attattttca tatgcttcca 1440

acttatatga tacttcctga gattttttca aaaactgcaa taaatttctt tgattttcta 1500

tttttctcat tacaattttt ctccagattt cttctttttt atcgtcaatc cagctcactt 1560

gctcattaat tcttgttgtt acttgaaaat gattatacag tcctaatgaa tgtaaaactg 1620

gctgatgttt ttcattacaa attatcagtg gaatattatg ttctgataat cttaactgta 1680

atattccgct aattttacat ctgcaatttt caactacaat tgccatgata tcatttaaag 1740

atactttatc agccttattt tcatcatctt catttatcat cacaagctgg ttatttaaaa 1800

ctgataattc attgactctt gttacatgga taatattaga catttttatt actcctttac 1860

tctaaagctt tatattcaaa cataactttc acaagttcac acaattcttc tgaatttcta 1920

1980

gtctgaattt ctattttctt atctgctcct attttaaatg ttgctacaaa accatattcc 2040

2100

aattttctta aattttccag cacttctaaa agtgaaattt cagcatgcgg aatatagtta 2160

aaatgtgcaa tatagtttcg tatatacaaa tcttttttct cttgttttaa tttttttact 2220

tttttatcag aatagatgct tcttttttct acattatctt tgtataattc tttataaaaa 2280

tttatatatt tttcaacaat ttgcccactt ttatatttta catttttact gttatcaaaa 2340

ttaaatattt cttcaatata atgattttca ggaaattcac ctttcaatct aaatcttaag 2400

2460

2520

tttccaattg ctttttcata ttctttataa tcttcatcat taaatttttc atctttttta 2580

2640

tttttattgc tgtattcttt caattctttt aaacttattt tatacttcgc tttatcagct 2700

attttttcaa gtaaatttaa catcccatat ttttttatat tataaaaagc tctatgcttt 2760

ataattttt ctccatcaaa atatatttta tttgtgtcaa atttcttcaa ttctttccta 2820

tctttttttt tattttcatt aaaatctaaa aattttccaa tttcattcgc ttctaattca 2880

aaatcttctg ttactctatt attatctaaa tttaaaagat ttataagttc aagttcatct 2940

gaaaaagttt cttcttttatt tgcactctga tatttttcaa gacttccctt caaattagtc 3000

3060

tttaatatct ttcctaattt tatctctctt acaaattcat ttatttcatg tggaatttct 3120

tattcctat tatgtttttc ataattttttt aaaattttat catatttttc tttattatct 3180

ttttttattt ttatttttaga aaatatatca ttattatcat tgttattatt actttctata 3240

3300

3360

3420

3480

3540

3600

aaagtatttc ttaaatcttc tattttatta tataatttcg taaaagaagg aacaaaagga 3660

3720

tcataataat taaatacatt tgcactattt aactgcttaa atatcttcaa tttcaatttt 3780

3840

aatgcaaata tatctttccc ttctaattcc aaattaaaat gcacaatccc atgtctaata 3900

ctgctaatag cttcatcaat atttgcaaaa aaatcttcta tctcattttt attatccata 3960

ttaaaatcat aactatagaa catttttaaa ttttctttta cttcattttg cttgttttca 4020

ttatatattt tatcaacttc tccagaaaca tatttttctt cgcccttatt attttttaca 4080

gtttttcctc tcattctacc tgtaatatca ttctcatttt cagtttcaag aatatttctc 4140

aatgaaaaat atgcaaccga agaaactcca attatatttc gtaaaaatgc ttcattttgt 4200

ctattcctag caataaaatc acttgttgca atctctccaa cttgtaaata ataattgtat 4260

ttcccacaat ttcttacata agtatccaat ttatttagta atttgttttc aattaatttt 4320

tttaaatttt gatattcaaa tattctctta attttatcgt tacttatgtt actcagtctt 4380

ttatacacat aatttttcaa aagctgactc atttcaattt ccacaaaatg acaaaaagca 4440

tattttatat ttttatcatt aagttcttct ttatccaaat aatattattata aaacacttgt 4500

4560

ttttgtattt cttcgtaaat aattttagca aaattttctt tatcattttt tcttccaatt 4620

attttgtgat agtattctct tattttatat ttttcatgtt tttttgaatt ttctattaaa 4680

aaaaataact tctcaatatc ttctttttta tacaatttat caaatgcttc ctgtacatta 4740

tttatataat cattacgctt tgctgattct ctataataat cataaataat atttcttttg 4800

ctcttccctc caactttttc aacattattt tcattaattt tctgataatt agccttattt 4860

tcttcaaatg aatattttaa agaatttatc ttattcaatt ttgcctcaac atcttttcta 4920

aatatttcta attcttcaga gttcacatct tcatttaaca atattttctt taaaactgaa 4980

aaactatttt tattttttaa atcatattct gaaatatctt cttcagaata atttttatcc 5040

tgtactgcat ttttctcttt cctattcttt aaatacagaa cactatcttt tagatgcaat 5100

actttatttg aaaaaaactt ttttaaattt tctcttctta ttctattttc ttcttcactt 5160

gcattatcag gattttttat atatatcc agtcttatac ttaaaagctc tgacaatctc 5220

tcactagtcc tattttcttc gctcgtactt tttactaatt ttccctcttc aatatatttt 5280

aaaacctcct aatatctata 5340

ttttttactc aatacctaat tctttttttca atgctttttg taaaatttgt gaaaaattca 5400

gatttttttc ctgtgccaat atatctaacc aaacaggaat tgttaaagtt ttctttttaa 5460

gtgcatttgt aacttttgcc acttcataca ctggatcaac agataaaata tacaaatact 5520

gattttcttt cagtttcaca tcctccactt ttgaaggctc aggaaattttt tttcttacat 5580

ccaaaaaatc agccaaatgc agacccaatg tctctctcaa attggaaaca gcctcctcca 5640

tgctatctcc aaatgtagca taataattta tctctccatc ttcaaactta tcaaaatcaa 5700

caatacaacc ataataagtc ccatcttcct tagttaccac tgctggataa aatacatcca 5760

ttttaattat ctccaatcta taccacgtgt taaatacgtg tttaaaaata tttataaaat 5820

tttttagcat ctctgctaaa ataaaacaat tatttcaaat ttttctattc cttaatcact 5880

cattgttagt gattcttttt ttacttggac aatttttcat ttaatttctt caattttttt 5940

6000

6060

6120

tggtactgtg accacacctt ccacacctgg gatcatccat tgataatgac tacctcttat 6180

acgcacaact tttccgccta attttctaaa tcttttttcg at 6222

<210> 78

<211> 6337

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 78

ctttctatct ttttcaaata aaattaggct ctagttagcc taatcgcata attatttatt 60

120

ttcagaacaa ccaagcaacc atattcaaaa aacaataaaa aatgagcaag aattgaaatt 180

ttattctcac tcagaagtta tttttattaa atatcacttt tcgatattgg ggtggtctat 240

atcaatttaa aagacagaat agataattct ttagagtttt agtccccttc gatattgggg 300

tggtctatat cagaagtcat ttaataaggc cactgttaaa agttttagtc cccttcgata 360

ttggggtggt ctatatccca tcctaatttc ttgctgatga gatatttatt tctaattttt 420

ctattttgtc tttattttca atactttcaa tcctattttt ctctttatta ataatataga 480

accaccctat actattatac catattttttt gatttttcaa aattccaata ttttgttttg 540

tgaaattttt tctcccattg tcacttctcc tgcaagtacc ttcatttttt gaaactgatc 600

660

ttctgccagt tcgatttttt cttttgtttt cgacctcatt atatataccg atttttgaag 720

ctgataatat cccttttcta tcaatttttt cctaaaagtc ctatattcaa atctctcaac 780

atctgtctgc ataggaaaat catacataag cagaccaaaa tactcaatac tcatagtcca 840

tcacgctcaa tgtcggaatt atcacttctt catcttttac aaaataattt cgtatactat 900

ccaaataata gtctaccgct tggaaaaaat catatttctt attgttaaat aataccttct 960

gctgtgctac aagaagtatt ttttgcctta ttttccttact taatttcact tcattcaaaa 1020

tatccttgta catataaaca agataatcca ccataggacg aaaaacctct attatatcat 1080

cagaaaaatt ataggcatta aactgtgact tatgatgtaa tcctaaactt ggatgaaatc 1140

cttttgctac aatctttgat gatattatag ctcttaaaat catatatcca taattaagtg 1200

cagaattcac tccatcttca tcaaatcttt taaaactatt actatacaat tcctgaaaat 1260

atatccttga agctattgct tcctgatgtt ctgcactcgc atcatctttt ttcaagtttt 1320

1380

1440

tttcccactc aatctgctca tttattcgta aagtcacttg aaaatgatta aataatccca 1500

gcgaatgaat ttcaggctga tgtttctcgt tgcaaataat aatcggaatg ttatttttcca 1560

ccagcctcaa ctgcaaaatc gcactaatct tacaatagca gttttcaata actatcgcag 1620

atatatcatt caaagaaatc ttatttttct catcattatt gtcttcatca accattataa 1680

gctgattatt cgatattgac aaatcatcag cccttgttat gtgaattata ttgggcattt 1740

taatcatact ccttataaat ttcattctta taacgtatca ttcgtatttt ctatttttgt 1800

taaaagttct attatcaagt tttttaatata atcagaatta taactttcta attctaaaac 1860

agaaactttt ttaggtttca ttaatctttc aagtatatca ttattaccga taagtttaaa 1920

ttttttcttt aattcatcat aatctaaatt cacatctttt ttaaatactt caaatacact 1980

tgcataagtt gaattattat aacgtgtact atatgataat aaattagaaa ctctatcaat 2040

ttgttctgca atactgtaat cagcaaacgg atttcttaca atatagaaat gtgaaatata 2100

gtttctaata ctttcatttt ccggcttatt aatttcagaa ttttcagaca aatcaattcc 2160

aaatccataa catattttct caaatttttt ataagattct tcatcaaaaa atttatagta 2220

tgctgttgtt gtataaaagc catcagatcc attacgctta ggataagctc tacttattcc 2280

agtattgtag ccacttaact taataattcc taattctctt agcccattta caatatagtg 2340

catatctctt tcaaatctag ccatttgaat agcaagtttc caatttatat ctatcaaata 2400

actttctatt ttattcaaat aattaaattc taccaaatct ctaatttttt tgtattcaga 2460

aactctatta taatcttttt caaatgattt atagttttta ttttgtatat ttttgcaaa 2520

aaagtcatca ttttctttca atttttttat atacttctct ttgtattctt tagaatatcc 2580

tgcatcaatt tcagatattt tattttttct 2640

aatattttta ccatcaatat taaataaaaa ttttgcatca gccattttaa tatcatttga 2700

aattaatcca taaattttat caaaatttgg atttccaata tttaaaaata aattcttttt 2760

ataaatatat aattcattct tacgttcttt aggataatat atttcttgaa atttattttc 2820

2880

2940

tatatactga tcaacctttt ttttcaaatc ctttttattt atgtttgata actttctttg 3000

3060

tacaattttt tctaattttt tctctaaaac atcacaacca ttaatatcat ctttaaattc 3120

3180

3240

attaagattc caattttcag ttatacattc atttctcaaa gtatttaatt gcattatttc 3300

3360

aaatctattt ctaattttat ttataaccgc attactattt aacagtgcaa atattgaaat 3420

3480

ttcgtaagtt ttattatcat taatgtcttt tattgtttc ttaatttctt gaatattcat 3540

tttaaaatct gaaaaatcaa aaagttcctc ataatttttt ctcaaatatc caatataaca 3600

3660

3720

agttttcttt aattcttgta aaaatatatt cttactttca ttttcttcta aatcatcttc 3780

3840

ttctgtttct atagtatcaa atggttcatt cttaggatta ttcctatata aatttaatat 3900

3960

aattttaata tcatttattt tagtaattat atttttttta tctttaaata ctacatctaa 4020

4080

tttattatag tcatcttgcg ttccttgtaa atctctttcc ttgctaatcg catgtaatat 4140

cctgtttctt tcatttgttc ctatctttgt aaatttccta ataaaattat ttgtaatgtt 4200

atttttatta tctataaaat ctaagtctct tattattttt atttttgaat ttaaaatttt 4260

tttatcaagt acgtaatttt tttctcgatc tcctccaaag aaatctatat tttcatcatt 4320

atttatattt tctctagaaa aaatcttatt taattccata ttggtagaag caaaaaaagt 4380

aatcaattct aaatccaatt cctctttagc gtgaagtcta gaaaaatcat cagtatttac 4440

tgttgtcata tctatatcat tatgtcttaa tttccctaaa tacataatat gctctaacgt 4500

atattgctta actcttttta aaattttttc agataatata ctttcattta aaattttttc 4560

tatttctatt ttttccattt tctttaatct gactttttgt tcatttacca atattttttc 4620

aattcttcct ttcaaatatc gatatatgat tttatatagt tctttttctt catcagattt 4680

ctttgaaaat tttttcgaat caaaattaac tttataatgt tttttaaata ttccaaaaat 4740

ttctgtatca caatttcctt tttttagttc tttttctaat ttttttatta attcatctat 4800

tttaaattct gctaaaattt tttctattttt ttcttttata ctattatttt ttatattttc 4860

tacaaaaaat tttacaattt tatctttttt attttctctt tctattttaa atttttcgtg 4920

cttatctaat agtacataag attttatata tgttctattt cttctctttt caagaaattc 4980

attattaact ttttttactt tttcaattct tttagtaata ttccaaaatt ctaactcttt 5040

tataacaaaa tcagctatat cttctactgt taaatctaca tttatattta aaattttttc 5100

5160

5220

catctttcaa taatttttct cttaaatgtt cttcataata 5280

tcgattttca aatacttttt ctgtttcatt ttcaattatt ttttctataa tcttatataa 5340

actcatgtta atatttttaa aaatttcgta aattgatttt tttgtttcta attcatcatt 5400

ttctattatt cttaatatta ttgaacaatc atttagtgtt ttattagtat actcatctct 5460

gatatctatc tctatttctt cttcattctc ttgtctcttt atttctattt ttttatcatc 5520

tttagttatt ccttgcctaa ttgcttcatc tattattttc ttttttgtaa tccccaatgc 5580

tttcaatttc tcagattttc catatgcttc tatatataat acaacttctt ctgtttccaa 5640

aaaatcatca ttatttttcta ttcttatgat tccttcttta cctttcaact taaatagaat 5700

atttcctgca tgaaattttc ttgtaaattc tttaagaata ttatcatttt ttttgtaatt 5760

aatatatttt ctaataaatt tattattatc aattttttct ttattattat ttttcattaat 5820

atttaaaatg tattgtttc catcatagtt ccttttaact tttactttcc gttttatttt 5880

aaaatctttt ttatcacgaa cttcatacca tctcttatgt ccaaataaat ttcccattcc 5940

aatctcctcg tttctacttt aatctaataa aatattttta aattaaatca attttacatc 6000

tttctaatca aaaatacaat tttccatttt tagtatacca catcaatatt aaatctcaaa 6060

aaaataagga gccgtcaaac atagctccct acttctattt actcataatc cccatctatc 6120

cttacttttc gtaaaatcaa tccttctttc gcctttagat ccaacttaat tttcccattt 6180

gaacctgttc taaatgttct gccttctgtt accaaatcaa taaatctttc atcctgataa 6240

tttgtttcaa attccacatt ttcccagctg ttaaacgaat tatttattac aacaataatt 6300

aaatgatcct cgattactct ttcatacaca attattt 6337

<210> 79

<211> 91

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 79

cgagguucug ucuuuugguc aggacaaccg ucuagcuaua agugcugcag gggugugaga 60

aacuccuauu gcuggacgau gucucuuuua u 91

<210> 80

<211> 16

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 80

uacgaggcau uagcac 16

<210> 81

<211> 101

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 81

ucagucgcac gcuauaggcc auaagucgac uuacauaucc gugcgugugc auuaugggcc 60

cauccacagg ucuauuccca cggauaauca cgacuuucca c 101

<210> 82

<211> 20

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 82

gaaagcuucg ugguuagcac 20

<210> 83

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 83

cccataattg ataggatcta tgaggt 26

<210> 84

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 84

aaatataaat acattaa 17

<210> 85

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 85

cccataattg ataggatcta tgaggt 26

<210> 86

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 86

atcggttata caggcta 17

<210> 87

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 87

cccataattg ataggatcta tgagga 26

<210> 88

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 88

acggcgccgg aaaacat 17

<210> 89

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 89

cccataattg ataggatcta tgaggc 26

<210> 90

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 90

agagccgcgc ctattgg 17

<210> 91

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 91

cccataattg ataggatcta tgaggt 26

<210> 92

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 92

aaaacggtcc cactaat 17

<210> 93

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 93

cccataattg ataggatcta tgaggt 26

<210> 94

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 94

aaaacggtcc cactaat 17

<210> 95

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 95

cccataattg ataggatcta tgaggt 26

<210> 96

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 96

tgagctgctg gcccgca 17

<210> 97

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 97

cccataattg ataggatcta tgagga 26

<210> 98

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 98

tggttccaat gcagtaa 17

<210> 99

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 99

cccataattg ataggatcta tgaggt 26

<210> 100

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 100

acctataacg gcaccta 17

<210> 101

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 101

cccataattg ataggatcta tgaggc 26

<210> 102

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 102

cccataattg ataggatcta tgaggt 26

<210> 103

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 103

cccataattg ataggatcta tgaggt 26

<210> 104

<211> 18

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 104

ttgccgccgt cctgcatg 18

<210> 105

<211> 27

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 105

cacgtagttt tgaagggaag ttagagg 27

<210> 106

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 106

accgaatatg agaactg 17

<210> 107

<211> 27

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 107

atcgtagttt tgaagggaag ttagagg 27

<210> 108

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 108

tacaaataaa catcaag 17

<210> 109

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 109

tcgtagtttt gaagggaagt tagagg 26

<210> 110

<211> 16

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 110

ccgctccggc cgtgat 16

<210> 111

<211> 27

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 111

atcgtagttt tgaagggaag ttagagg 27

<210> 112

<211> 27

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 112

atcgtagttt tgaagggaag ttagagg 27

<210> 113

<211> 27

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 113

atcgtagttt tgaagggaag ttagagg 27

<210> 114

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 114

gttagaattg aggatgtt gaagga 26

<210> 115

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 115

ttacacggcc tatggtc 17

<210> 116

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 116

gttagaattg aggatgtt gaaggt 26

<210> 117

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 117

cttatgcaca acccttt 17

<210> 118

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 118

gttagaattg aggatgtt gaagga 26

<210> 119

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 119

aactgaacgg cttgttt 17

<210> 120

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 120

gttagaattg aggatgtt gaagga 26

<210> 121

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 121

tcttactaat ttgccga 17

<210> 122

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 122

gttagaattg aggatgtt gaagga 26

<210> 123

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 123

gttagaattg aggatgtt gaagga 26

<210> 124

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 124

gttagaattg aggatgtt gaagga 26

<210> 125

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 125

aactgaacgg cttgttt 17

<210> 126

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 126

cccataattg ataggatcta tgaggt 26

<210> 127

<211> 27

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 127

atcgtagttt tgaagggaag ttagagg 27

<210> 128

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 128

gttagaattg aggatgtt gaagga 26

<210> 129

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 129

cccataattg ataggatcta tgaggt 26

<210> 130

<211> 27

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 130

atcgtagttt tgaagggaag ttagagg 27

<210> 131

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 131

gttagaattg aggatgtt gaagga 26

<210> 132

<211> 27

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 132

ccctctaactt cccttcaaaa ctacgat 27

<210> 133

<211> 17

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 133

cagttctcat attcggt 17

<210> 134

<211> 225

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 134

cctctaactt cccttcaaaa ctacgtgaca aaggttaaca tattgtaatt ccacttgatt

ttgtcatacc agggagaaaa ttggcatgga aatggatcaa tttcagcacc ccgattcggt 120

ttggacatgg aaatctaacc aacggggccg caaggcaagt ctgtggattc cctacctcga 180

tcaaataaga aaaattaagg ggactcgctg ggaatttgtt tataa 225

<210> 135

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 135

gtctcggcaa gcttggtcag tgttgggtga ttggcac 37

<210> 136

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 136

ccgcctgacg attcgtgaaa cggcattcgc tgcggc 36

<210> 137

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 137

cttcaacgaa gccacgccgc gaacggcgtg gagtg 35

<210> 138

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 138

gcttcaacga agccacgccg cgaacggcgt ggagtg 36

<210> 139

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 139

gtccaagaaa aaagaaatga tacgaggcat tagcac 36

<210> 140

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 140

gtgctaacca cgaagctttc cactaagctt tcgaac 36

<210> 141

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 141

gtgctaacca cgaagctttc cactaagctt tcgaac 36

<210> 142

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 142

gtgctaacca cgaagctttc cactaagctt tcgaac 36

<210> 143

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 143

gtgtcagtcg atcaagctgt tttcaccatc ggaacccc 38

<210> 144

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 144

gtgctaatcc cgaagctttc cactaagctt tcgaac 36

<210> 145

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 145

gtgccaacgc gctcaggatc tggcgcccac tgcgac 36

<210> 146

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 146

gtttcaatcc gcgcccccgt gagggggc 28

<210> 147

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 147

cggatcatcc ccgcatccgc gggggacac 29

<210> 148

<211> 31

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 148

gtttcaatcc gcgcccccgt gagaggggcga c 31

<210> 149

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 149

gtctattgcc aactatatct ggcttttctc aatac 35

<210> 150

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 150

gtctattgcc atctttatct ggcttttctc aatac 35

<210> 151

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 151

gttattgccc tctatcttgg gctcttctca tcaac 35

<210> 152

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 152

gttattgccc tctttcttgg gctatcttct ccagc 35

<210> 153

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 153

gtttggagaa cagcccgata tagagggcaa tagac 35

<210> 154

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 154

gttttgagaa tagcccgaca tagagggcaa tagac 35

<210> 155

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 155

gttatagtcc tcttacattt agaggtagtc tttaat 36

<210> 156

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 156

gttatagtcc tcttacattt agaggtagtc tttaat 36

<210> 157

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 157

gttcttaatc taaaatagtg aaatgtaaat 30

<210> 158

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 158

gcatttacat tacattattt tagattaaga ac 32

<210> 159

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 159

actacattat agctgattct gtaaggaaac tatagc 36

<210> 160

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 160

actacattat agctgattct gtaaggaaac tatagc 36

<210> 161

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 161

tagttccctt caatttttgggg attatcca 28

<210> 162

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 162

cctttcaaac gaagggcact tacaac 26

<210> 163

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 163

gttttaacta cttattgtga aatgtaaat 29

<210> 164

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 164

gttttaatta cttattgtga aatgtaaat 29

<210> 165

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 165

gtaagagact acctctatat gaaagaggac taaaac 36

<210> 166

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 166

gatttagagt acctcaaaac agaagaggac taaaac 36

<210> 167

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 167

gatttagagt agctcaaaaa agaagaggtc taaaac 36

<210> 168

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 168

gatttagagt acctcaaaac aaaagaggac taaaac 36

<210> 169

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 169

gttttagtcc ccttcgtttt tggggtagt 29

<210> 170

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 170

tcaatcctta ttttaatgga ttcactattc ttac 34

<210> 171

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 171

gttttaatag cacaaattgt attgtaaat 29

<210> 172

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 172

gtttcaatcc ttgttttaat ggataactta ctttaac 37

<210> 173

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 173

gtttcaatcc ttgttttaat ggatactcta ctttaac 37

<210> 174

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 174

gttttagtcc ccttcgatat tggggtggtc tatatc 36

<210> 175

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 175

acctctcccg ccagtaagcg gattgagac 29

<210> 176

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 176

gccgacac ctctcccgcc agtaagcgga ttgagac 37

<210> 177

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 177

gccgacac ctctcccgcc agtaagcgga ttgagac 37

<210> 178

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 178

gtcgctcccc gtggatcgaa ac 32

<210> 179

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 179

gtcgctcccc gtggatcgaa ac 32

<210> 180

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 180

ggttcagtcc gccgtcgtct tggcggtgat gtga 34

<210> 181

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 181

gccctctcct ccctccctgggg ccg 23

<210> 182

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 182

ggttcagtcc gccgtcgtct tggcggtgat gtga 34

<210> 183

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 183

ggttcagtcc gccgtcgtct tggcggtgat gtga 34

<210> 184

<211> 3994

<212> DNA

<213> Listeria weihenstephanensis

<400> 184

tggctgctga gctgtctctt aatttatgaa aaaataatta tgttttgcta atctgtcaag 60

ctgcagattc attaataatc tggtatagtt atgttgttgc aagcgaatag gcggatatat 120

tacctcaaaa cagaagagga ctaaaaccca aacgattggt gttacattat tttcatagat 180

ttagagtacc tcaaaacaga agaggactaa aacgcactct ccgacaataa tctcgtccat 240

tttgatttag agtacctcaa aacaaaagag gactaaaaca actctgtact tgtgaagtac 300

gttaaatccg atttagagta cctcaaaaca aaagaggact aaaacctctt ttgtggataa 360

gtattcgaaa taaagccata aaaactgtga tccaaagaac tggattattg gtttttatgg 420

ctttattcaa ttcttagtat tgtagatgaa ctgtcagcga atgttgtctt gcaacgtgcc 480

ttcttgtata atgaatatat tgataaataa tagaaatttc atacacgcat gaagaaacca 540

attaaagttt cagcaataat gaagcattag gtacatgact ataaaaccaa atggagctga 600

660

caagtaagga aggtcaactg gtttatgaag gtatcgatgg aaataagaca acagaaatta 720

tatttgataa gaaaaaagaa tcgttttata agagtatcct caataaaact gtgagaaaac 780

ctgatgaaaa aagaaaaaaa ataggcgtaa gcaggcaatt aataaagcga ttaataaaga 840

aataacagaa ttaatgttgg cgctgttaca tcaagaagtg ccaagccaaa agttacataa 900

tttaaagagt ctaaatacgg aatctttaac taaactattt aaaccgaagt tccaaaacat 960

gatttcttat ccgcctagca aaggtgccga acatgttcaa ttttgcctta cagatatagc 1020

ggtaccagcg attcgagatt tagatgaaat taagccagat tggggcattt ttttgaaaa 1080

tataagcaga caaccataca 1140

gaaatccatt gagcaaaaca aaatacagtc ccctgattcg ccaaggaaat tagtattgca 1200

aaaatatgtc acagcctttt tgaatggaga accgctggga ctcgatcttg tggcgaaaaa 1260

atataaactg gcagacttag cggagtcgtt taaagtagta gatttgaacg aggataaaag 1320

tgcaaactat aaaattaaag cgtgcttgca acaacatcag cgaaatattt tggatgaatt 1380

1440

tttcccaatc aaacgtgcac cgaatagaag taaacatgcg cgagcggact ttttgaaaaa 1500

1560

ggaacaagga aaaatgggagg catatgagct aacagatcct aaaacaaaag acttgcagga 1620

tattagatct ggtgaggcat ttagcttcaa atttattaat gcttgcgcct tcgcatccaa 1680

taatttgaag atgattttaa accctgaatg tgaaaaggat attttaggta agggcgattt 1740

taaaaagaat ttgccaaaca gtactacgca gtctgatgtt gtgaaaaaaa tgattccttt 1800

tttctcggat gagattcaaa atgtgaattt tgatgaagct atctgggcga ttagggggctc 1860

tattcagcaa attagaaatg aggtttacca ttgcaaaaag cattcttgga aaagcatact 1920

taaaataaaa ggctttgaat ttgaacctaa caatatgaaa tatacggatt ctgatatgca 1980

aaaattgatg gataaagata tcgccaaaat tccagacttc atcgaagaaa aacttaaaag 2040

tagtgggata ataaggttct acagtcatga taaattgcag tctatctggg aaatgaagca 2100

agggttttcg ttgttgacta ctaatgcgcc gtttgtccca agctttaaac gtgtctacgc 2160

2220

2280

ttattatcaa caatttatgc catggtttac agctgataat aatgctttcc gagatgctgc 2340

caattttgta ttgcgattaa ataaaaatag acagcaggat gcaaaagctt ttattaacat 2400

tagagaagtt gaagaaggtg agatgcctag agactatatg ggctatgtcc aaggtcaaat 2460

agcgatacat gaggattcaa ctgaggatac accgaatcat tttgaaaaat ttattagcca 2520

ggtttttatt aagggatttg atagtcatat gagatctgct gatttaaaat ttattaaaaa 2580

2640

agagccatca ttttgaaaa ataaagatga ctatattgca ttttggacat tctgcaaaat 2700

gctggatgct aggcatttaa gcgagctaag aaacgaaatg attaagtatg acggtcattt 2760

aactggagaa caagaaatca ttggtttagc attgcttgga gtggattcac gagagaatga 2820

ttggaagcaa ttttttagct cagaacggga atacgagaaa attatgaagg gctatgttgg 2880

agaggaattg tatcagcggg aaccgtaccg acaaagtgat ggcaaaacac cgattctttt 2940

tcgtggtgta gagcaagcga ggaagtatgg tactgaaaca gtgattcaac ggctttttga 3000

3060

aaccattgaa gagactattg agcgaagaaa agaattgcat aatgaatggg aaaaaaatcc 3120

3180

3240

gctaattgaa attctgggaa gatatgttgg ctatgtagca atagctgata gagactttca 3300

atgtatggcg aatcaatatt ttaagcattc aggaataact gagagagtgg aatattgggg 3360

cgataataga ctaaaaagta ttaaaaagct ggatacattc ttgaaaaaag aaggactgtt 3420

tgtttctgag aaaaatgcaa ggaatcatat agcgcattta aattatttat cactcaaatc 3480

3540

attaaagaat gccgtttcca agtcattaat cgatatttta gatagacatg gtatgagcgt 3600

taggttggtg ataaaaagct tagagccaaa 3660

aaaattgaga catctaggtg agaaaaaaat cgataatggt tatatagaaa caaatcaagt 3720

ttcagaagag tattgtggta tagtaaagag actattagaa atttgatata gaatgttgca 3780

3840

ctcaaaacag aagaggacta aaacaaatga ttgcaagccg ttacaaaaaa tatgatttag 3900

3960

3994

<210> 185

<211> 4053

<212> DNA

<213> Listeria newyorkensis

<400> 185

tgtattctga gttgtctctt aatttatgaa aaaataatta tgttttggta atctgtcaag 60

atgcagatcc attaataatc tggtatagtt atgttgttgc aagtgaatat attacctcaa 120

aataaaagag gactaaaact aattggtggc gtgatacgcc gtaatgcttg atttagagta 180

cctcaaaaca aaagaggact aaaactactt gtcgatatgg tatagctttt ttcagattta 240

gagtacctca aaacaaaaga ggactaaaac taaagcttct aaatggtggc gcgttacgcc 300

gatttagaat acctcaaaac aaaagaggac taaaacctag caagccggtc gccgcgctca 360

aagtaagatt tagagtacct caaaacaaaa gaggactaaa acctcttttg tggataagta 420

ttcgaaataa agccataaaa actgtgatcc aaagagctgg attattggtt tttatggctt 480

tattcaattc ttagtattgt agatgaacca tcagtgaatg ttgtcttgca acgtgccttc 540

ttgtataatg aatatattga taaataatag aaatttcata cacgcatgaa gaaaccaatt 600

aaattttcag cattaatgaa gcattaggta catgactata aaaccaaatg gagctgagta 660

gacagatgaa aatcacaaag atgagagtag atggaagaac tatcgtaatg gagaggacaa 720

780

ttgataagaa aaaagagtca ttttataaga gtatcctcaa taaaactgtg agaaaacccg 840

atgaaaaaga aaagaatagg cgtaagcagg caattaataa agcgattaat aaagaaataa 900

cagaattaat gttggcggtg ttacatcaag aagtgccaag ccaaaagtta cataatttaa 960

agagtctaaa tacggaatct ttaactaaac tatttaaacc gaagttccaa aacatgattt 1020

cttatccgcc tagcaaaggt gccgaacatg ttcagttttg ccttacagat atagcggtac 1080

cagcgattcg agatttagat gaaattaagc cagattgggg catttttttt gaaaaattga 1140

aaccctatac ggattgggca gaatcataca ttcactataa gcagacaacc atacagaaat 1200

1260

atgtcacagc ctttttgaat ggagaaccgc tgggactcga tcttgtggcg aaaaaatata 1320

aactggcaga cttagcggag tcgtttaaat tagtagattt gaacgaggat aaaagtgcaa 1380

actataaaat taaagcgtgc ttgcaacaac atcagcgaaa tattttggat gaattgaaag 1440

aagatccgga gttaaatcaa tatggtatag aagtgaagaa atatatacag cgatatttcc 1500

caatcaaacg tgcaccgaat agaagtaaac atgcacgagc ggactttttg aaaaaggaat 1560

taattgagtc tacagtggag caacaattta aaaatgctgt atatcattat gtactggaac 1620

aaggcaaaat ggaggcatat gagctaacag atcctaaaac aaaagacttg caggatatta 1680

gatctggtga ggcatttagc ttcaaattta ttaatgcttg cgccttcgca tcgaataatt 1740

tgaagatgat tttaaaccct gaatgtgaaa aggatatcct aggtaagggc aattttaaaa 1800

agaatttgcc aaacagtact acgcgatctg atgttgtgaa gaaaatgatt ccctttttct 1860

cggatgagct tcaaaatgtg aattttgatg aagctatctg ggcgattagg ggctctattc 1920

agcaaattag aaatgaggtt taccattgta aaaagcattc ttggaaaagc atacttaaaa 1980

taaaaggctt tgaatttgaa cctaacaata tgaaatatgc ggattctgat atgcaaaaat 2040

tgatggataa agatatcgcc aaaattccag agttcatcga agaaaaactt aaaagtagtg 2100

gagtagtaag gttctacagg catgatgagt tgcaatccat atgggaaatg aaacaaggat 2160

tttcgttgct gactactaat gcgccgtttg tcccaagctt taagcgtgtc tacgcaaaag 2220

ggcacgacta ccaaacttct aaaaatagat actataattt ggatttgact acttttgata 2280

ttttggaata tggagaagaa gattttcgtg cacgctattt cctgacgaag ctagtttatt 2340

atcagcaatt tatgccatgg tttacagctg ataataatgc tttccgagat gctgccaatt 2400

ttgtattgcg attaaataaa aatagacagc aggatgcaaa agcttttatt aacattagag 2460

aagttgaaga aggtgagatg cctagagact atatgggtta tgtccaaggt caaaatagcga 2520

2580

ttattaaggg ctttgatagg catatgagat ctgctaattt aaaatttatt aaaaatccaa 2640

gaaatcaggg gctagaacaa agtgagattg aggaaatgag ctttgatatt aaagtggagc 2700

cgtcattttt gaaaaataaa gatgactata ttgcattttg gatattctgc aaaatgcttg 2760

atgctaggca tttaagcgag ctaagaaacg aaatgattaa gtatgacggt catttaactg 2820

gagaacaaga aatcattggt ttagcattgc tcggagtgga ttcacgagag aatgattgga 2880

agcagttttt tagctcagaa cgggaatacg agaaaattat gaagggctat gttgtagagg 2940

aattgtatca gcgggaaccg taccgacaaa gtgatggcaa aacaccgatt ctttttcgtg 3000

3060 gtgtagagca agcgaggaag tatggtactg aaacagtgat

atcctgagtt caaagtgtca aaatgcaact tagcagagtg ggagcggcaa aaagaaacca 3120

ttgaagagac tattaagcga agaaaagaat tgcataatga atgggcaaaa aatccaaaaa 3180

3240

acaattggca taaaaataaa actacgcttg catacgttaa tgagctgcac catttgctaa 3300

3360

tggcgaatca atattttaag cattcaggaa taactgagag agtggaatat tggggcgata 3420

atagactaaa aagtattaaa aagctggata cattcttgaa aaaagaagga ctgtttgttt 3480

aaatctgagt 3540

gcacgttgct gtatttatcc gagaggttga gagaaatttt taagtatgat cgtaaattaa 3600

agaatgccgt ttccaagtca ttaatcgata ttttagatag acatggtatg agcgtcgtat 3660

ttgctaactt gaaagaaaat aaacataggt tggtgataaa aagcttagag ccaaaaaaat 3720

tgagacatct gggtgggaaa aaaatcgatg gtggttatat agaaacaaat caagtttcag 3780

3840

tctcaaaatg aaattggaag ttacaagagg acgtaatatg atgttactat taaaaatacc 3900

tcagaataga agaggactaa aacaaatgat tgcaagccgt tacaaaaaat atgattaga 3960

gtacctcaga acaaaagagg gctaaaacta gaataatcga ttgagacggt ctacaagtga 4020

tttagagtac ctcaaaacag aagaggacta aaa 4053

<210> 186

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<220>

<221> source

<223> /note="Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide"

<400> 186

auggccacuu uccagguggc aaagcccguu gacguuguga gcttctcaaa aag 53

<210> 187

<211> 32

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<220>

<221> modified_base

<222> (15)..(32)

<223> a, c, u, g, unknown or other

<400> 187

cugagaagug gcacnnnnnn nnnnnnnnnn nn 32

<210> 188

<211> 14

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 188

cugagaagug gcac 14

<210> 189

<211> 79

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 189

gucuagagga cagaauuuuu caacgggugu gccaauggcc acuuuccagg uggcaaagcc 60

cguugagcuu cucaaaaag 79

<210> 190

<211> 32

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<220>

<221> modified_base

<222> (15)..(32)

<223> a, c, u, g, unknown or other

<400> 190

cugagaagug gcacnnnnnn nnnnnnnnnn nn 32

<210> 191

<211> 39

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 191

acuuuccagg uggcaaagcc cguugagcuu cucaaaaag 39

<210> 192

<211> 53

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<220>

<221> source

<223> /note="Description of Combined DNA/RNA Molecule: Synthetic

oligonucleotide"

<400> 192

auggccacuu uccagguggc aaagcccguu gacguuguga gcttctcaaa aag 53

<210> 193

<211> 32

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<220>

<221> modified_base

<222> (15)..(32)

<223> a, c, u, g, unknown or other

<400> 193

cugagaagug gcacnnnnnn nnnnnnnnnn nn 32

<210> 194

<211> 111

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<220>

<221> modified_base

<222> (92)..(111)

<223> a, c, u, g, unknown or other

<400> 194

gucuagagga cagaauuuuu caacgggugu gccaauggcc acuuuccagg uggcaaagcc 60

cguugagcuu cucaaaucug agaaguggca cnnnnnnnnn nnnnnnnnnn n 111

<210> 195

<211> 52

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 195

aagtgctgca ggggtgtgag aaactcctat tgctggacga tgtctctttt at 52

<210> 196

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<220>

<221> modified_base

<222> (15)..(33)

<223> a, c, t, g, unknown or other

<400> 196

cgaggcatta gcacnnnnnn nnnnnnnnnn nnn 33

<210> 197

<211> 91

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 197

cgagguucug ucuuuugguc aggacaaccg ucuagcuaua agugcugcag gggugugaga 60

aacuccuauu gcuggacgau gucucuuuua u 91

<210> 198

<211> 14

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 198

cgaggcauua gcac 14

<210> 199

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 199

guucgaaagc uuaguggaaa gcuucguggu uagcac 36

<210> 200

<211> 101

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 200

ucagucgcac gcuauaggcc auaagucgac uuacauaucc gugcgugugc auuaugggcc 60

cauccacagg ucuauuccca cggauaauca cgacuuucca c 101

<210> 201

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 201

guucgaaagc uuaguggaaa gcuucguggu uagcac 36

<210> 202

<211> 18

<212> PRT

<213> Alicyclobacillus acidoterrestris

<400> 202

Leu Arg Val Met Ser Val Asp Leu Gly Leu Arg Thr Ser Ala Ser Ile

1 5 10 15

Ser Val

<210> 203

<211> 22

<212> PRT

<213> Alicyclobacillus acidoterrestris

<400> 203

Gln Arg Thr Leu Arg Gln Leu Arg Thr Gln Leu Ala Tyr Leu Arg Leu

1 5 10 15

Leu Val Arg Cys Gly Ser

twenty

<210> 204

<211> 12

<212> PRT

<213> Alicyclobacillus acidoterrestris

<400> 204

Cys Gln Leu Ile Leu Leu Glu Glu Leu Ser Glu Tyr

1 5 10

<210> 205

<211> 16

<212> PRT

<213> Alicyclobacillus acidoterrestris

<400> 205

His Gln Ile His Ala Asp Leu Asn Ala Ala Gln Asn Leu Gln Gln Arg

1 5 10 15

<210> 206

<211> 18

<212> PRT

<213> Alicyclobacillus contaminans

<400> 206

Val Arg Val Met Ser Val Asp Leu Gly Val Arg Tyr Gly Ala Ala Ile

1 5 10 15

Ser Val

<210> 207

<211> 22

<212> PRT

<213> Alicyclobacillus contaminans

<400> 207

Lys Gln Ala Leu Ala Ala Ile Arg Ala Glu Met Ser Ile Leu Arg Lys

1 5 10 15

Trp Leu Arg Val Ser Gln

twenty

<210> 208

<211> 12

<212> PRT

<213> Alicyclobacillus contaminans

<400> 208

Cys Asp Leu Ile Leu Phe Glu Asp Leu Ser Arg Tyr

1 5 10

<210> 209

<211> 16

<212> PRT

<213> Alicyclobacillus contaminans

<400> 209

Lys Cys Val His Ala Asp Ile Asn Ala Ala His Asn Leu Gln Arg Arg

1 5 10 15

<210> 210

<211> 18

<212> PRT

<213> Desulfovibrio inopinatus

<400> 210

Leu Arg Val Leu Ser Val Asp Leu Gly Met Arg Thr Phe Ala Ser Cys

1 5 10 15

Ser Val

<210> 211

<211> 22

<212> PRT

<213> Desulfovibrio inopinatus

<400> 211

Arg Ala Glu Ile Tyr Ala Leu Lys Arg Asp Ile Gln Arg Leu Lys Ser

1 5 10 15

Leu Leu Arg Leu Gly Glu

twenty

<210> 212

<211> 12

<212> PRT

<213> Desulfovibrio inopinatus

<400> 212

Cys Gln Leu Ile Leu Phe Glu Asp Leu Ala Arg Tyr

1 5 10

<210> 213

<211> 16

<212> PRT

<213> Desulfovibrio inopinatus

<400> 213

Cys Val Ile His Ala Asp Met Asn Ala Ala Gln Asn Leu Gln Arg Arg

1 5 10 15

<210> 214

<211> 18

<212> PRT

<213> Desulfonatronum thiodismutans

<400> 214

Leu Arg Val Leu Ser Val Asp Leu Gly Val Arg Ser Phe Ala Ala Cys

1 5 10 15

Ser Val

<210> 215

<211> 22

<212> PRT

<213> Desulfonatronum thiodismutans

<400> 215

Met Glu Glu Leu Arg Ser Leu Asn Gly Asp Ile Arg Arg Leu Lys Ala

1 5 10 15

Ile Leu Arg Leu Ser Val

twenty

<210> 216

<211> 12

<212> PRT

<213> Desulfonatronum thiodismutans

<400> 216

Cys Arg Leu Ile Leu Phe Glu Asp Leu Ala Arg Tyr

1 5 10

<210> 217

<211> 16

<212> PRT

<213> Desulfonatronum thiodismutans

<400> 217

His Val Ile His Ala Asp Ile Asn Ala Ala Gln Asn Leu Gln Arg Arg

1 5 10 15

<210> 218

<211> 18

<212> PRT

<213> Tuberibacillus calidus

<400> 218

Leu Arg Val Met Ser Val Asp Leu Gly Gln Arg Gln Ala Ala Ala Ile

1 5 10 15

Ser Ile

<210> 219

<211> 22

<212> PRT

<213> Tuberibacillus calidus

<400> 219

Asp Gln Ala Ile Arg Asp Leu Ser Arg Lys Leu Lys Phe Leu Lys Asn

1 5 10 15

Val Leu Asn Met Gln Lys

twenty

<210> 220

<211> 12

<212> PRT

<213> Tuberibacillus calidus

<400> 220

Cys Gln Leu Val Leu Phe Glu Asp Leu Ser Arg Tyr

1 5 10

<210> 221

<211> 16

<212> PRT

<213> Tuberibacillus calidus

<400> 221

Val Ile Thr His Ala Asp Ile Asn Ala Ala Gln Asn Leu Gln Lys Arg

1 5 10 15

<210> 222

<211> 18

<212> PRT

<213> Bacillus thermoamylovorans

<400> 222

Leu Arg Val Met Ser Ile Asp Leu Gly Gln Arg Gln Ala Ala Ala Ala

1 5 10 15

Ser Ile

<210> 223

<211> 22

<212> PRT

<213> Bacillus thermoamylovorans

<400> 223

Glu Asp Asn Leu Lys Leu Met Asn Gln Lys Leu Asn Phe Leu Arg Asn

1 5 10 15

Val Leu His Phe Gln Gln

twenty

<210> 224

<211> 12

<212> PRT

<213> Bacillus thermoamylovorans

<400> 224

Cys Gln Ile Ile Leu Phe Glu Asp Leu Ser Asn Tyr

1 5 10

<210> 225

<211> 16

<212> PRT

<213> Bacillus thermoamylovorans

<400> 225

Val Thr Thr His Ala Asp Ile Asn Ala Ala Gln Asn Leu Gln Lys Arg

1 5 10 15

<210> 226

<211> 18

<212> PRT

<213> Bacillus sp.

<400> 226

Phe Arg Val Met Ser Ile Asp Leu Gly Leu Arg Ala Ala Ala Ala Thr

1 5 10 15

Ser Ile

<210> 227

<211> 22

<212> PRT

<213> Bacillus sp.

<400> 227

Phe Gln Leu His Gln Arg Val Lys Phe Gln Ile Arg Val Leu Ala Gln

1 5 10 15

Ile Met Arg Met Ala Asn

twenty

<210> 228

<211> 12

<212> PRT

<213> Bacillus sp.

<400> 228

Cys Gln Val Ile Leu Phe Glu Asn Leu Ser Gln Tyr

1 5 10

<210> 229

<211> 16

<212> PRT

<213> Bacillus sp.

<400> 229

Val Phe Leu Gln Ala Asp Ile Asn Ala Ala His Asn Leu Gln Lys Arg

1 5 10 15

<210> 230

<211> 18

<212> PRT

<213> Methylobacterium nodulans

<400> 230

Leu Arg Val Leu Ser Ile Asp Leu Gly Val Arg Ser Phe Ala Thr Cys

1 5 10 15

Ser Val

<210> 231

<211> 22

<212> PRT

<213> Methylobacterium nodulans

<400> 231

Asp Ala Glu Leu Arg Gln Leu Arg Gly Gly Leu Asn Arg His Arg Gln

1 5 10 15

Leu Leu Arg Ala Ala Thr

twenty

<210> 232

<211> 12

<212> PRT

<213> Methylobacterium nodulans

<400> 232

Cys His Val Ile Leu Phe Glu Asp Leu Ser Arg Tyr

1 5 10

<210> 233

<211> 16

<212> PRT

<213> Methylobacterium nodulans

<400> 233

Ser Arg Ile His Ala Asp Ile Asn Ala Ala Gln Asn Leu Gln Arg Arg

1 5 10 15

<210> 234

<211> 18

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Candidatus Methanomethylophilus alvus sequence"

<400> 234

Leu Lys Ile Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Ile Tyr Val

1 5 10 15

Thr Met

<210> 235

<211> 22

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Candidatus Methanomethylophilus alvus sequence"

<400> 235

Arg Lys Ala Leu Asp Val Arg Glu Tyr Asp Asn Lys Glu Ala Arg Arg

1 5 10 15

Asn Trp Thr Lys Val Glu

twenty

<210> 236

<211> 13

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Candidatus Methanomethylophilus alvus sequence"

<400> 236

Asn Ala Ile Ile Val Met Glu Asp Leu Asn His Gly Phe

1 5 10

<210> 237

<211> 16

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Candidatus Methanomethylophilus alvus sequence"

<400> 237

Leu Pro Gln Asp Ser Asp Ala Asn Gly Ala Tyr Asn Ile Ala Leu Lys

1 5 10 15

<210> 238

<211> 18

<212> PRT

<213> Synergistes jonesii

<400> 238

Val Asn Ile Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Val Tyr Val

1 5 10 15

Ser Leu

<210> 239

<211> 22

<212> PRT

<213> Synergistes jonesii

<400> 239

His Ala Lys Leu Asn Gln Lys Glu Lys Glu Arg Asp Thr Ala Arg Lys

1 5 10 15

Ser Trp Lys Thr Ile Gly

twenty

<210> 240

<211> 13

<212> PRT

<213> Synergistes jonesii

<400> 240

Asn Ala Val Ile Val Met Glu Asp Leu Asn Ile Gly Phe

1 5 10

<210> 241

<211> 16

<212> PRT

<213> Synergistes jonesii

<400> 241

Leu Pro Ile Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Ala Leu Lys

1 5 10 15

<210> 242

<211> 18

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 242

Pro Tyr Val Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Leu Tyr Ile

1 5 10 15

Val Val

<210> 243

<211> 22

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 243

His Ser Leu Leu Asp Lys Lys Glu Lys Glu Arg Phe Glu Ala Arg Gln

1 5 10 15

Asn Trp Thr Ser Ile Glu

twenty

<210> 244

<211> 13

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 244

Asp Ala Val Ile Ala Leu Glu Asp Leu Asn Ser Gly Phe

1 5 10

<210> 245

<211> 16

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 245

Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Arg Lys

1 5 10 15

<210> 246

<211> 18

<212> PRT

<213> Francisella tularensis

<400> 246

Val His Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr

1 5 10 15

Thr Leu

<210> 247

<211> 22

<212> PRT

<213> Francisella tularensis

<400> 247

His Asp Lys Leu Ala Ala Ile Glu Lys Asp Arg Asp Ser Ala Arg Lys

1 5 10 15

Asp Trp Lys Lys Ile Asn

twenty

<210> 248

<211> 13

<212> PRT

<213> Francisella tularensis

<400> 248

Asn Ala Ile Val Val Phe Glu Asp Leu Asn Phe Gly Phe

1 5 10

<210> 249

<211> 16

<212> PRT

<213> Francisella tularensis

<400> 249

Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly Leu Lys

1 5 10 15

<210> 250

<211> 18

<212> PRT

<213> Moraxella caprae

<400> 250

Val Asn Val Ile Gly Ile Asp Arg Gly Glu Arg His Leu Leu Tyr Leu

1 5 10 15

Thr Val

<210> 251

<211> 22

<212> PRT

<213> Moraxella caprae

<400> 251

His Lys Ile Leu Asp Lys Arg Glu Ile Glu Arg Leu Asn Ala Arg Val

1 5 10 15

Gly Trp Gly Glu Ile Glu

twenty

<210> 252

<211> 13

<212> PRT

<213> Moraxella caprae

<400> 252

Asn Ala Ile Val Val Leu Glu Asp Leu Asn Phe Gly Phe

1 5 10

<210> 253

<211> 16

<212> PRT

<213> Moraxella caprae

<400> 253

Gln Pro Gln Asn Ala Asp Ala Asn Gly Ala Tyr His Ile Ala Leu Lys

1 5 10 15

<210> 254

<211> 18

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 254

Met His Ile Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Ile Tyr Leu

1 5 10 15

Cys Met

<210> 255

<211> 22

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 255

His Gln Leu Leu Lys Thr Arg Glu Asp Glu Asn Lys Ser Ala Arg Gln

1 5 10 15

Ser Trp Gln Thr Ile His

twenty

<210> 256

<211> 13

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 256

Asn Ala Ile Val Val Leu Glu Asp Leu Asn Phe Gly Phe

1 5 10

<210> 257

<211> 16

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 257

Met Pro Leu Asp Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Arg Lys

1 5 10 15

<210> 258

<211> 18

<212> PRT

<213> Prevotella albensis

<400> 258

Thr His Ile Ile Gly Ile Asp Arg Gly Glu Arg His Leu Leu Tyr Leu

1 5 10 15

Ser Leu

<210> 259

<211> 22

<212> PRT

<213> Prevotella albensis

<400> 259

His Asn Leu Leu Glu Lys Arg Glu Lys Glu Arg Thr Glu Ala Arg His

1 5 10 15

Ser Trp Ser Ser Ile Glu

twenty

<210> 260

<211> 13

<212> PRT

<213> Prevotella albensis

<400> 260

Asn Ala Ile Val Val Leu Glu Asp Leu Asn Gly Gly Phe

1 5 10

<210> 261

<211> 16

<212> PRT

<213> Prevotella albensis

<400> 261

Phe Pro Glu Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Arg Lys

1 5 10 15

<210> 262

<211> 18

<212> PRT

<213> Smithella sp.

<400> 262

Ile Asn Ile Ile Gly Ile Asp Arg Gly Glu Arg His Leu Leu Tyr Tyr

1 5 10 15

Ala Leu

<210> 263

<211> 22

<212> PRT

<213> Smithella sp.

<400> 263

His Asn Leu Leu Asp Lys Lys Glu Gly Asp Arg Ala Thr Ala Arg Gln

1 5 10 15

Glu Trp Gly Val Ile Glu

twenty

<210> 264

<211> 13

<212> PRT

<213> Smithella sp.

<400> 264

Asn Ala Ile Ile Val Met Glu Asp Leu Asn Phe Gly Phe

1 5 10

<210> 265

<211> 16

<212> PRT

<213> Smithella sp.

<400> 265

Met Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr His Ile Ala Leu Lys

1 5 10 15

<210> 266

<211> 18

<212> PRT

<213> Porphyromonas crevioricanis

<400> 266

Met His Val Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Leu Tyr Ile

1 5 10 15

Cys Val

<210> 267

<211> 22

<212> PRT

<213> Porphyromonas crevioricanis

<400> 267

His Asp Leu Leu Glu Ser Arg Asp Lys Asp Arg Gln Gln Glu Arg Arg

1 5 10 15

Asn Trp Gln Thr Ile Glu

twenty

<210> 268

<211> 13

<212> PRT

<213> Porphyromonas crevioricanis

<400> 268

Lys Ala Val Val Ala Leu Glu Asp Leu Asn Met Gly Phe

1 5 10

<210> 269

<211> 16

<212> PRT

<213> Porphyromonas crevioricanis

<400> 269

Leu Pro Lys Asp Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Leu Lys

1 5 10 15

<210> 270

<211> 18

<212> PRT

<213> Sulfolobus solfataricus

<400> 270

Gly Lys Val Val Ala Ile Asp Val Gly Val Glu Lys Leu Leu Ile Thr

1 5 10 15

Ser Asp

<210> 271

<211> 23

<212> PRT

<213> Sulfolobus solfataricus

<400> 271

Val Lys His Ile His Arg Glu Leu Ser Arg Lys Lys Phe Leu Ser Asn

1 5 10 15

Asn Trp Phe Lys Ala Lys Val

twenty

<210> 272

<211> 13

<212> PRT

<213> Sulfolobus solfataricus

<400> 272

Tyr Asp Val Val Val Met Glu Gly Ile His Ala Lys Gln

1 5 10

<210> 273

<211> 16

<212> PRT

<213> Sulfolobus solfataricus

<400> 273

Trp Ile Ala Asp Arg Asp Tyr Asn Ala Ser Leu Asn Ile Leu Arg Gly

1 5 10 15

<210> 274

<211> 18

<212> PRT

<213> Nostoc sp.

<400> 274

Leu Lys Thr Ile Gly Leu Asp Val Gly Leu Asn His Phe Leu Thr Asp

1 5 10 15

SerGlu

<210> 275

<211> 23

<212> PRT

<213> Nostoc sp.

<400> 275

Leu Lys Arg Leu Gln Arg Arg Leu Ser Lys Thr Lys Lys Gly Ser Asn

1 5 10 15

Asn Arg Val Lys Ala Arg Asn

twenty

<210> 276

<211> 13

<212> PRT

<213> Nostoc sp.

<400> 276

Ser Asp Leu Val Ala Tyr Glu Asp Leu Gln Val Arg Asn

1 5 10

<210> 277

<211> 16

<212> PRT

<213> Nostoc sp.

<400> 277

His Ile Gln Asp Arg Asp Trp Asn Ala Ala Arg Asn Ile Leu Glu Leu

1 5 10 15

<210> 278

<211> 18

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Candidatus Nitrososphaera gargensis sequence"

<400> 278

Ala Lys Pro Val Gly Ile Asp Val Gly Ile Ala Lys Phe Cys His His

1 5 10 15

Ser Asp

<210> 279

<211> 23

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Candidatus Nitrososphaera gargensis sequence"

<400> 279

Leu Arg Arg Ala His Arg Arg Val Ser Arg Arg Gln Ile Gly Ser Asn

1 5 10 15

Asn Arg Lys Lys Ala Lys Arg

twenty

<210> 280

<211> 13

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Candidatus Nitrososphaera gargensis sequence"

<400> 280

Tyr Asp Leu Ile Phe Leu Glu Arg Leu Arg Val Met Asn

1 5 10

<210> 281

<211> 16

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Candidatus Nitrososphaera gargensis sequence"

<400> 281

Ala Ile Leu Asp Arg Asp Tyr Asn Ser Ala Ile Asn Ile Leu Lys Arg

1 5 10 15

<210> 282

<211> 18

<212> PRT

<213> Helicobacter pylori

<400> 282

Lys Lys Ala Val Gly Leu Asp Met Gly Leu Arg Thr Leu Ile Val Thr

1 5 10 15

Ser Asp

<210> 283

<211> 23

<212> PRT

<213> Helicobacter pylori

<400> 283

Leu Thr Lys Ala Gln Arg Arg Leu Ser Lys Lys Val Lys Asp Ser Asn

1 5 10 15

Asn Arg Lys Lys Gln Ala Lys

twenty

<210> 284

<211> 13

<212> PRT

<213> Helicobacter pylori

<400> 284

Tyr Asp Leu Ile Gly Val Glu Thr Leu Asn Val Lys Ala

1 5 10

<210> 285

<211> 16

<212> PRT

<213> Helicobacter pylori

<400> 285

Thr Thr His His Arg Asp Tyr Asn Ala Ser Val Asn Ile Arg Asn Tyr

1 5 10 15

<210> 286

<211> 18

<212> PRT

<213> Flexibacter litoralis

<400> 286

Asn Gln Ala Val Gly Ile Asp Met Gly Ile Thr Phe Phe Cys Ile Asp

1 5 10 15

Ser Asn

<210> 287

<211> 23

<212> PRT

<213> Flexibacter litoralis

<400> 287

Leu Arg Ile Ala Asn Arg Ser Leu Ser Arg Lys Lys Lys Phe Ser Asn

1 5 10 15

Gly Trp Tyr Lys Lys Lys Val

twenty

<210> 288

<211> 13

<212> PRT

<213> Flexibacter litoralis

<400> 288

Asn Ser Leu Val Val Val Glu Asp Leu Lys Val Lys Asn

1 5 10

<210> 289

<211> 16

<212> PRT

<213> Flexibacter litoralis

<400> 289

His Glu Thr Asn Ala Asp Glu Asn Ala Ser Lys Asn Ile Leu Ser Glu

1 5 10 15

<210> 290

<211> 18

<212> PRT

<213> Escherichia coli

<400> 290

Ala Ser Met Val Gly Leu Asp Ala Gly Val Ala Lys Leu Ala Thr Leu

1 5 10 15

Ser Asp

<210> 291

<211> 23

<212> PRT

<213> Escherichia coli

<400> 291

Leu Ala Arg Leu Gln Arg Gln Leu Ser Arg Lys Val Lys Phe Ser Asn

1 5 10 15

Asn Trp Gln Lys Gln Lys Arg

twenty

<210> 292

<211> 13

<212> PRT

<213> Escherichia coli

<400> 292

His Ala Met Ile Val Ile Glu Asp Leu Lys Val Ser Asn

1 5 10

<210> 293

<211> 16

<212> PRT

<213> Escherichia coli

<400> 293

Tyr Thr Ala Asn Ala Asp Val Asn Gly Ala Arg Asn Ile Leu Ala Ala

1 5 10 15

<210> 294

<211> 18

<212> PRT

<213> Clostridium botulinum

<400> 294

Asn Lys Lys Val Gly Ile Asp Val Gly Leu Lys Glu Phe Ala Thr Thr

1 5 10 15

Ser Asp

<210> 295

<211> 23

<212> PRT

<213> Clostridium botulinum

<400> 295

Leu Ala Lys Leu Gln Lys Asp Leu Ser Arg Lys Lys Lys Asn Ser Asn

1 5 10 15

Asn Arg Lys Lys Ala Arg Leu

twenty

<210> 296

<211> 13

<212> PRT

<213> Clostridium botulinum

<400> 296

Asn Gln Ala Ile Val Ile Glu Asn Leu Lys Val Ser Asn

1 5 10

<210> 297

<211> 16

<212> PRT

<213> Clostridium botulinum

<400> 297

Met Ile Met Asp Arg Asp Leu Asn Ala Ser Lys Asn Leu Leu Asn Leu

1 5 10 15

<210> 298

<211> 18

<212> PRT

<213> Acidaminococcus sp.

<400> 298

Met Tyr Tyr Leu Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Tyr Ala

1 5 10 15

Val Thr

<210> 299

<211> 22

<212> PRT

<213> Acidaminococcus sp.

<400> 299

Ala Glu Arg Arg Ser Phe Arg Thr Ser Arg Arg Arg Leu Asp Arg Arg

1 5 10 15

Gln Gln Arg Val Lys Leu

twenty

<210> 300

<211> 13

<212> PRT

<213> Acidaminococcus sp.

<400> 300

Pro Lys Arg Ile Phe Ile Glu Met Ala Arg Asp Gly Glu

1 5 10

<210> 301

<211> 16

<212> PRT

<213> Acidaminococcus sp.

<400> 301

Leu His His Ala Lys Asp Ala Phe Leu Ala Ile Val Thr Gly Asn Val

1 5 10 15

<210> 302

<211> 18

<212> PRT

<213> Coprococcus catus

<400> 302

Glu Tyr Phe Leu Gly Leu Asp Met Gly Thr Gly Ser Leu Gly Trp Ala

1 5 10 15

Val Thr

<210> 303

<211> 22

<212> PRT

<213> Coprococcus catus

<400> 303

Glu Glu Arg Arg Met Phe Arg Thr Ala Arg Arg Arg Leu Asp Arg Arg

1 5 10 15

Asn Trp Arg Ile Gln Val

twenty

<210> 304

<211> 13

<212> PRT

<213> Coprococcus catus

<400> 304

Pro Lys Arg Val Phe Val Glu Met Ala Arg Glu Lys Gln

1 5 10

<210> 305

<211> 16

<212> PRT

<213> Coprococcus catus

<400> 305

Leu His His Ala Lys Asp Ala Tyr Leu Asn Ile Val Val Gly Asn Ala

1 5 10 15

<210> 306

<211> 18

<212> PRT

<213> Treponema denticola

<400> 306

Asp Tyr Phe Leu Gly Leu Asp Val Gly Thr Gly Ser Val Gly Trp Ala

1 5 10 15

Val Thr

<210> 307

<211> 22

<212> PRT

<213> Treponema denticola

<400> 307

Glu Val Arg Arg Leu His Arg Gly Ala Arg Arg Arg Ile Glu Arg Arg

1 5 10 15

Lys Lys Arg Ile Lys Leu

twenty

<210> 308

<211> 13

<212> PRT

<213> Treponema denticola

<400> 308

Pro Lys Lys Ile Phe Ile Glu Met Ala Lys Gly Ala Glu

1 5 10

<210> 309

<211> 16

<212> PRT

<213> Treponema denticola

<400> 309

Phe His His Ala His Asp Ala Tyr Leu Asn Ile Val Val Gly Asn Val

1 5 10 15

<210> 310

<211> 18

<212> PRT

<213> Mycoplasma mobile

<400> 310

Lys Val Val Leu Gly Leu Asp Leu Gly Ile Ala Ser Val Gly Trp Cys

1 5 10 15

Leu Thr

<210> 311

<211> 22

<212> PRT

<213> Mycoplasma mobile

<400> 311

Glu Thr Arg Arg Lys Lys Arg Gly Gln Arg Arg Arg Asn Arg Arg Leu

1 5 10 15

Phe Thr Arg Lys Arg Asp

twenty

<210> 312

<211> 13

<212> PRT

<213> Mycoplasma mobile

<400> 312

Ile Glu Lys Ile Val Val Glu Val Thr Arg Ser Ser Asn

1 5 10

<210> 313

<211> 16

<212> PRT

<213> Mycoplasma mobile

<400> 313

Gly His His Ala Glu Asp Ala Tyr Phe Ile Thr Ile Ile Ser Gln Tyr

1 5 10 15

<210> 314

<211> 18

<212> PRT

<213> Streptococcus thermophilus

<400> 314

Asp Leu Val Leu Gly Leu Asp Ile Gly Ile Gly Ser Val Gly Val Gly

1 5 10 15

Ile Leu

<210> 315

<211> 22

<212> PRT

<213> Streptococcus thermophilus

<400> 315

Leu Val Arg Arg Thr Asn Arg Gln Gly Arg Arg Leu Thr Arg Arg Lys

1 5 10 15

Lys His Arg Ile Val Arg

twenty

<210> 316

<211> 13

<212> PRT

<213> Streptococcus thermophilus

<400> 316

Phe Asp Asn Ile Val Ile Glu Met Ala Arg Glu Thr Asn

1 5 10

<210> 317

<211> 16

<212> PRT

<213> Streptococcus thermophilus

<400> 317

His His His Ala Val Asp Ala Leu Ile Ile Ala Ala Ser Ser Gln Leu

1 5 10 15

<210> 318

<211> 18

<212> PRT

<213> Campylobacter jejuni

<400> 318

Ala Arg Ile Leu Ala Phe Asp Ile Gly Ile Ser Ser Ile Gly Trp Ala

1 5 10 15

Phe Ser

<210> 319

<211> 22

<212> PRT

<213> Campylobacter jejuni

<400> 319

Leu Pro Arg Arg Leu Ala Arg Ser Ala Arg Lys Arg Leu Ala Arg Arg

1 5 10 15

Lys Ala Arg Leu Asn His

twenty

<210> 320

<211> 13

<212> PRT

<213> Campylobacter jejuni

<400> 320

Val His Lys Ile Asn Ile Glu Leu Ala Arg Glu Val Gly

1 5 10

<210> 321

<211> 16

<212> PRT

<213> Campylobacter jejuni

<400> 321

Leu His His Ala Ile Asp Ala Val Ile Ile Ala Tyr Ala Asn Asn Ser

1 5 10 15

<210> 322

<211> 18

<212> PRT

<213> Clostridium perfringens

<400> 322

Asn Tyr Ala Leu Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp Ala

1 5 10 15

Val Ile

<210> 323

<211> 22

<212> PRT

<213> Clostridium perfringens

<400> 323

Leu Pro Arg Arg Leu Ala Arg Gly Arg Arg Arg Leu Leu Arg Arg Lys

1 5 10 15

Ala Tyr Arg Val Glu Arg

twenty

<210> 324

<211> 13

<212> PRT

<213> Clostridium perfringens

<400> 324

Pro Val Arg Ile Asn Ile Glu Leu Ala Arg Asp Leu Ala

1 5 10

<210> 325

<211> 16

<212> PRT

<213> Clostridium perfringens

<400> 325

Lys His His Ala Leu Asp Ala Ala Val Val Gly Val Thr Thr Gln Gly

1 5 10 15

<210> 326

<211> 18

<212> PRT

<213> Akkermansia muciniphila

<400> 326

Ser Leu Thr Phe Ser Phe Asp Ile Gly Tyr Ala Ser Ile Gly Trp Ala

1 5 10 15

Val Ile

<210> 327

<211> 22

<212> PRT

<213> Akkermansia muciniphila

<400> 327

Phe Lys Arg Arg Glu Tyr Arg Arg Leu Arg Arg Asn Ile Arg Ser Arg

1 5 10 15

Arg Val Arg Ile Glu Arg

twenty

<210> 328

<211> 13

<212> PRT

<213> Akkermansia muciniphila

<400> 328

Ile Ser Arg Val Cys Val Glu Val Gly Lys Glu Leu Thr

1 5 10

<210> 329

<211> 16

<212> PRT

<213> Akkermansia muciniphila

<400> 329

Leu His His Ala Leu Asp Ala Cys Val Leu Gly Leu Ile Pro Tyr Ile

1 5 10 15

<210> 330

<211> 18

<212> PRT

<213> Bifidobacterium longum

<400> 330

Arg Tyr Arg Ile Gly Ile Asp Val Gly Leu Asn Ser Val Gly Leu Ala

1 5 10 15

Ala Val

<210> 331

<211> 22

<212> PRT

<213> Bifidobacterium longum

<400> 331

Asn Met Ser Gly Val Ala Arg Arg Thr Arg Arg Met Arg Arg Arg Lys

1 5 10 15

Arg Glu Arg Leu His Lys

twenty

<210> 332

<211> 13

<212> PRT

<213> Bifidobacterium longum

<400> 332

Pro Val Ser Val Asn Ile Glu His Val Arg Ser Ser Phe

1 5 10

<210> 333

<211> 16

<212> PRT

<213> Bifidobacterium longum

<400> 333

Arg His His Ala Val Asp Ala Ser Val Ile Ala Met Met Asn Thr Ala

1 5 10 15

<210> 334

<211> 18

<212> PRT

<213> Wolinella succinogenes

<400> 334

Val Ser Pro Ile Ser Val Asp Leu Gly Gly Lys Asn Thr Gly Phe Phe

1 5 10 15

Ser Phe

<210> 335

<211> 22

<212> PRT

<213> Wolinella succinogenes

<400> 335

Val Gly Arg Arg Ser Lys Arg His Ser Lys Arg Asn Asn Leu Arg Asn

1 5 10 15

Lys Leu Val Lys Arg Leu

twenty

<210> 336

<211> 13

<212> PRT

<213> Wolinella succinogenes

<400> 336

Lys Val Pro Ile Ile Leu Glu Gln Asn Ala Phe Glu Tyr

1 5 10

<210> 337

<211> 16

<212> PRT

<213> Wolinella succinogenes

<400> 337

Ser Ser His Ala Ile Asp Ala Val Met Ala Phe Val Ala Arg Tyr Gln

1 5 10 15

<210> 338

<211> 18

<212> PRT

<213> Legionella pneumophila

<400> 338

Leu Ser Pro Ile Gly Ile Asp Leu Gly Gly Lys Phe Thr Gly Val Cys

1 5 10 15

Leu Ser

<210> 339

<211> 22

<212> PRT

<213> Legionella pneumophila

<400> 339

Ala Gln Arg Arg Ala Thr Arg His Arg Val Arg Asn Lys Lys Arg Asn

1 5 10 15

Gln Phe Val Lys Arg Val

twenty

<210> 340

<211> 13

<212> PRT

<213> Legionella pneumophila

<400> 340

Leu Ile Pro Ile Tyr Leu Glu Gln Asn Arg Phe Glu Phe

1 5 10

<210> 341

<211> 15

<212> PRT

<213> Legionella pneumophila

<400> 341

Pro Ser His Ala Ile Asp Ala Thr Leu Thr Met Ser Ile Gly Leu

1 5 10 15

<210> 342

<211> 18

<212> PRT

<213> Francisella tularensis

<400> 342

Ile Leu Pro Ile Ala Ile Asp Leu Gly Val Lys Asn Thr Gly Val Phe

1 5 10 15

Ser Ala

<210> 343

<211> 22

<212> PRT

<213> Francisella tularensis

<400> 343

Asn Asn Arg Thr Ala Arg Arg His Gln Arg Arg Gly Ile Asp Arg Lys

1 5 10 15

Gln Leu Val Lys Arg Leu

twenty

<210> 344

<211> 13

<212> PRT

<213> Francisella tularensis

<400> 344

His Ile Pro Ile Ile Thr Glu Ser Asn Ala Phe Glu Phe

1 5 10

<210> 345

<211> 16

<212> PRT

<213> Francisella tularensis

<400> 345

Tyr Ser His Leu Ile Asp Ala Met Leu Ala Phe Cys Ile Ala Ala Asp

1 5 10 15

<210> 346

<211> 18

<212> PRT

<213> Streptococcus pyogenes

<400> 346

Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala

1 5 10 15

Val Ile

<210> 347

<211> 22

<212> PRT

<213> Streptococcus pyogenes

<400> 347

Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg

1 5 10 15

Lys Asn Arg Ile Cys Tyr

twenty

<210> 348

<211> 13

<212> PRT

<213> Streptococcus pyogenes

<400> 348

Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

1 5 10

<210> 349

<211> 16

<212> PRT

<213> Streptococcus pyogenes

<400> 349

Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala

1 5 10 15

<210> 350

<211> 18

<212> PRT

<213> Lactobacillus delbrueckii

<400> 350

Lys Val Ser Leu Gly Val Asp Thr Gly Gln Arg His Ile Gly Phe Ala

1 5 10 15

Ile Val

<210> 351

<211> 23

<212> PRT

<213> Lactobacillus delbrueckii

<400> 351

Tyr Thr Arg Lys Ile Tyr Arg Arg Ser Lys Arg Asn Arg Lys Thr Arg

1 5 10 15

Tyr Arg Gln Ala Arg Phe Leu

twenty

<210> 352

<211> 13

<212> PRT

<213> Lactobacillus delbrueckii

<400> 352

Asn Pro Asp Leu His Ile Glu Val Gly Lys Phe Asp Met

1 5 10

<210> 353

<211> 15

<212> PRT

<213> Lactobacillus delbrueckii

<400> 353

Lys Gly His Phe Met Asp Ala Ile Ala Ile Ser Gly Ile Lys Pro

1 5 10 15

<210> 354

<211> 18

<212> PRT

<213> Methanohalobium evestigatum

<400> 354

Pro Val Val Ala Gly Met Asp Ser Gly Ser Lys His Ile Gly Cys Ala

1 5 10 15

Ala Val

<210> 355

<211> 23

<212> PRT

<213> Methanohalobium evestigatum

<400> 355

Lys Asp Arg Ala Asp Tyr Arg Arg Asn Arg Arg Ser Arg Lys Thr Arg

1 5 10 15

Tyr Arg Lys Pro Arg Phe Asp

twenty

<210> 356

<211> 13

<212> PRT

<213> Methanohalobium evestigatum

<400> 356

Val Lys Lys Trp Ile Val Glu Thr Ala Ser Phe Asp Ile

1 5 10

<210> 357

<211> 15

<212> PRT

<213> Methanohalobium evestigatum

<400> 357

Lys Thr His Tyr Asn Asp Ala Val Ala Ile Cys Cys Asp Glu Asn

1 5 10 15

<210> 358

<211> 18

<212> PRT

<213> Clostridium botulinum

<400> 358

Pro Ile Thr Leu Gly Ile Asp Ser Gly Tyr Leu Asn Ile Gly Phe Ser

1 5 10 15

Ala Ile

<210> 359

<211> 22

<212> PRT

<213> Clostridium botulinum

<400> 359

Lys Glu Lys Ala Met Tyr Arg Arg Gln Arg Arg Ser Arg Leu Arg Tyr

1 5 10 15

Arg Lys Pro Arg Phe Asn

twenty

<210> 360

<211> 13

<212> PRT

<213> Clostridium botulinum

<400> 360

Ile Thr Asn Ile Ile Ile Glu Val Ala Asn Phe Asp Thr

1 5 10

<210> 361

<211> 15

<212> PRT

<213> Clostridium botulinum

<400> 361

Lys Thr His Tyr Asn Asp Ala Phe Cys Ile Ala Gly Ser Ser Asn

1 5 10 15

<210> 362

<211> 18

<212> PRT

<213> Geobacillus thermoleovorans

<400> 362

Pro Val Ser Leu Gly Val Asp Met Gly Thr Arg His Val Gly Ile Ser

1 5 10 15

Ala Thr

<210> 363

<211> 23

<212> PRT

<213> Geobacillus thermoleovorans

<400> 363

Ala Ile Arg Arg Gln Phe Arg Arg Ser Arg Arg Asn Arg Lys Thr Arg

1 5 10 15

Tyr Arg Glu Ala Arg Phe Leu

twenty

<210> 364

<211> 13

<212> PRT

<213> Geobacillus thermoleovorans

<400> 364

Val Thr Ser Val Thr Ile Glu Val Ala Ala Phe Asp Thr

1 5 10

<210> 365

<211> 15

<212> PRT

<213> Geobacillus thermoleovorans

<400> 365

Lys Ser His Met Val Asp Ala Arg Cys Ile Ser Gly Asn Pro Leu

1 5 10 15

<210> 366

<211> 18

<212> PRT

<213> Ammonifex degensii

<400> 366

Ser Leu Arg Ala Lys Val Asp Asp Gly Ser Arg Tyr Val Gly Ile Ala

1 5 10 15

Leu Val

<210> 367

<211> 23

<212> PRT

<213> Ammonifex degensii

<400> 367

Thr Leu Arg Arg Glu Tyr Arg Arg Gly Arg Arg Tyr Arg Ile Val Arg

1 5 10 15

His Arg Pro Cys Arg Asn Arg

twenty

<210> 368

<211> 13

<212> PRT

<213> Ammonifex degensii

<400> 368

Ile Ser Gly Val Asp Val Glu Leu Val Ser Ser Ser Gly Val

1 5 10

<210> 369

<211> 15

<212> PRT

<213> Ammonifex degensii

<400> 369

Lys Ser His Thr Asn Asp Ala Leu Ser Leu Phe Leu Pro Gly Gly

1 5 10 15

<210> 370

<211> 18

<212> PRT

<213> Polaromonas sp.

<400> 370

Pro Leu Arg Ile Lys Leu Asp Pro Gly Ser Lys Thr Thr Gly Val Ala

1 5 10 15

Leu Val

<210> 371

<211> 22

<212> PRT

<213> Polaromonas sp.

<400> 371

Thr Ala Arg Arg Gln Met Arg Arg Arg Arg Arg Ser Asn Leu Arg Cys

1 5 10 15

Arg Ala Pro Arg Phe Leu

twenty

<210> 372

<211> 13

<212> PRT

<213> Polaromonas sp.

<400> 372

Val Arg Ala Ile Ser Glu Leu Val Arg Phe Asp Met

1 5 10

<210> 373

<211> 15

<212> PRT

<213> Polaromonas sp.

<400> 373

Lys Thr His Ala Leu Asp Ala Ala Cys Val Gly Gln Val Arg Phe

1 5 10 15

<210> 374

<211> 18

<212> PRT

<213> Anabaena variabilis

<400> 374

Asp Leu Arg Ile Lys Leu Asp Pro Gly Ala Lys Ile Thr Gly Ile Ala

1 5 10 15

Leu Val

<210> 375

<211> 23

<212> PRT

<213> Anabaena variabilis

<400> 375

Ile Ser Arg Arg Gln Leu Arg Arg Thr Arg Arg Asn Arg Lys Thr Arg

1 5 10 15

Tyr Arg Lys Pro Arg Phe Leu

twenty

<210> 376

<211> 13

<212> PRT

<213> Anabaena variabilis

<400> 376

Ile Thr Ala Ile Ser Thr Glu Leu Val Lys Phe Asp Met

1 5 10

<210> 377

<211> 15

<212> PRT

<213> Anabaena variabilis

<400> 377

Lys Thr His Trp Leu Asp Ala Ala Cys Val Gly Gln Ser Thr Pro

1 5 10 15

<210> 378

<211> 18

<212> PRT

<213> Nostoc sp.

<400> 378

Pro Leu Arg Leu Lys Phe Asp Pro Gly Ala Lys Tyr Thr Gly Ile Ala

1 5 10 15

Leu Val

<210> 379

<211> 23

<212> PRT

<213> Nostoc sp.

<400> 379

Thr Ser Arg Arg Gln Leu Arg Arg Ser Arg Arg Ser Arg Lys Thr Arg

1 5 10 15

Tyr Arg Gln Pro Arg Phe Phe

twenty

<210> 380

<211> 13

<212> PRT

<213> Nostoc sp.

<400> 380

Ile Thr Ala Ile Ser Gln Glu Leu Val Lys Phe Asp Thr

1 5 10

<210> 381

<211> 15

<212> PRT

<213> Nostoc sp.

<400> 381

Lys Ser His Trp Leu Asp Ala Cys Cys Val Gly Ala Ser Thr Pro

1 5 10 15

<210> 382

<211> 18

<212> PRT

<213> Thermus thermophilus

<400> 382

Met Val Val Ala Gly Ile Asp Pro Gly Ile Thr His Leu Gly Leu Gly

1 5 10 15

Val Val

<210> 383

<211> 13

<212> PRT

<213> Thermus thermophilus

<400> 383

Pro Glu Ala Val Ala Val Glu Glu Gln Phe Phe Tyr Arg

1 5 10

<210> 384

<211> 15

<212> PRT

<213> Thermus thermophilus

<400> 384

Pro Ser His Leu Ala Asp Ala Leu Ala Ile Ala Leu Thr His Ala

1 5 10 15

<210> 385

<211> 18

<212> PRT

<213> Thermus thermophilus

<400> 385

Met Val Val Ala Gly Ile Asp Pro Gly Ile Thr His Leu Gly Leu Gly

1 5 10 15

Val Val

<210> 386

<211> 13

<212> PRT

<213> Thermus thermophilus

<400> 386

Pro Glu Ala Val Ala Val Glu Glu Gln Phe Phe Tyr Arg

1 5 10

<210> 387

<211> 15

<212> PRT

<213> Thermus thermophilus

<400> 387

Pro Ser His Leu Ala Asp Ala Leu Ala Ile Ala Leu Thr His Ala

1 5 10 15

<210> 388

<211> 18

<212> PRT

<213> Campylobacter jejuni

<400> 388

Ala Arg Ile Leu Ala Phe Asp Ile Gly Ile Ser Ser Ile Gly Trp Ala

1 5 10 15

Phe Ser

<210> 389

<211> 22

<212> PRT

<213> Campylobacter jejuni

<400> 389

Leu Pro Arg Arg Leu Ala Arg Ser Ala Arg Lys Arg Leu Ala Arg Arg

1 5 10 15

Lys Ala Arg Leu Asn His

twenty

<210> 390

<211> 13

<212> PRT

<213> Campylobacter jejuni

<400> 390

Val His Lys Ile Asn Ile Glu Leu Ala Arg Glu Val Gly

1 5 10

<210> 391

<211> 16

<212> PRT

<213> Campylobacter jejuni

<400> 391

Leu His His Ala Ile Asp Ala Val Ile Ile Ala Tyr Ala Asn Asn Ser

1 5 10 15

<210> 392

<211> 18

<212> PRT

<213> Clostridium perfringens

<400> 392

Asn Tyr Ala Leu Gly Leu Asp Ile Gly Ile Thr Ser Val Gly Trp Ala

1 5 10 15

Val Ile

<210> 393

<211> 22

<212> PRT

<213> Clostridium perfringens

<400> 393

Leu Pro Arg Arg Leu Ala Arg Gly Arg Arg Arg Leu Leu Arg Arg Lys

1 5 10 15

Ala Tyr Arg Val Glu Arg

twenty

<210> 394

<211> 13

<212> PRT

<213> Clostridium perfringens

<400> 394

Pro Val Arg Ile Asn Ile Glu Leu Ala Arg Asp Leu Ala

1 5 10

<210> 395

<211> 16

<212> PRT

<213> Clostridium perfringens

<400> 395

Lys His His Ala Leu Asp Ala Ala Val Val Gly Val Thr Thr Gln Gly

1 5 10 15

<210> 396

<211> 18

<212> PRT

<213> Akkermansia muciniphila

<400> 396

Ser Leu Thr Phe Ser Phe Asp Ile Gly Tyr Ala Ser Ile Gly Trp Ala

1 5 10 15

Val Ile

<210> 397

<211> 22

<212> PRT

<213> Akkermansia muciniphila

<400> 397

Phe Lys Arg Arg Glu Tyr Arg Arg Leu Arg Arg Asn Ile Arg Ser Arg

1 5 10 15

Arg Val Arg Ile Glu Arg

twenty

<210> 398

<211> 13

<212> PRT

<213> Akkermansia muciniphila

<400> 398

Ile Ser Arg Val Cys Val Glu Val Gly Lys Glu Leu Thr

1 5 10

<210> 399

<211> 16

<212> PRT

<213> Akkermansia muciniphila

<400> 399

Leu His His Ala Leu Asp Ala Cys Val Leu Gly Leu Ile Pro Tyr Ile

1 5 10 15

<210> 400

<211> 18

<212> PRT

<213> Bifidobacterium longum

<400> 400

Arg Tyr Arg Ile Gly Ile Asp Val Gly Leu Asn Ser Val Gly Leu Ala

1 5 10 15

Ala Val

<210> 401

<211> 22

<212> PRT

<213> Bifidobacterium longum

<400> 401

Asn Met Ser Gly Val Ala Arg Arg Thr Arg Arg Met Arg Arg Arg Lys

1 5 10 15

Arg Glu Arg Leu His Lys

twenty

<210> 402

<211> 13

<212> PRT

<213> Bifidobacterium longum

<400> 402

Pro Val Ser Val Asn Ile Glu His Val Arg Ser Ser Phe

1 5 10

<210> 403

<211> 16

<212> PRT

<213> Bifidobacterium longum

<400> 403

Arg His His Ala Val Asp Ala Ser Val Ile Ala Met Met Asn Thr Ala

1 5 10 15

<210> 404

<211> 18

<212> PRT

<213> Wolinella succinogenes

<400> 404

Val Ser Pro Ile Ser Val Asp Leu Gly Gly Lys Asn Thr Gly Phe Phe

1 5 10 15

Ser Phe

<210> 405

<211> 22

<212> PRT

<213> Wolinella succinogenes

<400> 405

Val Gly Arg Arg Ser Lys Arg His Ser Lys Arg Asn Asn Leu Arg Asn

1 5 10 15

Lys Leu Val Lys Arg Leu

twenty

<210> 406

<211> 13

<212> PRT

<213> Wolinella succinogenes

<400> 406

Lys Val Pro Ile Ile Leu Glu Gln Asn Ala Phe Glu Tyr

1 5 10

<210> 407

<211> 16

<212> PRT

<213> Wolinella succinogenes

<400> 407

Ser Ser His Ala Ile Asp Ala Val Met Ala Phe Val Ala Arg Tyr Gln

1 5 10 15

<210> 408

<211> 18

<212> PRT

<213> Legionella pneumophila

<400> 408

Leu Ser Pro Ile Gly Ile Asp Leu Gly Gly Lys Phe Thr Gly Val Cys

1 5 10 15

Leu Ser

<210> 409

<211> 22

<212> PRT

<213> Legionella pneumophila

<400> 409

Ala Gln Arg Arg Ala Thr Arg His Arg Val Arg Asn Lys Lys Arg Asn

1 5 10 15

Gln Phe Val Lys Arg Val

twenty

<210> 410

<211> 13

<212> PRT

<213> Legionella pneumophila

<400> 410

Leu Ile Pro Ile Tyr Leu Glu Gln Asn Arg Phe Glu Phe

1 5 10

<210> 411

<211> 15

<212> PRT

<213> Legionella pneumophila

<400> 411

Pro Ser His Ala Ile Asp Ala Thr Leu Thr Met Ser Ile Gly Leu

1 5 10 15

<210> 412

<211> 18

<212> PRT

<213> Francisella tularensis

<400> 412

Ile Leu Pro Ile Ala Ile Asp Leu Gly Val Lys Asn Thr Gly Val Phe

1 5 10 15

Ser Ala

<210> 413

<211> 22

<212> PRT

<213> Francisella tularensis

<400> 413

Asn Asn Arg Thr Ala Arg Arg His Gln Arg Arg Gly Ile Asp Arg Lys

1 5 10 15

Gln Leu Val Lys Arg Leu

twenty

<210> 414

<211> 13

<212> PRT

<213> Francisella tularensis

<400> 414

His Ile Pro Ile Ile Thr Glu Ser Asn Ala Phe Glu Phe

1 5 10

<210> 415

<211> 16

<212> PRT

<213> Francisella tularensis

<400> 415

Tyr Ser His Leu Ile Asp Ala Met Leu Ala Phe Cys Ile Ala Ala Asp

1 5 10 15

<210> 416

<211> 18

<212> PRT

<213> Streptococcus pyogenes

<400> 416

Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val Gly Trp Ala

1 5 10 15

Val Ile

<210> 417

<211> 22

<212> PRT

<213> Streptococcus pyogenes

<400> 417

Glu Ala Thr Arg Leu Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg

1 5 10 15

Lys Asn Arg Ile Cys Tyr

twenty

<210> 418

<211> 13

<212> PRT

<213> Streptococcus pyogenes

<400> 418

Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

1 5 10

<210> 419

<211> 16

<212> PRT

<213> Streptococcus pyogenes

<400> 419

Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala

1 5 10 15

<210> 420

<211> 18

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Candidatus Methanomethylophilus alvus sequence"

<400> 420

Leu Lys Ile Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Ile Tyr Val

1 5 10 15

Thr Met

<210> 421

<211> 22

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Candidatus Methanomethylophilus alvus sequence"

<400> 421

Arg Lys Ala Leu Asp Val Arg Glu Tyr Asp Asn Lys Glu Ala Arg Arg

1 5 10 15

Asn Trp Thr Lys Val Glu

twenty

<210> 422

<211> 13

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Candidatus Methanomethylophilus alvus sequence"

<400> 422

Asn Ala Ile Ile Val Met Glu Asp Leu Asn His Gly Phe

1 5 10

<210> 423

<211> 16

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Candidatus Methanomethylophilus alvus sequence"

<400> 423

Leu Pro Gln Asp Ser Asp Ala Asn Gly Ala Tyr Asn Ile Ala Leu Lys

1 5 10 15

<210> 424

<211> 18

<212> PRT

<213> Synergistes jonesii

<400> 424

Val Asn Ile Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Val Tyr Val

1 5 10 15

Ser Leu

<210> 425

<211> 22

<212> PRT

<213> Synergistes jonesii

<400> 425

His Ala Lys Leu Asn Gln Lys Glu Lys Glu Arg Asp Thr Ala Arg Lys

1 5 10 15

Ser Trp Lys Thr Ile Gly

twenty

<210> 426

<211> 13

<212> PRT

<213> Synergistes jonesii

<400> 426

Asn Ala Val Ile Val Met Glu Asp Leu Asn Ile Gly Phe

1 5 10

<210> 427

<211> 16

<212> PRT

<213> Synergistes jonesii

<400> 427

Leu Pro Ile Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Ala Leu Lys

1 5 10 15

<210> 428

<211> 18

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 428

Pro Tyr Val Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Leu Tyr Ile

1 5 10 15

Val Val

<210> 429

<211> 22

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 429

His Ser Leu Leu Asp Lys Lys Glu Lys Glu Arg Phe Glu Ala Arg Gln

1 5 10 15

Asn Trp Thr Ser Ile Glu

twenty

<210> 430

<211> 13

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 430

Asp Ala Val Ile Ala Leu Glu Asp Leu Asn Ser Gly Phe

1 5 10

<210> 431

<211> 16

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 431

Leu Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Arg Lys

1 5 10 15

<210> 432

<211> 18

<212> PRT

<213> Francisella tularensis

<400> 432

Val His Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr

1 5 10 15

Thr Leu

<210> 433

<211> 22

<212> PRT

<213> Francisella tularensis

<400> 433

His Asp Lys Leu Ala Ala Ile Glu Lys Asp Arg Asp Ser Ala Arg Lys

1 5 10 15

Asp Trp Lys Lys Ile Asn

twenty

<210> 434

<211> 13

<212> PRT

<213> Francisella tularensis

<400> 434

Asn Ala Ile Val Val Phe Glu Asp Leu Asn Phe Gly Phe

1 5 10

<210> 435

<211> 16

<212> PRT

<213> Francisella tularensis

<400> 435

Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly Leu Lys

1 5 10 15

<210> 436

<211> 18

<212> PRT

<213> Moraxella caprae

<400> 436

Val Asn Val Ile Gly Ile Asp Arg Gly Glu Arg His Leu Leu Tyr Leu

1 5 10 15

Thr Val

<210> 437

<211> 22

<212> PRT

<213> Moraxella caprae

<400> 437

His Lys Ile Leu Asp Lys Arg Glu Ile Glu Arg Leu Asn Ala Arg Val

1 5 10 15

Gly Trp Gly Glu Ile Glu

twenty

<210> 438

<211> 13

<212> PRT

<213> Moraxella caprae

<400> 438

Asn Ala Ile Val Val Leu Glu Asp Leu Asn Phe Gly Phe

1 5 10

<210> 439

<211> 16

<212> PRT

<213> Moraxella caprae

<400> 439

Gln Pro Gln Asn Ala Asp Ala Asn Gly Ala Tyr His Ile Ala Leu Lys

1 5 10 15

<210> 440

<211> 18

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 440

Met His Ile Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Ile Tyr Leu

1 5 10 15

Cys Met

<210> 441

<211> 22

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 441

His Gln Leu Leu Lys Thr Arg Glu Asp Glu Asn Lys Ser Ala Arg Gln

1 5 10 15

Ser Trp Gln Thr Ile His

twenty

<210> 442

<211> 13

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 442

Asn Ala Ile Val Val Leu Glu Asp Leu Asn Phe Gly Phe

1 5 10

<210> 443

<211> 16

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 443

Met Pro Leu Asp Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Arg Lys

1 5 10 15

<210> 444

<211> 18

<212> PRT

<213> Prevotella albensis

<400> 444

Thr His Ile Ile Gly Ile Asp Arg Gly Glu Arg His Leu Leu Tyr Leu

1 5 10 15

Ser Leu

<210> 445

<211> 22

<212> PRT

<213> Prevotella albensis

<400> 445

His Asn Leu Leu Glu Lys Arg Glu Lys Glu Arg Thr Glu Ala Arg His

1 5 10 15

Ser Trp Ser Ser Ile Glu

twenty

<210> 446

<211> 13

<212> PRT

<213> Prevotella albensis

<400> 446

Asn Ala Ile Val Val Leu Glu Asp Leu Asn Gly Gly Phe

1 5 10

<210> 447

<211> 16

<212> PRT

<213> Prevotella albensis

<400> 447

Phe Pro Glu Asn Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Arg Lys

1 5 10 15

<210> 448

<211> 18

<212> PRT

<213> Smithella sp.

<400> 448

Ile Asn Ile Ile Gly Ile Asp Arg Gly Glu Arg His Leu Leu Tyr Tyr

1 5 10 15

Ala Leu

<210> 449

<211> 22

<212> PRT

<213> Smithella sp.

<400> 449

His Asn Leu Leu Asp Lys Lys Glu Gly Asp Arg Ala Thr Ala Arg Gln

1 5 10 15

Glu Trp Gly Val Ile Glu

twenty

<210> 450

<211> 13

<212> PRT

<213> Smithella sp.

<400> 450

Asn Ala Ile Ile Val Met Glu Asp Leu Asn Phe Gly Phe

1 5 10

<210> 451

<211> 16

<212> PRT

<213> Smithella sp.

<400> 451

Met Pro Lys Asn Ala Asp Ala Asn Gly Ala Tyr His Ile Ala Leu Lys

1 5 10 15

<210> 452

<211> 18

<212> PRT

<213> Porphyromonas crevioricanis

<400> 452

Met His Val Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Leu Tyr Ile

1 5 10 15

Cys Val

<210> 453

<211> 22

<212> PRT

<213> Porphyromonas crevioricanis

<400> 453

His Asp Leu Leu Glu Ser Arg Asp Lys Asp Arg Gln Gln Glu Arg Arg

1 5 10 15

Asn Trp Gln Thr Ile Glu

twenty

<210> 454

<211> 13

<212> PRT

<213> Porphyromonas crevioricanis

<400> 454

Lys Ala Val Val Ala Leu Glu Asp Leu Asn Met Gly Phe

1 5 10

<210> 455

<211> 16

<212> PRT

<213> Porphyromonas crevioricanis

<400> 455

Leu Pro Lys Asp Ala Asp Ala Asn Gly Ala Tyr Asn Ile Ala Leu Lys

1 5 10 15

<210> 456

<211> 18

<212> PRT

<213> Alicyclobacillus acidoterrestris

<400> 456

Leu Arg Val Met Ser Val Asp Leu Gly Leu Arg Thr Ser Ala Ser Ile

1 5 10 15

Ser Val

<210> 457

<211> 22

<212> PRT

<213> Alicyclobacillus acidoterrestris

<400> 457

Gln Arg Thr Leu Arg Gln Leu Arg Thr Gln Leu Ala Tyr Leu Arg Leu

1 5 10 15

Leu Val Arg Cys Gly Ser

twenty

<210> 458

<211> 12

<212> PRT

<213> Alicyclobacillus acidoterrestris

<400> 458

Cys Gln Leu Ile Leu Leu Glu Glu Leu Ser Glu Tyr

1 5 10

<210> 459

<211> 16

<212> PRT

<213> Alicyclobacillus acidoterrestris

<400> 459

His Gln Ile His Ala Asp Leu Asn Ala Ala Gln Asn Leu Gln Gln Arg

1 5 10 15

<210> 460

<211> 18

<212> PRT

<213> Alicyclobacillus contaminans

<400> 460

Val Arg Val Met Ser Val Asp Leu Gly Val Arg Tyr Gly Ala Ala Ile

1 5 10 15

Ser Val

<210> 461

<211> 22

<212> PRT

<213> Alicyclobacillus contaminans

<400> 461

Lys Gln Ala Leu Ala Ala Ile Arg Ala Glu Met Ser Ile Leu Arg Lys

1 5 10 15

Trp Leu Arg Val Ser Gln

twenty

<210> 462

<211> 12

<212> PRT

<213> Alicyclobacillus contaminans

<400> 462

Cys Asp Leu Ile Leu Phe Glu Asp Leu Ser Arg Tyr

1 5 10

<210> 463

<211> 16

<212> PRT

<213> Alicyclobacillus contaminans

<400> 463

Lys Cys Val His Ala Asp Ile Asn Ala Ala His Asn Leu Gln Arg Arg

1 5 10 15

<210> 464

<211> 18

<212> PRT

<213> Desulfovibrio inopinatus

<400> 464

Leu Arg Val Leu Ser Val Asp Leu Gly Met Arg Thr Phe Ala Ser Cys

1 5 10 15

Ser Val

<210> 465

<211> 22

<212> PRT

<213> Desulfovibrio inopinatus

<400> 465

Arg Ala Glu Ile Tyr Ala Leu Lys Arg Asp Ile Gln Arg Leu Lys Ser

1 5 10 15

Leu Leu Arg Leu Gly Glu

twenty

<210> 466

<211> 12

<212> PRT

<213> Desulfovibrio inopinatus

<400> 466

Cys Gln Leu Ile Leu Phe Glu Asp Leu Ala Arg Tyr

1 5 10

<210> 467

<211> 16

<212> PRT

<213> Desulfovibrio inopinatus

<400> 467

Cys Val Ile His Ala Asp Met Asn Ala Ala Gln Asn Leu Gln Arg Arg

1 5 10 15

<210> 468

<211> 18

<212> PRT

<213> Desulfonatronum thiodismutans

<400> 468

Leu Arg Val Leu Ser Val Asp Leu Gly Val Arg Ser Phe Ala Ala Cys

1 5 10 15

Ser Val

<210> 469

<211> 22

<212> PRT

<213> Desulfonatronum thiodismutans

<400> 469

Met Glu Glu Leu Arg Ser Leu Asn Gly Asp Ile Arg Arg Leu Lys Ala

1 5 10 15

Ile Leu Arg Leu Ser Val

twenty

<210> 470

<211> 12

<212> PRT

<213> Desulfonatronum thiodismutans

<400> 470

Cys Arg Leu Ile Leu Phe Glu Asp Leu Ala Arg Tyr

1 5 10

<210> 471

<211> 16

<212> PRT

<213> Desulfonatronum thiodismutans

<400> 471

His Val Ile His Ala Asp Ile Asn Ala Ala Gln Asn Leu Gln Arg Arg

1 5 10 15

<210> 472

<211> 18

<212> PRT

<213> Tuberibacillus calidus

<400> 472

Leu Arg Val Met Ser Val Asp Leu Gly Gln Arg Gln Ala Ala Ala Ile

1 5 10 15

Ser Ile

<210> 473

<211> 22

<212> PRT

<213> Tuberibacillus calidus

<400> 473

Asp Gln Ala Ile Arg Asp Leu Ser Arg Lys Leu Lys Phe Leu Lys Asn

1 5 10 15

Val Leu Asn Met Gln Lys

twenty

<210> 474

<211> 12

<212> PRT

<213> Tuberibacillus calidus

<400> 474

Cys Gln Leu Val Leu Phe Glu Asp Leu Ser Arg Tyr

1 5 10

<210> 475

<211> 16

<212> PRT

<213> Tuberibacillus calidus

<400> 475

Val Ile Thr His Ala Asp Ile Asn Ala Ala Gln Asn Leu Gln Lys Arg

1 5 10 15

<210> 476

<211> 18

<212> PRT

<213> Bacillus thermoamylovorans

<400> 476

Leu Arg Val Met Ser Ile Asp Leu Gly Gln Arg Gln Ala Ala Ala Ala

1 5 10 15

Ser Ile

<210> 477

<211> 22

<212> PRT

<213> Bacillus thermoamylovorans

<400> 477

Glu Asp Asn Leu Lys Leu Met Asn Gln Lys Leu Asn Phe Leu Arg Asn

1 5 10 15

Val Leu His Phe Gln Gln

twenty

<210> 478

<211> 12

<212> PRT

<213> Bacillus thermoamylovorans

<400> 478

Cys Gln Ile Ile Leu Phe Glu Asp Leu Ser Asn Tyr

1 5 10

<210> 479

<211> 16

<212> PRT

<213> Bacillus thermoamylovorans

<400> 479

Val Thr Thr His Ala Asp Ile Asn Ala Ala Gln Asn Leu Gln Lys Arg

1 5 10 15

<210> 480

<211> 18

<212> PRT

<213> Bacillus sp.

<400> 480

Phe Arg Val Met Ser Ile Asp Leu Gly Leu Arg Ala Ala Ala Ala Thr

1 5 10 15

Ser Ile

<210> 481

<211> 22

<212> PRT

<213> Bacillus sp.

<400> 481

Phe Gln Leu His Gln Arg Val Lys Phe Gln Ile Arg Val Leu Ala Gln

1 5 10 15

Ile Met Arg Met Ala Asn

twenty

<210> 482

<211> 12

<212> PRT

<213> Bacillus sp.

<400> 482

Cys Gln Val Ile Leu Phe Glu Asn Leu Ser Gln Tyr

1 5 10

<210> 483

<211> 16

<212> PRT

<213> Bacillus sp.

<400> 483

Val Phe Leu Gln Ala Asp Ile Asn Ala Ala His Asn Leu Gln Lys Arg

1 5 10 15

<210> 484

<211> 18

<212> PRT

<213> Methylobacterium nodulans

<400> 484

Leu Arg Val Leu Ser Ile Asp Leu Gly Val Arg Ser Phe Ala Thr Cys

1 5 10 15

Ser Val

<210> 485

<211> 22

<212> PRT

<213> Methylobacterium nodulans

<400> 485

Asp Ala Glu Leu Arg Gln Leu Arg Gly Gly Leu Asn Arg His Arg Gln

1 5 10 15

Leu Leu Arg Ala Ala Thr

twenty

<210> 486

<211> 12

<212> PRT

<213> Methylobacterium nodulans

<400> 486

Cys His Val Ile Leu Phe Glu Asp Leu Ser Arg Tyr

1 5 10

<210> 487

<211> 16

<212> PRT

<213> Methylobacterium nodulans

<400> 487

Ser Arg Ile His Ala Asp Ile Asn Ala Ala Gln Asn Leu Gln Arg Arg

1 5 10 15

<210> 488

<211> 18

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 488

Lys Asn Ile Val Ser Ile Asp Gln Gly Glu Ala Gly Phe Ala Tyr Ala

1 5 10 15

Val Phe

<210> 489

<211> 22

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 489

His Ser Val Lys Lys Tyr Arg Gly Lys Lys Gln Arg Ile Gln Asn Phe

1 5 10 15

Asn Gln Lys Phe Asp Ser

twenty

<210> 490

<211> 13

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 490

Asn Ala Phe Pro Ile Leu Glu Lys Gln Val Gly Asn Leu

1 5 10

<210> 491

<211> 16

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 491

Lys Glu Gln His Ala Asp Val Asn Ala Ala Ile Asn Ile Gly Arg Arg

1 5 10 15

<210> 492

<211> 18

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 492

Asp His Ile Val Ala Ile Asp Leu Gly Glu Arg Ser Val Gly Phe Ala

1 5 10 15

Val Phe

<210> 493

<211> 22

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 493

Lys Ala Val Arg Ser His Arg Arg Arg Arg Gln Pro Asn Gln Lys Val

1 5 10 15

Asn Gln Thr Tyr Ser Thr

twenty

<210> 494

<211> 13

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 494

Asn Ala Phe Pro Val Leu Glu Phe Gln Ile Lys Asn Phe

1 5 10

<210> 495

<211> 16

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 495

Trp Thr Gly His Ala Asp Glu Asn Ala Ala Ile Asn Ile Gly Arg Arg

1 5 10 15

<210> 496

<211> 18

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Metagenome sequence"

<400> 496

Asp Arg Ile Val Ala Ile Asp Leu Gly Glu Arg Lys Ile Gly Tyr Ala

1 5 10 15

Ile Phe

<210> 497

<211> 22

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Metagenome sequence"

<400> 497

Lys Ala Val Gln Thr His Arg Asn Arg Arg Gln Pro Asn Tyr Arg Ile

1 5 10 15

Asp Gln Thr Tyr Ser Lys

twenty

<210> 498

<211> 13

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Metagenome sequence"

<400> 498

Gly Gly Phe Pro Val Leu Glu Ser Ser Val Arg Asn Phe

1 5 10

<210> 499

<211> 16

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Metagenome sequence"

<400> 499

His Glu Cys His Ala Asp Glu Asn Ala Ala Ile Asn Ile Gly Arg Lys

1 5 10 15

<210> 500

<211> 18

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 500

Asp His Leu Leu Ala Ile Asp Leu Gly Glu Lys Arg Val Gly Tyr Ala

1 5 10 15

Val Tyr

<210> 501

<211> 22

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 501

Lys Ala Val Arg Ser His Arg Gln Gln Arg Gln Pro Asn Gln Lys Val

1 5 10 15

Asn Gln Thr Tyr Ser Thr

twenty

<210> 502

<211> 13

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 502

Asn Ala Phe Pro Val Leu Glu Ser Ser Val Met Asn Phe

1 5 10

<210> 503

<211> 16

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 503

Phe Thr Gly His Ala Asp Glu Asn Ala Ala Ile Asn Ile Gly Trp Lys

1 5 10 15

<210> 504

<211> 15

<212> PRT

<213> Homo sapiens

<400> 504

Ile Pro Asp Trp Ile Val Asp Leu Arg His Glu Leu Thr His Lys

1 5 10 15

<210> 505

<211> 15

<212> PRT

<213> Homo sapiens

<400> 505

Ile Pro Asp Trp Ile Val Asp Leu Arg His Glu Leu Thr His Lys

1 5 10 15

<210> 506

<211> 15

<212> PRT

<213> Mycobacterium tuberculosis

<400> 506

Leu Gly Arg Phe Glu Ser Arg Val Arg Asn Thr Ala Ala His Glu

1 5 10 15

<210> 507

<211> 15

<212> PRT

<213> Mycobacterium tuberculosis

<400> 507

Leu Gly Arg Phe Glu Ser Arg Val Arg Asn Thr Ala Ala His Glu

1 5 10 15

<210> 508

<211> 15

<212> PRT

<213> Lactococcus lactis

<400> 508

Trp Ile Arg Ala Gly Trp Arg Ile Arg Asn Arg Ser Ala His Tyr

1 5 10 15

<210> 509

<211> 15

<212> PRT

<213> Lactococcus lactis

<400> 509

Trp Ile Arg Ala Gly Trp Phe Ile Arg Asn Arg Ser Ala His Tyr

1 5 10 15

<210> 510

<211> 15

<212> PRT

<213> Thermus thermophilus

<220>

<221> MOD_RES

<222> (5)..(5)

<223> Any amino acids

<400> 510

Leu Ala Leu Gly Xaa Val Asp Asp Arg Ser Leu Thr Val His Thr

1 5 10 15

<210> 511

<211> 15

<212> PRT

<213> Thermus thermophilus

<220>

<221> MOD_RES

<222> (5)..(5)

<223> Any amino acids

<400> 511

Leu Ala Leu Gly Xaa Val Asp Asp Arg Ser Leu Thr Val His Thr

1 5 10 15

<210> 512

<211> 22

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 512

Leu Lys Ser Met Leu Tyr Ser Met Arg Asn Ser Ser Phe His Phe Ser

1 5 10 15

Thr Glu Asn Val Asp Asn

twenty

<210> 513

<211> 14

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 513

Phe Arg Asn Glu Ile Asp His Phe His Tyr Phe Tyr Asp Arg

1 5 10

<210> 514

<211> 22

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 514

Leu Lys Asp Val Ile Tyr Ser Met Arg Asn Asp Ser Phe His Tyr Ala

1 5 10 15

Thr Glu Asn His Asn Asn

twenty

<210> 515

<211> 14

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 515

Leu Arg Asn Tyr Ile Glu His Phe Arg Tyr Tyr Ser Ser Phe

1 5 10

<210> 516

<211> 22

<212> PRT

<213> Clostridium aminophilum

<400> 516

Leu Arg Lys Ala Ile Tyr Ser Leu Arg Asn Glu Thr Phe His Phe Thr

1 5 10 15

Thr Leu Asn Lys Gly Ser

twenty

<210> 517

<211> 14

<212> PRT

<213> Clostridium aminophilum

<400> 517

Val Arg Lys Tyr Val Asp His Phe Lys Tyr Tyr Ala Thr Ser

1 5 10

<210> 518

<211> 22

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 518

Ile Ile Gln Ile Ile Tyr Ser Leu Arg Asn Lys Ser Phe His Phe Lys

1 5 10 15

Thr Tyr Asp His Gly Asp

twenty

<210> 519

<211> 14

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 519

Leu Arg Lys Tyr Val Asp His Phe Lys Tyr Tyr Ala Tyr Gly

1 5 10

<210> 520

<211> 22

<212> PRT

<213> Carnobacterium gallinarum

<400> 520

Leu Arg Gly Ser Val Gln Gln Ile Arg Asn Glu Ile Phe His Ser Phe

1 5 10 15

Asp Lys Asn Gln Lys Phe

twenty

<210> 521

<211> 14

<212> PRT

<213> Carnobacterium gallinarum

<400> 521

Ile Arg Asn Gln Thr Ala His Leu Ser Val Leu Gln Leu Glu

1 5 10

<210> 522

<211> 21

<212> PRT

<213> Carnobacterium gallinarum

<400> 522

Ile Arg Gly Ala Val Gln Arg Val Arg Asn Gln Ile Phe His Gln Gln

1 5 10 15

Ile Asn Lys Arg His

twenty

<210> 523

<211> 14

<212> PRT

<213> Carnobacterium gallinarum

<400> 523

Ile Arg Asn Asn Ile Ala His Leu His Val Leu Arg Asn Asp

1 5 10

<210> 524

<211> 20

<212> PRT

<213> Paludibacter propionicigenes

<400> 524

Ile Arg Gly Ala Val Gln Gln Ile Arg Asn Asn Val Asn His Tyr Lys

1 5 10 15

Lys Asp Ala Leu

twenty

<210> 525

<211> 14

<212> PRT

<213> Paludibacter propionicigenes

<400> 525

Ile Arg Asn His Ile Ala His Phe Asn Tyr Leu Thr Lys Asp

1 5 10

<210> 526

<211> 20

<212> PRT

<213> Listeria seeligeri

<400> 526

Leu Arg Gly Ala Ile Ala Pro Ile Arg Asn Glu Ile Ile His Leu Lys

1 5 10 15

Lys His Ser Trp

twenty

<210> 527

<211> 14

<212> PRT

<213> Listeria seeligeri

<400> 527

Lys Arg Asn Asn Ile Ser His Phe Asn Tyr Leu Asn Gly Gln

1 5 10

<210> 528

<211> 20

<212> PRT

<213> Listeria weihenstephanensis

<400> 528

Ile Arg Gly Ser Ile Gln Gln Ile Arg Asn Glu Val Tyr His Cys Lys

1 5 10 15

Lys His Ser Trp

twenty

<210> 529

<211> 14

<212> PRT

<213> Listeria weihenstephanensis

<400> 529

Ala Arg Asn His Ile Ala His Leu Asn Tyr Leu Ser Leu Lys

1 5 10

<210> 530

<211> 20

<212> PRT

<213> Listeria newyorkens

<400> 530

Ile Arg Gly Ser Ile Gln Gln Ile Arg Asn Glu Val Tyr His Cys Lys

1 5 10 15

Lys His Ser Trp

twenty

<210> 531

<211> 14

<212> PRT

<213> Listeria newyorkens

<400> 531

Ala Arg Asn His Ile Ala His Leu Asn Tyr Leu Ser Leu Lys

1 5 10

<210> 532

<211> 21

<212> PRT

<213> Leptotrichia wadei

<400> 532

Ile Ser Tyr Ser Ile Tyr Asn Val Arg Asn Gly Val Gly His Phe Asn

1 5 10 15

Lys Leu Ile Leu Gly

twenty

<210> 533

<211> 14

<212> PRT

<213> Leptotrichia wadei

<400> 533

Phe Arg Asn Tyr Ile Ala His Phe Leu His Leu His Thr Lys

1 5 10

<210> 534

<211> 21

<212> PRT

<213> Leptotrichia wadei

<400> 534

Met Leu Asn Ala Ile Thr Ser Ile Arg His Arg Val Val His Tyr Asn

1 5 10 15

Met Asn Thr Asn Ser

twenty

<210> 535

<211> 14

<212> PRT

<213> Leptotrichia wadei

<400> 535

Ile Arg Asn Tyr Ile Ala His Phe Asn Tyr Ile Pro Asp Ala

1 5 10

<210> 536

<211> 21

<212> PRT

<213> Leptotrichia wadei

<400> 536

Ile Asp Glu Ala Ile Ser Ser Ile Arg His Gly Ile Val His Phe Asn

1 5 10 15

Leu Glu Leu Glu Gly

twenty

<210> 537

<211> 14

<212> PRT

<213> Leptotrichia wadei

<400> 537

Ile Arg Asn Tyr Ile Ala His Phe Asn Tyr Ile Pro His Ala

1 5 10

<210> 538

<211> 22

<212> PRT

<213> Rhodobacter capsulatus

<400> 538

Leu Leu Arg Tyr Leu Arg Gly Cys Arg Asn Gln Thr Phe His Leu Gly

1 5 10 15

Ala Arg Ala Gly Phe Leu

twenty

<210> 539

<211> 21

<212> PRT

<213> Leptotrichia buccalis

<400> 539

Ile Asp Glu Ala Ile Ser Ser Ile Arg His Gly Ile Val His Phe Asn

1 5 10 15

Leu Glu Leu Glu Gly

twenty

<210> 540

<211> 14

<212> PRT

<213> Rhodobacter capsulatus

<400> 540

Thr Arg Lys Asp Leu Ala His Phe Asn Val Leu Asp Arg Ala

1 5 10

<210> 541

<211> 21

<212> PRT

<213> Leptotrichia sp.

<400> 541

Ile Asp Glu Ala Ile Ser Ser Ile Arg His Gly Ile Val His Phe Asn

1 5 10 15

Leu Glu Leu Glu Gly

twenty

<210> 542

<211> 14

<212> PRT

<213> Leptotrichia buccalis

<400> 542

Ile Arg Asn Tyr Ile Ala His Phe Asn Tyr Ile Pro His Ala

1 5 10

<210> 543

<211> 16

<212> PRT

<213> Leptotrichia sp.

<400> 543

Phe Gln Lys Glu Gly Tyr Leu Leu Arg Asn Lys Ile Leu His Asn Ser

1 5 10 15

<210> 544

<211> 14

<212> PRT

<213> Leptotrichia sp.

<400> 544

Ile Arg Asn Tyr Ile Ala His Phe Asn Tyr Ile Pro Asn Ala

1 5 10

<210> 545

<211> 15

<212> PRT

<213> Leptotrichia shahii

<400> 545

Phe Thr Lys Ile Gly Thr Asn Glu Arg Asn Arg Ile Leu His Ala

1 5 10 15

<210> 546

<211> 14

<212> PRT

<213> Leptotrichia sp.

<400> 546

Ile Arg Asn Tyr Ile Ser His Phe Tyr Ile Val Arg Asn Pro

1 5 10

<210> 547

<211> 14

<212> PRT

<213> Leptotrichia shahii

<400> 547

Ile Arg Asn Tyr Ile Ser His Phe Tyr Ile Val Arg Asn Pro

1 5 10

<210> 548

<211> 1129

<212> PRT

<213> Alicyclobacillus acidoterrestris

<400> 548

Met Ala Val Lys Ser Ile Lys Val Lys Leu Arg Leu Asp Asp Met Pro

1 5 10 15

Glu Ile Arg Ala Gly Leu Trp Lys Leu His Lys Glu Val Asn Ala Gly

20 25 30

Val Arg Tyr Tyr Thr Glu Trp Leu Ser Leu Leu Arg Gln Glu Asn Leu

35 40 45

Tyr Arg Arg Ser Pro Asn Gly Asp Gly Glu Gln Glu Cys Asp Lys Thr

50 55 60

Ala Glu Glu Cys Lys Ala Glu Leu Leu Glu Arg Leu Arg Ala Arg Gln

65 70 75 80

Val Glu Asn Gly His Arg Gly Pro Ala Gly Ser Asp Asp Glu Leu Leu

85 90 95

Gln Leu Ala Arg Gln Leu Tyr Glu Leu Leu Val Pro Gln Ala Ile Gly

100 105 110

Ala Lys Gly Asp Ala Gln Gln Ile Ala Arg Lys Phe Leu Ser Pro Leu

115 120 125

Ala Asp Lys Asp Ala Val Gly Gly Leu Gly Ile Ala Lys Ala Gly Asn

130 135 140

Lys Pro Arg Trp Val Arg Met Arg Glu Ala Gly Glu Pro Gly Trp Glu

145 150 155 160

Glu Glu Lys Glu Lys Ala Glu Thr Arg Lys Ser Ala Asp Arg Thr Ala

165 170 175

Asp Val Leu Arg Ala Leu Ala Asp Phe Gly Leu Lys Pro Leu Met Arg

180 185 190

Val Tyr Thr Asp Ser Glu Met Ser Ser Val Glu Trp Lys Pro Leu Arg

195 200 205

Lys Gly Gln Ala Val Arg Thr Trp Asp Arg Asp Met Phe Gln Gln Ala

210 215 220

Ile Glu Arg Met Met Ser Trp Glu Ser Trp Asn Gln Arg Val Gly Gln

225 230 235 240

Glu Tyr Ala Lys Leu Val Glu Gln Lys Asn Arg Phe Glu Gln Lys Asn

245 250 255

Phe Val Gly Gln Glu His Leu Val His Leu Val Asn Gln Leu Gln Gln

260 265 270

Asp Met Lys Glu Ala Ser Pro Gly Leu Glu Ser Lys Glu Gln Thr Ala

275 280 285

His Tyr Val Thr Gly Arg Ala Leu Arg Gly Ser Asp Lys Val Phe Glu

290 295 300

Lys Trp Gly Lys Leu Ala Pro Asp Ala Pro Phe Asp Leu Tyr Asp Ala

305 310 315 320

Glu Ile Lys Asn Val Gln Arg Arg Asn Thr Arg Arg Phe Gly Ser His

325 330 335

Asp Leu Phe Ala Lys Leu Ala Glu Pro Glu Tyr Gln Ala Leu Trp Arg

340 345 350

Glu Asp Ala Ser Phe Leu Thr Arg Tyr Ala Val Tyr Asn Ser Ile Leu

355 360 365

Arg Lys Leu Asn His Ala Lys Met Phe Ala Thr Phe Thr Leu Pro Asp

370 375 380

Ala Thr Ala His Pro Ile Trp Thr Arg Phe Asp Lys Leu Gly Gly Asn

385 390 395 400

Leu His Gln Tyr Thr Phe Leu Phe Asn Glu Phe Gly Glu Arg Arg His

405 410 415

Ala Ile Arg Phe His Lys Leu Leu Lys Val Glu Asn Gly Val Ala Arg

420 425 430

Glu Val Asp Asp Val Thr Val Pro Ile Ser Met Ser Glu Gln Leu Asp

435 440 445

Asn Leu Leu Pro Arg Asp Pro Asn Glu Pro Ile Ala Leu Tyr Phe Arg

450 455 460

Asp Tyr Gly Ala Glu Gln His Phe Thr Gly Glu Phe Gly Gly Ala Lys

465 470 475 480

Ile Gln Cys Arg Arg Asp Gln Leu Ala His Met His Arg Arg Arg Gly

485 490 495

Ala Arg Asp Val Tyr Leu Asn Val Ser Val Arg Val Gln Ser Gln Ser

500 505 510

Glu Ala Arg Gly Glu Arg Arg Pro Pro Tyr Ala Ala Val Phe Arg Leu

515 520 525

Val Gly Asp Asn His Arg Ala Phe Val His Phe Asp Lys Leu Ser Asp

530 535 540

Tyr Leu Ala Glu His Pro Asp Asp Gly Lys Leu Gly Ser Glu Gly Leu

545 550 555 560

Leu Ser Gly Leu Arg Val Met Ser Val Asp Leu Gly Leu Arg Thr Ser

565 570 575

Ala Ser Ile Ser Val Phe Arg Val Ala Arg Lys Asp Glu Leu Lys Pro

580 585 590

Asn Ser Lys Gly Arg Val Pro Phe Phe Phe Pro Ile Lys Gly Asn Asp

595 600 605

Asn Leu Val Ala Val His Glu Arg Ser Gln Leu Leu Lys Leu Pro Gly

610 615 620

Glu Thr Glu Ser Lys Asp Leu Arg Ala Ile Arg Glu Glu Glu Arg Gln Arg

625 630 635 640

Thr Leu Arg Gln Leu Arg Thr Gln Leu Ala Tyr Leu Arg Leu Leu Val

645 650 655

Arg Cys Gly Ser Glu Asp Val Gly Arg Arg Glu Arg Ser Trp Ala Lys

660 665 670

Leu Ile Glu Gln Pro Val Asp Ala Ala Asn His Met Thr Pro Asp Trp

675 680 685

Arg Glu Ala Phe Glu Asn Glu Leu Gln Lys Leu Lys Ser Leu His Gly

690 695 700

Ile Cys Ser Asp Lys Glu Trp Met Asp Ala Val Tyr Glu Ser Val Arg

705 710 715 720

Arg Val Trp Arg His Met Gly Lys Gln Val Arg Asp Trp Arg Lys Asp

725 730 735

Val Arg Ser Gly Glu Arg Pro Lys Ile Arg Gly Tyr Ala Lys Asp Val

740 745 750

Val Gly Gly Asn Ser Ile Glu Gln Ile Glu Tyr Leu Glu Arg Gln Tyr

755 760 765

Lys Phe Leu Lys Ser Trp Ser Phe Phe Gly Lys Val Ser Gly Gln Val

770 775 780

Ile Arg Ala Glu Lys Gly Ser Arg Phe Ala Ile Thr Leu Arg Glu His

785 790 795 800

Ile Asp His Ala Lys Glu Asp Arg Leu Lys Lys Leu Ala Asp Arg Ile

805 810 815

Ile Met Glu Ala Leu Gly Tyr Val Tyr Ala Leu Asp Glu Arg Gly Lys

820 825 830

Gly Lys Trp Val Ala Lys Tyr Pro Pro Cys Gln Leu Ile Leu Leu Glu

835 840 845

Glu Leu Ser Glu Tyr Gln Phe Asn Asn Asp Arg Pro Pro Ser Glu Asn

850 855 860

Asn Gln Leu Met Gln Trp Ser His Arg Gly Val Phe Gln Glu Leu Ile

865 870 875 880

Asn Gln Ala Gln Val His Asp Leu Leu Val Gly Thr Met Tyr Ala Ala

885 890 895

Phe Ser Ser Arg Phe Asp Ala Arg Thr Gly Ala Pro Gly Ile Arg Cys

900 905 910

Arg Arg Val Pro Ala Arg Cys Thr Gln Glu His Asn Pro Glu Pro Phe

915 920 925

Pro Trp Trp Leu Asn Lys Phe Val Val Glu His Thr Leu Asp Ala Cys

930 935 940

Pro Leu Arg Ala Asp Asp Leu Ile Pro Thr Gly Glu Gly Glu Ile Phe

945 950 955 960

Val Ser Pro Phe Ser Ala Glu Glu Gly Asp Phe His Gln Ile His Ala

965 970 975

Asp Leu Asn Ala Ala Gln Asn Leu Gln Gln Arg Leu Trp Ser Asp Phe

980 985 990

Asp Ile Ser Gln Ile Arg Leu Arg Cys Asp Trp Gly Glu Val Asp Gly

995 1000 1005

Glu Leu Val Leu Ile Pro Arg Leu Thr Gly Lys Arg Thr Ala Asp

1010 1015 1020

Ser Tyr Ser Asn Lys Val Phe Tyr Thr Asn Thr Gly Val Thr Tyr

1025 1030 1035

Tyr Glu Arg Glu Arg Gly Lys Lys Arg Arg Lys Val Phe Ala Gln

1040 1045 1050

Glu Lys Leu Ser Glu Glu Glu Ala Glu Leu Leu Val Glu Ala Asp

1055 1060 1065

Glu Ala Arg Glu Lys Ser Val Val Leu Met Arg Asp Pro Ser Gly

1070 1075 1080

Ile Ile Asn Arg Gly Asn Trp Thr Arg Gln Lys Glu Phe Trp Ser

1085 1090 1095

Met Val Asn Gln Arg Ile Glu Gly Tyr Leu Val Lys Gln Ile Arg

1100 1105 1110

Ser Arg Val Pro Leu Gln Asp Ser Ala Cys Glu Asn Thr Gly Asp

1115 1120 1125

ile

<210> 549

<211> 872

<212> PRT

<213> Alicyclobacillus contaminans

<400> 549

Met Gly Phe Asn Thr Ala Glu Leu Leu Arg Lys Val Glu Glu Glu Met

1 5 10 15

Arg Lys Thr Ser Val Gly Phe Asp Thr Asp Asn Pro Phe Ala His Arg

20 25 30

Ile Thr Arg Arg Ala Ile Arg Gly Trp Asp Arg Ile Ala Glu Ala Trp

35 40 45

Arg Arg Leu Pro Pro Asp Ala Pro Glu Ser Glu Tyr Ile Glu Ala Phe

50 55 60

Lys Asp Ile Gln Arg Lys Asn Pro Arg Lys Ile Gly Ser Glu Pro Leu

65 70 75 80

Phe Lys Asn Leu Ala Ala Pro Gly Val Arg Ser Glu Leu Leu Asn Asn

85 90 95

Pro Gln Val Leu Ile Thr Phe Ala Lys Tyr Asn Glu Leu Gln Arg Gln

100 105 110

Leu Ala Lys Ala Lys Gln Phe Ala Gln Lys Thr Leu Pro His Pro Val

115 120 125

Phe His Pro Val Trp Val Arg Tyr Asp Lys Leu Gly Gly Asn Leu His

130 135 140

His Tyr Gln Ile Glu Pro Ala Val His Ala Asn Asp Thr His Lys Val

145 150 155 160

Lys Phe Ser Ser Leu Leu Leu Pro Gln Glu Asp Gly Ser Tyr Ala Glu

165 170 175

Val Lys Asp Val Thr Val Ser Leu Ala Pro Ser Leu Gln Phe Pro Thr

180 185 190

Gly Leu Val His Pro Lys Val Thr Thr Pro Pro Arg Thr Gly Leu Val

195 200 205

Thr Val Met Asp Glu Glu Ala Gly Lys Pro Val Val Cys Tyr Arg Asp

210 215 220

Arg Gly His Asp Ala Leu Val Pro Val Ala Phe Gly Gly Ala Lys Leu

225 230 235 240

Gln Phe Asn Arg Ala His Leu Ser Ala Gly Tyr Arg Lys Gly Val Leu

245 250 255

Ser Ala Gly Gly Gly Gly Ser Ile Tyr Phe Asn Val Thr Leu Asp Val

260 265 270

Gln Val Pro Asn Glu Arg Asp Val Ser Lys Thr Phe Ser Phe Ser Arg

275 280 285

Asp Arg Asp Leu Val Ser Leu Lys Ala Glu Glu Leu Lys Arg Tyr Met

290 295 300

Glu Thr Lys Pro Leu Gly Met Pro Gly Val Arg Val Met Ser Val Asp

305 310 315 320

Leu Gly Val Arg Tyr Gly Ala Ala Ile Ser Val Phe Glu Val Lys Pro

325 330 335

Phe Ala Glu Val Arg Lys Asp Lys Leu His Tyr Pro Ile Thr Gly Cys

340 345 350

Glu Gly Phe Val Ala Glu His Glu Arg Ser Val Ile Leu Lys Leu Pro

355 360 365

Gly Glu Gly Val Arg Thr Ala Gly Lys Gln Ser Glu Arg Lys Gln Ala

370 375 380

Leu Ala Ala Ile Arg Ala Glu Met Ser Ile Leu Arg Lys Trp Leu Arg

385 390 395 400

Val Ser Gln Val Thr Glu Glu Asp Arg Ala Lys Ala Val Arg Gly Leu

405 410 415

Leu Glu Asp Glu Arg Gly Gly Gly Trp Thr Met Asp Pro Gly Glu Asp

420 425 430

Ser Asp His Gln Pro Leu Gln Gln Phe Leu His Glu Ala Arg Leu Ala

435 440 445

Val Gly Glu Leu Val Asn Leu Val His Leu Ser Pro Ala Glu Trp Glu

450 455 460

Arg Ala Val Ile Glu Arg His Arg Arg Leu Glu Arg Ile Thr Ala Ser

465 470 475 480

His Ile Arg Val Phe Gln Thr Met Arg Lys Val Trp Gly Lys Arg Arg

485 490 495

Asn Glu Asp Ala Ala His Thr Gly Gly Ile Ser Leu Ala His Ile Glu

500 505 510

His Leu Ile Gln Gln Arg Lys Leu Phe Ile Arg Trp Ser Thr His Ala

515 520 525

Arg Thr Tyr Gly Glu Val Arg Arg Leu Pro Lys His Glu Gly Phe Ala

530 535 540

Lys Arg Leu Gln Lys His Thr Asn His Val Lys Glu Asp Arg Ile Lys

545 550 555 560

Lys Leu Ala Asp Met Ile Val Met Ala Ala Arg Gly Tyr Arg Phe Leu

565 570 575

Asp Lys Arg Ala Arg Trp Val Lys Thr Arg His Ala Pro Cys Asp Leu

580 585 590

Ile Leu Phe Glu Asp Leu Ser Arg Tyr Arg Phe Thr Met Asp Arg Pro

595 600 605

Pro Thr Glu Asn Ser Gln Leu Met Asn Trp Ser His Arg Glu Leu Leu

610 615 620

Lys Thr Val Lys Met Gln Ala Ala Leu Phe Gly Ile Gly Val Gly Thr

625 630 635 640

Val Pro Ala Ala Phe Thr Ser Arg Phe Asp Ala Gln Thr Gly Ala Pro

645 650 655

Gly Leu Arg Cys Lys Arg Val Thr Lys Gln Asp Lys Glu Lys Thr Pro

660 665 670

Phe Trp Leu Ile Gln Phe Ala Glu Ile Thr Gly Val Asn Val Thr Asn

675 680 685

Val Glu Pro Gly Gln Leu Ile Pro Val Asp Gly Gly Glu Trp Phe Val

690 695 700

Ser Pro Lys Gly Pro Arg Ala Ala Asp Gly Leu Lys Cys Val His Ala

705 710 715 720

Asp Ile Asn Ala Ala His Asn Leu Gln Arg Arg Phe Trp Ile Pro Arg

725 730 735

Leu Pro Ser Val Lys Cys Arg Arg Tyr Val Glu Ala Glu Gly Phe Ala

740 745 750

Ala Val Pro Ser Ser Thr Ala Phe Met Lys Val His Gly Lys Gly Ala

755 760 765

Phe Val Ser Val Asp Gly Glu Phe Tyr Glu Tyr Gln Lys Gly Arg Arg

770 775 780

Val Ala Val Asn Arg Ala Asp Arg Thr Ser Ser Thr Leu Asp Glu Asp

785 790 795 800

Glu Gly Asp Ile Gly Glu Glu Met Leu Val Ser Ser Asn Gly Ala Gly

805 810 815

Glu Phe Val Arg Met Phe Tyr Asp Glu Ser Gly Tyr Val Gly Tyr Gly

820 825 830

Arg Trp Met Asp Ser Lys Val Phe Trp Gly Lys Val Arg Gln Ile Val

835 840 845

His Arg Ala Ile Gln Asp Gln Val Glu Lys Arg Ala Ala Ala Arg Gly

850 855 860

Glu Asn Gly Ala Thr Ser Ser Arg

865 870

<210> 550

<211> 1149

<212> PRT

<213> Desulfovibrio inopinatus

<400> 550

Met Pro Thr Arg Thr Ile Asn Leu Lys Leu Val Leu Gly Lys Asn Pro

1 5 10 15

Glu Asn Ala Thr Leu Arg Arg Ala Leu Phe Ser Thr His Arg Leu Val

20 25 30

Asn Gln Ala Thr Lys Arg Ile Glu Glu Phe Leu Leu Leu Cys Arg Gly

35 40 45

Glu Ala Tyr Arg Thr Val Asp Asn Glu Gly Lys Glu Ala Glu Ile Pro

50 55 60

Arg His Ala Val Gln Glu Glu Ala Leu Ala Phe Ala Lys Ala Ala Gln

65 70 75 80

Arg His Asn Gly Cys Ile Ser Thr Tyr Glu Asp Gln Glu Ile Leu Asp

85 90 95

Val Leu Arg Gln Leu Tyr Glu Arg Leu Val Pro Ser Val Asn Glu Asn

100 105 110

Asn Glu Ala Gly Asp Ala Gln Ala Ala Asn Ala Trp Val Ser Pro Leu

115 120 125

Met Ser Ala Glu Ser Glu Gly Gly Leu Ser Val Tyr Asp Lys Val Leu

130 135 140

Asp Pro Pro Pro Val Trp Met Lys Leu Lys Glu Glu Lys Ala Pro Gly

145 150 155 160

Trp Glu Ala Ala Ser Gln Ile Trp Ile Gln Ser Asp Glu Gly Gln Ser

165 170 175

Leu Leu Asn Lys Pro Gly Ser Pro Pro Arg Trp Ile Arg Lys Leu Arg

180 185 190

Ser Gly Gln Pro Trp Gln Asp Asp Phe Val Ser Asp Gln Lys Lys Lys

195 200 205

Gln Asp Glu Leu Thr Lys Gly Asn Ala Pro Leu Ile Lys Gln Leu Lys

210 215 220

Glu Met Gly Leu Leu Pro Leu Val Asn Pro Phe Phe Arg His Leu Leu

225 230 235 240

Asp Pro Glu Gly Lys Gly Val Ser Pro Trp Asp Arg Leu Ala Val Arg

245 250 255

Ala Ala Val Ala His Phe Ile Ser Trp Glu Ser Trp Asn His Arg Thr

260 265 270

Arg Ala Glu Tyr Asn Ser Leu Lys Leu Arg Arg Asp Glu Phe Glu Ala

275 280 285

Ala Ser Asp Glu Phe Lys Asp Asp Phe Thr Leu Leu Arg Gln Tyr Glu

290 295 300

Ala Lys Arg His Ser Thr Leu Lys Ser Ile Ala Leu Ala Asp Asp Ser

305 310 315 320

Asn Pro Tyr Arg Ile Gly Val Arg Ser Leu Arg Ala Trp Asn Arg Val

325 330 335

Arg Glu Glu Trp Ile Asp Lys Gly Ala Thr Glu Glu Gln Arg Val Thr

340 345 350

Ile Leu Ser Lys Leu Gln Thr Gln Leu Arg Gly Lys Phe Gly Asp Pro

355 360 365

Asp Leu Phe Asn Trp Leu Ala Gln Asp Arg His Val His Leu Trp Ser

370 375 380

Pro Arg Asp Ser Val Thr Pro Leu Val Arg Ile Asn Ala Val Asp Lys

385 390 395 400

Val Leu Arg Arg Arg Lys Pro Tyr Ala Leu Met Thr Phe Ala His Pro

405 410 415

Arg Phe His Pro Arg Trp Ile Leu Tyr Glu Ala Pro Gly Gly Ser Asn

420 425 430

Leu Arg Gln Tyr Ala Leu Asp Cys Thr Glu Asn Ala Leu His Ile Thr

435 440 445

Leu Pro Leu Leu Val Asp Asp Ala His Gly Thr Trp Ile Glu Lys Lys

450 455 460

Ile Arg Val Pro Leu Ala Pro Ser Gly Gln Ile Gln Asp Leu Thr Leu

465 470 475 480

Glu Lys Leu Glu Lys Lys Lys Asn Arg Leu Tyr Tyr Arg Ser Gly Phe

485 490 495

Gln Gln Phe Ala Gly Leu Ala Gly Gly Ala Glu Val Leu Phe His Arg

500 505 510

Pro Tyr Met Glu His Asp Glu Arg Ser Glu Glu Ser Leu Leu Glu Arg

515 520 525

Pro Gly Ala Val Trp Phe Lys Leu Thr Leu Asp Val Ala Thr Gln Ala

530 535 540

Pro Pro Asn Trp Leu Asp Gly Lys Gly Arg Val Arg Thr Pro Pro Glu

545 550 555 560

Val His His Phe Lys Thr Ala Leu Ser Asn Lys Ser Lys His Thr Arg

565 570 575

Thr Leu Gln Pro Gly Leu Arg Val Leu Ser Val Asp Leu Gly Met Arg

580 585 590

Thr Phe Ala Ser Cys Ser Val Phe Glu Leu Ile Glu Gly Lys Pro Glu

595 600 605

Thr Gly Arg Ala Phe Pro Val Ala Asp Glu Arg Ser Met Asp Ser Pro

610 615 620

Asn Lys Leu Trp Ala Lys His Glu Arg Ser Phe Lys Leu Thr Leu Pro

625 630 635 640

Gly Glu Thr Pro Ser Arg Lys Glu Glu Glu Glu Glu Arg Ser Ile Ala Arg

645 650 655

Ala Glu Ile Tyr Ala Leu Lys Arg Asp Ile Gln Arg Leu Lys Ser Leu

660 665 670

Leu Arg Leu Gly Glu Glu Asp Asn Asp Asn Arg Arg Asp Ala Leu Leu

675 680 685

Glu Gln Phe Phe Lys Gly Trp Gly Glu Glu Asp Val Val Pro Gly Gln

690 695 700

Ala Phe Pro Arg Ser Leu Phe Gln Gly Leu Gly Ala Ala Pro Phe Arg

705 710 715 720

Ser Thr Pro Glu Leu Trp Arg Gln His Cys Gln Thr Tyr Tyr Asp Lys

725 730 735

Ala Glu Ala Cys Leu Ala Lys His Ile Ser Asp Trp Arg Lys Arg Thr

740 745 750

Arg Pro Arg Pro Thr Ser Arg Glu Met Trp Tyr Lys Thr Arg Ser Tyr

755 760 765

His Gly Gly Lys Ser Ile Trp Met Leu Glu Tyr Leu Asp Ala Val Arg

770 775 780

Lys Leu Leu Leu Ser Trp Ser Leu Arg Gly Arg Thr Tyr Gly Ala Ile

785 790 795 800

Asn Arg Gln Asp Thr Ala Arg Phe Gly Ser Leu Ala Ser Arg Leu Leu

805 810 815

His His Ile Asn Ser Leu Lys Glu Asp Arg Ile Lys Thr Gly Ala Asp

820 825 830

Ser Ile Val Gln Ala Ala Arg Gly Tyr Ile Pro Leu Pro His Gly Lys

835 840 845

Gly Trp Glu Gln Arg Tyr Glu Pro Cys Gln Leu Ile Leu Phe Glu Asp

850 855 860

Leu Ala Arg Tyr Arg Phe Arg Val Asp Arg Pro Arg Arg Glu Asn Ser

865 870 875 880

Gln Leu Met Gln Trp Asn His Arg Ala Ile Val Ala Glu Thr Thr Met

885 890 895

Gln Ala Glu Leu Tyr Gly Gln Ile Val Glu Asn Thr Ala Ala Gly Phe

900 905 910

Ser Ser Arg Phe His Ala Ala Thr Gly Ala Pro Gly Val Arg Cys Arg

915 920 925

Phe Leu Leu Glu Arg Asp Phe Asp Asn Asp Leu Pro Lys Pro Tyr Leu

930 935 940

Leu Arg Glu Leu Ser Trp Met Leu Gly Asn Thr Lys Val Glu Ser Glu

945 950 955 960

Glu Glu Lys Leu Arg Leu Leu Ser Glu Lys Ile Arg Pro Gly Ser Leu

965 970 975

Val Pro Trp Asp Gly Gly Glu Gln Phe Ala Thr Leu His Pro Lys Arg

980 985 990

Gln Thr Leu Cys Val Ile His Ala Asp Met Asn Ala Ala Gln Asn Leu

995 1000 1005

Gln Arg Arg Phe Phe Gly Arg Cys Gly Glu Ala Phe Arg Leu Val

1010 1015 1020

Cys Gln Pro His Gly Asp Val Leu Arg Leu Ala Ser Thr Pro

1025 1030 1035

Gly Ala Arg Leu Leu Gly Ala Leu Gln Gln Leu Glu Asn Gly Gln

1040 1045 1050

Gly Ala Phe Glu Leu Val Arg Asp Met Gly Ser Thr Ser Gln Met

1055 1060 1065

Asn Arg Phe Val Met Lys Ser Leu Gly Lys Lys Lys Ile Lys Pro

1070 1075 1080

Leu Gln Asp Asn Asn Gly Asp Asp Glu Leu Glu Asp Val Leu Ser

1085 1090 1095

Val Leu Pro Glu Glu Asp Asp Thr Gly Arg Ile Thr Val Phe Arg

1100 1105 1110

Asp Ser Ser Gly Ile Phe Phe Pro Cys Asn Val Trp Ile Pro Ala

1115 1120 1125

Lys Gln Phe Trp Pro Ala Val Arg Ala Met Ile Trp Lys Val Met

1130 1135 1140

Ala Ser His Ser Leu Gly

1145

<210> 551

<211> 1194

<212> PRT

<213> Desulfonatronum thiodismutans

<400> 551

Met Val Leu Gly Arg Lys Asp Asp Thr Ala Glu Leu Arg Arg Ala Leu

1 5 10 15

Trp Thr Thr His Glu His Val Asn Leu Ala Val Ala Glu Val Glu Arg

20 25 30

Val Leu Leu Arg Cys Arg Gly Arg Ser Tyr Trp Thr Leu Asp Arg Arg

35 40 45

Gly Asp Pro Val His Val Pro Glu Ser Gln Val Ala Glu Asp Ala Leu

50 55 60

Ala Met Ala Arg Glu Ala Gln Arg Arg Asn Gly Trp Pro Val Val Gly

65 70 75 80

Glu Asp Glu Glu Ile Leu Leu Ala Leu Arg Tyr Leu Tyr Glu Gln Ile

85 90 95

Val Pro Ser Cys Leu Leu Asp Asp Leu Gly Lys Pro Leu Lys Gly Asp

100 105 110

Ala Gln Lys Ile Gly Thr Asn Tyr Ala Gly Pro Leu Phe Asp Ser Asp

115 120 125

Thr Cys Arg Arg Asp Glu Gly Lys Asp Val Ala Cys Cys Gly Pro Phe

130 135 140

His Glu Val Ala Gly Lys Tyr Leu Gly Ala Leu Pro Glu Trp Ala Thr

145 150 155 160

Pro Ile Ser Lys Gln Glu Phe Asp Gly Lys Asp Ala Ser His Leu Arg

165 170 175

Phe Lys Ala Thr Gly Gly Asp Asp Ala Phe Phe Arg Val Ser Ile Glu

180 185 190

Lys Ala Asn Ala Trp Tyr Glu Asp Pro Ala Asn Gln Asp Ala Leu Lys

195 200 205

Asn Lys Ala Tyr Asn Lys Asp Asp Trp Lys Lys Glu Lys Asp Lys Gly

210 215 220

Ile Ser Ser Trp Ala Val Lys Tyr Ile Gln Lys Gln Leu Gln Leu Gly

225 230 235 240

Gln Asp Pro Arg Thr Glu Val Arg Arg Lys Leu Trp Leu Glu Leu Gly

245 250 255

Leu Leu Pro Leu Phe Ile Pro Val Phe Asp Lys Thr Met Val Gly Asn

260 265 270

Leu Trp Asn Arg Leu Ala Val Arg Leu Ala Leu Ala His Leu Leu Ser

275 280 285

Trp Glu Ser Trp Asn His Arg Ala Val Gln Asp Gln Ala Leu Ala Arg

290 295 300

Ala Lys Arg Asp Glu Leu Ala Ala Leu Phe Leu Gly Met Glu Asp Gly

305 310 315 320

Phe Ala Gly Leu Arg Glu Tyr Glu Leu Arg Arg Asn Glu Ser Ile Lys

325 330 335

Gln His Ala Phe Glu Pro Val Asp Arg Pro Tyr Val Val Ser Gly Arg

340 345 350

Ala Leu Arg Ser Trp Thr Arg Val Arg Glu Glu Trp Leu Arg His Gly

355 360 365

Asp Thr Gln Glu Ser Arg Lys Asn Ile Cys Asn Arg Leu Gln Asp Arg

370 375 380

Leu Arg Gly Lys Phe Gly Asp Pro Asp Val Phe His Trp Leu Ala Glu

385 390 395 400

Asp Gly Gln Glu Ala Leu Trp Lys Glu Arg Asp Cys Val Thr Ser Phe

405 410 415

Ser Leu Leu Asn Asp Ala Asp Gly Leu Leu Glu Lys Arg Lys Gly Tyr

420 425 430

Ala Leu Met Thr Phe Ala Asp Ala Arg Leu His Pro Arg Trp Ala Met

435 440 445

Tyr Glu Ala Pro Gly Gly Ser Asn Leu Arg Thr Tyr Gln Ile Arg Lys

450 455 460

Thr Glu Asn Gly Leu Trp Ala Asp Val Val Leu Leu Ser Pro Arg Asn

465 470 475 480

Glu Ser Ala Ala Val Glu Glu Lys Thr Phe Asn Val Arg Leu Ala Pro

485 490 495

Ser Gly Gln Leu Ser Asn Val Ser Phe Asp Gln Ile Gln Lys Gly Ser

500 505 510

Lys Met Val Gly Arg Cys Arg Tyr Gln Ser Ala Asn Gln Gln Phe Glu

515 520 525

Gly Leu Leu Gly Gly Ala Glu Ile Leu Phe Asp Arg Lys Arg Ile Ala

530 535 540

Asn Glu Gln His Gly Ala Thr Asp Leu Ala Ser Lys Pro Gly His Val

545 550 555 560

Trp Phe Lys Leu Thr Leu Asp Val Arg Pro Gln Ala Pro Gln Gly Trp

565 570 575

Leu Asp Gly Lys Gly Arg Pro Ala Leu Pro Pro Glu Ala Lys His Phe

580 585 590

Lys Thr Ala Leu Ser Asn Lys Ser Lys Phe Ala Asp Gln Val Arg Pro

595 600 605

Gly Leu Arg Val Leu Ser Val Asp Leu Gly Val Arg Ser Phe Ala Ala

610 615 620

Cys Ser Val Phe Glu Leu Val Arg Gly Gly Pro Asp Gln Gly Thr Tyr

625 630 635 640

Phe Pro Ala Ala Asp Gly Arg Thr Val Asp Asp Pro Glu Lys Leu Trp

645 650 655

Ala Lys His Glu Arg Ser Phe Lys Ile Thr Leu Pro Gly Glu Asn Pro

660 665 670

Ser Arg Lys Glu Glu Ile Ala Arg Arg Ala Ala Met Glu Glu Leu Arg

675 680 685

Ser Leu Asn Gly Asp Ile Arg Arg Leu Lys Ala Ile Leu Arg Leu Ser

690 695 700

Val Leu Gln Glu Asp Asp Pro Arg Thr Glu His Leu Arg Leu Phe Met

705 710 715 720

Glu Ala Ile Val Asp Asp Pro Ala Lys Ser Ala Leu Asn Ala Glu Leu

725 730 735

Phe Lys Gly Phe Gly Asp Asp Arg Phe Arg Ser Thr Pro Asp Leu Trp

740 745 750

Lys Gln His Cys His Phe Phe His Asp Lys Ala Glu Lys Val Val Ala

755 760 765

Glu Arg Phe Ser Arg Trp Arg Thr Glu Thr Arg Pro Lys Ser Ser Ser

770 775 780

Trp Gln Asp Trp Arg Glu Arg Arg Gly Tyr Ala Gly Gly Lys Ser Tyr

785 790 795 800

Trp Ala Val Thr Tyr Leu Glu Ala Val Arg Gly Leu Ile Leu Arg Trp

805 810 815

Asn Met Arg Gly Arg Thr Tyr Gly Glu Val Asn Arg Gln Asp Lys Lys

820 825 830

Gln Phe Gly Thr Val Ala Ser Ala Leu Leu His His Ile Asn Gln Leu

835 840 845

Lys Glu Asp Arg Ile Lys Thr Gly Ala Asp Met Ile Ile Gln Ala Ala

850 855 860

Arg Gly Phe Val Pro Arg Lys Asn Gly Ala Gly Trp Val Gln Val His

865 870 875 880

Glu Pro Cys Arg Leu Ile Leu Phe Glu Asp Leu Ala Arg Tyr Arg Phe

885 890 895

Arg Thr Asp Arg Ser Arg Arg Glu Asn Ser Arg Leu Met Arg Trp Ser

900 905 910

His Arg Glu Ile Val Asn Glu Val Gly Met Gln Gly Glu Leu Tyr Gly

915 920 925

Leu His Val Asp Thr Thr Glu Ala Gly Phe Ser Ser Arg Tyr Leu Ala

930 935 940

Ser Ser Gly Ala Pro Gly Val Arg Cys Arg His Leu Val Glu Glu Asp

945 950 955 960

Phe His Asp Gly Leu Pro Gly Met His Leu Val Gly Glu Leu Asp Trp

965 970 975

Leu Leu Pro Lys Asp Lys Asp Arg Thr Ala Asn Glu Ala Arg Arg Leu

980 985 990

Leu Gly Gly Met Val Arg Pro Gly Met Leu Val Pro Trp Asp Gly Gly

995 1000 1005

Glu Leu Phe Ala Thr Leu Asn Ala Ala Ser Gln Leu His Val Ile

1010 1015 1020

His Ala Asp Ile Asn Ala Ala Gln Asn Leu Gln Arg Arg Phe Trp

1025 1030 1035

Gly Arg Cys Gly Glu Ala Ile Arg Ile Val Cys Asn Gln Leu Ser

1040 1045 1050

Val Asp Gly Ser Thr Arg Tyr Glu Met Ala Lys Ala Pro Lys Ala

1055 1060 1065

Arg Leu Leu Gly Ala Leu Gln Gln Leu Lys Asn Gly Asp Ala Pro

1070 1075 1080

Phe His Leu Thr Ser Ile Pro Asn Ser Gln Lys Pro Glu Asn Ser

1085 1090 1095

Tyr Val Met Thr Pro Thr Asn Ala Gly Lys Lys Tyr Arg Ala Gly

1100 1105 1110

Pro Gly Glu Lys Ser Ser Gly Glu Glu Asp Glu Leu Ala Leu Asp

1115 1120 1125

Ile Val Glu Gln Ala Glu Glu Leu Ala Gln Gly Arg Lys Thr Phe

1130 1135 1140

Phe Arg Asp Pro Ser Gly Val Phe Phe Ala Pro Asp Arg Trp Leu

1145 1150 1155

Pro Ser Glu Ile Tyr Trp Ser Arg Ile Arg Arg Arg Ile Trp Gln

1160 1165 1170

Val Thr Leu Glu Arg Asn Ser Ser Gly Arg Gln Glu Arg Ala Glu

1175 1180 1185

Met Asp Glu Met Pro Tyr

1190

<210> 552

<211> 1388

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 552

Met Ser Leu Asn Arg Ile Tyr Gln Gly Arg Val Ala Ala Val Glu Thr

1 5 10 15

Gly Thr Ala Leu Ala Lys Gly Asn Val Glu Trp Met Pro Ala Ala Gly

20 25 30

Gly Asp Glu Val Leu Trp Gln His His Glu Leu Phe Gln Ala Ala Ile

35 40 45

Asn Tyr Tyr Leu Val Ala Leu Leu Ala Leu Ala Asp Lys Asn Asn Pro

50 55 60

Val Leu Gly Pro Leu Ile Ser Gln Met Asp Asn Pro Gln Ser Pro Tyr

65 70 75 80

His Val Trp Gly Ser Phe Arg Arg Gln Gly Arg Gln Arg Thr Gly Leu

85 90 95

Ser Gln Ala Val Ala Pro Tyr Ile Thr Pro Gly Asn Asn Ala Pro Thr

100 105 110

Leu Asp Glu Val Phe Arg Ser Ile Leu Ala Gly Asn Pro Thr Asp Arg

115 120 125

Ala Thr Leu Asp Ala Ala Leu Met Gln Leu Leu Lys Ala Cys Asp Gly

130 135 140

Ala Gly Ala Ile Gln Gln Glu Gly Arg Ser Tyr Trp Pro Lys Phe Cys

145 150 155 160

Asp Pro Asp Ser Thr Ala Asn Phe Ala Gly Asp Pro Ala Met Leu Arg

165 170 175

Arg Glu Gln His Arg Leu Leu Leu Pro Gln Val Leu His Asp Pro Ala

180 185 190

Ile Thr His Asp Ser Pro Ala Leu Gly Ser Phe Asp Thr Tyr Ser Ile

195 200 205

Ala Thr Pro Asp Thr Arg Thr Pro Gln Leu Thr Gly Pro Lys Ala Arg

210 215 220

Ala Arg Leu Glu Gln Ala Ile Thr Leu Trp Arg Val Arg Leu Pro Glu

225 230 235 240

Ser Ala Ala Asp Phe Asp Arg Leu Ala Ser Ser Leu Lys Lys Ile Pro

245 250 255

Asp Asp Asp Ser Arg Leu Asn Leu Gln Gly Tyr Val Gly Ser Ser Ala

260 265 270

Lys Gly Glu Val Gln Ala Arg Leu Phe Ala Leu Leu Leu Phe Arg His

275 280 285

Leu Glu Arg Ser Ser Phe Thr Leu Gly Leu Leu Arg Ser Ala Thr Pro

290 295 300

Pro Pro Lys Asn Ala Glu Thr Pro Pro Pro Ala Gly Val Pro Leu Pro

305 310 315 320

Ala Ala Ser Ala Ala Asp Pro Val Arg Ile Ala Arg Gly Lys Arg Ser

325 330 335

Phe Val Phe Arg Ala Phe Thr Ser Leu Pro Cys Trp His Gly Gly Asp

340 345 350

Asn Ile His Pro Thr Trp Lys Ser Phe Asp Ile Ala Ala Phe Lys Tyr

355 360 365

Ala Leu Thr Val Ile Asn Gln Ile Glu Glu Lys Thr Lys Glu Arg Gln

370 375 380

Lys Glu Cys Ala Glu Leu Glu Thr Asp Phe Asp Tyr Met His Gly Arg

385 390 395 400

Leu Ala Lys Ile Pro Val Lys Tyr Thr Thr Gly Glu Ala Glu Pro Pro

405 410 415

Pro Ile Leu Ala Asn Asp Leu Arg Ile Pro Leu Leu Arg Glu Leu Leu

420 425 430

Gln Asn Ile Lys Val Asp Thr Ala Leu Thr Asp Gly Glu Ala Val Ser

435 440 445

Tyr Gly Leu Gln Arg Arg Thr Ile Arg Gly Phe Arg Glu Leu Arg Arg

450 455 460

Ile Trp Arg Gly His Ala Pro Ala Gly Thr Val Phe Ser Ser Glu Leu

465 470 475 480

Lys Glu Lys Leu Ala Gly Glu Leu Arg Gln Phe Gln Thr Asp Asn Ser

485 490 495

Thr Thr Ile Gly Ser Val Gln Leu Phe Asn Glu Leu Ile Gln Asn Pro

500 505 510

Lys Tyr Trp Pro Ile Trp Gln Ala Pro Asp Val Glu Thr Ala Arg Gln

515 520 525

Trp Ala Asp Ala Gly Phe Ala Asp Asp Pro Leu Ala Ala Leu Val Gln

530 535 540

Glu Ala Glu Leu Gln Glu Asp Ile Asp Ala Leu Lys Ala Pro Val Lys

545 550 555 560

Leu Thr Pro Ala Asp Pro Glu Tyr Ser Arg Arg Gln Tyr Asp Phe Asn

565 570 575

Ala Val Ser Lys Phe Gly Ala Gly Ser Arg Ser Ala Asn Arg His Glu

580 585 590

Pro Gly Gln Thr Glu Arg Gly His Asn Thr Phe Thr Thr Glu Ile Ala

595 600 605

Ala Arg Asn Ala Ala Asp Gly Asn Arg Trp Arg Ala Thr His Val Arg

610 615 620

Ile His Tyr Ser Ala Pro Arg Leu Leu Arg Asp Gly Leu Arg Arg Pro

625 630 635 640

Asp Thr Asp Gly Asn Glu Ala Leu Glu Ala Val Pro Trp Leu Gln Pro

645 650 655

Met Met Glu Ala Leu Ala Pro Leu Pro Thr Leu Pro Gln Asp Leu Thr

660 665 670

Gly Met Pro Val Phe Leu Met Pro Asp Val Thr Leu Ser Gly Glu Arg

675 680 685

Arg Ile Leu Leu Asn Leu Pro Val Thr Leu Glu Pro Ala Ala Leu Val

690 695 700

Glu Gln Leu Gly Asn Ala Gly Arg Trp Gln Asn Gln Phe Phe Gly Ser

705 710 715 720

Arg Glu Asp Pro Phe Ala Leu Arg Trp Pro Ala Asp Gly Ala Val Lys

725 730 735

Thr Ala Lys Gly Lys Thr His Ile Pro Trp His Gln Asp Arg Asp His

740 745 750

Phe Thr Val Leu Gly Val Asp Leu Gly Thr Arg Asp Ala Gly Ala Leu

755 760 765

Ala Leu Leu Asn Val Thr Ala Gln Lys Pro Ala Lys Pro Val His Arg

770 775 780

Ile Ile Gly Glu Ala Asp Gly Arg Thr Trp Tyr Ala Ser Leu Ala Asp

785 790 795 800

Ala Arg Met Ile Arg Leu Pro Gly Glu Asp Ala Arg Leu Phe Val Arg

805 810 815

Gly Lys Leu Val Gln Glu Pro Tyr Gly Glu Arg Gly Arg Asn Ala Ser

820 825 830

Leu Leu Glu Trp Glu Asp Ala Arg Asn Ile Ile Leu Arg Leu Gly Gln

835 840 845

Asn Pro Asp Glu Leu Leu Gly Ala Asp Pro Arg Arg His Ser Tyr Pro

850 855 860

Glu Ile Asn Asp Lys Leu Leu Val Ala Leu Arg Arg Ala Gln Ala Arg

865 870 875 880

Leu Ala Arg Leu Gln Asn Arg Ser Trp Arg Leu Arg Asp Leu Ala Glu

885 890 895

Ser Asp Lys Ala Leu Asp Glu Ile His Ala Glu Arg Ala Gly Glu Lys

900 905 910

Pro Ser Pro Leu Pro Pro Leu Ala Arg Asp Asp Ala Ile Lys Ser Thr

915 920 925

Asp Glu Ala Leu Leu Ser Gln Arg Asp Ile Ile Arg Arg Ser Phe Val

930 935 940

Gln Ile Ala Asn Leu Ile Leu Pro Leu Arg Gly Arg Arg Trp Glu Trp

945 950 955 960

Arg Pro His Val Glu Val Pro Asp Cys His Ile Leu Ala Gln Ser Asp

965 970 975

Pro Gly Thr Asp Asp Thr Lys Arg Leu Val Ala Gly Gln Arg Gly Ile

980 985 990

Ser His Glu Arg Ile Glu Gln Ile Glu Glu Leu Arg Arg Arg Cys Gln

995 1000 1005

Ser Leu Asn Arg Ala Leu Arg His Lys Pro Gly Glu Arg Pro Val

1010 1015 1020

Leu Gly Arg Pro Ala Lys Gly Glu Glu Ile Ala Asp Pro Cys Pro

1025 1030 1035

Ala Leu Leu Glu Lys Ile Asn Arg Leu Arg Asp Gln Arg Val Asp

1040 1045 1050

Gln Thr Ala His Ala Ile Leu Ala Ala Ala Leu Gly Val Arg Leu

1055 1060 1065

Arg Ala Pro Ser Lys Asp Arg Ala Glu Arg Arg His Arg Asp Ile

1070 1075 1080

His Gly Glu Tyr Glu Arg Phe Arg Ala Pro Ala Asp Phe Val Val

1085 1090 1095

Ile Glu Asn Leu Ser Arg Tyr Leu Ser Ser Gln Asp Arg Ala Arg

1100 1105 1110

Ser Glu Asn Thr Arg Leu Met Gln Trp Cys His Arg Gln Ile Val

1115 1120 1125

Gln Lys Leu Arg Gln Leu Cys Glu Thr Tyr Gly Ile Pro Val Leu

1130 1135 1140

Ala Val Pro Ala Ala Tyr Ser Ser Arg Phe Ser Ser Arg Asp Gly

1145 1150 1155

Ser Ala Gly Phe Arg Ala Val His Leu Thr Pro Asp His Arg His

1160 1165 1170

Arg Met Pro Trp Ser Arg Ile Leu Ala Arg Leu Lys Ala His Glu

1175 1180 1185

Glu Asp Gly Lys Arg Leu Glu Lys Thr Val Leu Asp Glu Ala Arg

1190 1195 1200

Ala Val Arg Gly Leu Phe Asp Arg Leu Asp Arg Phe Asn Ala Gly

1205 1210 1215

His Val Pro Gly Lys Pro Trp Arg Thr Leu Leu Ala Pro Leu Pro

1220 1225 1230

Gly Gly Pro Val Phe Val Pro Leu Gly Asp Ala Thr Pro Met Gln

1235 1240 1245

Ala Asp Leu Asn Ala Ala Ile Asn Ile Ala Leu Arg Gly Ile Ala

1250 1255 1260

Ala Pro Asp Arg His Asp Ile His His Arg Leu Arg Ala Glu Asn

1265 1270 1275

Lys Lys Arg Ile Leu Ser Leu Arg Leu Gly Thr Gln Arg Glu Lys

1280 1285 1290

Ala Arg Trp Pro Gly Gly Ala Pro Ala Val Thr Leu Ser Thr Pro

1295 1300 1305

Asn Asn Gly Ala Ser Pro Glu Asp Ser Asp Ala Leu Pro Glu Arg

1310 1315 1320

Val Ser Asn Leu Phe Val Asp Ile Ala Gly Val Ala Asn Phe Glu

1325 1330 1335

Arg Val Thr Ile Glu Gly Val Ser Gln Lys Phe Ala Thr Gly Arg

1340 1345 1350

Gly Leu Trp Ala Ser Val Lys Gln Arg Ala Trp Asn Arg Val Ala

1355 1360 1365

Arg Leu Asn Glu Thr Val Thr Asp Asn Asn Arg Asn Glu Glu Glu

1370 1375 1380

Asp Asp Ile Pro Met

1385

<210> 553

<211> 1132

<212> PRT

<213> Tuberibacillus calidus

<400> 553

Met Ala Thr Lys Ser Phe Ile Leu Lys Met Lys Thr Lys Asn Asn Pro

1 5 10 15

Gln Leu Arg Leu Ser Leu Trp Lys Thr His Glu Leu Phe Asn Phe Gly

20 25 30

Val Ala Tyr Tyr Met Asp Leu Leu Ser Leu Phe Arg Gln Lys Asp Leu

35 40 45

Tyr Met His Asn Asp Glu Asp Pro Asp His Pro Val Val Leu Lys Lys

50 55 60

Glu Glu Ile Gln Glu Arg Leu Trp Met Lys Val Arg Glu Thr Gln Gln

65 70 75 80

Lys Asn Gly Phe His Gly Glu Val Ser Lys Asp Glu Val Leu Glu Thr

85 90 95

Leu Arg Ala Leu Tyr Glu Glu Leu Val Pro Ser Ala Val Gly Lys Ser

100 105 110

Gly Glu Ala Asn Gln Ile Ser Asn Lys Tyr Leu Tyr Pro Leu Thr Asp

115 120 125

Pro Ala Ser Gln Ser Gly Lys Gly Thr Ala Asn Ser Gly Arg Lys Pro

130 135 140

Arg Trp Lys Lys Leu Lys Glu Ala Gly Asp Pro Ser Trp Lys Asp Ala

145 150 155 160

Tyr Glu Lys Trp Glu Lys Glu Arg Gln Glu Asp Pro Lys Leu Lys Ile

165 170 175

Leu Ala Ala Leu Gln Ser Phe Gly Leu Ile Pro Leu Phe Arg Pro Phe

180 185 190

Thr Glu Asn Asp His Lys Ala Val Ile Ser Val Lys Trp Met Pro Lys

195 200 205

Ser Lys Asn Gln Ser Val Arg Lys Phe Asp Lys Asp Met Phe Asn Gln

210 215 220

Ala Ile Glu Arg Phe Leu Ser Trp Glu Ser Trp Asn Glu Lys Val Ala

225 230 235 240

Glu Asp Tyr Glu Lys Thr Val Ser Ile Tyr Glu Ser Leu Gln Lys Glu

245 250 255

Leu Lys Gly Ile Ser Thr Lys Ala Phe Glu Ile Met Glu Arg Val Glu

260 265 270

Lys Ala Tyr Glu Ala His Leu Arg Glu Ile Thr Phe Ser Asn Ser Thr

275 280 285

Tyr Arg Ile Gly Asn Arg Ala Ile Arg Gly Trp Thr Glu Ile Val Lys

290 295 300

Lys Trp Met Lys Leu Asp Pro Ser Ala Pro Gln Gly Asn Tyr Leu Asp

305 310 315 320

Val Val Lys Asp Tyr Gln Arg Arg His Pro Arg Glu Ser Gly Asp Phe

325 330 335

Lys Leu Phe Glu Leu Leu Ser Arg Pro Glu Asn Gln Ala Ala Trp Arg

340 345 350

Glu Tyr Pro Glu Phe Leu Pro Leu Tyr Val Lys Tyr Arg His Ala Glu

355 360 365

Gln Arg Met Lys Thr Ala Lys Lys Gln Ala Thr Phe Thr Leu Cys Asp

370 375 380

Pro Ile Arg His Pro Leu Trp Val Arg Tyr Glu Glu Glu Arg Ser Gly Thr

385 390 395 400

Asn Leu Asn Lys Tyr Arg Leu Ile Met Asn Glu Lys Glu Lys Val Val

405 410 415

Gln Phe Asp Arg Leu Ile Cys Leu Asn Ala Asp Gly His Tyr Glu Glu

420 425 430

Gln Glu Asp Val Thr Val Pro Leu Ala Pro Ser Gln Gln Phe Asp Asp

435 440 445

Gln Ile Lys Phe Ser Ser Glu Asp Thr Gly Lys Gly Lys His Asn Phe

450 455 460

Ser Tyr Tyr His Lys Gly Ile Asn Tyr Glu Leu Lys Gly Thr Leu Gly

465 470 475 480

Gly Ala Arg Ile Gln Phe Asp Arg Glu His Leu Leu Arg Arg Gln Gly

485 490 495

Val Lys Ala Gly Asn Val Gly Arg Ile Phe Leu Asn Val Thr Leu Asn

500 505 510

Ile Glu Pro Met Gln Pro Phe Ser Arg Ser Gly Asn Leu Gln Thr Ser

515 520 525

Val Gly Lys Ala Leu Lys Val Tyr Val Asp Gly Tyr Pro Lys Val Val

530 535 540

Asn Phe Lys Pro Lys Glu Leu Thr Glu His Ile Lys Glu Ser Glu Lys

545 550 555 560

Asn Thr Leu Thr Leu Gly Val Glu Ser Leu Pro Thr Gly Leu Arg Val

565 570 575

Met Ser Val Asp Leu Gly Gln Arg Gln Ala Ala Ala Ile Ser Ile Phe

580 585 590

Glu Val Val Ser Glu Lys Pro Asp Asp Asn Lys Leu Phe Tyr Pro Val

595 600 605

Lys Asp Thr Asp Leu Phe Ala Val His Arg Thr Ser Phe Asn Ile Lys

610 615 620

Leu Pro Gly Glu Lys Arg Thr Glu Arg Arg Met Leu Glu Gln Gln Lys

625 630 635 640

Arg Asp Gln Ala Ile Arg Asp Leu Ser Arg Lys Leu Lys Phe Leu Lys

645 650 655

Asn Val Leu Asn Met Gln Lys Leu Glu Lys Thr Asp Glu Arg Glu Lys

660 665 670

Arg Val Asn Arg Trp Ile Lys Asp Arg Glu Arg Glu Glu Glu Asn Pro

675 680 685

Val Tyr Val Gln Glu Phe Glu Met Ile Ser Lys Val Leu Tyr Ser Pro

690 695 700

His Ser Val Trp Val Asp Gln Leu Lys Ser Ile His Arg Lys Leu Glu

705 710 715 720

Glu Gln Leu Gly Lys Glu Ile Ser Lys Trp Arg Gln Ser Ile Ser Gln

725 730 735

Gly Arg Gln Gly Val Tyr Gly Ile Ser Leu Lys Asn Ile Glu Asp Ile

740 745 750

Glu Lys Thr Arg Arg Leu Leu Phe Arg Trp Ser Met Arg Pro Glu Asn

755 760 765

Pro Gly Glu Val Lys Gln Leu Gln Pro Gly Glu Arg Phe Ala Ile Asp

770 775 780

Gln Gln Asn His Leu Asn His Leu Lys Asp Asp Arg Ile Lys Lys Leu

785 790 795 800

Ala Asn Gln Ile Val Met Thr Ala Leu Gly Tyr Arg Tyr Asp Gly Lys

805 810 815

Arg Lys Lys Trp Ile Ala Lys His Pro Ala Cys Gln Leu Val Leu Phe

820 825 830

Glu Asp Leu Ser Arg Tyr Ala Phe Tyr Asp Glu Arg Ser Arg Leu Glu

835 840 845

Asn Arg Asn Leu Met Arg Trp Ser Arg Arg Glu Ile Pro Lys Gln Val

850 855 860

Ala Gln Ile Gly Gly Leu Tyr Gly Leu Leu Val Gly Glu Val Gly Ala

865 870 875 880

Gln Tyr Ser Ser Arg Phe His Ala Lys Ser Gly Ala Pro Gly Ile Arg

885 890 895

Cys Arg Val Val Lys Glu His Glu Leu Tyr Ile Thr Glu Gly Gly Gln

900 905 910

Lys Val Arg Asn Gln Lys Phe Leu Asp Ser Leu Val Glu Asn Asn Ile

915 920 925

Ile Glu Pro Asp Asp Ala Arg Arg Leu Glu Pro Gly Asp Leu Ile Arg

930 935 940

Asp Gln Gly Gly Asp Lys Phe Ala Thr Leu Asp Glu Arg Gly Glu Leu

945 950 955 960

Val Ile Thr His Ala Asp Ile Asn Ala Ala Gln Asn Leu Gln Lys Arg

965 970 975

Phe Trp Thr Arg Thr His Gly Leu Tyr Arg Ile Arg Cys Glu Ser Arg

980 985 990

Glu Ile Lys Asp Ala Val Val Leu Val Pro Ser Asp Lys Asp Gln Lys

995 1000 1005

Glu Lys Met Glu Asn Leu Phe Gly Ile Gly Tyr Leu Gln Pro Phe

1010 1015 1020

Lys Gln Glu Asn Asp Val Tyr Lys Trp Val Lys Gly Glu Lys Ile

1025 1030 1035

Lys Gly Lys Lys Thr Ser Ser Gln Ser Asp Asp Lys Glu Leu Val

1040 1045 1050

Ser Glu Ile Leu Gln Glu Ala Ser Val Met Ala Asp Glu Leu Lys

1055 1060 1065

Gly Asn Arg Lys Thr Leu Phe Arg Asp Pro Ser Gly Tyr Val Phe

1070 1075 1080

Pro Lys Asp Arg Trp Tyr Thr Gly Gly Arg Tyr Phe Gly Thr Leu

1085 1090 1095

Glu His Leu Leu Lys Arg Lys Leu Ala Glu Arg Arg Leu Phe Asp

1100 1105 1110

Gly Gly Ser Ser Arg Arg Gly Leu Phe Asn Gly Thr Asp Ser Asn

1115 1120 1125

Thr Asn Val Glu

1130

<210> 554

<211> 1108

<212> PRT

<213> Bacillus thermoamylovorans

<400> 554

Met Ala Thr Arg Ser Phe Ile Leu Lys Ile Glu Pro Asn Glu Glu Val

1 5 10 15

Lys Lys Gly Leu Trp Lys Thr His Glu Val Leu Asn His Gly Ile Ala

20 25 30

Tyr Tyr Met Asn Ile Leu Lys Leu Ile Arg Gln Glu Ala Ile Tyr Glu

35 40 45

His His Glu Gln Asp Pro Lys Asn Pro Lys Lys Val Ser Lys Ala Glu

50 55 60

Ile Gln Ala Glu Leu Trp Asp Phe Val Leu Lys Met Gln Lys Cys Asn

65 70 75 80

Ser Phe Thr His Glu Val Asp Lys Asp Val Val Phe Asn Ile Leu Arg

85 90 95

Glu Leu Tyr Glu Glu Leu Val Pro Ser Ser Val Glu Lys Lys Gly Glu

100 105 110

Ala Asn Gln Leu Ser Asn Lys Phe Leu Tyr Pro Leu Val Asp Pro Asn

115 120 125

Ser Gln Ser Gly Lys Gly Thr Ala Ser Ser Gly Arg Lys Pro Arg Trp

130 135 140

Tyr Asn Leu Lys Ile Ala Gly Asp Pro Ser Trp Glu Glu Glu Lys Lys

145 150 155 160

Lys Trp Glu Glu Asp Lys Lys Lys Asp Pro Leu Ala Lys Ile Leu Gly

165 170 175

Lys Leu Ala Glu Tyr Gly Leu Ile Pro Leu Phe Ile Pro Phe Thr Asp

180 185 190

Ser Asn Glu Pro Ile Val Lys Glu Ile Lys Trp Met Glu Lys Ser Arg

195 200 205

Asn Gln Ser Val Arg Arg Leu Asp Lys Asp Met Phe Ile Gln Ala Leu

210 215 220

Glu Arg Phe Leu Ser Trp Glu Ser Trp Asn Leu Lys Val Lys Glu Glu

225 230 235 240

Tyr Glu Lys Val Glu Lys Glu His Lys Thr Leu Glu Glu Arg Ile Lys

245 250 255

Glu Asp Ile Gln Ala Phe Lys Ser Leu Glu Gln Tyr Glu Lys Glu Arg

260 265 270

Gln Glu Gln Leu Leu Arg Asp Thr Leu Asn Thr Asn Glu Tyr Arg Leu

275 280 285

Ser Lys Arg Gly Leu Arg Gly Trp Arg Glu Ile Ile Gln Lys Trp Leu

290 295 300

Lys Met Asp Glu Asn Glu Pro Ser Glu Lys Tyr Leu Glu Val Phe Lys

305 310 315 320

Asp Tyr Gln Arg Lys His Pro Arg Glu Ala Gly Asp Tyr Ser Val Tyr

325 330 335

Glu Phe Leu Ser Lys Lys Glu Asn His Phe Ile Trp Arg Asn His Pro

340 345 350

Glu Tyr Pro Tyr Leu Tyr Ala Thr Phe Cys Glu Ile Asp Lys Lys Lys

355 360 365

Lys Asp Ala Lys Gln Gln Ala Thr Phe Thr Leu Ala Asp Pro Ile Asn

370 375 380

His Pro Leu Trp Val Arg Phe Glu Glu Arg Ser Gly Ser Asn Leu Asn

385 390 395 400

Lys Tyr Arg Ile Leu Thr Glu Gln Leu His Thr Glu Lys Leu Lys Lys

405 410 415

Lys Leu Thr Val Gln Leu Asp Arg Leu Ile Tyr Pro Thr Glu Ser Gly

420 425 430

Gly Trp Glu Glu Lys Gly Lys Val Asp Ile Val Leu Leu Pro Ser Arg

435 440 445

Gln Phe Tyr Asn Gln Ile Phe Leu Asp Ile Glu Glu Lys Gly Lys His

450 455 460

Ala Phe Thr Tyr Lys Asp Glu Ser Ile Lys Phe Pro Leu Lys Gly Thr

465 470 475 480

Leu Gly Gly Ala Arg Val Gln Phe Asp Arg Asp His Leu Arg Arg Tyr

485 490 495

Pro His Lys Val Glu Ser Gly Asn Val Gly Arg Ile Tyr Phe Asn Met

500 505 510

Thr Val Asn Ile Glu Pro Thr Glu Ser Pro Val Ser Lys Ser Leu Lys

515 520 525

Ile His Arg Asp Asp Phe Pro Lys Phe Val Asn Phe Lys Pro Lys Glu

530 535 540

Leu Thr Glu Trp Ile Lys Asp Ser Lys Gly Lys Lys Leu Lys Ser Gly

545 550 555 560

Ile Glu Ser Leu Glu Ile Gly Leu Arg Val Met Ser Ile Asp Leu Gly

565 570 575

Gln Arg Gln Ala Ala Ala Ala Ser Ile Phe Glu Val Val Asp Gln Lys

580 585 590

Pro Asp Ile Glu Gly Lys Leu Phe Phe Pro Ile Lys Gly Thr Glu Leu

595 600 605

Tyr Ala Val His Arg Ala Ser Phe Asn Ile Lys Leu Pro Gly Glu Thr

610 615 620

Leu Val Lys Ser Arg Glu Val Leu Arg Lys Ala Arg Glu Asp Asn Leu

625 630 635 640

Lys Leu Met Asn Gln Lys Leu Asn Phe Leu Arg Asn Val Leu His Phe

645 650 655

Gln Gln Phe Glu Asp Ile Thr Glu Arg Glu Lys Arg Val Thr Lys Trp

660 665 670

Ile Ser Arg Gln Glu Asn Ser Asp Val Pro Leu Val Tyr Gln Asp Glu

675 680 685

Leu Ile Gln Ile Arg Glu Leu Met Tyr Lys Pro Tyr Lys Asp Trp Val

690 695 700

Ala Phe Leu Lys Gln Leu His Lys Arg Leu Glu Val Glu Ile Gly Lys

705 710 715 720

Glu Val Lys His Trp Arg Lys Ser Leu Ser Asp Gly Arg Lys Gly Leu

725 730 735

Tyr Gly Ile Ser Leu Lys Asn Ile Asp Glu Ile Asp Arg Thr Arg Lys

740 745 750

Phe Leu Leu Arg Trp Ser Leu Arg Pro Thr Glu Pro Gly Glu Val Arg

755 760 765

Arg Leu Glu Pro Gly Gln Arg Phe Ala Ile Asp Gln Leu Asn His Leu

770 775 780

Asn Ala Leu Lys Glu Asp Arg Leu Lys Lys Met Ala Asn Thr Ile Ile

785 790 795 800

Met His Ala Leu Gly Tyr Cys Tyr Asp Val Arg Lys Lys Lys Trp Gln

805 810 815

Ala Lys Asn Pro Ala Cys Gln Ile Ile Leu Phe Glu Asp Leu Ser Asn

820 825 830

Tyr Asn Pro Tyr Glu Glu Arg Ser Arg Phe Glu Asn Ser Lys Leu Met

835 840 845

Lys Trp Ser Arg Arg Glu Ile Pro Arg Gln Val Ala Leu Gln Gly Glu

850 855 860

Ile Tyr Gly Leu Gln Val Gly Glu Val Gly Ala Gln Phe Ser Ser Arg

865 870 875 880

Phe His Ala Lys Thr Gly Ser Pro Gly Ile Arg Cys Ser Val Val Thr

885 890 895

Lys Glu Lys Leu Gln Asp Asn Arg Phe Phe Lys Asn Leu Gln Arg Glu

900 905 910

Gly Arg Leu Thr Leu Asp Lys Ile Ala Val Leu Lys Glu Gly Asp Leu

915 920 925

Tyr Pro Asp Lys Gly Gly Glu Lys Phe Ile Ser Leu Ser Lys Asp Arg

930 935 940

Lys Leu Val Thr Thr His Ala Asp Ile Asn Ala Ala Gln Asn Leu Gln

945 950 955 960

Lys Arg Phe Trp Thr Arg Thr His Gly Phe Tyr Lys Val Tyr Cys Lys

965 970 975

Ala Tyr Gln Val Asp Gly Gln Thr Val Tyr Ile Pro Glu Ser Lys Asp

980 985 990

Gln Lys Gln Lys Ile Ile Glu Glu Phe Gly Glu Gly Tyr Phe Ile Leu

995 1000 1005

Lys Asp Gly Val Tyr Glu Trp Gly Asn Ala Gly Lys Leu Lys Ile

1010 1015 1020

Lys Lys Gly Ser Ser Lys Gln Ser Ser Ser Glu Leu Val Asp Ser

1025 1030 1035

Asp Ile Leu Lys Asp Ser Phe Asp Leu Ala Ser Glu Leu Lys Gly

1040 1045 1050

Glu Lys Leu Met Leu Tyr Arg Asp Pro Ser Gly Asn Val Phe Pro

1055 1060 1065

Ser Asp Lys Trp Met Ala Ala Gly Val Phe Phe Gly Lys Leu Glu

1070 1075 1080

Arg Ile Leu Ile Ser Lys Leu Thr Asn Gln Tyr Ser Ile Ser Thr

1085 1090 1095

Ile Glu Asp Asp Ser Ser Lys Gln Ser Met

1100 1105

<210> 555

<211> 864

<212> PRT

<213> Brevibacillus sp.

<400> 555

Met Pro Lys Ile Leu Arg Gly His Lys Trp Ile Ser Leu Leu Glu Gln

1 5 10 15

Tyr Glu Glu Asn Arg Glu Arg Glu Leu Arg Glu Asn Met Thr Ala Ala

20 25 30

Asn Asp Lys Tyr Arg Ile Thr Lys Arg Gln Met Lys Gly Trp Asn Glu

35 40 45

Leu Tyr Glu Leu Trp Ser Thr Phe Pro Ala Ser Ala Ser His Glu Gln

50 55 60

Tyr Lys Glu Ala Leu Lys Arg Val Gln Gln Arg Leu Arg Gly Arg Phe

65 70 75 80

Gly Asp Ala His Phe Phe Gln Tyr Leu Met Glu Glu Glu Lys Asn Arg Leu

85 90 95

Ile Trp Lys Gly Asn Pro Gln Arg Ile His Tyr Phe Val Ala Arg Asn

100 105 110

Glu Leu Thr Lys Arg Leu Glu Glu Ala Lys Gln Ser Ala Thr Met Thr

115 120 125

Leu Pro Asn Ala Arg Lys His Pro Leu Trp Val Arg Phe Asp Ala Arg

130 135 140

Gly Gly Asn Leu Gln Asp Tyr Tyr Leu Thr Ala Glu Ala Asp Lys Pro

145 150 155 160

Arg Ser Arg Arg Phe Val Thr Phe Ser Gln Leu Ile Trp Pro Ser Glu

165 170 175

Ser Gly Trp Met Glu Lys Lys Asp Val Glu Val Glu Leu Ala Leu Ser

180 185 190

Arg Gln Phe Tyr Gln Gln Val Lys Leu Leu Lys Asn Asp Lys Gly Lys

195 200 205

Gln Lys Ile Glu Phe Lys Asp Lys Gly Ser Gly Ser Thr Phe Asn Gly

210 215 220

His Leu Gly Gly Ala Lys Leu Gln Leu Glu Arg Gly Asp Leu Glu Lys

225 230 235 240

Glu Glu Lys Asn Phe Glu Asp Gly Glu Ile Gly Ser Val Tyr Leu Asn

245 250 255

Val Val Ile Asp Phe Glu Pro Leu Gln Glu Val Lys Asn Gly Arg Val

260 265 270

Gln Ala Pro Tyr Gly Gln Val Leu Gln Leu Ile Arg Arg Pro Asn Glu

275 280 285

Phe Pro Lys Val Thr Thr Tyr Lys Ser Glu Gln Leu Val Glu Trp Ile

290 295 300

Lys Ala Ser Pro Gln His Ser Ala Gly Val Glu Ser Leu Ala Ser Gly

305 310 315 320

Phe Arg Val Met Ser Ile Asp Leu Gly Leu Arg Ala Ala Ala Ala Thr

325 330 335

Ser Ile Phe Ser Val Glu Glu Ser Ser Asp Lys Asn Ala Ala Asp Phe

340 345 350

Ser Tyr Trp Ile Glu Gly Thr Pro Leu Val Ala Val His His Arg Ser

355 360 365

Tyr Met Leu Arg Leu Pro Gly Glu Gln Val Glu Lys Gln Val Met Glu

370 375 380

Lys Arg Asp Glu Arg Phe Gln Leu His Gln Arg Val Lys Phe Gln Ile

385 390 395 400

Arg Val Leu Ala Gln Ile Met Arg Met Ala Asn Lys Gln Tyr Gly Asp

405 410 415

Arg Trp Asp Glu Leu Asp Ser Leu Lys Gln Ala Val Glu Gln Lys Lys

420 425 430

Ser Pro Leu Asp Gln Thr Asp Arg Thr Phe Trp Glu Gly Ile Val Cys

435 440 445

Asp Leu Thr Lys Val Leu Pro Arg Asn Glu Ala Asp Trp Glu Gln Ala

450 455 460

Val Val Gln Ile His Arg Lys Ala Glu Glu Tyr Val Gly Lys Ala Val

465 470 475 480

Gln Ala Trp Arg Lys Arg Phe Ala Ala Asp Glu Arg Lys Gly Ile Ala

485 490 495

Gly Leu Ser Met Trp Asn Ile Glu Glu Leu Glu Gly Leu Arg Lys Leu

500 505 510

Leu Ile Ser Trp Ser Arg Arg Ser Arg Asn Pro Gln Glu Val Asn Arg

515 520 525

Phe Glu Arg Gly His Thr Ser His Gln Arg Leu Leu Thr His Ile Gln

530 535 540

Asn Val Lys Glu Asp Arg Leu Lys Gln Leu Ser His Ala Ile Val Met

545 550 555 560

Thr Ala Leu Gly Tyr Val Tyr Asp Glu Arg Lys Gln Glu Trp Cys Ala

565 570 575

Glu Tyr Pro Ala Cys Gln Val Ile Leu Phe Glu Asn Leu Ser Gln Tyr

580 585 590

Arg Ser Asn Leu Asp Arg Ser Thr Lys Glu Asn Ser Thr Leu Met Lys

595 600 605

Trp Ala His Arg Ser Ile Pro Lys Tyr Val His Met Gln Ala Glu Pro

610 615 620

Tyr Gly Ile Gln Ile Gly Asp Val Arg Ala Glu Tyr Ser Ser Arg Phe

625 630 635 640

Tyr Ala Lys Thr Gly Thr Pro Gly Ile Arg Cys Lys Lys Val Arg Gly

645 650 655

Gln Asp Leu Gln Gly Arg Arg Phe Glu Asn Leu Gln Lys Arg Leu Val

660 665 670

Asn Glu Gln Phe Leu Thr Glu Glu Gln Val Lys Gln Leu Arg Pro Gly

675 680 685

Asp Ile Val Pro Asp Asp Ser Gly Glu Leu Phe Met Thr Leu Thr Asp

690 695 700

Gly Ser Gly Ser Lys Glu Val Val Phe Leu Gln Ala Asp Ile Asn Ala

705 710 715 720

Ala His Asn Leu Gln Lys Arg Phe Trp Gln Arg Tyr Asn Glu Leu Phe

725 730 735

Lys Val Ser Cys Arg Val Ile Val Arg Asp Glu Glu Glu Tyr Leu Val

740 745 750

Pro Lys Thr Lys Ser Val Gln Ala Lys Leu Gly Lys Gly Leu Phe Val

755 760 765

Lys Lys Ser Asp Thr Ala Trp Lys Asp Val Tyr Val Trp Asp Ser Gln

770 775 780

Ala Lys Leu Lys Gly Lys Thr Thr Phe Thr Glu Glu Ser Glu Ser Pro

785 790 795 800

Glu Gln Leu Glu Asp Phe Gln Glu Ile Ile Glu Glu Ala Glu Glu Ala

805 810 815

Lys Gly Thr Tyr Arg Thr Leu Phe Arg Asp Pro Ser Gly Val Phe Phe

820 825 830

Pro Glu Ser Val Trp Tyr Pro Gln Lys Asp Phe Trp Gly Glu Val Lys

835 840 845

Arg Lys Leu Tyr Gly Lys Leu Arg Glu Arg Phe Leu Thr Lys Ala Arg

850 855 860

<210> 556

<211> 1108

<212> PRT

<213> Bacillus sp.

<400> 556

Met Ala Ile Arg Ser Ile Lys Leu Lys Leu Lys Thr His Thr Gly Pro

1 5 10 15

Glu Ala Gln Asn Leu Arg Lys Gly Ile Trp Arg Thr His Arg Leu Leu

20 25 30

Asn Glu Gly Val Ala Tyr Tyr Met Lys Met Leu Leu Leu Phe Arg Gln

35 40 45

Glu Ser Thr Gly Glu Arg Pro Lys Glu Glu Leu Gln Glu Glu Leu Ile

50 55 60

Cys His Ile Arg Glu Gln Gln Gln Arg Asn Gln Ala Asp Lys Asn Thr

65 70 75 80

Gln Ala Leu Pro Leu Asp Lys Ala Leu Glu Ala Leu Arg Gln Leu Tyr

85 90 95

Glu Leu Leu Val Pro Ser Ser Val Gly Gln Ser Gly Asp Ala Gln Ile

100 105 110

Ile Ser Arg Lys Phe Leu Ser Pro Leu Val Asp Pro Asn Ser Glu Gly

115 120 125

Gly Lys Gly Thr Ser Lys Ala Gly Ala Lys Pro Thr Trp Gln Lys Lys

130 135 140

Lys Glu Ala Asn Asp Pro Thr Trp Glu Gln Asp Tyr Glu Lys Trp Lys

145 150 155 160

Lys Arg Arg Glu Glu Asp Pro Thr Ala Ser Val Ile Thr Thr Leu Glu

165 170 175

Glu Tyr Gly Ile Arg Pro Ile Phe Pro Leu Tyr Thr Asn Thr Val Thr

180 185 190

Asp Ile Ala Trp Leu Pro Leu Gln Ser Asn Gln Phe Val Arg Thr Trp

195 200 205

Asp Arg Asp Met Leu Gln Gln Ala Ile Glu Arg Leu Leu Ser Trp Glu

210 215 220

Ser Trp Asn Lys Arg Val Gln Glu Glu Tyr Ala Lys Leu Lys Glu Lys

225 230 235 240

Met Ala Gln Leu Asn Glu Gln Leu Glu Gly Gly Gln Glu Trp Ile Ser

245 250 255

Leu Leu Glu Gln Tyr Glu Glu Asn Arg Glu Arg Glu Leu Arg Glu Asn

260 265 270

Met Thr Ala Ala Asn Asp Lys Tyr Arg Ile Thr Lys Arg Gln Met Lys

275 280 285

Gly Trp Asn Glu Leu Tyr Glu Leu Trp Ser Thr Phe Pro Ala Ser Ala

290 295 300

Ser His Glu Gln Tyr Lys Glu Ala Leu Lys Arg Val Gln Gln Arg Leu

305 310 315 320

Arg Gly Arg Phe Gly Asp Ala His Phe Phe Gln Tyr Leu Met Glu Glu

325 330 335

Lys Asn Arg Leu Ile Trp Lys Gly Asn Pro Gln Arg Ile His Tyr Phe

340 345 350

Val Ala Arg Asn Glu Leu Thr Lys Arg Leu Glu Glu Ala Lys Gln Ser

355 360 365

Ala Thr Met Thr Leu Pro Asn Ala Arg Lys His Pro Leu Trp Val Arg

370 375 380

Phe Asp Ala Arg Gly Gly Asn Leu Gln Asp Tyr Tyr Leu Thr Ala Glu

385 390 395 400

Ala Asp Lys Pro Arg Ser Arg Arg Phe Val Thr Phe Ser Gln Leu Ile

405 410 415

Trp Pro Ser Glu Ser Gly Trp Met Glu Lys Lys Asp Val Glu Val Glu

420 425 430

Leu Ala Leu Ser Arg Gln Phe Tyr Gln Gln Val Lys Leu Leu Lys Asn

435 440 445

Asp Lys Gly Lys Gln Lys Ile Glu Phe Lys Asp Lys Gly Ser Gly Ser

450 455 460

Thr Phe Asn Gly His Leu Gly Gly Ala Lys Leu Gln Leu Glu Arg Gly

465 470 475 480

Asp Leu Glu Lys Glu Glu Lys Asn Phe Glu Asp Gly Glu Ile Gly Ser

485 490 495

Val Tyr Leu Asn Val Val Ile Asp Phe Glu Pro Leu Gln Glu Val Lys

500 505 510

Asn Gly Arg Val Gln Ala Pro Tyr Gly Gln Val Leu Gln Leu Ile Arg

515 520 525

Arg Pro Asn Glu Phe Pro Lys Val Thr Thr Tyr Lys Ser Glu Gln Leu

530 535 540

Val Glu Trp Ile Lys Ala Ser Pro Gln His Ser Ala Gly Val Glu Ser

545 550 555 560

Leu Ala Ser Gly Phe Arg Val Met Ser Ile Asp Leu Gly Leu Arg Ala

565 570 575

Ala Ala Ala Thr Ser Ile Phe Ser Val Glu Glu Ser Ser Asp Lys Asn

580 585 590

Ala Ala Asp Phe Ser Tyr Trp Ile Glu Gly Thr Pro Leu Val Ala Val

595 600 605

His Gln Arg Ser Tyr Met Leu Arg Leu Pro Gly Glu Gln Val Glu Lys

610 615 620

Gln Val Met Glu Lys Arg Asp Glu Arg Phe Gln Leu His Gln Arg Val

625 630 635 640

Lys Phe Gln Ile Arg Val Leu Ala Gln Ile Met Arg Met Ala Asn Lys

645 650 655

Gln Tyr Gly Asp Arg Trp Asp Glu Leu Asp Ser Leu Lys Gln Ala Val

660 665 670

Glu Gln Lys Lys Ser Pro Leu Asp Gln Thr Asp Arg Thr Phe Trp Glu

675 680 685

Gly Ile Val Cys Asp Leu Thr Lys Val Leu Pro Arg Asn Glu Ala Asp

690 695 700

Trp Glu Gln Ala Val Val Gln Ile His Arg Lys Ala Glu Glu Tyr Val

705 710 715 720

Gly Lys Ala Val Gln Ala Trp Arg Lys Arg Phe Ala Ala Asp Glu Arg

725 730 735

Lys Gly Ile Ala Gly Leu Ser Met Trp Asn Ile Glu Glu Leu Glu Gly

740 745 750

Leu Arg Lys Leu Leu Ile Ser Trp Ser Arg Arg Thr Arg Asn Pro Gln

755 760 765

Glu Val Asn Arg Phe Glu Arg Gly His Thr Ser His Gln Arg Leu Leu

770 775 780

Thr His Ile Gln Asn Val Lys Glu Asp Arg Leu Lys Gln Leu Ser His

785 790 795 800

Ala Ile Val Met Thr Ala Leu Gly Tyr Val Tyr Asp Glu Arg Lys Gln

805 810 815

Glu Trp Cys Ala Glu Tyr Pro Ala Cys Gln Val Ile Leu Phe Glu Asn

820 825 830

Leu Ser Gln Tyr Arg Ser Asn Leu Asp Arg Ser Thr Lys Glu Asn Ser

835 840 845

Thr Leu Met Lys Trp Ala His Arg Ser Ile Pro Lys Tyr Val His Met

850 855 860

Gln Ala Glu Pro Tyr Gly Ile Gln Ile Gly Asp Val Arg Ala Glu Tyr

865 870 875 880

Ser Ser Arg Phe Tyr Ala Lys Thr Gly Thr Pro Gly Ile Arg Cys Lys

885 890 895

Lys Val Arg Gly Gln Asp Leu Gln Gly Arg Arg Phe Glu Asn Leu Gln

900 905 910

Lys Arg Leu Val Asn Glu Gln Phe Leu Thr Glu Glu Glu Gln Val Lys Gln

915 920 925

Leu Arg Pro Gly Asp Ile Val Pro Asp Asp Ser Gly Glu Leu Phe Met

930 935 940

Thr Leu Thr Asp Gly Ser Gly Ser Lys Glu Val Val Phe Leu Gln Ala

945 950 955 960

Asp Ile Asn Ala Ala His Asn Leu Gln Lys Arg Phe Trp Gln Arg Tyr

965 970 975

Asn Glu Leu Phe Lys Val Ser Cys Arg Val Ile Val Arg Asp Glu Glu

980 985 990

Glu Tyr Leu Val Pro Lys Thr Lys Ser Val Gln Ala Lys Leu Gly Lys

995 1000 1005

Gly Leu Phe Val Lys Lys Ser Asp Thr Ala Trp Lys Asp Val Tyr

1010 1015 1020

Val Trp Asp Ser Gln Ala Lys Leu Lys Gly Lys Thr Thr Phe Thr

1025 1030 1035

Glu Glu Ser Glu Ser Pro Glu Gln Leu Glu Asp Phe Gln Glu Ile

1040 1045 1050

Ile Glu Glu Ala Glu Glu Ala Lys Gly Thr Tyr Arg Thr Leu Phe

1055 1060 1065

Arg Asp Pro Ser Gly Val Phe Phe Pro Glu Ser Val Trp Tyr Pro

1070 1075 1080

Gln Lys Asp Phe Trp Gly Glu Val Lys Arg Lys Leu Tyr Gly Lys

1085 1090 1095

Leu Arg Glu Arg Phe Leu Thr Lys Ala Arg

1100 1105

<210> 557

<211> 1489

<212> PRT

<213> Desulfatirhabdium butyrativorans

<400> 557

Met Pro Leu Ser Asn Asn Pro Pro Val Thr Gln Arg Ala Tyr Thr Leu

1 5 10 15

Arg Leu Arg Gly Ala Asp Pro Ser Asp Leu Ser Trp Arg Glu Ala Leu

20 25 30

Trp His Thr His Glu Ala Val Asn Lys Gly Ala Lys Val Phe Gly Asp

35 40 45

Trp Leu Leu Thr Leu Arg Gly Gly Leu Asp His Thr Leu Ala Asp Thr

50 55 60

Lys Val Lys Gly Gly Lys Gly Lys Pro Asp Arg Asp Pro Thr Pro Glu

65 70 75 80

Glu Arg Lys Ala Arg Arg Ile Leu Leu Ala Leu Ser Trp Leu Ser Val

85 90 95

Glu Ser Lys Leu Gly Ala Pro Ser Ser Tyr Ile Val Ala Ser Gly Asp

100 105 110

Glu Pro Ala Lys Asp Arg Asn Asp Asn Val Val Ser Ala Leu Glu Glu

115 120 125

Ile Leu Gln Ser Arg Lys Val Ala Lys Ser Glu Ile Asp Asp Trp Lys

130 135 140

Arg Asp Cys Ser Ala Ser Leu Ser Ala Ala Ile Arg Asp Asp Ala Val

145 150 155 160

Trp Val Asn Arg Ser Lys Val Phe Asp Glu Ala Val Lys Ser Val Gly

165 170 175

Ser Ser Leu Thr Arg Glu Glu Ala Trp Asp Met Leu Glu Arg Phe Phe

180 185 190

Gly Ser Arg Asp Ala Tyr Leu Thr Pro Met Lys Asp Pro Glu Asp Lys

195 200 205

Ser Ser Glu Thr Glu Gln Glu Asp Lys Ala Lys Asp Leu Val Gln Lys

210 215 220

Ala Gly Gln Trp Leu Ser Ser Arg Tyr Gly Thr Ser Glu Gly Ala Asp

225 230 235 240

Phe Cys Arg Met Ser Asp Ile Tyr Gly Lys Ile Ala Ala Trp Ala Asp

245 250 255

Asn Ala Ser Gln Gly Gly Ser Ser Thr Val Asp Asp Leu Val Ser Glu

260 265 270

Leu Arg Gln His Phe Asp Thr Lys Glu Ser Lys Ala Thr Asn Gly Leu

275 280 285

Asp Trp Ile Ile Gly Leu Ser Ser Tyr Thr Gly His Thr Pro Asn Pro

290 295 300

Val His Glu Leu Leu Arg Gln Asn Thr Ser Leu Asn Lys Ser His Leu

305 310 315 320

Asp Asp Leu Lys Lys Lys Ala Asn Thr Arg Ala Glu Ser Cys Lys Ser

325 330 335

Lys Ile Gly Ser Lys Gly Gln Arg Pro Tyr Ser Asp Ala Ile Leu Asn

340 345 350

Asp Val Glu Ser Val Cys Gly Phe Thr Tyr Arg Val Asp Lys Asp Gly

355 360 365

Gln Pro Val Ser Val Ala Asp Tyr Ser Lys Tyr Asp Val Asp Tyr Lys

370 375 380

Trp Gly Thr Ala Arg His Tyr Ile Phe Ala Val Met Leu Asp His Ala

385 390 395 400

Ala Arg Arg Ile Ser Leu Ala His Lys Trp Ile Lys Arg Ala Glu Ala

405 410 415

Glu Arg His Lys Phe Glu Glu Asp Ala Lys Arg Ile Ala Asn Val Pro

420 425 430

Ala Arg Ala Arg Glu Trp Leu Asp Ser Phe Cys Lys Glu Arg Ser Val

435 440 445

Thr Ser Gly Ala Val Glu Pro Tyr Arg Ile Arg Arg Arg Ala Val Asp

450 455 460

Gly Trp Lys Glu Val Val Ala Ala Trp Ser Lys Ser Asp Cys Lys Ser

465 470 475 480

Thr Glu Asp Arg Ile Ala Ala Ala Arg Ala Leu Gln Asp Asp Ser Glu

485 490 495

Ile Asp Lys Phe Gly Asp Ile Gln Leu Phe Glu Ala Leu Ala Glu Asp

500 505 510

Asp Ala Leu Cys Val Trp His Lys Asp Gly Glu Ala Thr Asn Glu Pro

515 520 525

Asp Phe Gln Pro Leu Ile Asp Tyr Ser Leu Ala Ile Glu Ala Glu Phe

530 535 540

Lys Lys Arg Gln Phe Lys Val Pro Ala Tyr Arg His Pro Asp Glu Leu

545 550 555 560

Leu His Pro Val Phe Cys Asp Phe Gly Lys Ser Arg Trp Lys Ile Asn

565 570 575

Tyr Asp Val His Lys Asn Val Gln Ala Pro Phe Tyr Arg Gly Leu Cys

580 585 590

Leu Thr Leu Trp Thr Gly Ser Glu Ile Lys Pro Val Pro Leu Cys Trp

595 600 605

Gln Ser Lys Arg Leu Thr Arg Asp Leu Ala Leu Gly Asn Asn His Arg

610 615 620

Asn Asp Ala Ala Ser Ala Val Thr Arg Ala Asp Arg Leu Gly Arg Ala

625 630 635 640

Ala Ser Asn Val Thr Lys Ser Asp Met Val Asn Ile Thr Gly Leu Phe

645 650 655

Glu Gln Ala Asp Trp Asn Gly Arg Leu Gln Ala Pro Arg Gln Gln Leu

660 665 670

Glu Ala Ile Ala Val Val Arg Asp Asn Pro Arg Leu Ser Glu Gln Glu

675 680 685

Arg Asn Leu Arg Met Cys Gly Met Ile Glu His Ile Arg Trp Leu Val

690 695 700

Thr Phe Ser Val Lys Leu Gln Pro Gln Gly Pro Trp Cys Ala Tyr Ala

705 710 715 720

Glu Gln His Gly Leu Asn Thr Asn Pro Gln Tyr Trp Pro His Ala Asp

725 730 735

Thr Asn Arg Asp Arg Lys Val His Ala Arg Leu Ile Leu Pro Arg Leu

740 745 750

Pro Gly Leu Arg Val Leu Ser Val Asp Leu Gly His Arg Tyr Ala Ala

755 760 765

Ala Cys Ala Val Trp Glu Ala Val Asn Thr Glu Thr Val Lys Glu Ala

770 775 780

Cys Gln Asn Val Gly Arg Asp Met Pro Lys Glu His Asp Leu Tyr Leu

785 790 795 800

His Ile Lys Val Lys Lys Gln Gly Ile Gly Lys Gln Thr Glu Val Asp

805 810 815

Lys Thr Thr Ile Tyr Arg Arg Ile Gly Ala Asp Thr Leu Pro Asp Gly

820 825 830

Arg Pro His Pro Ala Pro Trp Ala Arg Leu Asp Arg Gln Phe Leu Ile

835 840 845

Lys Leu Gln Gly Glu Glu Lys Asp Ala Arg Glu Ala Ser Asn Glu Glu

850 855 860

Ile Trp Ala Leu His Gln Met Glu Cys Lys Leu Asp Arg Thr Lys Pro

865 870 875 880

Leu Ile Asp Arg Leu Ile Ala Ser Gly Trp Gly Leu Leu Lys Arg Gln

885 890 895

Met Ala Arg Leu Asp Ala Leu Lys Glu Leu Gly Trp Ile Pro Ala Pro

900 905 910

Asp Ser Ser Glu Asn Leu Ser Arg Glu Asp Gly Glu Ala Lys Asp Tyr

915 920 925

Arg Glu Ser Leu Ala Val Asp Asp Leu Met Phe Ser Ala Val Arg Thr

930 935 940

Leu Arg Leu Ala Leu Gln Arg His Gly Asn Arg Ala Arg Ile Ala Tyr

945 950 955 960

Tyr Leu Ile Ser Glu Val Lys Ile Arg Pro Gly Gly Ile Gln Glu Lys

965 970 975

Leu Asp Glu Asn Gly Arg Ile Asp Leu Leu Gln Asp Ala Leu Ala Leu

980 985 990

Trp His Glu Leu Phe Ser Ser Pro Gly Trp Arg Asp Glu Ala Ala Lys

995 1000 1005

Gln Leu Trp Asp Ser Arg Ile Ala Thr Leu Ala Gly Tyr Lys Ala

1010 1015 1020

Pro Glu Glu Asn Gly Asp Asn Val Ser Asp Val Ala Tyr Arg Lys

1025 1030 1035

Lys Gln Gln Val Tyr Arg Glu Gln Leu Arg Asn Val Ala Lys Thr

1040 1045 1050

Leu Ser Gly Asp Val Ile Thr Cys Lys Glu Leu Ser Asp Ala Trp

1055 1060 1065

Lys Glu Arg Trp Glu Asp Glu Asp Gln Arg Trp Lys Lys Leu Leu

1070 1075 1080

Arg Trp Phe Lys Asp Trp Val Leu Pro Ser Gly Thr Gln Ala Asn

1085 1090 1095

Asn Ala Thr Ile Arg Asn Val Gly Gly Leu Ser Leu Ser Arg Leu

1100 1105 1110

Ala Thr Ile Thr Glu Phe Arg Arg Lys Val Gln Val Gly Phe Phe

1115 1120 1125

Thr Arg Leu Arg Pro Asp Gly Thr Arg His Glu Ile Gly Glu Gln

1130 1135 1140

Phe Gly Gln Lys Thr Leu Asp Ala Leu Glu Leu Leu Arg Glu Gln

1145 1150 1155

Arg Val Lys Gln Leu Ala Ser Arg Ile Ala Glu Ala Ala Leu Gly

1160 1165 1170

Ile Gly Ser Glu Gly Gly Lys Gly Trp Asp Gly Gly Lys Arg Pro

1175 1180 1185

Arg Gln Arg Ile Asn Asp Ser Arg Phe Ala Pro Cys His Ala Val

1190 1195 1200

Val Ile Glu Asn Leu Ala Asn Tyr Arg Pro Asp Glu Thr Arg Thr

1205 1210 1215

Arg Leu Glu Asn Arg Arg Leu Met Thr Trp Ser Ala Ser Lys Val

1220 1225 1230

His Lys Tyr Leu Ser Glu Ala Cys Gln Leu Asn Gly Leu Tyr Leu

1235 1240 1245

Cys Thr Val Ser Ala Trp Tyr Thr Ser Arg Gln Asp Ser Arg Thr

1250 1255 1260

Gly Ala Pro Gly Ile Arg Cys Gln Asp Val Ser Val Arg Glu Phe

1265 1270 1275

Met Gln Ser Pro Phe Trp Arg Lys Gln Val Lys Gln Ala Glu Ala

1280 1285 1290

Lys His Asp Glu Asn Lys Gly Asp Ala Arg Glu Arg Phe Leu Cys

1295 1300 1305

Glu Leu Asn Lys Thr Trp Lys Ala Lys Thr Pro Ala Glu Trp Lys

1310 1315 1320

Lys Ala Gly Phe Val Arg Ile Pro Leu Arg Gly Gly Glu Ile Phe

1325 1330 1335

Val Ser Ala Asp Ser Lys Ser Pro Ser Ala Lys Gly Ile His Ala

1340 1345 1350

Asp Leu Asn Ala Ala Ala Asn Ile Gly Leu Arg Ala Leu Thr Asp

1355 1360 1365

Pro Asp Trp Pro Gly Lys Trp Trp Tyr Val Pro Cys Asp Pro Val

1370 1375 1380

Ser Phe Glu Ser Lys Met Asp Tyr Val Lys Gly Cys Ala Ala Val

1385 1390 1395

Lys Val Gly Gln Pro Leu Arg Gln Pro Ala Gln Thr Asn Ala Asp

1400 1405 1410

Gly Ala Ala Ser Lys Ile Arg Lys Gly Lys Lys Asn Arg Thr Ala

1415 1420 1425

Gly Thr Ser Lys Glu Lys Val Tyr Leu Trp Arg Asp Ile Ser Ala

1430 1435 1440

Phe Pro Leu Glu Ser Asn Glu Ile Gly Glu Trp Lys Glu Thr Ser

1445 1450 1455

Ala Tyr Gln Asn Asp Val Gln Tyr Arg Val Ile Arg Met Leu Lys

1460 1465 1470

Glu His Ile Lys Ser Leu Asp Asn Arg Thr Gly Asp Asn Val Glu

1475 1480 1485

gly

<210> 558

<211> 277

<212> PRT

<213> Alicyclobacillus herbarius

<400> 558

Met Leu Lys Gln Ala Val Leu Gly Asn Gly Pro Leu Ile Asn Trp Glu

1 5 10 15

Lys Asn Val Lys Arg Gly Lys Gly Met Ala Thr Lys Ser Ile Lys Val

20 25 30

Lys Leu Arg Leu Gly Lys His Pro Asp Ile Arg Ala Gly Ile Trp Gln

35 40 45

Leu His Lys Ala Ala Asn Ala Gly Val Arg Tyr Tyr Thr Glu Trp Leu

50 55 60

Ser Leu Met Arg Gln Lys Asn Leu Tyr Thr Arg Gly Pro Lys Gly Glu

65 70 75 80

Gln Gln Leu Tyr Arg Ser Gly Glu Gln Cys Arg Arg Glu Leu Leu Gln

85 90 95

Arg Leu Arg Glu Arg Gln Arg Leu Asn Gly Arg Thr Asp Glu Pro Gly

100 105 110

Thr Asp Glu Glu Leu Leu Lys Val Ala Arg Gln Ile Tyr Glu Val Leu

115 120 125

Val Pro Gln Ser Ile Gly Lys Ser Gly Asp Ala Gln Gln Leu Ala Ser

130 135 140

Asn Phe Leu Ser Pro Leu Val Asp Pro Asn Ser Lys Gly Gly Gln Gly

145 150 155 160

Gln Ser Asn Ala Gly Arg Lys Pro Ala Trp Gln Lys Met Arg Asp Glu

165 170 175

Gly Asn Pro Gly Trp Val Ala Ala Lys Glu Arg Tyr Glu Gln Arg Lys

180 185 190

Ala Thr Asp Pro Thr Lys Lys Met Ile Glu Met Leu Asp Gly Leu Gly

195 200 205

Leu Lys Pro Leu Phe Ser Val Phe Thr Glu Thr Tyr Thr Thr Gly Val

210 215 220

Lys Trp Lys Asp Leu Ser Lys Arg Gln Gly Val Arg Thr Trp Asp Arg

225 230 235 240

Asp Met Phe Gln Ser Leu Ser Glu Arg Ser Gly Val Ile Asp Val Gly

245 250 255

Ser His Thr Val His His Ile Asp Leu Ala Thr Ala Ser Asp Ala Gln

260 265 270

Ile Gln Tyr Glu Leu

275

<210> 559

<211> 277

<212> PRT

<213> Alicyclobacillus contaminans

<400> 559

Met Ser Val Lys Ser Ile Lys Phe Lys Leu Met Ile Gly Gly Pro Gln

1 5 10 15

Tyr Thr Arg Ile Arg Arg Gly Ile Tyr Lys Thr His Glu Val Phe Asn

20 25 30

Glu Gly Val Arg Tyr Tyr Gln Glu Trp Leu Leu Leu Met Arg Gln Gly

35 40 45

Asp Val Tyr Arg Tyr Gln Asp Asp Lys Pro Glu Ile Val Leu Ser Ala

50 55 60

Glu His Cys Lys Arg Glu Leu Leu Arg Arg Leu Arg Gln Val Gln Lys

65 70 75 80

Glu Asn Val Gly Arg Thr Ser His Thr Asp Glu Glu Leu Leu Gln Val

85 90 95

Met Arg Ala Leu Tyr Glu Leu Ile Val Pro Ser Ala Val Gly Lys Lys

100 105 110

Gly Asp Ala Ala Ser Leu Ser Arg Lys Phe Leu Ser Pro Leu Ala Trp

115 120 125

Lys Asp Ser Lys Gly Leu Thr Gly Glu Ser Lys Ala Gly Asn Lys Pro

130 135 140

Arg Trp Lys Arg Leu Gln Glu Gln Gly Leu Pro Tyr Glu Glu Glu Tyr

145 150 155 160

Asn Arg Trp Leu Arg Glu Lys Glu Ser Asp Pro Ala Lys His Ile Pro

165 170 175

Ala Gln Leu Ala Ser Met Gly Leu Lys Pro Phe Leu Lys Val Phe Thr

180 185 190

Glu Ser Thr Glu Gly Ile Ala Trp Leu Pro Leu Ala Lys Asp Gln Gly

195 200 205

Val Arg Thr Trp Asp Arg Asp Met Phe Gln Gln Ala Ile Glu Gly Leu

210 215 220

Leu Ser Trp Glu Ser Trp Asn Arg Arg Val Arg Glu Glu Tyr Asp Ala

225 230 235 240

Leu Ser Ala Arg Val Tyr Ala Tyr His Ala Lys His Phe Ala Asp Gln

245 250 255

Pro Gly Trp Ala Val Tyr Trp Pro Gln Ser Gln Pro Arg Gln Lys Gly

260 265 270

Trp Val Lys Met Lys

275

<210> 560

<211> 218

<212> PRT

<213> Citrobacter freundii

<400> 560

Met Tyr Glu Leu Trp Ser Thr Phe Pro Ala Ser Ala Ser His Glu Gln

1 5 10 15

Tyr Lys Glu Ala Leu Lys Arg Val Gln Gln Arg Leu Arg Gly Arg Phe

20 25 30

Gly Asp Ala His Phe Phe Gln Tyr Leu Met Glu Glu Glu Lys Asn Arg Leu

35 40 45

Ile Trp Lys Gly Asn Pro Gln Arg Ile His Tyr Phe Val Ala Arg Asn

50 55 60

Glu Leu Thr Lys Arg Leu Glu Glu Ala Lys Gln Ser Ala Thr Met Thr

65 70 75 80

Leu Pro Asn Ala Arg Lys His Pro Leu Trp Val Arg Phe Asp Ala Arg

85 90 95

Gly Gly Asn Leu Gln Asp Tyr Tyr Leu Thr Ala Glu Ala Asp Lys Pro

100 105 110

Arg Ser Arg Arg Phe Val Thr Phe Ser Gln Leu Ile Trp Pro Ser Glu

115 120 125

Ser Gly Trp Met Glu Lys Lys Asp Val Glu Val Glu Leu Ala Leu Ser

130 135 140

Arg Gln Phe Tyr Gln Gln Val Lys Leu Leu Lys Asn Asp Lys Gly Lys

145 150 155 160

Gln Lys Ile Glu Phe Lys Asp Lys Gly Ser Gly Ser Thr Phe Asn Gly

165 170 175

His Leu Gly Gly Ala Lys Leu Gln Leu Glu Arg Gly Asp Leu Glu Lys

180 185 190

Glu Glu Lys Thr Ser Arg Thr Gly Lys Ser Ala Ala Phe Thr Leu Thr

195 200 205

Leu Ser Leu Ile Ser Asn Leu Cys Lys Lys

210 215

<210> 561

<211> 218

<212> PRT

<213> Citrobacter freundii

<400> 561

Leu Tyr Glu Leu Trp Ser Thr Phe Pro Ala Ser Ala Ser His Glu Gln

1 5 10 15

Tyr Lys Glu Ala Leu Lys Arg Val Gln Gln Arg Leu Arg Gly Arg Phe

20 25 30

Gly Asp Ala His Phe Phe Gln Tyr Leu Met Glu Glu Glu Lys Asn Arg Leu

35 40 45

Ile Trp Lys Gly Asn Pro Gln Arg Ile His Tyr Phe Val Ala Arg Asn

50 55 60

Glu Leu Thr Lys Arg Leu Glu Glu Ala Lys Gln Ser Ala Thr Met Thr

65 70 75 80

Leu Pro Asn Ala Arg Lys His Pro Leu Trp Val Arg Phe Asp Ala Arg

85 90 95

Gly Gly Asn Leu Gln Asp Tyr Tyr Leu Thr Ala Glu Ala Asp Lys Pro

100 105 110

Arg Ser Arg Arg Phe Val Thr Phe Ser Gln Leu Ile Trp Pro Ser Glu

115 120 125

Ser Gly Trp Met Glu Lys Lys Asp Val Glu Val Glu Leu Ala Leu Ser

130 135 140

Arg Gln Phe Tyr Gln Gln Val Lys Leu Leu Lys Asn Asp Lys Gly Lys

145 150 155 160

Gln Lys Ile Glu Phe Lys Asp Lys Gly Ser Gly Ser Thr Phe Asn Gly

165 170 175

His Leu Gly Gly Ala Lys Leu Gln Leu Glu Arg Gly Asp Leu Glu Lys

180 185 190

Glu Glu Lys Thr Ser Arg Thr Gly Lys Ser Ala Ala Phe Thr Leu Thr

195 200 205

Leu Ser Leu Ile Ser Asn Leu Cys Lys Lys

210 215

<210> 562

<211> 482

<212> PRT

<213> Brevibacillus agri

<400> 562

Met Glu Lys Arg Asp Glu Arg Phe Gln Leu His Gln Arg Val Lys Phe

1 5 10 15

Gln Ile Arg Val Leu Ala Gln Ile Met Arg Met Ala Asn Lys Gln Tyr

20 25 30

Gly Asp Arg Trp Asp Glu Leu Asp Ser Leu Lys Gln Ala Val Glu Gln

35 40 45

Lys Lys Ser Pro Leu Asp Gln Thr Asp Arg Thr Phe Trp Glu Gly Ile

50 55 60

Val Cys Asp Leu Thr Lys Val Leu Pro Arg Asn Glu Ala Asp Trp Glu

65 70 75 80

Gln Ala Val Val Gln Ile His Arg Lys Ala Glu Glu Tyr Val Gly Lys

85 90 95

Ala Val Gln Ala Trp Arg Lys Arg Phe Ala Ala Asp Glu Arg Lys Gly

100 105 110

Ile Ala Gly Leu Ser Met Trp Asn Ile Glu Glu Leu Glu Gly Leu Arg

115 120 125

Lys Leu Leu Ile Ser Trp Ser Arg Arg Thr Arg Asn Pro Gln Glu Val

130 135 140

Asn Arg Phe Glu Arg Gly His Thr Ser His Gln Arg Leu Leu Thr His

145 150 155 160

Ile Gln Asn Val Lys Glu Asp Arg Leu Lys Gln Leu Ser His Ala Ile

165 170 175

Val Met Thr Ala Leu Gly Tyr Val Tyr Asp Glu Arg Lys Gln Glu Trp

180 185 190

Cys Ala Glu Tyr Pro Ala Cys Gln Val Ile Leu Phe Glu Asn Leu Ser

195 200 205

Gln Tyr Arg Ser Asn Leu Asp Arg Ser Thr Lys Glu Asn Ser Thr Leu

210 215 220

Met Lys Trp Ala His Arg Ser Ile Pro Lys Tyr Val His Met Gln Ala

225 230 235 240

Glu Pro Tyr Gly Ile Gln Ile Gly Asp Val Arg Ala Glu Tyr Ser Ser

245 250 255

Arg Phe Tyr Ala Lys Thr Gly Thr Pro Gly Ile Arg Cys Lys Lys Val

260 265 270

Arg Gly Gln Asp Leu Gln Gly Arg Arg Phe Glu Asn Leu Gln Lys Arg

275 280 285

Leu Val Asn Glu Gln Phe Leu Thr Glu Glu Gln Val Lys Gln Leu Arg

290 295 300

Pro Gly Asp Ile Val Pro Asp Asp Ser Gly Glu Leu Phe Met Thr Leu

305 310 315 320

Thr Asp Gly Ser Gly Ser Lys Glu Val Val Phe Leu Gln Ala Asp Ile

325 330 335

Asn Ala Ala His Asn Leu Gln Lys Arg Phe Trp Gln Arg Tyr Asn Glu

340 345 350

Leu Phe Lys Val Ser Cys Arg Val Ile Val Arg Asp Glu Glu Glu Tyr

355 360 365

Leu Val Pro Lys Thr Lys Ser Val Gln Ala Lys Leu Gly Lys Gly Leu

370 375 380

Phe Val Lys Lys Ser Asp Thr Ala Trp Lys Asp Val Tyr Val Trp Asp

385 390 395 400

Ser Gln Ala Lys Leu Lys Gly Lys Thr Thr Phe Thr Glu Glu Ser Glu

405 410 415

Ser Pro Glu Gln Leu Glu Asp Phe Gln Glu Ile Ile Glu Glu Ala Glu

420 425 430

Glu Ala Lys Gly Thr Tyr Arg Thr Leu Phe Arg Asp Pro Ser Gly Val

435 440 445

Phe Phe Pro Glu Ser Val Trp Tyr Pro Gln Lys Asp Phe Trp Gly Glu

450 455 460

Val Lys Arg Lys Leu Tyr Gly Lys Leu Arg Glu Arg Phe Leu Thr Lys

465 470 475 480

Ala Arg

<210> 563

<211> 584

<212> PRT

<213> Brevibacillus agri

<400> 563

Met Ala Ile Arg Ser Ile Lys Leu Lys Leu Lys Thr His Thr Gly Pro

1 5 10 15

Glu Ala Gln Asn Leu Arg Lys Gly Ile Trp Arg Thr His Arg Leu Leu

20 25 30

Asn Glu Gly Val Ala Tyr Tyr Met Lys Met Leu Leu Leu Phe Arg Gln

35 40 45

Glu Ser Thr Gly Glu Arg Pro Lys Glu Glu Leu Gln Glu Glu Leu Ile

50 55 60

Cys His Ile Arg Glu Gln Gln Gln Arg Asn Gln Ala Asp Lys Asn Thr

65 70 75 80

Gln Ala Leu Pro Leu Asp Lys Ala Leu Glu Ala Leu Arg Gln Leu Tyr

85 90 95

Glu Leu Leu Val Pro Ser Ser Val Gly Gln Ser Gly Asp Ala Gln Ile

100 105 110

Ile Ser Arg Lys Phe Leu Ser Pro Leu Val Asp Pro Asn Ser Glu Gly

115 120 125

Gly Lys Gly Thr Ser Lys Ala Gly Ala Lys Pro Thr Trp Gln Lys Lys

130 135 140

Lys Glu Ala Asn Asp Pro Thr Trp Glu Gln Asp Tyr Glu Lys Trp Lys

145 150 155 160

Lys Arg Arg Glu Glu Asp Pro Thr Ala Ser Val Ile Thr Thr Leu Glu

165 170 175

Glu Tyr Gly Ile Arg Pro Ile Phe Pro Leu Tyr Thr Asn Thr Val Thr

180 185 190

Asp Ile Ala Trp Leu Pro Leu Gln Ser Asn Gln Phe Val Arg Thr Trp

195 200 205

Asp Arg Asp Met Leu Gln Gln Ala Ile Glu Arg Leu Leu Ser Trp Glu

210 215 220

Ser Trp Asn Lys Arg Val Gln Glu Glu Tyr Ala Lys Leu Lys Glu Lys

225 230 235 240

Met Ala Gln Leu Asn Glu Gln Leu Glu Gly Gly Gln Glu Trp Ile Ser

245 250 255

Leu Leu Glu Gln Tyr Glu Glu Asn Arg Glu Arg Glu Leu Arg Glu Asn

260 265 270

Met Thr Ala Ala Asn Asp Lys Tyr Arg Ile Thr Lys Arg Gln Met Lys

275 280 285

Gly Trp Asn Glu Leu Tyr Glu Leu Trp Ser Thr Phe Pro Ala Ser Ala

290 295 300

Ser His Glu Gln Tyr Lys Glu Ala Leu Lys Arg Val Gln Gln Arg Leu

305 310 315 320

Arg Gly Arg Phe Gly Asp Ala His Phe Phe Gln Tyr Leu Met Glu Glu

325 330 335

Lys Asn Arg Leu Ile Trp Lys Gly Asn Pro Gln Arg Ile His Tyr Phe

340 345 350

Val Ala Arg Asn Glu Leu Thr Lys Arg Leu Glu Glu Ala Lys Gln Ser

355 360 365

Ala Thr Met Thr Leu Pro Asn Ala Arg Lys His Pro Leu Trp Val Arg

370 375 380

Phe Asp Ala Arg Gly Gly Asn Leu Gln Asp Tyr Tyr Leu Thr Ala Glu

385 390 395 400

Ala Asp Lys Pro Arg Ser Arg Arg Phe Val Thr Phe Ser Gln Leu Ile

405 410 415

Trp Pro Ser Glu Ser Gly Trp Met Glu Lys Lys Asp Val Glu Val Glu

420 425 430

Leu Ala Leu Ser Arg Gln Phe Tyr Gln Gln Val Lys Leu Leu Lys Asn

435 440 445

Asp Lys Gly Lys Gln Lys Ile Glu Phe Lys Asp Lys Gly Ser Gly Ser

450 455 460

Thr Phe Asn Gly His Leu Gly Gly Ala Lys Leu Gln Leu Glu Arg Gly

465 470 475 480

Asp Leu Glu Lys Glu Glu Lys Asn Phe Glu Asp Gly Glu Ile Gly Ser

485 490 495

Val Tyr Leu Asn Val Val Ile Asp Phe Glu Pro Leu Gln Glu Val Lys

500 505 510

Asn Gly Arg Val Gln Ala Pro Tyr Gly Gln Val Leu Gln Leu Ile Arg

515 520 525

Arg Pro Asn Glu Phe Pro Lys Val Thr Thr Tyr Lys Ser Glu Gln Leu

530 535 540

Val Glu Trp Ile Lys Ala Ser Pro Gln His Ser Ala Gly Val Glu Ser

545 550 555 560

Leu Ala Ser Gly Phe Arg Val Met Ser Ile Asp Leu Gly Leu Arg Ala

565 570 575

Ala Ala Ala Thr Ser Ile Phe Leu

580

<210> 564

<211> 259

<212> PRT

<213> Brevibacillus sp.

<400> 564

Met Ala Ile Arg Ser Ile Lys Leu Lys Leu Lys Thr His Thr Gly Pro

1 5 10 15

Glu Ala Gln Asn Leu Arg Lys Gly Ile Trp Arg Thr His Arg Leu Leu

20 25 30

Asn Glu Gly Val Ala Tyr Tyr Met Lys Met Leu Leu Leu Phe Arg Gln

35 40 45

Glu Ser Thr Gly Glu Arg Pro Lys Glu Glu Leu Gln Glu Glu Leu Ile

50 55 60

Cys His Ile Arg Glu Gln Gln Gln Arg Asn Gln Ala Asp Lys Asn Thr

65 70 75 80

Gln Ala Leu Pro Leu Asp Lys Ala Leu Glu Ala Leu Arg Gln Leu Tyr

85 90 95

Glu Leu Leu Val Pro Ser Ser Val Gly Gln Ser Gly Asp Ala Gln Ile

100 105 110

Ile Ser Arg Lys Phe Leu Ser Pro Leu Val Asp Pro Asn Ser Glu Gly

115 120 125

Gly Lys Gly Thr Ser Lys Ala Gly Ala Lys Pro Thr Trp Gln Lys Lys

130 135 140

Lys Glu Ala Asn Asp Pro Thr Trp Glu Gln Asp Tyr Glu Lys Trp Lys

145 150 155 160

Lys Arg Arg Glu Glu Asp Pro Thr Ala Ser Val Ile Thr Thr Leu Glu

165 170 175

Glu Tyr Gly Ile Arg Pro Ile Phe Pro Leu Tyr Thr Asn Thr Val Thr

180 185 190

Asp Ile Ala Trp Leu Pro Leu Gln Ser Asn Gln Phe Val Arg Thr Trp

195 200 205

Asp Arg Asp Met Leu Gln Gln Ala Ile Glu Arg Leu Leu Ser Trp Glu

210 215 220

Ser Trp Asn Lys Arg Val Gln Glu Glu Tyr Ala Lys Leu Lys Glu Lys

225 230 235 240

Met Ala Gln Leu Asn Glu Gln Leu Glu Gly Gly Gln Glu Trp Cys Thr

245 250 255

Leu Ser Arg

<210> 565

<211> 658

<212> PRT

<213> Methylobacterium nodulans

<400> 565

Met Leu Thr Lys Gln Asp Lys Gln Gln Lys Ile Thr Tyr Cys Thr Asn

1 5 10 15

Met Asn Glu Val Phe Glu Ala Lys Leu Gly Ser Ala Asp Leu Leu Leu

20 25 30

Asn Trp Asp His Leu Arg Gly Arg Ile Arg Asp Arg Val Asp Ala Gly

35 40 45

Asp Ile Gly Ser Ala Phe Leu Lys Leu Ala Leu Asp Val Ala His Val

50 55 60

Leu Pro Asp Gly Val Asp Asp Gln Leu Ala Arg Ala Ala Phe His Phe

65 70 75 80

Gln Ser Ala Lys Gly Ala Lys Ser Lys His Ala Asp Ser Val Gln Ala

85 90 95

Gly Leu Arg Val Leu Ser Ile Asp Leu Gly Val Arg Ser Phe Ala Thr

100 105 110

Cys Ser Val Phe Glu Leu Lys Asp Thr Ala Pro Thr Thr Gly Val Ala

115 120 125

Phe Pro Leu Ala Glu Phe Arg Leu Trp Ala Val His Glu Arg Ser Phe

130 135 140

Thr Leu Glu Leu Pro Gly Glu Asn Val Gly Ala Ala Gly Gln Gln Trp

145 150 155 160

Arg Ala Gln Ala Asp Ala Glu Leu Arg Gln Leu Arg Gly Gly Leu Asn

165 170 175

Arg His Arg Gln Leu Leu Arg Ala Ala Thr Val Gln Lys Gly Glu Arg

180 185 190

Asp Ala Tyr Leu Thr Asp Leu Arg Glu Ala Trp Ser Ala Lys Glu Leu

195 200 205

Trp Pro Phe Glu Ala Ser Leu Leu Ser Glu Leu Glu Arg Cys Ser Thr

210 215 220

Val Ala Asp Pro Leu Trp Gln Asp Thr Cys Lys Arg Ala Ala Arg Leu

225 230 235 240

Tyr Arg Thr Glu Phe Gly Ala Val Val Ser Glu Trp Arg Ser Arg Thr

245 250 255

Arg Ser Arg Glu Asp Arg Lys Tyr Ala Gly Lys Ser Met Trp Ser Val

260 265 270

Gln His Leu Thr Asp Val Arg Arg Phe Leu Gln Ser Trp Ser Leu Ala

275 280 285

Gly Arg Ala Ser Gly Asp Ile Arg Arg Leu Asp Arg Glu Arg Gly Gly

290 295 300

Val Phe Ala Lys Asp Leu Leu Asp His Ile Asp Ala Leu Lys Asp Asp

305 310 315 320

Arg Leu Lys Thr Gly Ala Asp Leu Ile Val Gln Ala Ala Arg Gly Phe

325 330 335

Gln Arg Asn Glu Phe Gly Tyr Trp Val Gln Lys His Ala Pro Cys His

340 345 350

Val Ile Leu Phe Glu Asp Leu Ser Arg Tyr Arg Met Arg Thr Asp Arg

355 360 365

Pro Arg Arg Glu Asn Ser Gln Leu Met Gln Trp Ala His Arg Gly Val

370 375 380

Pro Asp Met Val Gly Met Gln Gly Glu Ile Tyr Gly Ile Gln Asp Arg

385 390 395 400

Arg Asp Pro Asp Ser Ala Arg Lys His Ala Arg Gln Pro Leu Ala Ala

405 410 415

Phe Cys Leu Asp Thr Pro Ala Ala Phe Ser Ser Arg Tyr His Ala Ser

420 425 430

Thr Met Thr Pro Gly Ile Arg Cys His Pro Leu Arg Lys Arg Glu Phe

435 440 445

Glu Asp Gln Gly Phe Leu Glu Leu Leu Lys Arg Glu Asn Glu Gly Leu

450 455 460

Asp Leu Asn Gly Tyr Lys Pro Gly Asp Leu Val Pro Leu Pro Gly Gly

465 470 475 480

Glu Val Phe Val Cys Leu Asn Ala Asn Gly Leu Ser Arg Ile His Ala

485 490 495

Asp Ile Asn Ala Ala Gln Asn Leu Gln Arg Arg Phe Trp Thr Gln His

500 505 510

Gly Asp Ala Phe Arg Leu Pro Cys Gly Lys Ser Ala Val Gln Gly Gln

515 520 525

Ile Arg Trp Ala Pro Leu Ser Met Gly Lys Arg Gln Ala Gly Ala Leu

530 535 540

Gly Gly Phe Gly Tyr Leu Glu Pro Thr Gly His Asp Ser Gly Ser Cys

545 550 555 560

Gln Trp Arg Lys Thr Thr Glu Ala Glu Trp Arg Arg Leu Ser Gly Ala

565 570 575

Gln Lys Asp Arg Asp Glu Ala Ala Ala Ala Glu Asp Glu Glu Leu Gln

580 585 590

Gly Leu Glu Glu Glu Glu Leu Leu Glu Arg Ser Gly Glu Arg Val Val Phe

595 600 605

Phe Arg Asp Pro Ser Gly Val Val Leu Pro Thr Asp Leu Trp Phe Pro

610 615 620

Ser Ala Ala Phe Trp Ser Ile Val Arg Ala Lys Thr Val Gly Arg Leu

625 630 635 640

Arg Ser His Leu Asp Ala Gln Ala Glu Ala Ser Tyr Ala Val Ala Ala

645 650 655

Gly Leu

<210> 566

<211> 464

<212> PRT

<213> Methylobacterium nodulans

<400> 566

Met Pro Val Arg Ser Leu Lys Leu Lys Ile Val Val Pro Arg His Pro

1 5 10 15

Ser Glu Leu Glu Lys Ala Gln Ala Leu Trp Ser Thr His Arg Leu Val

20 25 30

Asn Glu Ala Val Ser Phe Tyr Glu Gln Lys Leu Leu Leu Leu Arg Gly

35 40 45

Glu Thr Tyr Ser Thr Ser Asp Gly Ser Val Pro Gln Asp Glu Val Arg

50 55 60

Arg Gln Leu Leu Glu Gln Ala Arg Glu Ala Gln Ala Arg Asn Gly Gly

65 70 75 80

Ser Gly Gly Ser Asp Asp Glu Ile Val Arg Leu Cys Arg Ser Leu Tyr

85 90 95

Glu Ala Ile Val Leu Ala Asp Asp Ala Asn Ala Gln Leu Ala Asn Ala

100 105 110

Phe Leu Gly Pro Leu Thr Asp Pro Asn Ser Ala Gly Phe Leu Glu Ala

115 120 125

Phe Asn Lys Val Asp Arg Pro Ala Pro Ser Trp Leu Asp Gln Val Pro

130 135 140

Ala Ser Asp Pro Ile Asp Pro Ala Val Leu Ala Glu Ala Asn Ala Trp

145 150 155 160

Leu Asp Thr Asp Ala Gly Arg Ala Trp Leu Val Asp Thr Gly Ala Pro

165 170 175

Pro Arg Trp Arg Ser Leu Ala Ala Lys Gln Asp Pro Ile Trp Pro Arg

180 185 190

Glu Phe Ala Arg Lys Leu Gly Glu Leu Arg Lys Glu Ala Ala Ser Gly

195 200 205

Thr Ser Ala Ile Ile Lys Ala Leu Lys Arg Asp Phe Gly Val Leu Pro

210 215 220

Leu Phe Gln Pro Ser Leu Ala Pro Arg Ile Leu Gly Ser Arg Ser Ser

225 230 235 240

Leu Thr Pro Trp Asp Arg Leu Ala Phe Arg Leu Ala Val Gly His Leu

245 250 255

Leu Ser Trp Glu Ser Trp Cys Thr Arg Ala Arg Asp Glu His Thr Ala

260 265 270

Arg Val Gln Arg Leu Glu Gln Phe Ser Ser Ala His Leu Lys Gly Asp

275 280 285

Leu Ala Thr Lys Val Ser Thr Leu Arg Glu Tyr Glu Arg Ala Arg Lys

290 295 300

Glu Gln Ile Ala Gln Leu Gly Leu Pro Met Gly Glu Arg Asp Phe Leu

305 310 315 320

Ile Thr Val Arg Met Thr Arg Gly Trp Asp Asp Leu Arg Glu Lys Trp

325 330 335

Arg Arg Ser Gly Asp Lys Gly Gln Glu Ala Leu His Ala Ile Ile Ala

340 345 350

Thr Glu Gln Thr Arg Lys Arg Gly Arg Phe Gly Asp Pro Asp Leu Phe

355 360 365

Arg Trp Leu Ala Arg Pro Glu Asn His His Val Trp Ala Asp Gly His

370 375 380

Ala Asp Ala Val Gly Val Leu Ala Arg Val Asn Ala Met Glu Arg Leu

385 390 395 400

Val Glu Arg Ser Arg Asp Thr Ala Leu Met Thr Leu Pro Asp Pro Val

405 410 415

Ala His Pro Arg Ser Ala Gln Trp Glu Ala Glu Gly Gly Ser Asn Leu

420 425 430

Arg Asn Tyr Gln Leu Glu Ala Val Gly Gly Glu Leu Gln Ile Thr Leu

435 440 445

Pro Leu Leu Lys Ala Ala Asp Asp Gly Arg Cys Ile Asp Thr Pro Leu

450 455 460

<210> 567

<211> 370

<212> PRT

<213> Methylobacterium nodulans

<400> 567

Met Tyr Glu Ala Ile Val Leu Ala Asp Asp Ala Asn Ala Gln Leu Ala

1 5 10 15

Asn Ala Phe Leu Gly Pro Leu Thr Asp Pro Asn Ser Ala Gly Phe Leu

20 25 30

Glu Ala Phe Asn Lys Val Asp Arg Pro Ala Pro Ser Trp Leu Asp Gln

35 40 45

Val Pro Ala Ser Asp Pro Ile Asp Pro Ala Val Leu Ala Glu Ala Asn

50 55 60

Ala Trp Leu Asp Thr Asp Ala Gly Arg Ala Trp Leu Val Asp Thr Gly

65 70 75 80

Ala Pro Pro Arg Trp Arg Ser Leu Ala Ala Lys Gln Asp Pro Ile Trp

85 90 95

Pro Arg Glu Phe Ala Arg Lys Leu Gly Glu Leu Arg Lys Glu Ala Ala

100 105 110

Ser Gly Thr Ser Ala Ile Ile Lys Ala Leu Lys Arg Asp Phe Gly Val

115 120 125

Leu Pro Leu Phe Gln Pro Ser Leu Ala Pro Arg Ile Leu Gly Ser Arg

130 135 140

Ser Ser Leu Thr Pro Trp Asp Arg Leu Ala Phe Arg Leu Ala Val Gly

145 150 155 160

His Leu Leu Ser Trp Glu Ser Trp Cys Thr Arg Ala Arg Asp Glu His

165 170 175

Thr Ala Arg Val Gln Arg Leu Glu Gln Phe Ser Ser Ala His Leu Lys

180 185 190

Gly Asp Leu Ala Thr Lys Val Ser Thr Leu Arg Glu Tyr Glu Arg Ala

195 200 205

Arg Lys Glu Gln Ile Ala Gln Leu Gly Leu Pro Met Gly Glu Arg Asp

210 215 220

Phe Leu Ile Thr Val Arg Met Thr Arg Gly Trp Asp Asp Leu Arg Glu

225 230 235 240

Lys Trp Arg Arg Ser Gly Asp Lys Gly Gln Glu Ala Leu His Ala Ile

245 250 255

Ile Ala Thr Glu Gln Thr Arg Lys Arg Gly Arg Phe Gly Asp Pro Asp

260 265 270

Leu Phe Arg Trp Leu Ala Arg Pro Glu Asn His His Val Trp Ala Asp

275 280 285

Gly His Ala Asp Ala Val Gly Val Leu Ala Arg Val Asn Ala Met Glu

290 295 300

Arg Leu Val Glu Arg Ser Arg Asp Thr Ala Leu Met Thr Leu Pro Asp

305 310 315 320

Pro Val Ala His Pro Arg Ser Ala Gln Trp Glu Ala Glu Gly Gly Ser

325 330 335

Asn Leu Arg Asn Tyr Gln Leu Glu Ala Val Gly Gly Glu Leu Gln Ile

340 345 350

Thr Leu Pro Leu Leu Lys Ala Ala Asp Asp Gly Arg Cys Ile Asp Thr

355 360 365

ProLeu

370

<210> 568

<211> 1050

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 568

Met Arg Ser Asn Tyr His Gly Gly Arg Asn Ala Arg Gln Trp Arg Lys

1 5 10 15

Gln Ile Ser Gly Leu Ala Arg Arg Thr Lys Glu Thr Val Phe Thr Tyr

20 25 30

Lys Phe Pro Leu Glu Thr Asp Ala Ala Glu Ile Asp Phe Asp Lys Ala

35 40 45

Val Gln Thr Tyr Gly Ile Ala Glu Gly Val Gly His Gly Ser Leu Ile

50 55 60

Gly Leu Val Cys Ala Phe His Leu Ser Gly Phe Arg Leu Phe Ser Lys

65 70 75 80

Ala Gly Glu Ala Met Ala Phe Arg Asn Arg Ser Arg Tyr Pro Thr Asp

85 90 95

Ala Phe Ala Glu Lys Leu Ser Ala Ile Met Gly Ile Gln Leu Pro Thr

100 105 110

Leu Ser Pro Glu Gly Leu Asp Leu Ile Phe Gln Ser Pro Pro Arg Ser

115 120 125

Arg Asp Gly Ile Ala Pro Val Trp Ser Glu Asn Glu Val Arg Asn Arg

130 135 140

Leu Tyr Thr Asn Trp Thr Gly Arg Gly Pro Ala Asn Lys Pro Asp Glu

145 150 155 160

His Leu Leu Glu Ile Ala Gly Glu Ile Ala Lys Gln Val Phe Pro Lys

165 170 175

Phe Gly Gly Trp Asp Asp Leu Ala Ser Asp Pro Asp Lys Ala Leu Ala

180 185 190

Ala Ala Asp Lys Tyr Phe Gln Ser Gln Gly Asp Phe Pro Ser Ile Ala

195 200 205

Ser Leu Pro Ala Ala Ile Met Leu Ser Pro Ala Asn Ser Thr Val Asp

210 215 220

Phe Glu Gly Asp Tyr Ile Ala Ile Asp Pro Ala Ala Glu Thr Leu Leu

225 230 235 240

His Gln Ala Val Ser Arg Cys Ala Ala Arg Leu Gly Arg Glu Arg Pro

245 250 255

Asp Leu Asp Gln Asn Lys Gly Pro Phe Val Ser Ser Leu Gln Asp Ala

260 265 270

Leu Val Ser Ser Gln Asn Asn Gly Leu Ser Trp Leu Phe Gly Val Gly

275 280 285

Phe Gln His Trp Lys Glu Lys Ser Pro Lys Glu Leu Ile Asp Glu Tyr

290 295 300

Lys Val Pro Ala Asp Gln His Gly Ala Val Thr Gln Val Lys Ser Phe

305 310 315 320

Val Asp Ala Ile Pro Leu Asn Pro Leu Phe Asp Thr Thr His Tyr Gly

325 330 335

Glu Phe Arg Ala Ser Val Ala Gly Lys Val Arg Ser Trp Val Ala Asn

340 345 350

Tyr Trp Lys Arg Leu Leu Asp Leu Lys Ser Leu Leu Ala Thr Glu

355 360 365

Phe Thr Leu Pro Glu Ser Ile Ser Asp Pro Lys Ala Val Ser Leu Phe

370 375 380

Ser Gly Leu Leu Val Asp Pro Gln Gly Leu Lys Lys Val Ala Asp Ser

385 390 395 400

Leu Pro Ala Arg Leu Val Ser Ala Glu Glu Ala Ile Asp Arg Leu Met

405 410 415

Gly Val Gly Ile Pro Thr Ala Ala Asp Ile Ala Gln Val Glu Arg Val

420 425 430

Ala Asp Glu Ile Gly Ala Phe Ile Gly Gln Val Gln Gln Phe Asn Asn

435 440 445

Gln Val Lys Gln Lys Leu Glu Asn Leu Gln Asp Ala Asp Asp Glu Glu

450 455 460

Phe Leu Lys Gly Leu Lys Ile Glu Leu Pro Ser Gly Asp Lys Glu Pro

465 470 475 480

Pro Ala Ile Asn Arg Ile Ser Gly Gly Ala Pro Asp Ala Ala Ala Glu

485 490 495

Ile Ser Glu Leu Glu Glu Lys Leu Gln Arg Leu Leu Asp Ala Arg Ser

500 505 510

Glu His Phe Gln Thr Ile Ser Glu Trp Ala Glu Glu Asn Ala Val Thr

515 520 525

Leu Asp Pro Ile Ala Ala Met Val Glu Leu Glu Arg Leu Arg Leu Ala

530 535 540

Glu Arg Gly Ala Thr Gly Asp Pro Glu Glu Tyr Ala Leu Arg Leu Leu

545 550 555 560

Leu Gln Arg Ile Gly Arg Leu Ala Asn Arg Val Ser Pro Val Ser Ala

565 570 575

Gly Ser Ile Arg Glu Leu Leu Lys Pro Val Phe Met Glu Glu Arg Glu

580 585 590

Phe Asn Leu Phe Phe His Asn Arg Leu Gly Ser Leu Tyr Arg Ser Pro

595 600 605

Tyr Ser Thr Ser Arg His Gln Pro Phe Ser Ile Asp Val Gly Lys Ala

610 615 620

Lys Ala Ile Asp Trp Ile Ala Gly Leu Asp Gln Ile Ser Ser Asp Ile

625 630 635 640

Glu Lys Ala Leu Ser Gly Ala Gly Glu Ala Leu Gly Asp Gln Leu Arg

645 650 655

Asp Trp Ile Asn Leu Ala Gly Phe Ala Ile Ser Gln Arg Leu Arg Gly

660 665 670

Leu Pro Asp Thr Val Pro Asn Ala Leu Ala Gln Val Arg Cys Pro Asp

675 680 685

Asp Val Arg Ile Pro Pro Leu Leu Ala Met Leu Leu Glu Glu Asp Asp

690 695 700

Ile Ala Arg Asp Val Cys Leu Lys Ala Phe Asn Leu Tyr Val Ser Ala

705 710 715 720

Ile Asn Gly Cys Leu Phe Gly Ala Leu Arg Glu Gly Phe Ile Val Arg

725 730 735

Thr Arg Phe Gln Arg Ile Gly Thr Asp Gln Ile His Tyr Val Pro Lys

740 745 750

Asp Lys Ala Trp Glu Tyr Pro Asp Arg Leu Asn Thr Ala Lys Gly Pro

755 760 765

Ile Asn Ala Ala Val Ser Ser Asp Trp Ile Glu Lys Asp Gly Ala Val

770 775 780

Ile Lys Pro Val Glu Thr Val Arg Asn Leu Ser Ser Thr Gly Phe Ala

785 790 795 800

Gly Ala Gly Val Ser Glu Tyr Leu Val Gln Ala Pro His Asp Trp Tyr

805 810 815

Thr Pro Leu Asp Leu Arg Asp Val Ala His Leu Val Thr Gly Leu Pro

820 825 830

Val Glu Lys Asn Ile Thr Lys Leu Lys Arg Leu Thr Asn Arg Thr Ala

835 840 845

Phe Arg Met Val Gly Ala Ser Ser Phe Lys Thr His Leu Asp Ser Val

850 855 860

Leu Leu Ser Asp Lys Ile Lys Leu Gly Asp Phe Thr Ile Ile Ile Asp

865 870 875 880

Gln His Tyr Arg Gln Ser Val Thr Tyr Gly Gly Lys Val Lys Ile Ser

885 890 895

Tyr Glu Pro Glu Arg Leu Gln Val Glu Ala Ala Val Pro Val Val Asp

900 905 910

Thr Arg Asp Arg Thr Val Pro Glu Pro Asp Thr Leu Phe Asp His Ile

915 920 925

Val Ala Ile Asp Leu Gly Glu Arg Ser Val Gly Phe Ala Val Phe Asp

930 935 940

Ile Lys Ser Cys Leu Arg Thr Gly Glu Val Lys Pro Ile His Asp Asn

945 950 955 960

Asn Gly Asn Pro Val Val Gly Thr Val Ala Val Pro Ser Ile Arg Arg

965 970 975

Leu Met Lys Ala Val Arg Ser His Arg Arg Arg Arg Gln Pro Asn Gln

980 985 990

Lys Val Asn Gln Thr Tyr Ser Thr Ala Leu Gln Asn Tyr Arg Glu Asn

995 1000 1005

Val Ile Gly Asp Val Cys Asn Arg Ile Asp Thr Leu Met Glu Arg

1010 1015 1020

Tyr Asn Ala Phe Pro Val Leu Glu Phe Gln Ile Lys Asn Phe Gln

1025 1030 1035

Ala Gly Ala Lys Gln Leu Glu Ile Val Tyr Gly Ser

1040 1045 1050

<210> 569

<211> 1222

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 569

Met Lys Lys Phe Glu Leu Lys Gln Asn Phe Arg Asn Asn Tyr Ser Gly

1 5 10 15

Lys Thr Leu Arg Asn Phe Arg Gln Thr Leu Ala Gln Ile Ala Asn Lys

20 25 30

Lys Ser Ser Asp Ser Ile Leu Thr Ile Lys Phe Lys Leu Asp Cys Ser

35 40 45

Lys Thr Gly Lys Leu Pro Lys Tyr Glu Asn Leu Ile Ser Leu Tyr Asp

50 55 60

Thr Ile Glu Asp Ile Lys Lys Gly Thr Leu Ser Tyr Tyr Leu Phe Thr

65 70 75 80

Leu Ile Val Ser Gly Phe Lys Phe Phe Gly Ser Ala Ser Gln Ala Lys

85 90 95

Ala Phe Ser Thr Lys Asp Ile Phe Lys Asp Asn Asp Phe Tyr Asn Gln

100 105 110

Phe Lys Ile Gln Ser His Leu Asp Leu Pro Asp Phe Val Pro Ser Lys

115 120 125

Ile Tyr Gln Arg Leu Lys Lys Asn Val Arg Ser Thr Asn Gly Lys Asp

130 135 140

Asn Ala Phe Lys Ala Ser Val Ile Val Ala Glu Tyr Arg Lys Glu Ile

145 150 155 160

Gly Lys Leu Lys Asn Lys Asp Glu Ser Ser Glu His Gln Cys Glu Glu

165 170 175

Leu Phe Lys Lys Ile Gly Thr Ala Leu Glu Thr Arg Phe Ser Ser Trp

180 185 190

Gln Asp Leu Ile Asn Asn Cys Ser Thr Gly Cys Glu Ile Ile Asp Glu

195 200 205

Ile Leu Asn Asp Ser Phe Gly Thr Leu Pro Ser Ile Lys Lys Met Val

210 215 220

Leu Ala Ser Thr Thr Gln Ser Ser Asp Gly Glu Gln Asp Gly Ile Ala

225 230 235 240

Ile Ala Tyr Asp Pro Asp Ser Thr Phe Ile Lys Ser Asp Glu Leu Leu

245 250 255

Asn Pro Tyr Phe Ala Val Ala Thr Ile Leu Lys Ser Met Pro Pro Glu

260 265 270

Ile Gln Gln Asp Lys Lys Ser Ala Tyr Val Lys Ala Asn Leu Thr Thr

275 280 285

Pro Thr His Asn Ala Leu Ser Trp Ile Phe Gly Lys Gly Leu Thr Leu

290 295 300

Phe Gln Thr Glu Ser Thr Glu Lys Leu Cys Ala Met Phe Asn Val Ser

305 310 315 320

Asp Lys Arg Val Ile Glu Gln Val Gln Asp Ala Ala Lys Ala Val Lys

325 330 335

Leu Pro Ala Glu Leu Asp Leu Asn His Cys Thr Leu Lys Phe Gln Asp

340 345 350

Phe Arg Ser Ser Leu Gly Gly His Leu Asp Ser Trp Thr Thr Asn Tyr

355 360 365

Leu Lys Arg Leu Asp Glu Leu Asn Asp Leu Leu Leu Asn Leu Pro Lys

370 375 380

Asn Leu Ser Leu Pro Asp Ile Phe Met Ile Asp Gly Lys Asp Phe Ile

385 390 395 400

Glu Tyr Ser Gly Cys Asn Arg Asp Glu Ile Gln Gln Met Ile Asp Phe

405 410 415

Val Val Asn Glu Gln Asn Arg Ile Lys Leu Gln Glu Ser Leu Asn Ala

420 425 430

Leu Leu Gly Lys Gly Asn Asn Gln Ile Cys Ser Asp Asp Ile Ser Thr

435 440 445

Val Lys Asp Phe Ser Glu Ile Val Asn Ser Leu His Ser Phe Val Gln

450 455 460

Gln Ile Asp Asn Ser Leu Glu Gln Ser Ser Asn Glu Ala Asn Ser Ile

465 470 475 480

Phe Ser Glu Leu Lys Lys Lys Ile Glu Lys Asn Glu Lys Trp Asp Ile

485 490 495

Trp Lys Asn Asn Leu Lys Lys Ile Pro Lys Leu Asn Lys Leu Ser Gly

500 505 510

Gly Val Pro Asp Ala Trp Lys Glu Ile Arg Glu Ile Glu Gln Lys Phe

515 520 525

His Glu Ile Ser Glu Asn Gln Lys Lys His Phe Thr Glu Val Met Glu

530 535 540

Trp Ile Asp Ala Gly Asn Gly Thr Ile Asp Ile Phe Glu Ser Arg Phe

545 550 555 560

Lys Tyr Asp Glu Leu Leu Lys Lys Ser Lys Lys Asn Asn Leu Gln Ser

565 570 575

Ala Asp Glu Leu Ala Phe Arg Ser Val Leu Asn Lys Leu Gly Arg Phe

580 585 590

Ala Arg Gln Gly Asn Asp Leu Val Cys Glu Lys Ile Lys Asn Trp Phe

595 600 605

Lys Glu Gln Asn Ile Phe Asp Ser Ser Lys Asp Phe Asn Arg Tyr Phe

610 615 620

Ile Asn Gln Lys Gly Phe Ile Phe Lys His Pro Ser Ser Lys Lys Asp

625 630 635 640

Asn Ser Pro Tyr Asn Leu Ser Ala Asn Leu Leu Glu Lys Arg Tyr Glu

645 650 655

Val Thr Asn Thr Val Gly Ala Leu Leu Glu Gln Cys Glu Ser Asp Pro

660 665 670

Ala Ile Val Asn Asp Pro Phe Ser Met Arg Ser Leu Val Glu Phe Arg

675 680 685

Ala Leu Trp Phe Ser Ile Asn Ile Ser Gly Ile Ser Lys Glu Gln His

690 695 700

Ile Pro Thr Lys Ile Ala Gln Pro Lys Leu Asp Asp Ser Thr Tyr Gln

705 710 715 720

Glu Ser Val Ser Pro Thr Leu Lys Tyr Arg Leu Glu Lys Glu Gln Ile

725 730 735

Thr Ser Ser Glu Leu Asn Ser Ile Phe Thr Val Tyr Lys Ser Leu Leu

740 745 750

Ser Gly Leu Ser Ile Arg Leu Ser Arg Asn Ser Phe Tyr Leu Arg Thr

755 760 765

Lys Phe Ser Trp Ile Gly Asn Asn Ser Leu Ile Tyr Cys Pro Lys Glu

770 775 780

Thr Thr Trp Lys Ile Pro Ala Ala Tyr Phe Lys Ser Asp Leu Trp Asn

785 790 795 800

Glu Tyr Lys Asp Lys Gln Ile Leu Ile Val Asn Glu Glu Tyr Asp Val

805 810 815

Asp Val Val Lys Thr Phe Glu Ser Val Tyr Lys Ile Val Lys Ser Lys

820 825 830

Asp Asn Asn Glu Lys Asn Arg Ile Leu Pro Leu Leu Lys Gln Leu Pro

835 840 845

His Asp Trp Met Phe Lys Leu Pro Phe Gly Ala Ser Asn Ala Glu Lys

850 855 860

Cys Lys Val Leu Lys Leu Glu Lys Asn Asn Lys Lys Phe Lys Pro Leu

865 870 875 880

Ser Val Ser Lys Asp Ser Leu Ala Arg Leu Ser Gly Pro Ser Thr Tyr

885 890 895

Phe Asn Gln Ile Asp Glu Ile Met Met Asn Asp Glu Ser Glu Leu Ser

900 905 910

Glu Met Thr Leu Leu Ala Asp Glu Pro Val Arg Gln Gln Met Ser Asn

915 920 925

Gly Lys Ile Glu Ile Ile Pro Asp Asp Tyr Val Met Ser Leu Ala Ile

930 935 940

Pro Ile Thr Arg Ser Leu Lys Lys Gly Asn Thr Glu Ser Phe Pro Phe

945 950 955 960

Lys Asn Ile Val Ser Ile Asp Gln Gly Glu Ala Gly Phe Ala Tyr Ala

965 970 975

Val Phe Lys Leu Ser Asp Cys Gly Asn Glu Arg Ala Glu Pro Ile Ala

980 985 990

Thr Gly Leu Ile Pro Ile Pro Ser Ile Arg Arg Leu Ile His Ser Val

995 1000 1005

Lys Lys Tyr Arg Gly Lys Lys Gln Arg Ile Gln Asn Phe Asn Gln

1010 1015 1020

Lys Phe Asp Ser Thr Met Phe Thr Leu Arg Glu Asn Val Thr Gly

1025 1030 1035

Asp Ile Cys Gly Leu Ile Val Ala Leu Met Lys Lys Tyr Asn Ala

1040 1045 1050

Phe Pro Ile Leu Glu Lys Gln Val Gly Asn Leu Glu Ser Gly Ser

1055 1060 1065

Lys Gln Leu Met Leu Val Tyr Lys Ala Val Asn Ser Lys Phe Leu

1070 1075 1080

Ala Ala Lys Val Asp Met Gln Asn Asp Gln Arg Arg Ser Trp Trp

1085 1090 1095

Tyr Gln Gly Asn Ser Trp Asn Thr Pro Ile Leu Arg Ile Ser Asn

1100 1105 1110

Pro Asn Gln Ser Asn Asn Lys Asn Ile Val Lys Asn Ile Asn Gly

1115 1120 1125

Lys Lys Tyr Glu Glu Leu Lys Ile Tyr Pro Gly Tyr Ser Val Ser

1130 1135 1140

Ala Tyr Met Thr Ser Cys Ile Cys His Val Cys Gly Arg Asn Ala

1145 1150 1155

Leu Glu Leu Leu Lys Asn Asp Asp Ser Thr Gly Lys Val Lys Lys

1160 1165 1170

Tyr Gln Ile Asn Gln Asp Gly Glu Val Thr Ile Gly Gly Glu Val

1175 1180 1185

Ile Lys Leu Tyr Arg Lys Pro Asp Arg Leu Thr Pro Val Lys Asn

1190 1195 1200

Leu Ala Lys Lys Gly Asn Arg Glu Arg Thr Tyr Ala Ser Ile Asn

1205 1210 1215

Glu Arg Ala Pro

1220

<210> 570

<211> 1255

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 570

Met Arg Ser Asn Tyr His Gly Gly Arg Asn Ala Arg Gln Trp Arg Lys

1 5 10 15

Gln Ile Ser Gly Leu Ala Arg Arg Thr Lys Glu Thr Val Phe Thr Tyr

20 25 30

Lys Phe Pro Leu Glu Thr Asp Ala Ala Glu Ile Asp Phe Asp Lys Ala

35 40 45

Val Gln Thr Tyr Gly Ile Ala Glu Gly Val Gly His Gly Ser Leu Ile

50 55 60

Gly Leu Val Cys Ala Phe His Leu Ser Gly Phe Arg Leu Phe Ser Lys

65 70 75 80

Ala Gly Glu Ala Met Ala Phe Arg Asn Arg Ser Arg Tyr Pro Thr Asp

85 90 95

Ala Phe Ala Glu Lys Leu Ser Ala Ile Met Gly Ile Gln Leu Pro Thr

100 105 110

Leu Ser Pro Glu Gly Leu Asp Leu Ile Phe Gln Ser Pro Pro Arg Ser

115 120 125

Arg Asp Gly Ile Ala Pro Val Trp Ser Glu Asn Glu Val Arg Asn Arg

130 135 140

Leu Tyr Thr Asn Trp Thr Gly Arg Gly Pro Ala Asn Lys Pro Asp Glu

145 150 155 160

His Leu Leu Glu Ile Ala Gly Glu Ile Ala Lys Gln Val Phe Pro Lys

165 170 175

Phe Gly Gly Trp Asp Asp Leu Ala Ser Asp Pro Asp Lys Ala Leu Ala

180 185 190

Ala Ala Asp Lys Tyr Phe Gln Ser Gln Gly Asp Phe Pro Ser Ile Ala

195 200 205

Ser Leu Pro Ala Ala Ile Met Leu Ser Pro Ala Asn Ser Thr Val Asp

210 215 220

Phe Glu Gly Asp Tyr Ile Ala Ile Asp Pro Ala Ala Glu Thr Leu Leu

225 230 235 240

His Gln Ala Val Ser Arg Cys Ala Ala Arg Leu Gly Arg Glu Arg Pro

245 250 255

Asp Leu Asp Gln Asn Lys Gly Pro Phe Val Ser Ser Leu Gln Asp Ala

260 265 270

Leu Val Ser Ser Gln Asn Asn Gly Leu Ser Trp Leu Phe Gly Val Gly

275 280 285

Phe Gln His Trp Lys Glu Lys Ser Pro Lys Glu Leu Ile Asp Glu Tyr

290 295 300

Lys Val Pro Ala Asp Gln His Gly Ala Val Thr Gln Val Lys Ser Phe

305 310 315 320

Val Asp Ala Ile Pro Leu Asn Pro Leu Phe Asp Thr Thr His Tyr Gly

325 330 335

Glu Phe Arg Ala Ser Val Ala Gly Lys Val Arg Ser Trp Val Ala Asn

340 345 350

Tyr Trp Lys Arg Leu Leu Asp Leu Lys Ser Leu Leu Ala Thr Glu

355 360 365

Phe Thr Leu Pro Glu Ser Ile Ser Asp Pro Lys Ala Val Ser Leu Phe

370 375 380

Ser Gly Leu Leu Val Asp Pro Gln Gly Leu Lys Lys Val Ala Asp Ser

385 390 395 400

Leu Pro Ala Arg Leu Val Ser Ala Glu Glu Ala Ile Asp Arg Leu Met

405 410 415

Gly Val Gly Ile Pro Thr Ala Ala Asp Ile Ala Gln Val Glu Arg Val

420 425 430

Ala Asp Glu Ile Gly Ala Phe Ile Gly Gln Val Gln Gln Phe Asn Asn

435 440 445

Gln Val Lys Gln Lys Leu Glu Asn Leu Gln Asp Ala Asp Asp Glu Glu

450 455 460

Phe Leu Lys Gly Leu Lys Ile Glu Leu Pro Ser Gly Asp Lys Glu Pro

465 470 475 480

Pro Ala Ile Asn Arg Ile Ser Gly Gly Ala Pro Asp Ala Ala Ala Glu

485 490 495

Ile Ser Glu Leu Glu Glu Lys Leu Gln Arg Leu Leu Asp Ala Arg Ser

500 505 510

Glu His Phe Gln Thr Ile Ser Glu Trp Ala Glu Glu Asn Ala Val Thr

515 520 525

Leu Asp Pro Ile Ala Ala Met Val Glu Leu Glu Arg Leu Arg Leu Ala

530 535 540

Glu Arg Gly Ala Thr Gly Asp Pro Glu Glu Tyr Ala Leu Arg Leu Leu

545 550 555 560

Leu Gln Arg Ile Gly Arg Leu Ala Asn Arg Val Ser Pro Val Ser Ala

565 570 575

Gly Ser Ile Arg Glu Leu Leu Lys Pro Val Phe Met Glu Glu Arg Glu

580 585 590

Phe Asn Leu Phe Phe His Asn Arg Leu Gly Ser Leu Tyr Arg Ser Pro

595 600 605

Tyr Ser Thr Ser Arg His Gln Pro Phe Ser Ile Asp Val Gly Lys Ala

610 615 620

Lys Ala Ile Asp Trp Ile Ala Gly Leu Asp Gln Ile Ser Ser Asp Ile

625 630 635 640

Glu Lys Ala Leu Ser Gly Ala Gly Glu Ala Leu Gly Asp Gln Leu Arg

645 650 655

Asp Trp Ile Asn Leu Ala Gly Phe Ala Ile Ser Gln Arg Leu Arg Gly

660 665 670

Leu Pro Asp Thr Val Pro Asn Ala Leu Ala Gln Val Arg Cys Pro Asp

675 680 685

Asp Val Arg Ile Pro Pro Leu Leu Ala Met Leu Leu Glu Glu Asp Asp

690 695 700

Ile Ala Arg Asp Val Cys Leu Lys Ala Phe Asn Leu Tyr Val Ser Ala

705 710 715 720

Ile Asn Gly Cys Leu Phe Gly Ala Leu Arg Glu Gly Phe Ile Val Arg

725 730 735

Thr Arg Phe Gln Arg Ile Gly Thr Asp Gln Ile His Tyr Val Pro Lys

740 745 750

Asp Lys Ala Trp Glu Tyr Pro Asp Arg Leu Asn Thr Ala Lys Gly Pro

755 760 765

Ile Asn Ala Ala Val Ser Ser Asp Trp Ile Glu Lys Asp Gly Ala Val

770 775 780

Ile Lys Pro Val Glu Thr Val Arg Asn Leu Ser Ser Thr Gly Phe Ala

785 790 795 800

Gly Ala Gly Val Ser Glu Tyr Leu Val Gln Ala Pro His Asp Trp Tyr

805 810 815

Thr Pro Leu Asp Leu Arg Asp Val Ala His Leu Val Thr Gly Leu Pro

820 825 830

Val Glu Lys Asn Ile Thr Lys Leu Lys Arg Leu Thr Asn Arg Thr Ala

835 840 845

Phe Arg Met Val Gly Ala Ser Ser Phe Lys Thr His Leu Asp Ser Val

850 855 860

Leu Leu Ser Asp Lys Ile Lys Leu Gly Asp Phe Thr Ile Ile Ile Asp

865 870 875 880

Gln His Tyr Arg Gln Ser Val Thr Tyr Gly Gly Lys Val Lys Ile Ser

885 890 895

Tyr Glu Pro Glu Arg Leu Gln Val Glu Ala Ala Val Pro Val Val Asp

900 905 910

Thr Arg Asp Arg Thr Val Pro Glu Pro Asp Thr Leu Phe Asp His Ile

915 920 925

Val Ala Ile Asp Leu Gly Glu Arg Ser Val Gly Phe Ala Val Phe Asp

930 935 940

Ile Lys Ser Cys Leu Arg Thr Gly Glu Val Lys Pro Ile His Asp Asn

945 950 955 960

Asn Gly Asn Pro Val Val Gly Thr Val Ala Val Pro Ser Ile Arg Arg

965 970 975

Leu Met Lys Ala Val Arg Ser His Arg Arg Arg Arg Gln Pro Asn Gln

980 985 990

Lys Val Asn Gln Thr Tyr Ser Thr Ala Leu Gln Asn Tyr Arg Glu Asn

995 1000 1005

Val Ile Gly Asp Val Cys Asn Arg Ile Asp Thr Leu Met Glu Arg

1010 1015 1020

Tyr Asn Ala Phe Pro Val Leu Glu Phe Gln Ile Lys Asn Phe Gln

1025 1030 1035

Ala Gly Ala Lys Gln Leu Glu Ile Val Tyr Gly Ser Val Leu His

1040 1045 1050

Arg Tyr Thr Phe Ser Gly Val Asp Ala His Lys Ala Lys Arg Arg

1055 1060 1065

Glu Tyr Trp Tyr Asn Gly Glu Leu Trp Glu His Pro Tyr Leu Met

1070 1075 1080

Ala Lys Lys Trp Asn Glu Glu Thr Asn Ser Met Ser Gly Ala Pro

1085 1090 1095

Lys Pro Val Ser Leu Phe Pro Gly Val Thr Val Asn Ala Ala Arg

1100 1105 1110

Thr Ser Gln Ile Cys His Gln Cys Gln Arg Asn Pro Met Ser His

1115 1120 1125

Leu Arg Gly Leu Thr Gly Thr Ile Glu Ile Ser Ser Asp Gly Leu

1130 1135 1140

Leu Glu Leu Asp Asp Gly Thr Ile Arg Leu Phe Glu Thr Ser Asp

1145 1150 1155

Tyr Asp Glu Asp Lys Phe Lys Gln Ser Arg Arg Glu Lys Arg Arg

1160 1165 1170

Leu Asp Ala Asn Val Leu Leu Ser Gly Arg His Arg Ala Glu Tyr

1175 1180 1185

Ile Tyr Thr Val Ala Lys Arg Asn Leu Arg Arg Pro Pro Lys Asn

1190 1195 1200

Val Met Thr Lys Asp Thr Thr Gln Ser Arg Tyr Thr Cys Leu Tyr

1205 1210 1215

Lys Asn Cys Ser Trp Thr Gly His Ala Asp Glu Asn Ala Ala Ile

1220 1225 1230

Asn Ile Gly Arg Arg Tyr Leu Ala Glu Arg Ile Asp Met Pro Ala

1235 1240 1245

Ser Lys Thr Lys Ala Ala Val

1250 1255

<210> 571

<211> 1258

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Metagenome sequence"

<400> 571

Met Asn Ala Arg Asp Trp Arg Lys His Val Gly Val Leu Ala Gln Gln

1 5 10 15

His Lys Glu Thr Thr Arg Thr Tyr Thr Phe Pro Leu Asp Thr Thr Gly

20 25 30

Ser Ala Ile Asp Phe Asp Ala Ala Leu Gln Ala Tyr Asn Ala Val Glu

35 40 45

Gly Val Gly Tyr Gly Ser Leu Leu Gly Leu Ala Cys Ala Val His Leu

50 55 60

Ser Gly Phe Arg Leu Phe Ser Thr Gly Lys Glu Ala Ala Thr Phe Arg

65 70 75 80

Asn Arg Ala Arg Tyr Pro Asn Ala Ala Phe Gln Ala Ala Leu Arg Lys

85 90 95

Glu Leu Gly Thr Thr Ile Thr Thr Leu Thr Pro Glu Thr Leu Asp Arg

100 105 110

Leu Phe Ser Ser Arg Pro Lys Arg Arg Asn Gly Val Pro Leu Pro Trp

115 120 125

Asn Gln Asp Ser Ile Arg Asp Arg Leu Tyr Thr Asn Trp Val Lys Pro

130 135 140

Arg Pro Gly Asp Thr Pro Asp Ala Val Leu Phe Gln Ile Ala Thr Gly

145 150 155 160

Ile Ala Gln Glu Ile Thr Glu Asp Val Ser Ser Trp Thr Asp Leu Ala

165 170 175

Lys Asn Ser Asp Arg Gly Leu Lys Ala Ala His Arg Tyr Phe Ala Arg

180 185 190

Val Gly Gly Phe Pro Ala Phe Asp Asn Leu Thr Pro Pro Ala Thr Val

195 200 205

Gln Pro Thr Asp Thr Thr Ile Asp Tyr Asp Pro Asn Ala Pro Phe His

210 215 220

Leu Val Ser His Ala Asp Gln Thr Leu Ile His Gln Ser Ile Ser Leu

225 230 235 240

Cys Ala His Arg Ile Arg Gln Glu Asp Pro Ala Leu Asp Pro Asn Lys

245 250 255

Ser Gly Phe Ile Lys Gln Leu Gln Asn Asn Phe Leu Ser Gln Thr Phe

260 265 270

Tyr Gly Leu Ser Trp Leu Phe Gly Ala Gly Tyr Val His Phe Arg Glu

275 280 285

Cys Thr Ala Asn Asp Leu Ala Ile Gln Tyr Gly Ile Pro Asn Asn Cys

290 295 300

Arg Asp Gly Ile His Gln Ile Lys Ser Phe Ala Asp Ala Ile Leu Pro

305 310 315 320

Asn Thr Phe Phe Glu Lys Lys His Tyr Arg Lys Asp Ser Arg Ser Val

325 330 335

Gly Lys Lys Ala Lys Ser Trp Ile Ser Asn Tyr Trp Gln Arg Leu Leu

340 345 350

Gln Leu Gln Thr Trp Val Asp Asp His Thr Trp Val Thr Leu Pro Gln

355 360 365

Glu Leu Thr Glu Ala Gln Phe Lys Pro Leu Phe Arg Gly Leu Leu Val

370 375 380

Asp Ala Val Glu Leu Met Ala Ile Ala Glu Arg Leu Pro Gln Arg Leu

385 390 395 400

Ala Asp Cys Arg Asp Ser Leu Asp Cys Leu Met Gly Lys Gly Pro Gln

405 410 415

Ala Ala Thr Lys Asn Asp Val Glu Ile Val Glu Lys Val Arg Glu Glu

420 425 430

Ile Glu Ser Phe Val Gly Gln Ile Glu Gln Leu Gly Asn Gln Leu Arg

435 440 445

His Gln Leu Glu Asn Glu Asn Asn Asp Gln Val His Arg Asp Asn Leu

450 455 460

His Gln Leu Lys Asn Arg Leu Pro Leu Asp Leu Arg Arg Pro Gln Ala

465 470 475 480

Leu Asn Lys Ile Ser Gly Gly Val Pro Asp Val Ala Lys Ser Ile Arg

485 490 495

Gly Leu Glu Thr Gln Leu Asp Gln Val Leu Lys Glu Arg Arg Ser His

500 505 510

Phe Gly Arg Leu Thr Lys Trp Ala Lys Glu Cys Gly Ile Thr Leu Asp

515 520 525

Pro Leu Gln Pro Leu Ile Glu Ser Glu Lys Gln Arg Val Ala Glu Arg

530 535 540

Gly Ser Ala His Asp Ala Lys Glu Leu Ala Ile Arg Leu Leu Leu Gln

545 550 555 560

Arg Ile Gly Arg Leu Gly His Arg Leu Ser Pro Thr Asn Ala Thr Ala

565 570 575

Ile Gln Glu Leu Leu Arg Pro Val Phe Ala Val Lys Arg Glu Phe Asn

580 585 590

Leu Phe Phe His Asn His Met Gly Ala Leu Tyr Arg Ser Pro Tyr Ser

595 600 605

Thr Ser Arg His Gln Pro Phe Gln Ile Asn Val Asp Val Ala His Gly

610 615 620

Thr Asp Trp Ile Gly Thr Ile Glu Thr Leu Ile Gln Asn Leu Phe Thr

625 630 635 640

Gln Ile Gln Asp Asp Ala Leu Leu Arg Asp Leu Val Gln Leu Glu Gly

645 650 655

Phe Val Phe Ser His Lys Leu Arg Ala Leu Pro Gly Val Ile Pro Ser

660 665 670

Glu Leu Ala Arg Pro Asn Asn Leu Gln Gln Met Gly Leu Pro Ala Leu

675 680 685

Leu Leu Val Leu Leu Gln Ala Asp Gln Val His Arg Glu Thr Val Leu

690 695 700

Arg Val Phe Asn Leu Tyr Gly Ser Ala Ile Asn Gly Tyr Leu Phe Gln

705 710 715 720

Ala Leu Arg Pro Gly Phe Ile Val Arg Ala Gly Phe Gln Arg Leu Glu

725 730 735

Thr Lys Lys Leu Arg Tyr Val Pro Lys Ala Gln Ser Trp Gln Tyr Pro

740 745 750

Asp Arg Leu His His Ala Lys Ser Ala Ile Lys Asn Ser Leu Ser Ala

755 760 765

Gly Trp Ile Lys Lys Asn His Gln Gly Ala Ile Leu Pro Gln Lys Thr

770 775 780

Leu Thr Ala Leu Val Lys Gln Lys Ser Leu Lys Asp Thr Gly Val Pro

785 790 795 800

Glu Tyr Leu Val Gln Ala Pro His Asp Trp Tyr Val Pro Ile Asp Leu

805 810 815

Arg Gly Pro Ala Ile Pro Ile Glu Gly Leu Thr Val Gly Thr Glu Gly

820 825 830

Pro Glu Leu Thr Gln Leu Gly Pro Met Lys Asp Asp Cys Ala Phe Arg

835 840 845

Ala Ile Gly Pro Ser Ser Phe Lys Ser Lys Ile Asp Ala Gly Leu Leu

850 855 860

Pro Gln Asp Val Lys Tyr Gly Asp Met Thr Leu Ile Phe Asp Gln His

865 870 875 880

Tyr Gln Gln Ser Ile Ser Phe Ala Asn Gly Thr Phe Ser Ile Gln Tyr

885 890 895

Gln Pro Thr Ser Leu Gln Val Lys Ala Ala Ile Pro Val Val Asp Lys

900 905 910

Arg Pro Arg Asp Thr Arg Asn Asn Ser His Leu Tyr Asp Arg Ile Val

915 920 925

Ala Ile Asp Leu Gly Glu Arg Lys Ile Gly Tyr Ala Ile Phe Asp Leu

930 935 940

Lys Gln Val Leu Lys Ser Glu Gln Leu Glu Pro Met Arg Glu Asp Gly

945 950 955 960

Lys Pro Leu Ile Gly Ser Ile Ser Ile Arg Ser Ile Arg Gly Leu Met

965 970 975

Lys Ala Val Gln Thr His Arg Asn Arg Arg Gln Pro Asn Tyr Arg Ile

980 985 990

Asp Gln Thr Tyr Ser Lys Ala Leu Met His Tyr Arg Glu Ser Val Ile

995 1000 1005

Gly Asp Val Cys Asn Ala Ile Asp Thr Leu Cys Ala Arg Tyr Gly

1010 1015 1020

Gly Phe Pro Val Leu Glu Ser Ser Val Arg Asn Phe Glu Val Gly

1025 1030 1035

Ser Ala Gln Leu Lys Thr Val Tyr Gly Ser Val Ser Arg Arg Tyr

1040 1045 1050

Thr Trp Ser Ala Val Asp Ala His Lys Asn Gln Arg Gln Gln Tyr

1055 1060 1065

Trp Leu Gly Gly Thr Lys Asp Lys Ile Pro Ile Trp Thr His Pro

1070 1075 1080

Tyr Leu Met Thr Arg Glu Trp Asp Glu Lys Asn Ser Lys Trp Ser

1085 1090 1095

Asn Arg Ser Lys Pro Leu Lys Met His Pro Gly Val Glu Val His

1100 1105 1110

Pro Ala Gly Thr Ser Gln Ile Cys His Gln Cys Lys Arg Asn Pro

1115 1120 1125

Ile Gly Ala Leu Trp Asn Val Ala Asp Thr Val Val Leu Asp Asp

1130 1135 1140

Gln Gly Gln Leu Asp Leu Asp Asp Gly Thr Ile Arg Leu Asn Ser

1145 1150 1155

Gly Tyr Ile Asp Thr Thr Glu Ile Lys Arg Ala Arg Arg Lys Lys

1160 1165 1170

Ile Arg Leu Pro Glu Asn Lys Pro Leu Thr Gly Ser His Lys Thr

1175 1180 1185

Ser His Val Arg Ala Val Ala Arg Arg Asn Leu Arg Gln Pro Pro

1190 1195 1200

Lys Ser Thr Arg Ala Lys Asp Thr Thr Gln Ser Arg Tyr Thr Cys

1205 1210 1215

Leu Tyr Val Asp Cys Gly His Glu Cys His Ala Asp Glu Asn Ala

1220 1225 1230

Ala Ile Asn Ile Gly Arg Lys Tyr Leu Gln Glu Arg Ile His Ile

1235 1240 1245

Glu Ala Ser Arg Gln Ala Leu Ser Thr Arg

1250 1255

<210> 572

<211> 1269

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

marine metagenome sequence"

<400> 572

Met Val Ala Gly Leu Lys Lys Ile Lys Arg Asp Gly Val Thr Met Lys

1 5 10 15

Ser Asn Tyr His Gly Gly Val Lys Ala Arg Ala Trp Arg Lys Arg Ile

20 25 30

Gly Gly Leu Ala Arg Arg Gln Lys Glu Thr Val Phe Thr Tyr Lys Phe

35 40 45

Pro Leu Glu Thr Glu Glu Glu Ala Gly Ile Asp Phe Asp Lys Ala Val Gln

50 55 60

Thr Tyr Gly Ile Ala Glu Gly Ile Ser Gln Gly Ser Leu Ile Gly Leu

65 70 75 80

Val Cys Ala Phe His Leu Ser Gly Phe Arg Leu Phe Ser Lys Ala Asp

85 90 95

Glu Thr Lys Ala Phe Cys Asn Gln Gly Arg Tyr Pro Asn Gln Ala Phe

100 105 110

Ala Glu Lys Leu Arg Asn Glu Leu Ser Val Thr Leu Pro Lys Leu Ser

115 120 125

Pro Gln Ser Leu Asp Val Leu Phe Gln Ser Ser Pro Lys Ser Lys Asn

130 135 140

Gly Val Ala Pro Glu Trp Ser Lys Asn Ala Ile Arg Asn Arg Leu Tyr

145 150 155 160

Thr Asn Trp Thr Gly Lys Gly Ala Gly Thr Asn Pro Asp Glu His Leu

165 170 175

Leu Glu Ile Ala Glu Asp Ile Ala Ala Glu Ile Asp Ser Asp Leu Asp

180 185 190

Gly Trp Lys Asp Leu Glu Glu His Pro Glu Lys Gly Leu Ser Ala Ala

195 200 205

Asp Arg Tyr Phe Gln Ala Gln Gly Asp Phe Pro Ser Leu Thr Gly Leu

210 215 220

Pro Pro Ser Val Pro Leu Thr Pro Gln Asn Ser Thr Val Ala Phe Glu

225 230 235 240

Gly Asp Pro Val Cys Leu Asn Pro Ser Asp Asn Thr Leu Leu His Gln

245 250 255

Ala Val Ala Arg Cys Ala Gly Arg Ile Leu Gln Glu Gln Pro Asn Leu

260 265 270

Ser Pro Asp Lys Asn Arg Phe Ile Asn Gln Leu Gln Asp Glu Leu Val

275 280 285

Ser Ser Gln Asn Asn Gly Leu Ser Trp Leu Phe Gly Val Gly Phe Lys

290 295 300

Tyr Trp Lys Glu Met Ser Val Asp Gln Leu Ala Asp Asp Tyr Lys Val

305 310 315 320

Lys Ser Thr Asp Leu Asp Ala Leu Lys Gln Val Lys Ser Phe Ile Asp

325 330 335

Ala Ile Pro Leu Asn Pro Leu Phe Asp Thr Pro His Tyr Gly Glu Phe

340 345 350

Arg Ala Ser Val Ala Gly Lys Met Arg Ser Trp Val Lys Asn Tyr Trp

355 360 365

Lys Arg Leu Leu Asp Leu Lys Ser Gln Leu Gly Thr Ala Asn Ile Asn

370 375 380

Leu Pro Glu Gly Leu Asp Glu Gln Arg Ala Glu Asn Leu Phe Ser Gly

385 390 395 400

Leu Leu Ile Asp Ser Lys Gly Leu Arg Gln Val Thr Asp Lys Leu Pro

405 410 415

Ser Arg Leu Lys Lys Ala Glu Asp Thr Ile Asp Arg Leu Met Gly Asp

420 425 430

Gly Asn Pro Thr Ser Asp Asp Ile Glu Gln Val Glu Thr Val Ala Ala

435 440 445

Glu Ile Ser Ala Phe Ile Gly Gln Val Glu Gln Phe Asn Asn Gln Leu

450 455 460

Glu Gln Arg Leu Glu Asn Pro Leu Glu Gly Asp Asp Glu Thr Phe Leu

465 470 475 480

Lys Gln Leu Lys Ile Asp Leu Pro Ala Glu Phe Lys Lys Pro Pro Ala

485 490 495

Ile Asn Arg Ile Ser Gly Gly Ser Pro Asp Pro Thr Ala Glu Ile Ala

500 505 510

Glu Leu Glu Glu Lys Leu Asp Arg Leu Met Ser Ala Arg Lys Glu His

515 520 525

Tyr Glu Thr Ile Ala Glu Trp Ala Ser Ala Asn Lys Val Thr Leu Asp

530 535 540

Pro Met Glu Ala Met Thr Thr Leu Glu Ala Gln Arg Leu Thr Glu Arg

545 550 555 560

Gly Ala Glu Gly Asp Gln Glu Glu Phe Ala Leu Arg Leu Leu Leu Gln

565 570 575

Arg Ile Gly Arg Leu Ala Asn Arg Leu Ser Pro Gln Gly Ala Thr Ala

580 585 590

Ile Arg Asp Leu Leu Arg Pro Val Phe Thr Glu Lys Arg Glu Phe Asn

595 600 605

Leu Phe Phe His Asn Arg Met Gly Ser Leu Tyr Arg Ser Pro Tyr Ser

610 615 620

Thr Ser Arg His Gln Pro Phe Thr Ile Asp Val Ala Val Ala Lys Asn

625 630 635 640

Thr Asp Trp Met Asp Ala Leu Asp Gly Ile Ala Glu Thr Ile Met Lys

645 650 655

Gly Leu Ser Gln Ala Gly Asp Glu Leu Ser Leu Arg Leu Arg Asp Trp

660 665 670

Ile Asn Ile Ser Gly Phe Ser Leu Ser Gln Arg Leu Arg Gly Leu Pro

675 680 685

Asp Thr Val Pro Gly Glu Leu Ala Leu Val Arg Ser Ala Asp Asp Val

690 695 700

Arg Ile Pro Pro Met Leu Ala Leu Gln Leu Glu Glu Asp Glu Val Ser

705 710 715 720

Arg Glu Val Cys Leu Lys Ala Phe Asn Leu Tyr Val Ser Ala Ile Asn

725 730 735

Gly Cys Leu Phe Arg Ala Leu Arg Glu Gly Phe Ile Val Arg Thr Lys

740 745 750

Phe Gln Arg Leu Glu Arg Asp Val Leu Ser Tyr Val Pro Lys Thr Lys

755 760 765

Leu Trp Asn Tyr Pro Gln Arg Leu Asp Thr Ala Arg Gly Pro Ile His

770 775 780

Ser Ala Leu Ala Ala Ala Trp Ile Asn Lys Glu Gly Ser Val Ile Asp

785 790 795 800

Pro Val Glu Thr Val Thr Ala Leu Ser Asp Thr Gly Phe Ser Asp Asp

805 810 815

Gly Ile Pro Glu Tyr Leu Val Gln Ala Pro His Asp Trp Tyr Thr Pro

820 825 830

Ile Asp Leu Arg Asp Ile Ser Lys Pro Val Ser Gly Leu Pro Val Lys

835 840 845

Lys Asn Ile Thr Gly Leu Lys Arg Gln Lys Lys Gln Thr Ala Phe Arg

850 855 860

Met Val Gly Pro Ser Ser Phe Lys Ser His Leu Asp Ser Thr Leu Leu

865 870 875 880

Ser Glu Glu Val Lys Leu Gly Asp Phe Thr Leu Ile Phe Asp Gln Tyr

885 890 895

Tyr Lys Gln Arg Val Ser Tyr Asn Gly Arg Val Lys Ile Thr Phe Glu

900 905 910

Pro Asp Arg Leu His Val Glu Ala Ala Val Pro Val Ile Asp Lys Arg

915 920 925

Val Arg Pro Ser Thr Glu Glu Asp Ala Leu Phe Asp His Leu Leu Ala

930 935 940

Ile Asp Leu Gly Glu Lys Arg Val Gly Tyr Ala Val Tyr Asp Ile Lys

945 950 955 960

Ala Cys Leu Arg Thr Gly Asp Ile Lys Pro Leu Glu Asp Gly Asp Gly

965 970 975

Lys Pro Ile Val Gly Ser Val Ala Val Pro Ser Ile Arg Arg Leu Met

980 985 990

Lys Ala Val Arg Ser His Arg Gln Gln Arg Gln Pro Asn Gln Lys Val

995 1000 1005

Asn Gln Thr Tyr Ser Thr Ala Leu Met Asn Tyr Arg Glu Asn Val

1010 1015 1020

Ile Gly Asp Val Cys Asn Arg Ile Asp Thr Leu Met Glu Lys Tyr

1025 1030 1035

Asn Ala Phe Pro Val Leu Glu Ser Ser Val Met Asn Phe Glu Ala

1040 1045 1050

Gly Ser Arg Gln Leu Glu Met Val Tyr Gly Ser Val Leu His Arg

1055 1060 1065

Tyr Thr Tyr Ser Lys Ile Asp Ala His Thr Ala Lys Arg Lys Glu

1070 1075 1080

Tyr Trp Tyr Thr Gly Glu Tyr Trp Asp His Pro Tyr Leu Met Ala

1085 1090 1095

His Lys Trp Asn Glu Arg Thr Arg Ser Tyr Ser Gly Ser Leu Ser

1100 1105 1110

Ala Leu Thr Leu Tyr Pro Gly Val Met Val His Pro Ala Gly Thr

1115 1120 1125

Ser Gln Arg Cys His Gln Cys Lys Arg Asn Pro Met Val Glu Ile

1130 1135 1140

Lys Gln Leu Thr Gly Gln Val Glu Ile Asn Ala Asp Gly Ser Leu

1145 1150 1155

Glu Leu Asp Asp Gly Thr Ile Cys Leu Tyr Glu Gly Tyr Asp Tyr

1160 1165 1170

Ser Pro Glu Glu Tyr Lys Lys Ala Lys Arg Glu Lys Arg Arg Leu

1175 1180 1185

Asp Pro Asn Val Pro Leu Ser Gly Arg His Gln Ala Lys His Val

1190 1195 1200

Ser Ala Val Ala Lys Arg Asn Leu Arg Arg Pro Thr Val Ser Met

1205 1210 1215

Met Ser Gly Asp Thr Thr Gln Ala Arg Tyr Val Cys Leu Tyr Thr

1220 1225 1230

Asp Cys Asp Phe Thr Gly His Ala Asp Glu Asn Ala Ala Ile Asn

1235 1240 1245

Ile Gly Trp Lys Tyr Leu Thr Glu Arg Ile Ala Leu Ser Glu Ser

1250 1255 1260

Lys Asp Lys Ala Gly Val

1265

<210> 573

<211> 1285

<212> PRT

<213> Rhodobacter capsulatus

<400> 573

Met Gln Ile Gly Lys Val Gln Gly Arg Thr Ile Ser Glu Phe Gly Asp

1 5 10 15

Pro Ala Gly Gly Leu Lys Arg Lys Ile Ser Thr Asp Gly Lys Asn Arg

20 25 30

Lys Glu Leu Pro Ala His Leu Ser Ser Asp Pro Lys Ala Leu Ile Gly

35 40 45

Gln Trp Ile Ser Gly Ile Asp Lys Ile Tyr Arg Lys Pro Asp Ser Arg

50 55 60

Lys Ser Asp Gly Lys Ala Ile His Ser Pro Thr Pro Ser Lys Met Gln

65 70 75 80

Phe Asp Ala Arg Asp Asp Leu Gly Glu Ala Phe Trp Lys Leu Val Ser

85 90 95

Glu Ala Gly Leu Ala Gln Asp Ser Asp Tyr Asp Gln Phe Lys Arg Arg

100 105 110

Leu His Pro Tyr Gly Asp Lys Phe Gln Pro Ala Asp Ser Gly Ala Lys

115 120 125

Leu Lys Phe Glu Ala Asp Pro Pro Glu Pro Gln Ala Phe His Gly Arg

130 135 140

Trp Tyr Gly Ala Met Ser Lys Arg Gly Asn Asp Ala Lys Glu Leu Ala

145 150 155 160

Ala Ala Leu Tyr Glu His Leu His Val Asp Glu Lys Arg Ile Asp Gly

165 170 175

Gln Pro Lys Arg Asn Pro Lys Thr Asp Lys Phe Ala Pro Gly Leu Val

180 185 190

Val Ala Arg Ala Leu Gly Ile Glu Ser Ser Val Leu Pro Arg Gly Met

195 200 205

Ala Arg Leu Ala Arg Asn Trp Gly Glu Glu Glu Ile Gln Thr Tyr Phe

210 215 220

Val Val Asp Val Ala Ala Ser Val Lys Glu Val Ala Lys Ala Ala Val

225 230 235 240

Ser Ala Ala Gln Ala Phe Asp Pro Arg Gln Val Ser Gly Arg Ser

245 250 255

Leu Ser Pro Lys Val Gly Phe Ala Leu Ala Glu His Leu Glu Arg Val

260 265 270

Thr Gly Ser Lys Arg Cys Ser Phe Asp Pro Ala Ala Gly Pro Ser Val

275 280 285

Leu Ala Leu His Asp Glu Val Lys Lys Thr Tyr Lys Arg Leu Cys Ala

290 295 300

Arg Gly Lys Asn Ala Ala Arg Ala Phe Pro Ala Asp Lys Thr Glu Leu

305 310 315 320

Leu Ala Leu Met Arg His Thr His Glu Asn Arg Val Arg Asn Gln Met

325 330 335

Val Arg Met Gly Arg Val Ser Glu Tyr Arg Gly Gln Gln Ala Gly Asp

340 345 350

Leu Ala Gln Ser His Tyr Trp Thr Ser Ala Gly Gln Thr Glu Ile Lys

355 360 365

Glu Ser Glu Ile Phe Val Arg Leu Trp Val Gly Ala Phe Ala Leu Ala

370 375 380

Gly Arg Ser Met Lys Ala Trp Ile Asp Pro Met Gly Lys Ile Val Asn

385 390 395 400

Thr Glu Lys Asn Asp Arg Asp Leu Thr Ala Ala Val Asn Ile Arg Gln

405 410 415

Val Ile Ser Asn Lys Glu Met Val Ala Glu Ala Met Ala Arg Arg Gly

420 425 430

Ile Tyr Phe Gly Glu Thr Pro Glu Leu Asp Arg Leu Gly Ala Glu Gly

435 440 445

Asn Glu Gly Phe Val Phe Ala Leu Leu Arg Tyr Leu Arg Gly Cys Arg

450 455 460

Asn Gln Thr Phe His Leu Gly Ala Arg Ala Gly Phe Leu Lys Glu Ile

465 470 475 480

Arg Lys Glu Leu Glu Lys Thr Arg Trp Gly Lys Ala Lys Glu Ala Glu

485 490 495

His Val Val Leu Thr Asp Lys Thr Val Ala Ala Ile Arg Ala Ile Ile

500 505 510

Asp Asn Asp Ala Lys Ala Leu Gly Ala Arg Leu Leu Ala Asp Leu Ser

515 520 525

Gly Ala Phe Val Ala His Tyr Ala Ser Lys Glu His Phe Ser Thr Leu

530 535 540

Tyr Ser Glu Ile Val Lys Ala Val Lys Asp Ala Pro Glu Val Ser Ser

545 550 555 560

Gly Leu Pro Arg Leu Lys Leu Leu Leu Lys Arg Ala Asp Gly Val Arg

565 570 575

Gly Tyr Val His Gly Leu Arg Asp Thr Arg Lys His Ala Phe Ala Thr

580 585 590

Lys Leu Pro Pro Pro Pro Ala Pro Arg Glu Leu Asp Asp Pro Ala Thr

595 600 605

Lys Ala Arg Tyr Ile Ala Leu Leu Arg Leu Tyr Asp Gly Pro Phe Arg

610 615 620

Ala Tyr Ala Ser Gly Ile Thr Gly Thr Ala Leu Ala Gly Pro Ala Ala

625 630 635 640

Arg Ala Lys Glu Ala Ala Thr Ala Leu Ala Gln Ser Val Asn Val Thr

645 650 655

Lys Ala Tyr Ser Asp Val Met Glu Gly Arg Ser Ser Arg Leu Arg Pro

660 665 670

Pro Asn Asp Gly Glu Thr Leu Arg Glu Tyr Leu Ser Ala Leu Thr Gly

675 680 685

Glu Thr Ala Thr Glu Phe Arg Val Gln Ile Gly Tyr Glu Ser Asp Ser

690 695 700

Glu Asn Ala Arg Lys Gln Ala Glu Phe Ile Glu Asn Tyr Arg Arg Asp

705 710 715 720

Met Leu Ala Phe Met Phe Glu Asp Tyr Ile Arg Ala Lys Gly Phe Asp

725 730 735

Trp Ile Leu Lys Ile Glu Pro Gly Ala Thr Ala Met Thr Arg Ala Pro

740 745 750

Val Leu Pro Glu Pro Ile Asp Thr Arg Gly Gln Tyr Glu His Trp Gln

755 760 765

Ala Ala Leu Tyr Leu Val Met His Phe Val Pro Ala Ser Asp Val Ser

770 775 780

Asn Leu Leu His Gln Leu Arg Lys Trp Glu Ala Leu Gln Gly Lys Tyr

785 790 795 800

Glu Leu Val Gln Asp Gly Asp Ala Thr Asp Gln Ala Asp Ala Arg Arg

805 810 815

Glu Ala Leu Asp Leu Val Lys Arg Phe Arg Asp Val Leu Val Leu Phe

820 825 830

Leu Lys Thr Gly Glu Ala Arg Phe Glu Gly Arg Ala Ala Pro Phe Asp

835 840 845

Leu Lys Pro Phe Arg Ala Leu Phe Ala Asn Pro Ala Thr Phe Asp Arg

850 855 860

Leu Phe Met Ala Thr Pro Thr Thr Ala Arg Pro Ala Glu Asp Asp Pro

865 870 875 880

Glu Gly Asp Gly Ala Ser Glu Pro Glu Leu Arg Val Ala Arg Thr Leu

885 890 895

Arg Gly Leu Arg Gln Ile Ala Arg Tyr Asn His Met Ala Val Leu Ser

900 905 910

Asp Leu Phe Ala Lys His Lys Val Arg Asp Glu Glu Val Ala Arg Leu

915 920 925

Ala Glu Ile Glu Asp Glu Thr Gln Glu Lys Ser Gln Ile Val Ala Ala

930 935 940

Gln Glu Leu Arg Thr Asp Leu His Asp Lys Val Met Lys Cys His Pro

945 950 955 960

Lys Thr Ile Ser Pro Glu Glu Arg Gln Ser Tyr Ala Ala Ala Ile Lys

965 970 975

Thr Ile Glu Glu His Arg Phe Leu Val Gly Arg Val Tyr Leu Gly Asp

980 985 990

His Leu Arg Leu His Arg Leu Met Met Asp Val Ile Gly Arg Leu Ile

995 1000 1005

Asp Tyr Ala Gly Ala Tyr Glu Arg Asp Thr Gly Thr Phe Leu Ile

1010 1015 1020

Asn Ala Ser Lys Gln Leu Gly Ala Gly Ala Asp Trp Ala Val Thr

1025 1030 1035

Ile Ala Gly Ala Ala Asn Thr Asp Ala Arg Thr Gln Thr Arg Lys

1040 1045 1050

Asp Leu Ala His Phe Asn Val Leu Asp Arg Ala Asp Gly Thr Pro

1055 1060 1065

Asp Leu Thr Ala Leu Val Asn Arg Ala Arg Glu Met Met Ala Tyr

1070 1075 1080

Asp Arg Lys Arg Lys Asn Ala Val Pro Arg Ser Ile Leu Asp Met

1085 1090 1095

Leu Ala Arg Leu Gly Leu Thr Leu Lys Trp Gln Met Lys Asp His

1100 1105 1110

Leu Leu Gln Asp Ala Thr Ile Thr Gln Ala Ala Ile Lys His Leu

1115 1120 1125

Asp Lys Val Arg Leu Thr Val Gly Gly Pro Ala Ala Val Thr Glu

1130 1135 1140

Ala Arg Phe Ser Gln Asp Tyr Leu Gln Met Val Ala Ala Val Phe

1145 1150 1155

Asn Gly Ser Val Gln Asn Pro Lys Pro Arg Arg Arg Asp Asp Gly

1160 1165 1170

Asp Ala Trp His Lys Pro Pro Lys Pro Ala Thr Ala Gln Ser Gln

1175 1180 1185

Pro Asp Gln Lys Pro Asn Lys Ala Pro Ser Ala Gly Ser Arg

1190 1195 1200

Leu Pro Pro Pro Gln Val Gly Glu Val Tyr Glu Gly Val Val Val

1205 1210 1215

Lys Val Ile Asp Thr Gly Ser Leu Gly Phe Leu Ala Val Glu Gly

1220 1225 1230

Val Ala Gly Asn Ile Gly Leu His Ile Ser Arg Leu Arg Arg Ile

1235 1240 1245

Arg Glu Asp Ala Ile Ile Val Gly Arg Arg Tyr Arg Phe Arg Val

1250 1255 1260

Glu Ile Tyr Val Pro Pro Lys Ser Asn Thr Ser Lys Leu Asn Ala

1265 1270 1275

Ala Asp Leu Val Arg Ile Asp

1280 1285

<210> 574

<211> 1340

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 574

Met Gln Ile Ser Lys Val Asn His Lys His Val Ala Val Gly Gln Lys

1 5 10 15

Asp Arg Glu Arg Ile Thr Gly Phe Ile Tyr Asn Asp Pro Val Gly Asp

20 25 30

Glu Lys Ser Leu Glu Asp Val Val Ala Lys Arg Ala Asn Asp Thr Lys

35 40 45

Val Leu Phe Asn Val Phe Asn Thr Lys Asp Leu Tyr Asp Ser Gln Glu

50 55 60

Ser Asp Lys Ser Glu Lys Asp Lys Glu Ile Ile Ser Lys Gly Ala Lys

65 70 75 80

Phe Val Ala Lys Ser Phe Asn Ser Ala Ile Thr Ile Leu Lys Lys Gln

85 90 95

Asn Lys Ile Tyr Ser Thr Leu Thr Ser Gln Gln Val Ile Lys Glu Leu

100 105 110

Lys Asp Lys Phe Gly Gly Ala Arg Ile Tyr Asp Asp Asp Ile Glu Glu

115 120 125

Ala Leu Thr Glu Thr Leu Lys Lys Ser Phe Arg Lys Glu Asn Val Arg

130 135 140

Asn Ser Ile Lys Val Leu Ile Glu Asn Ala Ala Gly Ile Arg Ser Ser

145 150 155 160

Leu Ser Lys Asp Glu Glu Glu Leu Ile Gln Glu Tyr Phe Val Lys Gln

165 170 175

Leu Val Glu Glu Tyr Thr Lys Thr Lys Leu Gln Lys Asn Val Val Lys

180 185 190

Ser Ile Lys Asn Gln Asn Met Val Ile Gln Pro Asp Ser Asp Ser Gln

195 200 205

Val Leu Ser Leu Ser Glu Ser Arg Arg Glu Lys Gln Ser Ser Ala Val

210 215 220

Ser Ser Asp Thr Leu Val Asn Cys Lys Glu Lys Asp Val Leu Lys Ala

225 230 235 240

Phe Leu Thr Asp Tyr Ala Val Leu Asp Glu Asp Glu Arg Asn Ser Leu

245 250 255

Leu Trp Lys Leu Arg Asn Leu Val Asn Leu Tyr Phe Tyr Gly Ser Glu

260 265 270

Ser Ile Arg Asp Tyr Ser Tyr Thr Lys Glu Lys Ser Val Trp Lys Glu

275 280 285

His Asp Glu Gln Lys Ala Asn Lys Thr Leu Phe Ile Asp Glu Ile Cys

290 295 300

His Ile Thr Lys Ile Gly Lys Asn Gly Lys Glu Gln Lys Val Leu Asp

305 310 315 320

Tyr Glu Glu Asn Arg Ser Arg Cys Arg Lys Gln Asn Ile Asn Tyr Tyr

325 330 335

Arg Ser Ala Leu Asn Tyr Ala Lys Asn Asn Thr Ser Gly Ile Phe Glu

340 345 350

Asn Glu Asp Ser Asn His Phe Trp Ile His Leu Ile Glu Asn Glu Val

355 360 365

Glu Arg Leu Tyr Asn Gly Ile Glu Asn Gly Glu Glu Phe Lys Phe Glu

370 375 380

Thr Gly Tyr Ile Ser Glu Lys Val Trp Lys Ala Val Ile Asn His Leu

385 390 395 400

Ser Ile Lys Tyr Ile Ala Leu Gly Lys Ala Val Tyr Asn Tyr Ala Met

405 410 415

Lys Glu Leu Ser Ser Pro Gly Asp Ile Glu Pro Gly Lys Ile Asp Asp

420 425 430

Ser Tyr Ile Asn Gly Ile Thr Ser Phe Asp Tyr Glu Ile Ile Lys Ala

435 440 445

Glu Glu Ser Leu Gln Arg Asp Ile Ser Met Asn Val Val Phe Ala Thr

450 455 460

Asn Tyr Leu Ala Cys Ala Thr Val Asp Thr Asp Lys Asp Phe Leu Leu

465 470 475 480

Phe Ser Lys Glu Asp Ile Arg Ser Cys Thr Lys Lys Asp Gly Asn Leu

485 490 495

Cys Lys Asn Ile Met Gln Phe Trp Gly Gly Tyr Ser Thr Trp Lys Asn

500 505 510

Phe Cys Glu Glu Tyr Leu Lys Asp Asp Lys Asp Ala Leu Glu Leu Leu

515 520 525

Tyr Ser Leu Lys Ser Met Leu Tyr Ser Met Arg Asn Ser Ser Phe His

530 535 540

Phe Ser Thr Glu Asn Val Asp Asn Gly Ser Trp Asp Thr Glu Leu Ile

545 550 555 560

Gly Lys Leu Phe Glu Glu Asp Cys Asn Arg Ala Ala Arg Ile Glu Lys

565 570 575

Glu Lys Phe Tyr Asn Asn Asn Leu His Met Phe Tyr Ser Ser Ser Ser Leu

580 585 590

Leu Glu Lys Val Leu Glu Arg Leu Tyr Ser Ser His His Glu Arg Ala

595 600 605

Ser Gln Val Pro Ser Phe Asn Arg Val Phe Val Arg Lys Asn Phe Pro

610 615 620

Ser Ser Leu Ser Glu Gln Arg Ile Thr Pro Lys Phe Thr Asp Ser Lys

625 630 635 640

Asp Glu Gln Ile Trp Gln Ser Ala Val Tyr Tyr Leu Cys Lys Glu Ile

645 650 655

Tyr Tyr Asn Asp Phe Leu Gln Ser Lys Glu Ala Tyr Lys Leu Phe Arg

660 665 670

Glu Gly Val Lys Asn Leu Asp Lys Asn Asp Ile Asn Asn Gln Lys Ala

675 680 685

Ala Asp Ser Phe Lys Gln Ala Val Val Tyr Tyr Gly Lys Ala Ile Gly

690 695 700

Asn Ala Thr Leu Ser Gln Val Cys Gln Ala Ile Met Thr Glu Tyr Asn

705 710 715 720

Arg Gln Asn Asn Asp Gly Leu Lys Lys Lys Ser Ala Tyr Ala Glu Lys

725 730 735

Gln Asn Ser Asn Lys Tyr Lys His Tyr Pro Leu Phe Leu Lys Gln Val

740 745 750

Leu Gln Ser Ala Phe Trp Glu Tyr Leu Asp Glu Asn Lys Glu Ile Tyr

755 760 765

Gly Phe Ile Ser Ala Gln Ile His Lys Ser Asn Val Glu Ile Lys Ala

770 775 780

Glu Asp Phe Ile Ala Asn Tyr Ser Ser Gln Gln Tyr Lys Lys Leu Val

785 790 795 800

Asp Lys Val Lys Lys Thr Pro Glu Leu Gln Lys Trp Tyr Thr Leu Gly

805 810 815

Arg Leu Ile Asn Pro Arg Gln Ala Asn Gln Phe Leu Gly Ser Ile Arg

820 825 830

Asn Tyr Val Gln Phe Val Lys Asp Ile Gln Arg Arg Ala Lys Glu Asn

835 840 845

Gly Asn Pro Ile Arg Asn Tyr Tyr Glu Val Leu Glu Ser Asp Ser Ile

850 855 860

Ile Lys Ile Leu Glu Met Cys Thr Lys Leu Asn Gly Thr Thr Ser Asn

865 870 875 880

Asp Ile His Asp Tyr Phe Arg Asp Glu Asp Glu Tyr Ala Glu Tyr Ile

885 890 895

Ser Gln Phe Val Asn Phe Gly Asp Val His Ser Gly Ala Ala Leu Asn

900 905 910

Ala Phe Cys Asn Ser Glu Ser Glu Gly Lys Lys Asn Gly Ile Tyr Tyr

915 920 925

Asp Gly Ile Asn Pro Ile Val Asn Arg Asn Trp Val Leu Cys Lys Leu

930 935 940

Tyr Gly Ser Pro Asp Leu Ile Ser Lys Ile Ile Ser Arg Val Asn Glu

945 950 955 960

Asn Met Ile His Asp Phe His Lys Gln Glu Asp Leu Ile Arg Glu Tyr

965 970 975

Gln Ile Lys Gly Ile Cys Ser Asn Lys Lys Glu Gln Gln Asp Leu Arg

980 985 990

Thr Phe Gln Val Leu Lys Asn Arg Val Glu Leu Arg Asp Ile Val Glu

995 1000 1005

Tyr Ser Glu Ile Ile Asn Glu Leu Tyr Gly Gln Leu Ile Lys Trp

1010 1015 1020

Cys Tyr Leu Arg Glu Arg Asp Leu Met Tyr Phe Gln Leu Gly Phe

1025 1030 1035

His Tyr Leu Cys Leu Asn Asn Ala Ser Ser Lys Glu Ala Asp Tyr

1040 1045 1050

Ile Lys Ile Asn Val Asp Asp Arg Asn Ile Ser Gly Ala Ile Leu

1055 1060 1065

Tyr Gln Ile Ala Ala Met Tyr Ile Asn Gly Leu Pro Val Tyr Tyr

1070 1075 1080

Lys Lys Asp Asp Met Tyr Val Ala Leu Lys Ser Gly Lys Lys Ala

1085 1090 1095

Ser Asp Glu Leu Asn Ser Asn Glu Gln Thr Ser Lys Lys Ile Asn

1100 1105 1110

Tyr Phe Leu Lys Tyr Gly Asn Asn Ile Leu Gly Asp Lys Lys Asp

1115 1120 1125

Gln Leu Tyr Leu Ala Gly Leu Glu Leu Phe Glu Asn Val Ala Glu

1130 1135 1140

His Glu Asn Ile Ile Ile Phe Arg Asn Glu Ile Asp His Phe His

1145 1150 1155

Tyr Phe Tyr Asp Arg Asp Arg Ser Met Leu Asp Leu Tyr Ser Glu

1160 1165 1170

Val Phe Asp Arg Phe Phe Thr Tyr Asp Met Lys Leu Arg Lys Asn

1175 1180 1185

Val Val Asn Met Leu Tyr Asn Ile Leu Leu Asp His Asn Ile Val

1190 1195 1200

Ser Ser Phe Val Phe Glu Thr Gly Glu Lys Lys Val Gly Arg Gly

1205 1210 1215

Asp Ser Glu Val Ile Lys Pro Ser Ala Lys Ile Arg Leu Arg Ala

1220 1225 1230

Asn Asn Gly Val Ser Ser Asp Val Phe Thr Tyr Lys Val Gly Ser

1235 1240 1245

Lys Asp Glu Leu Lys Ile Ala Thr Leu Pro Ala Lys Asn Glu Glu

1250 1255 1260

Phe Leu Leu Asn Val Ala Arg Leu Ile Tyr Tyr Pro Asp Met Glu

1265 1270 1275

Ala Val Ser Glu Asn Met Val Arg Glu Gly Val Val Lys Val Glu

1280 1285 1290

Lys Ser Asn Asp Lys Lys Gly Lys Ile Ser Arg Gly Ser Asn Thr

1295 1300 1305

Arg Ser Ser Asn Gln Ser Lys Tyr Asn Asn Lys Ser Lys Asn Arg

1310 1315 1320

Met Asn Tyr Ser Met Gly Ser Ile Phe Glu Lys Met Asp Leu Lys

1325 1330 1335

Phe Asp

1340

<210> 575

<211> 1437

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 575

Met Lys Ile Ser Lys Val Arg Glu Glu Asn Arg Gly Ala Lys Leu Thr

1 5 10 15

Val Asn Ala Lys Thr Ala Val Val Ser Glu Asn Arg Ser Gln Glu Gly

20 25 30

Ile Leu Tyr Asn Asp Pro Ser Arg Tyr Gly Lys Ser Arg Lys Asn Asp

35 40 45

Glu Asp Arg Asp Arg Tyr Ile Glu Ser Arg Leu Lys Ser Ser Gly Lys

50 55 60

Leu Tyr Arg Ile Phe Asn Glu Asp Lys Asn Lys Arg Glu Thr Asp Glu

65 70 75 80

Leu Gln Trp Phe Leu Ser Glu Ile Val Lys Lys Ile Asn Arg Arg Asn

85 90 95

Gly Leu Val Leu Ser Asp Met Leu Ser Val Asp Asp Arg Ala Phe Glu

100 105 110

Lys Ala Phe Glu Lys Tyr Ala Glu Leu Ser Tyr Thr Asn Arg Arg Asn

115 120 125

Lys Val Ser Gly Ser Pro Ala Phe Glu Thr Cys Gly Val Asp Ala Ala

130 135 140

Thr Ala Glu Arg Leu Lys Gly Ile Ile Ser Glu Thr Asn Phe Ile Asn

145 150 155 160

Arg Ile Lys Asn Asn Ile Asp Asn Lys Val Ser Glu Asp Ile Ile Asp

165 170 175

Arg Ile Ile Ala Lys Tyr Leu Lys Lys Ser Leu Cys Arg Glu Arg Val

180 185 190

Lys Arg Gly Leu Lys Lys Leu Leu Met Asn Ala Phe Asp Leu Pro Tyr

195 200 205

Ser Asp Pro Asp Ile Asp Val Gln Arg Asp Phe Ile Asp Tyr Val Leu

210 215 220

Glu Asp Phe Tyr His Val Arg Ala Lys Ser Gln Val Ser Arg Ser Ile

225 230 235 240

Lys Asn Met Asn Met Pro Val Gln Pro Glu Gly Asp Gly Lys Phe Ala

245 250 255

Ile Thr Val Ser Lys Gly Gly Thr Glu Ser Gly Asn Lys Arg Ser Ala

260 265 270

Glu Lys Glu Ala Phe Lys Lys Phe Leu Ser Asp Tyr Ala Ser Leu Asp

275 280 285

Glu Arg Val Arg Asp Asp Met Leu Arg Arg Met Arg Arg Leu Val Val

290 295 300

Leu Tyr Phe Tyr Gly Ser Asp Asp Ser Lys Leu Ser Asp Val Asn Glu

305 310 315 320

Lys Phe Asp Val Trp Glu Asp His Ala Ala Arg Arg Val Asp Asn Arg

325 330 335

Glu Phe Ile Lys Leu Pro Leu Glu Asn Lys Leu Ala Asn Gly Lys Thr

340 345 350

Asp Lys Asp Ala Glu Arg Ile Arg Lys Asn Thr Val Lys Glu Leu Tyr

355 360 365

Arg Asn Gln Asn Ile Gly Cys Tyr Arg Gln Ala Val Lys Ala Val Glu

370 375 380

Glu Asp Asn Asn Gly Arg Tyr Phe Asp Asp Lys Met Leu Asn Met Phe

385 390 395 400

Phe Ile His Arg Ile Glu Tyr Gly Val Glu Lys Ile Tyr Ala Asn Leu

405 410 415

Lys Gln Val Thr Glu Phe Lys Ala Arg Thr Gly Tyr Leu Ser Glu Lys

420 425 430

Ile Trp Lys Asp Leu Ile Asn Tyr Ile Ser Ile Lys Tyr Ile Ala Met

435 440 445

Gly Lys Ala Val Tyr Asn Tyr Ala Met Asp Glu Leu Asn Ala Ser Asp

450 455 460

Lys Lys Glu Ile Glu Leu Gly Lys Ile Ser Glu Glu Tyr Leu Ser Gly

465 470 475 480

Ile Ser Ser Phe Asp Tyr Glu Leu Ile Lys Ala Glu Glu Met Leu Gln

485 490 495

Arg Glu Thr Ala Val Tyr Val Ala Phe Ala Ala Arg His Leu Ser Ser

500 505 510

Gln Thr Val Glu Leu Asp Ser Glu Asn Ser Asp Phe Leu Leu Leu Lys

515 520 525

Pro Lys Gly Thr Met Asp Lys Asn Asp Lys Asn Lys Leu Ala Ser Asn

530 535 540

Asn Ile Leu Asn Phe Leu Lys Asp Lys Glu Thr Leu Arg Asp Thr Ile

545 550 555 560

Leu Gln Tyr Phe Gly Gly His Ser Leu Trp Thr Asp Phe Pro Phe Asp

565 570 575

Lys Tyr Leu Ala Gly Gly Lys Asp Asp Val Asp Phe Leu Thr Asp Leu

580 585 590

Lys Asp Val Ile Tyr Ser Met Arg Asn Asp Ser Phe His Tyr Ala Thr

595 600 605

Glu Asn His Asn Asn Gly Lys Trp Asn Lys Glu Leu Ile Ser Ala Met

610 615 620

Phe Glu His Glu Thr Glu Arg Met Thr Val Val Met Lys Asp Lys Phe

625 630 635 640

Tyr Ser Asn Asn Leu Pro Met Phe Tyr Lys Asn Asp Asp Leu Lys Lys

645 650 655

Leu Leu Ile Asp Leu Tyr Lys Asp Asn Val Glu Arg Ala Ser Gln Val

660 665 670

Pro Ser Phe Asn Lys Val Phe Val Arg Lys Asn Phe Pro Ala Leu Val

675 680 685

Arg Asp Lys Asp Asn Leu Gly Ile Glu Leu Asp Leu Lys Ala Asp Ala

690 695 700

Asp Lys Gly Glu Asn Glu Leu Lys Phe Tyr Asn Ala Leu Tyr Tyr Met

705 710 715 720

Phe Lys Glu Ile Tyr Tyr Asn Ala Phe Leu Asn Asp Lys Asn Val Arg

725 730 735

Glu Arg Phe Ile Thr Lys Ala Thr Lys Val Ala Asp Asn Tyr Asp Arg

740 745 750

Asn Lys Glu Arg Asn Leu Lys Asp Arg Ile Lys Ser Ala Gly Ser Asp

755 760 765

Glu Lys Lys Lys Leu Arg Glu Gln Leu Gln Asn Tyr Ile Ala Glu Asn

770 775 780

Asp Phe Gly Gln Arg Ile Lys Asn Ile Val Gln Val Asn Pro Asp Tyr

785 790 795 800

Thr Leu Ala Gln Ile Cys Gln Leu Ile Met Thr Glu Tyr Asn Gln Gln

805 810 815

Asn Asn Gly Cys Met Gln Lys Lys Ser Ala Ala Arg Lys Asp Ile Asn

820 825 830

Lys Asp Ser Tyr Gln His Tyr Lys Met Leu Leu Leu Val Asn Leu Arg

835 840 845

Lys Ala Phe Leu Glu Phe Ile Lys Glu Asn Tyr Ala Phe Val Leu Lys

850 855 860

Pro Tyr Lys His Asp Leu Cys Asp Lys Ala Asp Phe Val Pro Asp Phe

865 870 875 880

Ala Lys Tyr Val Lys Pro Tyr Ala Gly Leu Ile Ser Arg Val Ala Gly

885 890 895

Ser Ser Glu Leu Gln Lys Trp Tyr Ile Val Ser Arg Phe Leu Ser Pro

900 905 910

Ala Gln Ala Asn His Met Leu Gly Phe Leu His Ser Tyr Lys Gln Tyr

915 920 925

Val Trp Asp Ile Tyr Arg Arg Ala Ser Glu Thr Gly Thr Glu Ile Asn

930 935 940

His Ser Ile Ala Glu Asp Lys Ile Ala Gly Val Asp Ile Thr Asp Val

945 950 955 960

Asp Ala Val Ile Asp Leu Ser Val Lys Leu Cys Gly Thr Ile Ser Ser

965 970 975

Glu Ile Ser Asp Tyr Phe Lys Asp Asp Glu Val Tyr Ala Glu Tyr Ile

980 985 990

Ser Ser Tyr Leu Asp Phe Glu Tyr Asp Gly Gly Asn Tyr Lys Asp Ser

995 1000 1005

Leu Asn Arg Phe Cys Asn Ser Asp Ala Val Asn Asp Gln Lys Val

1010 1015 1020

Ala Leu Tyr Tyr Asp Gly Glu His Pro Lys Leu Asn Arg Asn Ile

1025 1030 1035

Ile Leu Ser Lys Leu Tyr Gly Glu Arg Arg Phe Leu Glu Lys Ile

1040 1045 1050

Thr Asp Arg Val Ser Arg Ser Asp Ile Val Glu Tyr Tyr Lys Leu

1055 1060 1065

Lys Lys Glu Thr Ser Gln Tyr Gln Thr Lys Gly Ile Phe Asp Ser

1070 1075 1080

Glu Asp Glu Gln Lys Asn Ile Lys Lys Phe Gln Glu Met Lys Asn

1085 1090 1095

Ile Val Glu Phe Arg Asp Leu Met Asp Tyr Ser Glu Ile Ala Asp

1100 1105 1110

Glu Leu Gln Gly Gln Leu Ile Asn Trp Ile Tyr Leu Arg Glu Arg

1115 1120 1125

Asp Leu Met Asn Phe Gln Leu Gly Tyr His Tyr Ala Cys Leu Asn

1130 1135 1140

Asn Asp Ser Asn Lys Gln Ala Thr Tyr Val Thr Leu Asp Tyr Gln

1145 1150 1155

Gly Lys Lys Asn Arg Lys Ile Asn Gly Ala Ile Leu Tyr Gln Ile

1160 1165 1170

Cys Ala Met Tyr Ile Asn Gly Leu Pro Leu Tyr Tyr Val Asp Lys

1175 1180 1185

Asp Ser Ser Glu Trp Thr Val Ser Asp Gly Lys Glu Ser Thr Gly

1190 1195 1200

Ala Lys Ile Gly Glu Phe Tyr Arg Tyr Ala Lys Ser Phe Glu Asn

1205 1210 1215

Thr Ser Asp Cys Tyr Ala Ser Gly Leu Glu Ile Phe Glu Asn Ile

1220 1225 1230

Ser Glu His Asp Asn Ile Thr Glu Leu Arg Asn Tyr Ile Glu His

1235 1240 1245

Phe Arg Tyr Tyr Ser Ser Phe Asp Arg Ser Phe Leu Gly Ile Tyr

1250 1255 1260

Ser Glu Val Phe Asp Arg Phe Phe Thr Tyr Asp Leu Lys Tyr Arg

1265 1270 1275

Lys Asn Val Pro Thr Ile Leu Tyr Asn Ile Leu Leu Gln His Phe

1280 1285 1290

Val Asn Val Arg Phe Glu Phe Val Ser Gly Lys Lys Met Ile Gly

1295 1300 1305

Ile Asp Lys Lys Asp Arg Lys Ile Ala Lys Glu Lys Glu Cys Ala

1310 1315 1320

Arg Ile Thr Ile Arg Glu Lys Asn Gly Val Tyr Ser Glu Gln Phe

1325 1330 1335

Thr Tyr Lys Leu Lys Asn Gly Thr Val Tyr Val Asp Ala Arg Asp

1340 1345 1350

Lys Arg Tyr Leu Gln Ser Ile Ile Arg Leu Leu Phe Tyr Pro Glu

1355 1360 1365

Lys Val Asn Met Asp Glu Met Ile Glu Val Lys Glu Lys Lys Lys

1370 1375 1380

Pro Ser Asp Asn Asn Thr Gly Lys Gly Tyr Ser Lys Arg Asp Arg

1385 1390 1395

Gln Gln Asp Arg Lys Glu Tyr Asp Lys Tyr Lys Glu Lys Lys Lys

1400 1405 1410

Lys Glu Gly Asn Phe Leu Ser Gly Met Gly Gly Asn Ile Asn Trp

1415 1420 1425

Asp Glu Ile Asn Ala Gln Leu Lys Asn

1430 1435

<210> 576

<211> 1385

<212> PRT

<213> Clostridium aminophilum

<400> 576

Met Lys Phe Ser Lys Val Asp His Thr Arg Ser Ala Val Gly Ile Gln

1 5 10 15

Lys Ala Thr Asp Ser Val His Gly Met Leu Tyr Thr Asp Pro Lys Lys

20 25 30

Gln Glu Val Asn Asp Leu Asp Lys Arg Phe Asp Gln Leu Asn Val Lys

35 40 45

Ala Lys Arg Leu Tyr Asn Val Phe Asn Gln Ser Lys Ala Glu Glu Asp

50 55 60

Asp Asp Glu Lys Arg Phe Gly Lys Val Val Lys Lys Leu Asn Arg Glu

65 70 75 80

Leu Lys Asp Leu Leu Phe His Arg Glu Val Ser Arg Tyr Asn Ser Ile

85 90 95

Gly Asn Ala Lys Tyr Asn Tyr Tyr Gly Ile Lys Ser Asn Pro Glu Glu

100 105 110

Ile Val Ser Asn Leu Gly Met Val Glu Ser Leu Lys Gly Glu Arg Asp

115 120 125

Pro Gln Lys Val Ile Ser Lys Leu Leu Leu Tyr Tyr Leu Arg Lys Gly

130 135 140

Leu Lys Pro Gly Thr Asp Gly Leu Arg Met Ile Leu Glu Ala Ser Cys

145 150 155 160

Gly Leu Arg Lys Leu Ser Gly Asp Glu Lys Glu Leu Lys Val Phe Leu

165 170 175

Gln Thr Leu Asp Glu Asp Phe Glu Lys Lys Thr Phe Lys Lys Asn Leu

180 185 190

Ile Arg Ser Ile Glu Asn Gln Asn Met Ala Val Gln Pro Ser Asn Glu

195 200 205

Gly Asp Pro Ile Ile Gly Ile Thr Gln Gly Arg Phe Asn Ser Gln Lys

210 215 220

Asn Glu Glu Lys Ser Ala Ile Glu Arg Met Met Ser Met Tyr Ala Asp

225 230 235 240

Leu Asn Glu Asp His Arg Glu Asp Val Leu Arg Lys Leu Arg Arg Leu

245 250 255

Asn Val Leu Tyr Phe Asn Val Asp Thr Glu Lys Thr Glu Glu Pro Thr

260 265 270

Leu Pro Gly Glu Val Asp Thr Asn Pro Val Phe Glu Val Trp His Asp

275 280 285

His Glu Lys Gly Lys Glu Asn Asp Arg Gln Phe Ala Thr Phe Ala Lys

290 295 300

Ile Leu Thr Glu Asp Arg Glu Thr Arg Lys Lys Glu Lys Leu Ala Val

305 310 315 320

Lys Glu Ala Leu Asn Asp Leu Lys Ser Ala Ile Arg Asp His Asn Ile

325 330 335

Met Ala Tyr Arg Cys Ser Ile Lys Val Thr Glu Gln Asp Lys Asp Gly

340 345 350

Leu Phe Phe Glu Asp Gln Arg Ile Asn Arg Phe Trp Ile His His Ile

355 360 365

Glu Ser Ala Val Glu Arg Ile Leu Ala Ser Ile Asn Pro Glu Lys Leu

370 375 380

Tyr Lys Leu Arg Ile Gly Tyr Leu Gly Glu Lys Val Trp Lys Asp Leu

385 390 395 400

Leu Asn Tyr Leu Ser Ile Lys Tyr Ile Ala Val Gly Lys Ala Val Phe

405 410 415

His Phe Ala Met Glu Asp Leu Gly Lys Thr Gly Gln Asp Ile Glu Leu

420 425 430

Gly Lys Leu Ser Asn Ser Val Ser Gly Gly Leu Thr Ser Phe Asp Tyr

435 440 445

Glu Gln Ile Arg Ala Asp Glu Thr Leu Gln Arg Gln Leu Ser Val Glu

450 455 460

Val Ala Phe Ala Ala Asn Asn Leu Phe Arg Ala Val Val Gly Gln Thr

465 470 475 480

Gly Lys Lys Ile Glu Gln Ser Lys Ser Glu Glu Asn Glu Glu Asp Phe

485 490 495

Leu Leu Trp Lys Ala Glu Lys Ile Ala Glu Ser Ile Lys Lys Glu Gly

500 505 510

Glu Gly Asn Thr Leu Lys Ser Ile Leu Gln Phe Phe Gly Gly Ala Ser

515 520 525

Ser Trp Asp Leu Asn His Phe Cys Ala Ala Tyr Gly Asn Glu Ser Ser

530 535 540

Ala Leu Gly Tyr Glu Thr Lys Phe Ala Asp Asp Leu Arg Lys Ala Ile

545 550 555 560

Tyr Ser Leu Arg Asn Glu Thr Phe His Phe Thr Thr Leu Asn Lys Gly

565 570 575

Ser Phe Asp Trp Asn Ala Lys Leu Ile Gly Asp Met Phe Ser His Glu

580 585 590

Ala Ala Thr Gly Ile Ala Val Glu Arg Thr Arg Phe Tyr Ser Asn Asn

595 600 605

Leu Pro Met Phe Tyr Arg Glu Ser Asp Leu Lys Arg Ile Met Asp His

610 615 620

Leu Tyr Asn Thr Tyr His Pro Arg Ala Ser Gln Val Pro Ser Phe Asn

625 630 635 640

Ser Val Phe Val Arg Lys Asn Phe Arg Leu Phe Leu Ser Asn Thr Leu

645 650 655

Asn Thr Asn Thr Ser Phe Asp Thr Glu Val Tyr Gln Lys Trp Glu Ser

660 665 670

Gly Val Tyr Tyr Leu Phe Lys Glu Ile Tyr Tyr Asn Ser Phe Leu Pro

675 680 685

Ser Gly Asp Ala His His Leu Phe Phe Glu Gly Leu Arg Arg Ile Arg

690 695 700

Lys Glu Ala Asp Asn Leu Pro Ile Val Gly Lys Glu Ala Lys Lys Arg

705 710 715 720

Asn Ala Val Gln Asp Phe Gly Arg Arg Cys Asp Glu Leu Lys Asn Leu

725 730 735

Ser Leu Ser Ala Ile Cys Gln Met Ile Met Thr Glu Tyr Asn Glu Gln

740 745 750

Asn Asn Gly Asn Arg Lys Val Lys Ser Thr Arg Glu Asp Lys Arg Lys

755 760 765

Pro Asp Ile Phe Gln His Tyr Lys Met Leu Leu Leu Arg Thr Leu Gln

770 775 780

Glu Ala Phe Ala Ile Tyr Ile Arg Arg Glu Glu Phe Lys Phe Ile Phe

785 790 795 800

Asp Leu Pro Lys Thr Leu Tyr Val Met Lys Pro Val Glu Glu Phe Leu

805 810 815

Pro Asn Trp Lys Ser Gly Met Phe Asp Ser Leu Val Glu Arg Val Lys

820 825 830

Gln Ser Pro Asp Leu Gln Arg Trp Tyr Val Leu Cys Lys Phe Leu Asn

835 840 845

Gly Arg Leu Leu Asn Gln Leu Ser Gly Val Ile Arg Ser Tyr Ile Gln

850 855 860

Phe Ala Gly Asp Ile Gln Arg Arg Ala Lys Ala Asn His Asn Arg Leu

865 870 875 880

Tyr Met Asp Asn Thr Gln Arg Val Glu Tyr Tyr Ser Asn Val Leu Glu

885 890 895

Val Val Asp Phe Cys Ile Lys Gly Thr Ser Arg Phe Ser Asn Val Phe

900 905 910

Ser Asp Tyr Phe Arg Asp Glu Asp Ala Tyr Ala Asp Tyr Leu Asp Asn

915 920 925

Tyr Leu Gln Phe Lys Asp Glu Lys Ile Ala Glu Val Ser Ser Phe Ala

930 935 940

Ala Leu Lys Thr Phe Cys Asn Glu Glu Glu Val Lys Ala Gly Ile Tyr

945 950 955 960

Met Asp Gly Glu Asn Pro Val Met Gln Arg Asn Ile Val Met Ala Lys

965 970 975

Leu Phe Gly Pro Asp Glu Val Leu Lys Asn Val Val Pro Lys Val Thr

980 985 990

Arg Glu Glu Ile Glu Glu Tyr Tyr Gln Leu Glu Lys Gln Ile Ala Pro

995 1000 1005

Tyr Arg Gln Asn Gly Tyr Cys Lys Ser Glu Glu Asp Gln Lys Lys

1010 1015 1020

Leu Leu Arg Phe Gln Arg Ile Lys Asn Arg Val Glu Phe Gln Thr

1025 1030 1035

Ile Thr Glu Phe Ser Glu Ile Ile Asn Glu Leu Leu Gly Gln Leu

1040 1045 1050

Ile Ser Trp Ser Phe Leu Arg Glu Arg Asp Leu Leu Tyr Phe Gln

1055 1060 1065

Leu Gly Phe His Tyr Leu Cys Leu His Asn Asp Thr Glu Lys Pro

1070 1075 1080

Ala Glu Tyr Lys Glu Ile Ser Arg Glu Asp Gly Thr Val Ile Arg

1085 1090 1095

Asn Ala Ile Leu His Gln Val Ala Ala Met Tyr Val Gly Gly Leu

1100 1105 1110

Pro Val Tyr Thr Leu Ala Asp Lys Lys Leu Ala Ala Phe Glu Lys

1115 1120 1125

Gly Glu Ala Asp Cys Lys Leu Ser Ile Ser Lys Asp Thr Ala Gly

1130 1135 1140

Ala Gly Lys Lys Ile Lys Asp Phe Phe Arg Tyr Ser Lys Tyr Val

1145 1150 1155

Leu Ile Lys Asp Arg Met Leu Thr Asp Gln Asn Gln Lys Tyr Thr

1160 1165 1170

Ile Tyr Leu Ala Gly Leu Glu Leu Phe Glu Asn Thr Asp Glu His

1175 1180 1185

Asp Asn Ile Thr Asp Val Arg Lys Tyr Val Asp His Phe Lys Tyr

1190 1195 1200

Tyr Ala Thr Ser Asp Glu Asn Ala Met Ser Ile Leu Asp Leu Tyr

1205 1210 1215

Ser Glu Ile His Asp Arg Phe Phe Thr Tyr Asp Met Lys Tyr Gln

1220 1225 1230

Lys Asn Val Ala Asn Met Leu Glu Asn Ile Leu Leu Arg His Phe

1235 1240 1245

Val Leu Ile Arg Pro Glu Phe Phe Thr Gly Ser Lys Lys Val Gly

1250 1255 1260

Glu Gly Lys Lys Ile Thr Cys Lys Ala Arg Ala Gln Ile Glu Ile

1265 1270 1275

Ala Glu Asn Gly Met Arg Ser Glu Asp Phe Thr Tyr Lys Leu Ser

1280 1285 1290

Asp Gly Lys Lys Asn Ile Ser Thr Cys Met Ile Ala Ala Arg Asp

1295 1300 1305

Gln Lys Tyr Leu Asn Thr Val Ala Arg Leu Leu Tyr Tyr Pro His

1310 1315 1320

Glu Ala Lys Lys Ser Ile Val Asp Thr Arg Glu Lys Lys Asn Asn

1325 1330 1335

Lys Lys Thr Asn Arg Gly Asp Gly Thr Phe Asn Lys Gln Lys Gly

1340 1345 1350

Thr Ala Arg Lys Glu Lys Asp Asn Gly Pro Arg Glu Phe Asn Asp

1355 1360 1365

Thr Gly Phe Ser Asn Thr Pro Phe Ala Gly Phe Asp Pro Phe Arg

1370 1375 1380

Asn Ser

1385

<210> 577

<211> 1334

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 577

Met Lys Ile Ser Lys Val Asp His Thr Arg Met Ala Val Ala Lys Gly

1 5 10 15

Asn Gln His Arg Arg Asp Glu Ile Ser Gly Ile Leu Tyr Lys Asp Pro

20 25 30

Thr Lys Thr Gly Ser Ile Asp Phe Asp Glu Arg Phe Lys Lys Leu Asn

35 40 45

Cys Ser Ala Lys Ile Leu Tyr His Val Phe Asn Gly Ile Ala Glu Gly

50 55 60

Ser Asn Lys Tyr Lys Asn Ile Val Asp Lys Val Asn Asn Asn Asn Leu Asp

65 70 75 80

Arg Val Leu Phe Thr Gly Lys Ser Tyr Asp Arg Lys Ser Ile Ile Asp

85 90 95

Ile Asp Thr Val Leu Arg Asn Val Glu Lys Ile Asn Ala Phe Asp Arg

100 105 110

Ile Ser Thr Glu Glu Arg Glu Gln Ile Ile Asp Asp Leu Leu Glu Ile

115 120 125

Gln Leu Arg Lys Gly Leu Arg Lys Gly Lys Ala Gly Leu Arg Glu Val

130 135 140

Leu Leu Ile Gly Ala Gly Val Ile Val Arg Thr Asp Lys Lys Gln Glu

145 150 155 160

Ile Ala Asp Phe Leu Glu Ile Leu Asp Glu Asp Phe Asn Lys Thr Asn

165 170 175

Gln Ala Lys Asn Ile Lys Leu Ser Ile Glu Asn Gln Gly Leu Val Val

180 185 190

Ser Pro Val Ser Arg Gly Glu Glu Arg Ile Phe Asp Val Ser Gly Ala

195 200 205

Gln Lys Gly Lys Ser Ser Lys Lys Ala Gln Glu Lys Glu Ala Leu Ser

210 215 220

Ala Phe Leu Leu Asp Tyr Ala Asp Leu Asp Lys Asn Val Arg Phe Glu

225 230 235 240

Tyr Leu Arg Lys Ile Arg Arg Leu Ile Asn Leu Tyr Phe Tyr Val Lys

245 250 255

Asn Asp Asp Val Met Ser Leu Thr Glu Ile Pro Ala Glu Val Asn Leu

260 265 270

Glu Lys Asp Phe Asp Ile Trp Arg Asp His Glu Gln Arg Lys Glu Glu

275 280 285

Asn Gly Asp Phe Val Gly Cys Pro Asp Ile Leu Leu Ala Asp Arg Asp

290 295 300

Val Lys Lys Ser Asn Ser Lys Gln Val Lys Ile Ala Glu Arg Gln Leu

305 310 315 320

Arg Glu Ser Ile Arg Glu Lys Asn Ile Lys Arg Tyr Arg Phe Ser Ile

325 330 335

Lys Thr Ile Glu Lys Asp Asp Gly Thr Tyr Phe Phe Ala Asn Lys Gln

340 345 350

Ile Ser Val Phe Trp Ile His Arg Ile Glu Asn Ala Val Glu Arg Ile

355 360 365

Leu Gly Ser Ile Asn Asp Lys Lys Leu Tyr Arg Leu Arg Leu Gly Tyr

370 375 380

Leu Gly Glu Lys Val Trp Lys Asp Ile Leu Asn Phe Leu Ser Ile Lys

385 390 395 400

Tyr Ile Ala Val Gly Lys Ala Val Phe Asn Phe Ala Met Asp Asp Leu

405 410 415

Gln Glu Lys Asp Arg Asp Ile Glu Pro Gly Lys Ile Ser Glu Asn Ala

420 425 430

Val Asn Gly Leu Thr Ser Phe Asp Tyr Glu Gln Ile Lys Ala Asp Glu

435 440 445

Met Leu Gln Arg Glu Val Ala Val Asn Val Ala Phe Ala Ala Asn Asn

450 455 460

Leu Ala Arg Val Thr Val Asp Ile Pro Gln Asn Gly Glu Lys Glu Asp

465 470 475 480

Ile Leu Leu Trp Asn Lys Ser Asp Ile Lys Lys Tyr Lys Lys Asn Ser

485 490 495

Lys Lys Gly Ile Leu Lys Ser Ile Leu Gln Phe Phe Gly Gly Ala Ser

500 505 510

Thr Trp Asn Met Lys Met Phe Glu Ile Ala Tyr His Asp Gln Pro Gly

515 520 525

Asp Tyr Glu Glu Asn Tyr Leu Tyr Asp Ile Ile Gln Ile Ile Tyr Ser

530 535 540

Leu Arg Asn Lys Ser Phe His Phe Lys Thr Tyr Asp His Gly Asp Lys

545 550 555 560

Asn Trp Asn Arg Glu Leu Ile Gly Lys Met Ile Glu His Asp Ala Glu

565 570 575

Arg Val Ile Ser Val Glu Arg Glu Lys Phe His Ser Asn Asn Leu Pro

580 585 590

Met Phe Tyr Lys Asp Ala Asp Leu Lys Lys Ile Leu Asp Leu Leu Tyr

595 600 605

Ser Asp Tyr Ala Gly Arg Ala Ser Gln Val Pro Ala Phe Asn Thr Val

610 615 620

Leu Val Arg Lys Asn Phe Pro Glu Phe Leu Arg Lys Asp Met Gly Tyr

625 630 635 640

Lys Val His Phe Asn Asn Pro Glu Val Glu Asn Gln Trp His Ser Ala

645 650 655

Val Tyr Tyr Leu Tyr Lys Glu Ile Tyr Tyr Asn Leu Phe Leu Arg Asp

660 665 670

Lys Glu Val Lys Asn Leu Phe Tyr Thr Ser Leu Lys Asn Ile Arg Ser

675 680 685

Glu Val Ser Asp Lys Lys Gln Lys Leu Ala Ser Asp Asp Phe Ala Ser

690 695 700

Arg Cys Glu Glu Ile Glu Asp Arg Ser Leu Pro Glu Ile Cys Gln Ile

705 710 715 720

Ile Met Thr Glu Tyr Asn Ala Gln Asn Phe Gly Asn Arg Lys Val Lys

725 730 735

Ser Gln Arg Val Ile Glu Lys Asn Lys Asp Ile Phe Arg His Tyr Lys

740 745 750

Met Leu Leu Ile Lys Thr Leu Ala Gly Ala Phe Ser Leu Tyr Leu Lys

755 760 765

Gln Glu Arg Phe Ala Phe Ile Gly Lys Ala Thr Pro Ile Pro Tyr Glu

770 775 780

Thr Thr Asp Val Lys Asn Phe Leu Pro Glu Trp Lys Ser Gly Met Tyr

785 790 795 800

Ala Ser Phe Val Glu Glu Ile Lys Asn Asn Leu Asp Leu Gln Glu Trp

805 810 815

Tyr Ile Val Gly Arg Phe Leu Asn Gly Arg Met Leu Asn Gln Leu Ala

820 825 830

Gly Ser Leu Arg Ser Tyr Ile Gln Tyr Ala Glu Asp Ile Glu Arg Arg

835 840 845

Ala Ala Glu Asn Arg Asn Lys Leu Phe Ser Lys Pro Asp Glu Lys Ile

850 855 860

Glu Ala Cys Lys Lys Ala Val Arg Val Leu Asp Leu Cys Ile Lys Ile

865 870 875 880

Ser Thr Arg Ile Ser Ala Glu Phe Thr Asp Tyr Phe Asp Ser Glu Asp

885 890 895

Asp Tyr Ala Asp Tyr Leu Glu Lys Tyr Leu Lys Tyr Gln Asp Asp Ala

900 905 910

Ile Lys Glu Leu Ser Gly Ser Ser Tyr Ala Ala Leu Asp His Phe Cys

915 920 925

Asn Lys Asp Asp Leu Lys Phe Asp Ile Tyr Val Asn Ala Gly Gln Lys

930 935 940

Pro Ile Leu Gln Arg Asn Ile Val Met Ala Lys Leu Phe Gly Pro Asp

945 950 955 960

Asn Ile Leu Ser Glu Val Met Glu Lys Val Thr Glu Ser Ala Ile Arg

965 970 975

Glu Tyr Tyr Asp Tyr Leu Lys Lys Val Ser Gly Tyr Arg Val Arg Gly

980 985 990

Lys Cys Ser Thr Glu Lys Glu Gln Glu Asp Leu Leu Lys Phe Gln Arg

995 1000 1005

Leu Lys Asn Ala Val Glu Phe Arg Asp Val Thr Glu Tyr Ala Glu

1010 1015 1020

Val Ile Asn Glu Leu Leu Gly Gln Leu Ile Ser Trp Ser Tyr Leu

1025 1030 1035

Arg Glu Arg Asp Leu Leu Tyr Phe Gln Leu Gly Phe His Tyr Met

1040 1045 1050

Cys Leu Lys Asn Lys Ser Phe Lys Pro Ala Glu Tyr Val Asp Ile

1055 1060 1065

Arg Arg Asn Asn Gly Thr Ile Ile His Asn Ala Ile Leu Tyr Gln

1070 1075 1080

Ile Val Ser Met Tyr Ile Asn Gly Leu Asp Phe Tyr Ser Cys Asp

1085 1090 1095

Lys Glu Gly Lys Thr Leu Lys Pro Ile Glu Thr Gly Lys Gly Val

1100 1105 1110

Gly Ser Lys Ile Gly Gln Phe Ile Lys Tyr Ser Gln Tyr Leu Tyr

1115 1120 1125

Asn Asp Pro Ser Tyr Lys Leu Glu Ile Tyr Asn Ala Gly Leu Glu

1130 1135 1140

Val Phe Glu Asn Ile Asp Glu His Asp Asn Ile Thr Asp Leu Arg

1145 1150 1155

Lys Tyr Val Asp His Phe Lys Tyr Tyr Ala Tyr Gly Asn Lys Met

1160 1165 1170

Ser Leu Leu Asp Leu Tyr Ser Glu Phe Phe Asp Arg Phe Phe Thr

1175 1180 1185

Tyr Asp Met Lys Tyr Gln Lys Asn Val Val Asn Val Leu Glu Asn

1190 1195 1200

Ile Leu Leu Arg His Phe Val Ile Phe Tyr Pro Lys Phe Gly Ser

1205 1210 1215

Gly Lys Lys Asp Val Gly Ile Arg Asp Cys Lys Lys Glu Arg Ala

1220 1225 1230

Gln Ile Glu Ile Ser Glu Gln Ser Leu Thr Ser Glu Asp Phe Met

1235 1240 1245

Phe Lys Leu Asp Asp Lys Ala Gly Glu Glu Ala Lys Lys Phe Pro

1250 1255 1260

Ala Arg Asp Glu Arg Tyr Leu Gln Thr Ile Ala Lys Leu Leu Tyr

1265 1270 1275

Tyr Pro Asn Glu Ile Glu Asp Met Asn Arg Phe Met Lys Lys Gly

1280 1285 1290

Glu Thr Ile Asn Lys Lys Val Gln Phe Asn Arg Lys Lys Lys Ile

1295 1300 1305

Thr Arg Lys Gln Lys Asn Asn Ser Ser Asn Glu Val Leu Ser Ser

1310 1315 1320

Thr Met Gly Tyr Leu Phe Lys Asn Ile Lys Leu

1325 1330

<210> 578

<211> 1175

<212> PRT

<213> Carnobacterium gallinarum

<400> 578

Met Arg Ile Thr Lys Val Lys Ile Lys Leu Asp Asn Lys Leu Tyr Gln

1 5 10 15

Val Thr Met Gln Lys Glu Glu Lys Tyr Gly Thr Leu Lys Leu Asn Glu

20 25 30

Glu Ser Arg Lys Ser Thr Ala Glu Ile Leu Arg Leu Lys Lys Ala Ser

35 40 45

Phe Asn Lys Ser Phe His Ser Lys Thr Ile Asn Ser Gln Lys Glu Asn

50 55 60

Lys Asn Ala Thr Ile Lys Lys Asn Gly Asp Tyr Ile Ser Gln Ile Phe

65 70 75 80

Glu Lys Leu Val Gly Val Asp Thr Asn Lys Asn Ile Arg Lys Pro Lys

85 90 95

Met Ser Leu Thr Asp Leu Lys Asp Leu Pro Lys Lys Asp Leu Ala Leu

100 105 110

Phe Ile Lys Arg Lys Phe Lys Asn Asp Asp Ile Val Glu Ile Lys Asn

115 120 125

Leu Asp Leu Ile Ser Leu Phe Tyr Asn Ala Leu Gln Lys Val Pro Gly

130 135 140

Glu His Phe Thr Asp Glu Ser Trp Ala Asp Phe Cys Gln Glu Met Met

145 150 155 160

Pro Tyr Arg Glu Tyr Lys Asn Lys Phe Ile Glu Arg Lys Ile Ile Leu

165 170 175

Leu Ala Asn Ser Ile Glu Gln Asn Lys Gly Phe Ser Ile Asn Pro Glu

180 185 190

Thr Phe Ser Lys Arg Lys Arg Val Leu His Gln Trp Ala Ile Glu Val

195 200 205

Gln Glu Arg Gly Asp Phe Ser Ile Leu Asp Glu Lys Leu Ser Lys Leu

210 215 220

Ala Glu Ile Tyr Asn Phe Lys Lys Met Cys Lys Arg Val Gln Asp Glu

225 230 235 240

Leu Asn Asp Leu Glu Lys Ser Met Lys Lys Gly Lys Asn Pro Glu Lys

245 250 255

Glu Lys Glu Ala Tyr Lys Lys Gln Lys Asn Phe Lys Ile Lys Thr Ile

260 265 270

Trp Lys Asp Tyr Pro Tyr Lys Thr His Ile Gly Leu Ile Glu Lys Ile

275 280 285

Lys Glu Asn Glu Glu Leu Asn Gln Phe Asn Ile Glu Ile Gly Lys Tyr

290 295 300

Phe Glu His Tyr Phe Pro Ile Lys Lys Glu Arg Cys Thr Glu Asp Glu

305 310 315 320

Pro Tyr Tyr Leu Asn Ser Glu Thr Ile Ala Thr Thr Val Asn Tyr Gln

325 330 335

Leu Lys Asn Ala Leu Ile Ser Tyr Leu Met Gln Ile Gly Lys Tyr Lys

340 345 350

Gln Phe Gly Leu Glu Asn Gln Val Leu Asp Ser Lys Lys Leu Gln Glu

355 360 365

Ile Gly Ile Tyr Glu Gly Phe Gln Thr Lys Phe Met Asp Ala Cys Val

370 375 380

Phe Ala Thr Ser Ser Leu Lys Asn Ile Ile Glu Pro Met Arg Ser Gly

385 390 395 400

Asp Ile Leu Gly Lys Arg Glu Phe Lys Glu Ala Ile Ala Thr Ser Ser

405 410 415

Phe Val Asn Tyr His His Phe Phe Pro Tyr Phe Pro Phe Glu Leu Lys

420 425 430

Gly Met Lys Asp Arg Glu Ser Glu Leu Ile Pro Phe Gly Glu Gln Thr

435 440 445

Glu Ala Lys Gln Met Gln Asn Ile Trp Ala Leu Arg Gly Ser Val Gln

450 455 460

Gln Ile Arg Asn Glu Ile Phe His Ser Phe Asp Lys Asn Gln Lys Phe

465 470 475 480

Asn Leu Pro Gln Leu Asp Lys Ser Asn Phe Glu Phe Asp Ala Ser Glu

485 490 495

Asn Ser Thr Gly Lys Ser Gln Ser Tyr Ile Glu Thr Asp Tyr Lys Phe

500 505 510

Leu Phe Glu Ala Glu Lys Asn Gln Leu Glu Gln Phe Phe Ile Glu Arg

515 520 525

Ile Lys Ser Ser Gly Ala Leu Glu Tyr Tyr Pro Leu Lys Ser Leu Glu

530 535 540

Lys Leu Phe Ala Lys Lys Glu Met Lys Phe Ser Leu Gly Ser Gln Val

545 550 555 560

Val Ala Phe Ala Pro Ser Tyr Lys Lys Leu Val Lys Lys Gly His Ser

565 570 575

Tyr Gln Thr Ala Thr Glu Gly Thr Ala Asn Tyr Leu Gly Leu Ser Tyr

580 585 590

Tyr Asn Arg Tyr Glu Leu Lys Glu Glu Ser Phe Gln Ala Gln Tyr Tyr

595 600 605

Leu Leu Lys Leu Ile Tyr Gln Tyr Val Phe Leu Pro Asn Phe Ser Gln

610 615 620

Gly Asn Ser Pro Ala Phe Arg Glu Thr Val Lys Ala Ile Leu Arg Ile

625 630 635 640

Asn Lys Asp Glu Ala Arg Lys Lys Met Lys Lys Asn Lys Lys Phe Leu

645 650 655

Arg Lys Tyr Ala Phe Glu Gln Val Arg Glu Met Glu Phe Lys Glu Thr

660 665 670

Pro Asp Gln Tyr Met Ser Tyr Leu Gln Ser Glu Met Arg Glu Glu Lys

675 680 685

Val Arg Lys Ala Glu Lys Asn Asp Lys Gly Phe Glu Lys Asn Ile Thr

690 695 700

Met Asn Phe Glu Lys Leu Leu Met Gln Ile Phe Val Lys Gly Phe Asp

705 710 715 720

Val Phe Leu Thr Thr Phe Ala Gly Lys Glu Leu Leu Leu Ser Ser Glu

725 730 735

Glu Lys Val Ile Lys Glu Thr Glu Ile Ser Leu Ser Lys Lys Ile Asn

740 745 750

Glu Arg Glu Lys Thr Leu Lys Ala Ser Ile Gln Val Glu His Gln Leu

755 760 765

Val Ala Thr Asn Ser Ala Ile Ser Tyr Trp Leu Phe Cys Lys Leu Leu

770 775 780

Asp Ser Arg His Leu Asn Glu Leu Arg Asn Glu Met Ile Lys Phe Lys

785 790 795 800

Gln Ser Arg Ile Lys Phe Asn His Thr Gln His Ala Glu Leu Ile Gln

805 810 815

Asn Leu Leu Pro Ile Val Glu Leu Thr Ile Leu Ser Asn Asp Tyr Asp

820 825 830

Glu Lys Asn Asp Ser Gln Asn Val Asp Val Ser Ala Tyr Phe Glu Asp

835 840 845

Lys Ser Leu Tyr Glu Thr Ala Pro Tyr Val Gln Thr Asp Asp Arg Thr

850 855 860

Arg Val Ser Phe Arg Pro Ile Leu Lys Leu Glu Lys Tyr His Thr Lys

865 870 875 880

Ser Leu Ile Glu Ala Leu Leu Lys Asp Asn Pro Gln Phe Arg Val Ala

885 890 895

Ala Thr Asp Ile Gln Glu Trp Met His Lys Arg Glu Glu Ile Gly Glu

900 905 910

Leu Val Glu Lys Arg Lys Asn Leu His Thr Glu Trp Ala Glu Gly Gln

915 920 925

Gln Thr Leu Gly Ala Glu Lys Arg Glu Glu Tyr Arg Asp Tyr Cys Lys

930 935 940

Lys Ile Asp Arg Phe Asn Trp Lys Ala Asn Lys Val Thr Leu Thr Tyr

945 950 955 960

Leu Ser Gln Leu His Tyr Leu Ile Thr Asp Leu Leu Gly Arg Met Val

965 970 975

Gly Phe Ser Ala Leu Phe Glu Arg Asp Leu Val Tyr Phe Ser Arg Ser

980 985 990

Phe Ser Glu Leu Gly Gly Glu Thr Tyr His Ile Ser Asp Tyr Lys Asn

995 1000 1005

Leu Ser Gly Val Leu Arg Leu Asn Ala Glu Val Lys Pro Ile Lys

1010 1015 1020

Ile Lys Asn Ile Lys Val Ile Asp Asn Glu Glu Asn Pro Tyr Lys

1025 1030 1035

Gly Asn Glu Pro Glu Val Lys Pro Phe Leu Asp Arg Leu His Ala

1040 1045 1050

Tyr Leu Glu Asn Val Ile Gly Ile Lys Ala Val His Gly Lys Ile

1055 1060 1065

Arg Asn Gln Thr Ala His Leu Ser Val Leu Gln Leu Glu Leu Ser

1070 1075 1080

Met Ile Glu Ser Met Asn Asn Leu Arg Asp Leu Met Ala Tyr Asp

1085 1090 1095

Arg Lys Leu Lys Asn Ala Val Thr Lys Ser Met Ile Lys Ile Leu

1100 1105 1110

Asp Lys His Gly Met Ile Leu Lys Leu Lys Ile Asp Glu Asn His

1115 1120 1125

Lys Asn Phe Glu Ile Glu Ser Leu Ile Pro Lys Glu Ile Ile His

1130 1135 1140

Leu Lys Asp Lys Ala Ile Lys Thr Asn Gln Val Ser Glu Glu Tyr

1145 1150 1155

Cys Gln Leu Val Leu Ala Leu Leu Thr Thr Asn Pro Gly Asn Gln

1160 1165 1170

Leu Asn

1175

<210> 579

<211> 1164

<212> PRT

<213> Carnobacterium gallinarum

<400> 579

Met Arg Met Thr Lys Val Lys Ile Asn Gly Ser Pro Val Ser Met Asn

1 5 10 15

Arg Ser Lys Leu Asn Gly His Leu Val Trp Asn Gly Thr Thr Asn Thr

20 25 30

Val Asn Ile Leu Thr Lys Lys Glu Gln Ser Phe Ala Ala Ser Phe Leu

35 40 45

Asn Lys Thr Leu Val Lys Ala Asp Gln Val Lys Gly Tyr Lys Val Leu

50 55 60

Ala Glu Asn Ile Phe Ile Ile Phe Glu Gln Leu Glu Lys Ser Asn Ser

65 70 75 80

Glu Lys Pro Ser Val Tyr Leu Asn Asn Ile Arg Arg Leu Lys Glu Ala

85 90 95

Gly Leu Lys Arg Phe Phe Lys Ser Lys Tyr His Glu Glu Ile Lys Tyr

100 105 110

Thr Ser Glu Lys Asn Gln Ser Val Pro Thr Lys Leu Asn Leu Ile Pro

115 120 125

Leu Phe Phe Asn Ala Val Asp Arg Ile Gln Glu Asp Lys Phe Asp Glu

130 135 140

Lys Asn Trp Ser Tyr Phe Cys Lys Glu Met Ser Pro Tyr Leu Asp Tyr

145 150 155 160

Lys Lys Ser Tyr Leu Asn Arg Lys Lys Glu Ile Leu Ala Asn Ser Ile

165 170 175

Gln Gln Asn Arg Gly Phe Ser Met Pro Thr Ala Glu Glu Pro Asn Leu

180 185 190

Leu Ser Lys Arg Lys Gln Leu Phe Gln Gln Trp Ala Met Lys Phe Gln

195 200 205

Glu Ser Pro Leu Ile Gln Gln Asn Asn Phe Ala Val Glu Gln Phe Asn

210 215 220

Lys Glu Phe Ala Asn Lys Ile Asn Glu Leu Ala Ala Val Tyr Asn Val

225 230 235 240

Asp Glu Leu Cys Thr Ala Ile Thr Glu Lys Leu Met Asn Phe Asp Lys

245 250 255

Asp Lys Ser Asn Lys Thr Arg Asn Phe Glu Ile Lys Lys Leu Trp Lys

260 265 270

Gln His Pro His Asn Lys Asp Lys Ala Leu Ile Lys Leu Phe Asn Gln

275 280 285

Glu Gly Asn Glu Ala Leu Asn Gln Phe Asn Ile Glu Leu Gly Lys Tyr

290 295 300

Phe Glu His Tyr Phe Pro Lys Thr Gly Lys Lys Glu Ser Ala Glu Ser

305 310 315 320

Tyr Tyr Leu Asn Pro Gln Thr Ile Ile Lys Thr Val Gly Tyr Gln Leu

325 330 335

Arg Asn Ala Phe Val Gln Tyr Leu Leu Gln Val Gly Lys Leu His Gln

340 345 350

Tyr Asn Lys Gly Val Leu Asp Ser Gln Thr Leu Gln Glu Ile Gly Met

355 360 365

Tyr Glu Gly Phe Gln Thr Lys Phe Met Asp Ala Cys Val Phe Ala Ser

370 375 380

Ser Ser Leu Arg Asn Ile Ile Gln Ala Thr Thr Asn Glu Asp Ile Leu

385 390 395 400

Thr Arg Glu Lys Phe Lys Lys Glu Leu Glu Lys Asn Val Glu Leu Lys

405 410 415

His Asp Leu Phe Phe Lys Thr Glu Ile Val Glu Glu Arg Asp Glu Asn

420 425 430

Pro Ala Lys Lys Ile Ala Met Thr Pro Asn Glu Leu Asp Leu Trp Ala

435 440 445

Ile Arg Gly Ala Val Gln Arg Val Arg Asn Gln Ile Phe His Gln Gln

450 455 460

Ile Asn Lys Arg His Glu Pro Asn Gln Leu Lys Val Gly Ser Phe Glu

465 470 475 480

Asn Gly Asp Leu Gly Asn Val Ser Tyr Gln Lys Thr Ile Tyr Gln Lys

485 490 495

Leu Phe Asp Ala Glu Ile Lys Asp Ile Glu Ile Tyr Phe Ala Glu Lys

500 505 510

Ile Lys Ser Ser Gly Ala Leu Glu Gln Tyr Ser Met Lys Asp Leu Glu

515 520 525

Lys Leu Phe Ser Asn Lys Glu Leu Thr Leu Ser Leu Gly Gly Gln Val

530 535 540

Val Ala Phe Ala Pro Ser Tyr Lys Lys Leu Tyr Lys Gln Gly Tyr Phe

545 550 555 560

Tyr Gln Asn Glu Lys Thr Ile Glu Leu Glu Gln Phe Thr Asp Tyr Asp

565 570 575

Phe Ser Asn Asp Val Phe Lys Ala Asn Tyr Tyr Leu Ile Lys Leu Ile

580 585 590

Tyr His Tyr Val Phe Leu Pro Gln Phe Ser Gln Ala Asn Asn Lys Leu

595 600 605

Phe Lys Asp Thr Val His Tyr Val Ile Gln Gln Asn Lys Glu Leu Asn

610 615 620

Thr Thr Glu Lys Asp Lys Lys Asn Asn Lys Lys Ile Arg Lys Tyr Ala

625 630 635 640

Phe Glu Gln Val Lys Leu Met Lys Asn Glu Ser Pro Glu Lys Tyr Met

645 650 655

Gln Tyr Leu Gln Arg Glu Met Gln Glu Glu Arg Thr Ile Lys Glu Ala

660 665 670

Lys Lys Thr Asn Glu Glu Lys Pro Asn Tyr Asn Phe Glu Lys Leu Leu

675 680 685

Ile Gln Ile Phe Ile Lys Gly Phe Asp Thr Phe Leu Arg Asn Phe Asp

690 695 700

Leu Asn Leu Asn Pro Ala Glu Glu Leu Val Gly Thr Val Lys Glu Lys

705 710 715 720

Ala Glu Gly Leu Arg Lys Arg Lys Glu Arg Ile Ala Lys Ile Leu Asn

725 730 735

Val Asp Glu Gln Ile Lys Thr Gly Asp Glu Glu Ile Ala Phe Trp Ile

740 745 750

Phe Ala Lys Leu Leu Asp Ala Arg His Leu Ser Glu Leu Arg Asn Glu

755 760 765

Met Ile Lys Phe Lys Gln Ser Val Lys Lys Gly Leu Ile Lys Asn

770 775 780

Gly Asp Leu Ile Glu Gln Met Gln Pro Ile Leu Glu Leu Cys Ile Leu

785 790 795 800

Ser Asn Asp Ser Glu Ser Met Glu Lys Glu Ser Phe Asp Lys Ile Glu

805 810 815

Val Phe Leu Glu Lys Val Glu Leu Ala Lys Asn Glu Pro Tyr Met Gln

820 825 830

Glu Asp Lys Leu Thr Pro Val Lys Phe Arg Phe Met Lys Gln Leu Glu

835 840 845

Lys Tyr Gln Thr Arg Asn Phe Ile Glu Asn Leu Val Ile Glu Asn Pro

850 855 860

Glu Phe Lys Val Ser Glu Lys Ile Val Leu Asn Trp His Glu Glu Lys

865 870 875 880

Glu Lys Ile Ala Asp Leu Val Asp Lys Arg Thr Lys Leu His Glu Glu

885 890 895

Trp Ala Ser Lys Ala Arg Glu Ile Glu Glu Tyr Asn Glu Lys Ile Lys

900 905 910

Lys Asn Lys Ser Lys Lys Leu Asp Lys Pro Ala Glu Phe Ala Lys Phe

915 920 925

Ala Glu Tyr Lys Ile Ile Cys Glu Ala Ile Glu Asn Phe Asn Arg Leu

930 935 940

Asp His Lys Val Arg Leu Thr Tyr Leu Lys Asn Leu His Tyr Leu Met

945 950 955 960

Ile Asp Leu Met Gly Arg Met Val Gly Phe Ser Val Leu Phe Glu Arg

965 970 975

Asp Phe Val Tyr Met Gly Arg Ser Tyr Ser Ala Leu Lys Lys Gln Ser

980 985 990

Ile Tyr Leu Asn Asp Tyr Asp Thr Phe Ala Asn Ile Arg Asp Trp Glu

995 1000 1005

Val Asn Glu Asn Lys His Leu Phe Gly Thr Ser Ser Ser Asp Leu

1010 1015 1020

Thr Phe Gln Glu Thr Ala Glu Phe Lys Asn Leu Lys Lys Pro Met

1025 1030 1035

Glu Asn Gln Leu Lys Ala Leu Leu Gly Val Thr Asn His Ser Phe

1040 1045 1050

Glu Ile Arg Asn Asn Ile Ala His Leu His Val Leu Arg Asn Asp

1055 1060 1065

Gly Lys Gly Glu Gly Val Ser Leu Leu Ser Cys Met Asn Asp Leu

1070 1075 1080

Arg Lys Leu Met Ser Tyr Asp Arg Lys Leu Lys Asn Ala Val Thr

1085 1090 1095

Lys Ala Ile Ile Lys Ile Leu Asp Lys His Gly Met Ile Leu Lys

1100 1105 1110

Leu Thr Asn Asn Asp His Thr Lys Pro Phe Glu Ile Glu Ser Leu

1115 1120 1125

Lys Pro Lys Lys Ile Ile His Leu Glu Lys Ser Asn His Ser Phe

1130 1135 1140

Pro Met Asp Gln Val Ser Gln Glu Tyr Cys Asp Leu Val Lys Lys

1145 1150 1155

Met Leu Val Phe Thr Asn

1160

<210> 580

<211> 1154

<212> PRT

<213> Paludibacter propionicigenes

<400> 580

Met Arg Val Ser Lys Val Lys Val Lys Asp Gly Gly Lys Asp Lys Met

1 5 10 15

Val Leu Val His Arg Lys Thr Thr Gly Ala Gln Leu Val Tyr Ser Gly

20 25 30

Gln Pro Val Ser Asn Glu Thr Ser Asn Ile Leu Pro Glu Lys Lys Arg

35 40 45

Gln Ser Phe Asp Leu Ser Thr Leu Asn Lys Thr Ile Ile Lys Phe Asp

50 55 60

Thr Ala Lys Lys Gln Lys Leu Asn Val Asp Gln Tyr Lys Ile Val Glu

65 70 75 80

Lys Ile Phe Lys Tyr Pro Lys Gln Glu Leu Pro Lys Gln Ile Lys Ala

85 90 95

Glu Glu Ile Leu Pro Phe Leu Asn His Lys Phe Gln Glu Pro Val Lys

100 105 110

Tyr Trp Lys Asn Gly Lys Glu Glu Ser Phe Asn Leu Thr Leu Leu Ile

115 120 125

Val Glu Ala Val Gln Ala Gln Asp Lys Arg Lys Leu Gln Pro Tyr Tyr

130 135 140

Asp Trp Lys Thr Trp Tyr Ile Gln Thr Lys Ser Asp Leu Leu Lys Lys

145 150 155 160

Ser Ile Glu Asn Asn Arg Ile Asp Leu Thr Glu Asn Leu Ser Lys Arg

165 170 175

Lys Lys Ala Leu Leu Ala Trp Glu Thr Glu Phe Thr Ala Ser Gly Ser

180 185 190

Ile Asp Leu Thr His Tyr His Lys Val Tyr Met Thr Asp Val Leu Cys

195 200 205

Lys Met Leu Gln Asp Val Lys Pro Leu Thr Asp Asp Lys Gly Lys Ile

210 215 220

Asn Thr Asn Ala Tyr His Arg Gly Leu Lys Lys Ala Leu Gln Asn His

225 230 235 240

Gln Pro Ala Ile Phe Gly Thr Arg Glu Val Pro Asn Glu Ala Asn Arg

245 250 255

Ala Asp Asn Gln Leu Ser Ile Tyr His Leu Glu Val Val Lys Tyr Leu

260 265 270

Glu His Tyr Phe Pro Ile Lys Thr Ser Lys Arg Arg Asn Thr Ala Asp

275 280 285

Asp Ile Ala His Tyr Leu Lys Ala Gln Thr Leu Lys Thr Thr Ile Glu

290 295 300

Lys Gln Leu Val Asn Ala Ile Arg Ala Asn Ile Ile Gln Gln Gly Lys

305 310 315 320

Thr Asn His His Glu Leu Lys Ala Asp Thr Thr Ser Asn Asp Leu Ile

325 330 335

Arg Ile Lys Thr Asn Glu Ala Phe Val Leu Asn Leu Thr Gly Thr Cys

340 345 350

Ala Phe Ala Ala Asn Asn Ile Arg Asn Met Val Asp Asn Glu Gln Thr

355 360 365

Asn Asp Ile Leu Gly Lys Gly Asp Phe Ile Lys Ser Leu Leu Lys Asp

370 375 380

Asn Thr Asn Ser Gln Leu Tyr Ser Phe Phe Phe Gly Glu Gly Leu Ser

385 390 395 400

Thr Asn Lys Ala Glu Lys Glu Thr Gln Leu Trp Gly Ile Arg Gly Ala

405 410 415

Val Gln Gln Ile Arg Asn Asn Val Asn His Tyr Lys Lys Asp Ala Leu

420 425 430

Lys Thr Val Phe Asn Ile Ser Asn Phe Glu Asn Pro Thr Ile Thr Asp

435 440 445

Pro Lys Gln Gln Thr Asn Tyr Ala Asp Thr Ile Tyr Lys Ala Arg Phe

450 455 460

Ile Asn Glu Leu Glu Lys Ile Pro Glu Ala Phe Ala Gln Gln Leu Lys

465 470 475 480

Thr Gly Gly Ala Val Ser Tyr Tyr Thr Ile Glu Asn Leu Lys Ser Leu

485 490 495

Leu Thr Thr Phe Gln Phe Ser Leu Cys Arg Ser Thr Ile Pro Phe Ala

500 505 510

Pro Gly Phe Lys Lys Val Phe Asn Gly Gly Ile Asn Tyr Gln Asn Ala

515 520 525

Lys Gln Asp Glu Ser Phe Tyr Glu Leu Met Leu Glu Gln Tyr Leu Arg

530 535 540

Lys Glu Asn Phe Ala Glu Glu Ser Tyr Asn Ala Arg Tyr Phe Met Leu

545 550 555 560

Lys Leu Ile Tyr Asn Asn Leu Phe Leu Pro Gly Phe Thr Asp Arg

565 570 575

Lys Ala Phe Ala Asp Ser Val Gly Phe Val Gln Met Gln Asn Lys Lys

580 585 590

Gln Ala Glu Lys Val Asn Pro Arg Lys Lys Glu Ala Tyr Ala Phe Glu

595 600 605

Ala Val Arg Pro Met Thr Ala Ala Asp Ser Ile Ala Asp Tyr Met Ala

610 615 620

Tyr Val Gln Ser Glu Leu Met Gln Glu Gln Asn Lys Lys Glu Glu Lys

625 630 635 640

Val Ala Glu Glu Thr Arg Ile Asn Phe Glu Lys Phe Val Leu Gln Val

645 650 655

Phe Ile Lys Gly Phe Asp Ser Phe Leu Arg Ala Lys Glu Phe Asp Phe

660 665 670

Val Gln Met Pro Gln Pro Gln Leu Thr Ala Thr Ala Ser Asn Gln Gln

675 680 685

Lys Ala Asp Lys Leu Asn Gln Leu Glu Ala Ser Ile Thr Ala Asp Cys

690 695 700

Lys Leu Thr Pro Gln Tyr Ala Lys Ala Asp Asp Ala Thr His Ile Ala

705 710 715 720

Phe Tyr Val Phe Cys Lys Leu Leu Asp Ala Ala His Leu Ser Asn Leu

725 730 735

Arg Asn Glu Leu Ile Lys Phe Arg Glu Ser Val Asn Glu Phe Lys Phe

740 745 750

His His Leu Leu Glu Ile Ile Glu Ile Cys Leu Leu Ser Ala Asp Val

755 760 765

Val Pro Thr Asp Tyr Arg Asp Leu Tyr Ser Ser Glu Ala Asp Cys Leu

770 775 780

Ala Arg Leu Arg Pro Phe Ile Glu Gln Gly Ala Asp Ile Thr Asn Trp

785 790 795 800

Ser Asp Leu Phe Val Gln Ser Asp Lys His Ser Pro Val Ile His Ala

805 810 815

Asn Ile Glu Leu Ser Val Lys Tyr Gly Thr Thr Lys Leu Leu Glu Gln

820 825 830

Ile Ile Asn Lys Asp Thr Gln Phe Lys Thr Thr Glu Ala Asn Phe Thr

835 840 845

Ala Trp Asn Thr Ala Gln Lys Ser Ile Glu Gln Leu Ile Lys Gln Arg

850 855 860

Glu Asp His His Glu Gln Trp Val Lys Ala Lys Asn Ala Asp Asp Lys

865 870 875 880

Glu Lys Gln Glu Arg Lys Arg Glu Lys Ser Asn Phe Ala Gln Lys Phe

885 890 895

Ile Glu Lys His Gly Asp Asp Tyr Leu Asp Ile Cys Asp Tyr Ile Asn

900 905 910

Thr Tyr Asn Trp Leu Asp Asn Lys Met His Phe Val His Leu Asn Arg

915 920 925

Leu His Gly Leu Thr Ile Glu Leu Leu Gly Arg Met Ala Gly Phe Val

930 935 940

Ala Leu Phe Asp Arg Asp Phe Gln Phe Phe Asp Glu Gln Gln Ile Ala

945 950 955 960

Asp Glu Phe Lys Leu His Gly Phe Val Asn Leu His Ser Ile Asp Lys

965 970 975

Lys Leu Asn Glu Val Pro Thr Lys Lys Ile Lys Glu Ile Tyr Asp Ile

980 985 990

Arg Asn Lys Ile Ile Gln Ile Asn Gly Asn Lys Ile Asn Glu Ser Val

995 1000 1005

Arg Ala Asn Leu Ile Gln Phe Ile Ser Ser Lys Arg Asn Tyr Tyr

1010 1015 1020

Asn Asn Ala Phe Leu His Val Ser Asn Asp Glu Ile Lys Glu Lys

1025 1030 1035

Gln Met Tyr Asp Ile Arg Asn His Ile Ala His Phe Asn Tyr Leu

1040 1045 1050

Thr Lys Asp Ala Ala Asp Phe Ser Leu Ile Asp Leu Ile Asn Glu

1055 1060 1065

Leu Arg Glu Leu Leu His Tyr Asp Arg Lys Leu Lys Asn Ala Val

1070 1075 1080

Ser Lys Ala Phe Ile Asp Leu Phe Asp Lys His Gly Met Ile Leu

1085 1090 1095

Lys Leu Lys Leu Asn Ala Asp His Lys Leu Lys Val Glu Ser Leu

1100 1105 1110

Glu Pro Lys Lys Ile Tyr His Leu Gly Ser Ser Ala Lys Asp Lys

1115 1120 1125

Pro Glu Tyr Gln Tyr Cys Thr Asn Gln Val Met Met Ala Tyr Cys

1130 1135 1140

Asn Met Cys Arg Ser Leu Leu Glu Met Lys Lys

1145 1150

<210> 581

<211> 1120

<212> PRT

<213> Listeria seeligeri

<400> 581

Met Trp Ile Ser Ile Lys Thr Leu Ile His His Leu Gly Val Leu Phe

1 5 10 15

Phe Cys Asp Tyr Met Tyr Asn Arg Arg Glu Lys Lys Ile Ile Glu Val

20 25 30

Lys Thr Met Arg Ile Thr Lys Val Glu Val Asp Arg Lys Lys Val Leu

35 40 45

Ile Ser Arg Asp Lys Asn Gly Gly Lys Leu Val Tyr Glu Asn Glu Met

50 55 60

Gln Asp Asn Thr Glu Gln Ile Met His His Lys Lys Ser Ser Phe Tyr

65 70 75 80

Lys Ser Val Val Asn Lys Thr Ile Cys Arg Pro Glu Gln Lys Gln Met

85 90 95

Lys Lys Leu Val His Gly Leu Leu Gln Glu Asn Ser Gln Glu Lys Ile

100 105 110

Lys Val Ser Asp Val Thr Lys Leu Asn Ile Ser Asn Phe Leu Asn His

115 120 125

Arg Phe Lys Lys Ser Leu Tyr Tyr Phe Pro Glu Asn Ser Pro Asp Lys

130 135 140

Ser Glu Glu Tyr Arg Ile Glu Ile Asn Leu Ser Gln Leu Leu Glu Asp

145 150 155 160

Ser Leu Lys Lys Gln Gln Gly Thr Phe Ile Cys Trp Glu Ser Phe Ser

165 170 175

Lys Asp Met Glu Leu Tyr Ile Asn Trp Ala Glu Asn Tyr Ile Ser Ser

180 185 190

Lys Thr Lys Leu Ile Lys Lys Ser Ile Arg Asn Asn Arg Ile Gln Ser

195 200 205

Thr Glu Ser Arg Ser Gly Gln Leu Met Asp Arg Tyr Met Lys Asp Ile

210 215 220

Leu Asn Lys Asn Lys Pro Phe Asp Ile Gln Ser Val Ser Glu Lys Tyr

225 230 235 240

Gln Leu Glu Lys Leu Thr Ser Ala Leu Lys Ala Thr Phe Lys Glu Ala

245 250 255

Lys Lys Asn Asp Lys Glu Ile Asn Tyr Lys Leu Lys Ser Thr Leu Gln

260 265 270

Asn His Glu Arg Gln Ile Ile Glu Glu Leu Lys Glu Asn Ser Glu Leu

275 280 285

Asn Gln Phe Asn Ile Glu Ile Arg Lys His Leu Glu Thr Tyr Phe Pro

290 295 300

Ile Lys Lys Thr Asn Arg Lys Val Gly Asp Ile Arg Asn Leu Glu Ile

305 310 315 320

Gly Glu Ile Gln Lys Ile Val Asn His Arg Leu Lys Asn Lys Ile Val

325 330 335

Gln Arg Ile Leu Gln Glu Gly Lys Leu Ala Ser Tyr Glu Ile Glu Ser

340 345 350

Thr Val Asn Ser Asn Ser Leu Gln Lys Ile Lys Ile Glu Glu Ala Phe

355 360 365

Ala Leu Lys Phe Ile Asn Ala Cys Leu Phe Ala Ser Asn Asn Leu Arg

370 375 380

Asn Met Val Tyr Pro Val Cys Lys Lys Asp Ile Leu Met Ile Gly Glu

385 390 395 400

Phe Lys Asn Ser Phe Lys Glu Ile Lys His Lys Lys Phe Ile Arg Gln

405 410 415

Trp Ser Gln Phe Phe Ser Gln Glu Ile Thr Val Asp Asp Ile Glu Leu

420 425 430

Ala Ser Trp Gly Leu Arg Gly Ala Ile Ala Pro Ile Arg Asn Glu Ile

435 440 445

Ile His Leu Lys Lys His Ser Trp Lys Lys Phe Phe Asn Asn Pro Thr

450 455 460

Phe Lys Val Lys Lys Ser Lys Ile Ile Asn Gly Lys Thr Lys Asp Val

465 470 475 480

Thr Ser Glu Phe Leu Tyr Lys Glu Thr Leu Phe Lys Asp Tyr Phe Tyr

485 490 495

Ser Glu Leu Asp Ser Val Pro Glu Leu Ile Ile Asn Lys Met Glu Ser

500 505 510

Ser Lys Ile Leu Asp Tyr Tyr Ser Ser Asp Gln Leu Asn Gln Val Phe

515 520 525

Thr Ile Pro Asn Phe Glu Leu Ser Leu Leu Thr Ser Ala Val Pro Phe

530 535 540

Ala Pro Ser Phe Lys Arg Val Tyr Leu Lys Gly Phe Asp Tyr Gln Asn

545 550 555 560

Gln Asp Glu Ala Gln Pro Asp Tyr Asn Leu Lys Leu Asn Ile Tyr Asn

565 570 575

Glu Lys Ala Phe Asn Ser Glu Ala Phe Gln Ala Gln Tyr Ser Leu Phe

580 585 590

Lys Met Val Tyr Tyr Gln Val Phe Leu Pro Gln Phe Thr Thr Asn Asn

595 600 605

Asp Leu Phe Lys Ser Ser Val Asp Phe Ile Leu Thr Leu Asn Lys Glu

610 615 620

Arg Lys Gly Tyr Ala Lys Ala Phe Gln Asp Ile Arg Lys Met Asn Lys

625 630 635 640

Asp Glu Lys Pro Ser Glu Tyr Met Ser Tyr Ile Gln Ser Gln Leu Met

645 650 655

Leu Tyr Gln Lys Lys Gln Glu Glu Lys Glu Lys Ile Asn His Phe Glu

660 665 670

Lys Phe Ile Asn Gln Val Phe Ile Lys Gly Phe Asn Ser Phe Ile Glu

675 680 685

Lys Asn Arg Leu Thr Tyr Ile Cys His Pro Thr Lys Asn Thr Val Pro

690 695 700

Glu Asn Asp Asn Ile Glu Ile Pro Phe His Thr Asp Met Asp Asp Ser

705 710 715 720

Asn Ile Ala Phe Trp Leu Met Cys Lys Leu Leu Asp Ala Lys Gln Leu

725 730 735

Ser Glu Leu Arg Asn Glu Met Ile Lys Phe Ser Cys Ser Leu Gln Ser

740 745 750

Thr Glu Glu Ile Ser Thr Phe Thr Lys Ala Arg Glu Val Ile Gly Leu

755 760 765

Ala Leu Leu Asn Gly Glu Lys Gly Cys Asn Asp Trp Lys Glu Leu Phe

770 775 780

Asp Asp Lys Glu Ala Trp Lys Lys Asn Met Ser Leu Tyr Val Ser Glu

785 790 795 800

Glu Leu Leu Gln Ser Leu Pro Tyr Thr Gln Glu Asp Gly Gln Thr Pro

805 810 815

Val Ile Asn Arg Ser Ile Asp Leu Val Lys Lys Tyr Gly Thr Glu Thr

820 825 830

Ile Leu Glu Lys Leu Phe Ser Ser Ser Asp Asp Tyr Lys Val Ser Ala

835 840 845

Lys Asp Ile Ala Lys Leu His Glu Tyr Asp Val Thr Glu Lys Ile Ala

850 855 860

Gln Gln Glu Ser Leu His Lys Gln Trp Ile Glu Lys Pro Gly Leu Ala

865 870 875 880

Arg Asp Ser Ala Trp Thr Lys Lys Tyr Gln Asn Val Ile Asn Asp Ile

885 890 895

Ser Asn Tyr Gln Trp Ala Lys Thr Lys Val Glu Leu Thr Gln Val Arg

900 905 910

His Leu His Gln Leu Thr Ile Asp Leu Leu Ser Arg Leu Ala Gly Tyr

915 920 925

Met Ser Ile Ala Asp Arg Asp Phe Gln Phe Ser Ser Asn Tyr Ile Leu

930 935 940

Glu Arg Glu Asn Ser Glu Tyr Arg Val Thr Ser Trp Ile Leu Leu Ser

945 950 955 960

Glu Asn Lys Asn Lys Asn Lys Tyr Asn Asp Tyr Glu Leu Tyr Asn Leu

965 970 975

Lys Asn Ala Ser Ile Lys Val Ser Ser Lys Asn Asp Pro Gln Leu Lys

980 985 990

Val Asp Leu Lys Gln Leu Arg Leu Thr Leu Glu Tyr Leu Glu Leu Phe

995 1000 1005

Asp Asn Arg Leu Lys Glu Lys Arg Asn Asn Ile Ser His Phe Asn

1010 1015 1020

Tyr Leu Asn Gly Gln Leu Gly Asn Ser Ile Leu Glu Leu Phe Asp

1025 1030 1035

Asp Ala Arg Asp Val Leu Ser Tyr Asp Arg Lys Leu Lys Asn Ala

1040 1045 1050

Val Ser Lys Ser Leu Lys Glu Ile Leu Ser Ser His Gly Met Glu

1055 1060 1065

Val Thr Phe Lys Pro Leu Tyr Gln Thr Asn His His Leu Lys Ile

1070 1075 1080

Asp Lys Leu Gln Pro Lys Lys Ile His His Leu Gly Glu Lys Ser

1085 1090 1095

Thr Val Ser Ser Asn Gln Val Ser Asn Glu Tyr Cys Gln Leu Val

1100 1105 1110

Arg Thr Leu Leu Thr Met Lys

1115 1120

<210> 582

<211> 970

<212> PRT

<213> Listeria weihenstephanensis

<400> 582

Met Leu Ala Leu Leu His Gln Glu Val Pro Ser Gln Lys Leu His Asn

1 5 10 15

Leu Lys Ser Leu Asn Thr Glu Ser Leu Thr Lys Leu Phe Lys Pro Lys

20 25 30

Phe Gln Asn Met Ile Ser Tyr Pro Ser Lys Gly Ala Glu His Val

35 40 45

Gln Phe Cys Leu Thr Asp Ile Ala Val Pro Ala Ile Arg Asp Leu Asp

50 55 60

Glu Ile Lys Pro Asp Trp Gly Ile Phe Phe Glu Lys Leu Lys Pro Tyr

65 70 75 80

Thr Asp Trp Ala Glu Ser Tyr Ile His Tyr Lys Gln Thr Thr Ile Gln

85 90 95

Lys Ser Ile Glu Gln Asn Lys Ile Gln Ser Pro Asp Ser Pro Arg Lys

100 105 110

Leu Val Leu Gln Lys Tyr Val Thr Ala Phe Leu Asn Gly Glu Pro Leu

115 120 125

Gly Leu Asp Leu Val Ala Lys Lys Tyr Lys Leu Ala Asp Leu Ala Glu

130 135 140

Ser Phe Lys Val Val Asp Leu Asn Glu Asp Lys Ser Ala Asn Tyr Lys

145 150 155 160

Ile Lys Ala Cys Leu Gln Gln His Gln Arg Asn Ile Leu Asp Glu Leu

165 170 175

Lys Glu Asp Pro Glu Leu Asn Gln Tyr Gly Ile Glu Val Lys Lys Tyr

180 185 190

Ile Gln Arg Tyr Phe Pro Ile Lys Arg Ala Pro Asn Arg Ser Lys His

195 200 205

Ala Arg Ala Asp Phe Leu Lys Lys Glu Leu Ile Glu Ser Thr Val Glu

210 215 220

Gln Gln Phe Lys Asn Ala Val Tyr His Tyr Val Leu Glu Gln Gly Lys

225 230 235 240

Met Glu Ala Tyr Glu Leu Thr Asp Pro Lys Thr Lys Asp Leu Gln Asp

245 250 255

Ile Arg Ser Gly Glu Ala Phe Ser Phe Lys Phe Ile Asn Ala Cys Ala

260 265 270

Phe Ala Ser Asn Asn Leu Lys Met Ile Leu Asn Pro Glu Cys Glu Lys

275 280 285

Asp Ile Leu Gly Lys Gly Asp Phe Lys Lys Asn Leu Pro Asn Ser Thr

290 295 300

Thr Gln Ser Asp Val Val Lys Lys Met Ile Pro Phe Phe Ser Asp Glu

305 310 315 320

Ile Gln Asn Val Asn Phe Asp Glu Ala Ile Trp Ala Ile Arg Gly Ser

325 330 335

Ile Gln Gln Ile Arg Asn Glu Val Tyr His Cys Lys Lys His Ser Trp

340 345 350

Lys Ser Ile Leu Lys Ile Lys Gly Phe Glu Phe Glu Pro Asn Asn Met

355 360 365

Lys Tyr Thr Asp Ser Asp Met Gln Lys Leu Met Asp Lys Asp Ile Ala

370 375 380

Lys Ile Pro Asp Phe Ile Glu Glu Lys Leu Lys Ser Ser Gly Ile Ile

385 390 395 400

Arg Phe Tyr Ser His Asp Lys Leu Gln Ser Ile Trp Glu Met Lys Gln

405 410 415

Gly Phe Ser Leu Leu Thr Thr Asn Ala Pro Phe Val Pro Ser Phe Lys

420 425 430

Arg Val Tyr Ala Lys Gly His Asp Tyr Gln Thr Ser Lys Asn Arg Tyr

435 440 445

Tyr Asp Leu Gly Leu Thr Thr Phe Asp Ile Leu Glu Tyr Gly Glu Glu

450 455 460

Asp Phe Arg Ala Arg Tyr Phe Leu Thr Lys Leu Val Tyr Tyr Gln Gln

465 470 475 480

Phe Met Pro Trp Phe Thr Ala Asp Asn Asn Ala Phe Arg Asp Ala Ala

485 490 495

Asn Phe Val Leu Arg Leu Asn Lys Asn Arg Gln Gln Asp Ala Lys Ala

500 505 510

Phe Ile Asn Ile Arg Glu Val Glu Glu Gly Glu Met Pro Arg Asp Tyr

515 520 525

Met Gly Tyr Val Gln Gly Gln Ile Ala Ile His Glu Asp Ser Thr Glu

530 535 540

Asp Thr Pro Asn His Phe Glu Lys Phe Ile Ser Gln Val Phe Ile Lys

545 550 555 560

Gly Phe Asp Ser His Met Arg Ser Ala Asp Leu Lys Phe Ile Lys Asn

565 570 575

Pro Arg Asn Gln Gly Leu Glu Gln Ser Glu Ile Glu Glu Met Ser Phe

580 585 590

Asp Ile Lys Val Glu Pro Ser Phe Leu Lys Asn Lys Asp Asp Tyr Ile

595 600 605

Ala Phe Trp Thr Phe Cys Lys Met Leu Asp Ala Arg His Leu Ser Glu

610 615 620

Leu Arg Asn Glu Met Ile Lys Tyr Asp Gly His Leu Thr Gly Glu Gln

625 630 635 640

Glu Ile Ile Gly Leu Ala Leu Leu Gly Val Asp Ser Arg Glu Asn Asp

645 650 655

Trp Lys Gln Phe Phe Ser Ser Glu Arg Glu Tyr Glu Lys Ile Met Lys

660 665 670

Gly Tyr Val Gly Glu Glu Leu Tyr Gln Arg Glu Pro Tyr Arg Gln Ser

675 680 685

Asp Gly Lys Thr Pro Ile Leu Phe Arg Gly Val Glu Gln Ala Arg Lys

690 695 700

Tyr Gly Thr Glu Thr Val Ile Gln Arg Leu Phe Asp Ala Ser Pro Glu

705 710 715 720

Phe Lys Val Ser Lys Cys Asn Ile Thr Glu Trp Glu Arg Gln Lys Glu

725 730 735

Thr Ile Glu Glu Thr Ile Glu Arg Arg Lys Glu Leu His Asn Glu Trp

740 745 750

Glu Lys Asn Pro Lys Lys Pro Gln Asn Asn Ala Phe Phe Lys Glu Tyr

755 760 765

Lys Glu Cys Cys Asp Ala Ile Asp Ala Tyr Asn Trp His Lys Asn Lys

770 775 780

Thr Thr Leu Val Tyr Val Asn Glu Leu His His Leu Leu Ile Glu Ile

785 790 795 800

Leu Gly Arg Tyr Val Gly Tyr Val Ala Ile Ala Asp Arg Asp Phe Gln

805 810 815

Cys Met Ala Asn Gln Tyr Phe Lys His Ser Gly Ile Thr Glu Arg Val

820 825 830

Glu Tyr Trp Gly Asp Asn Arg Leu Lys Ser Ile Lys Lys Leu Asp Thr

835 840 845

Phe Leu Lys Lys Glu Gly Leu Phe Val Ser Glu Lys Asn Ala Arg Asn

850 855 860

His Ile Ala His Leu Asn Tyr Leu Ser Leu Lys Ser Glu Cys Thr Leu

865 870 875 880

Leu Tyr Leu Ser Glu Arg Leu Arg Glu Ile Phe Lys Tyr Asp Arg Lys

885 890 895

Leu Lys Asn Ala Val Ser Lys Ser Leu Ile Asp Ile Leu Asp Arg His

900 905 910

Gly Met Ser Val Val Phe Ala Asn Leu Lys Glu Asn Lys His Arg Leu

915 920 925

Val Ile Lys Ser Leu Glu Pro Lys Lys Leu Arg His Leu Gly Glu Lys

930 935 940

Lys Ile Asp Asn Gly Tyr Ile Glu Thr Asn Gln Val Ser Glu Glu Tyr

945 950 955 960

Cys Gly Ile Val Lys Arg Leu Leu Glu Ile

965 970

<210> 583

<211> 1051

<212> PRT

<213> Listeria newyorkensis

<400> 583

Met Lys Ile Thr Lys Met Arg Val Asp Gly Arg Thr Ile Val Met Glu

1 5 10 15

Arg Thr Ser Lys Glu Gly Gln Leu Gly Tyr Glu Gly Ile Asp Gly Asn

20 25 30

Lys Thr Thr Glu Ile Ile Phe Asp Lys Lys Lys Glu Ser Phe Tyr Lys

35 40 45

Ser Ile Leu Asn Lys Thr Val Arg Lys Pro Asp Glu Lys Glu Lys Asn

50 55 60

Arg Arg Lys Gln Ala Ile Asn Lys Ala Ile Asn Lys Glu Ile Thr Glu

65 70 75 80

Leu Met Leu Ala Val Leu His Gln Glu Val Pro Ser Gln Lys Leu His

85 90 95

Asn Leu Lys Ser Leu Asn Thr Glu Ser Leu Thr Lys Leu Phe Lys Pro

100 105 110

Lys Phe Gln Asn Met Ile Ser Tyr Pro Pro Ser Lys Gly Ala Glu His

115 120 125

Val Gln Phe Cys Leu Thr Asp Ile Ala Val Pro Ala Ile Arg Asp Leu

130 135 140

Asp Glu Ile Lys Pro Asp Trp Gly Ile Phe Phe Glu Lys Leu Lys Pro

145 150 155 160

Tyr Thr Asp Trp Ala Glu Ser Tyr Ile His Tyr Lys Gln Thr Thr Ile

165 170 175

Gln Lys Ser Ile Glu Gln Asn Lys Ile Gln Ser Pro Asp Ser Pro Arg

180 185 190

Lys Leu Val Leu Gln Lys Tyr Val Thr Ala Phe Leu Asn Gly Glu Pro

195 200 205

Leu Gly Leu Asp Leu Val Ala Lys Lys Tyr Lys Leu Ala Asp Leu Ala

210 215 220

Glu Ser Phe Lys Leu Val Asp Leu Asn Glu Asp Lys Ser Ala Asn Tyr

225 230 235 240

Lys Ile Lys Ala Cys Leu Gln Gln His Gln Arg Asn Ile Leu Asp Glu

245 250 255

Leu Lys Glu Asp Pro Glu Leu Asn Gln Tyr Gly Ile Glu Val Lys Lys

260 265 270

Tyr Ile Gln Arg Tyr Phe Pro Ile Lys Arg Ala Pro Asn Arg Ser Lys

275 280 285

His Ala Arg Ala Asp Phe Leu Lys Lys Glu Leu Ile Glu Ser Thr Val

290 295 300

Glu Gln Gln Phe Lys Asn Ala Val Tyr His Tyr Val Leu Glu Gln Gly

305 310 315 320

Lys Met Glu Ala Tyr Glu Leu Thr Asp Pro Lys Thr Lys Asp Leu Gln

325 330 335

Asp Ile Arg Ser Gly Glu Ala Phe Ser Phe Lys Phe Ile Asn Ala Cys

340 345 350

Ala Phe Ala Ser Asn Asn Leu Lys Met Ile Leu Asn Pro Glu Cys Glu

355 360 365

Lys Asp Ile Leu Gly Lys Gly Asn Phe Lys Lys Asn Leu Pro Asn Ser

370 375 380

Thr Thr Arg Ser Asp Val Val Lys Lys Met Ile Pro Phe Phe Ser Asp

385 390 395 400

Glu Leu Gln Asn Val Asn Phe Asp Glu Ala Ile Trp Ala Ile Arg Gly

405 410 415

Ser Ile Gln Gln Ile Arg Asn Glu Val Tyr His Cys Lys Lys His Ser

420 425 430

Trp Lys Ser Ile Leu Lys Ile Lys Gly Phe Glu Phe Glu Pro Asn Asn

435 440 445

Met Lys Tyr Ala Asp Ser Asp Met Gln Lys Leu Met Asp Lys Asp Ile

450 455 460

Ala Lys Ile Pro Glu Phe Ile Glu Glu Lys Leu Lys Ser Ser Gly Val

465 470 475 480

Val Arg Phe Tyr Arg His Asp Glu Leu Gln Ser Ile Trp Glu Met Lys

485 490 495

Gln Gly Phe Ser Leu Leu Thr Thr Asn Ala Pro Phe Val Pro Ser Phe

500 505 510

Lys Arg Val Tyr Ala Lys Gly His Asp Tyr Gln Thr Ser Lys Asn Arg

515 520 525

Tyr Tyr Asn Leu Asp Leu Thr Thr Phe Asp Ile Leu Glu Tyr Gly Glu

530 535 540

Glu Asp Phe Arg Ala Arg Tyr Phe Leu Thr Lys Leu Val Tyr Tyr Gln

545 550 555 560

Gln Phe Met Pro Trp Phe Thr Ala Asp Asn Asn Ala Phe Arg Asp Ala

565 570 575

Ala Asn Phe Val Leu Arg Leu Asn Lys Asn Arg Gln Gln Asp Ala Lys

580 585 590

Ala Phe Ile Asn Ile Arg Glu Val Glu Glu Gly Glu Met Pro Arg Asp

595 600 605

Tyr Met Gly Tyr Val Gln Gly Gln Ile Ala Ile His Glu Asp Ser Ile

610 615 620

Glu Asp Thr Pro Asn His Phe Glu Lys Phe Ile Ser Gln Val Phe Ile

625 630 635 640

Lys Gly Phe Asp Arg His Met Arg Ser Ala Asn Leu Lys Phe Ile Lys

645 650 655

Asn Pro Arg Asn Gln Gly Leu Glu Gln Ser Glu Ile Glu Glu Met Ser

660 665 670

Phe Asp Ile Lys Val Glu Pro Ser Phe Leu Lys Asn Lys Asp Asp Tyr

675 680 685

Ile Ala Phe Trp Ile Phe Cys Lys Met Leu Asp Ala Arg His Leu Ser

690 695 700

Glu Leu Arg Asn Glu Met Ile Lys Tyr Asp Gly His Leu Thr Gly Glu

705 710 715 720

Gln Glu Ile Ile Gly Leu Ala Leu Leu Gly Val Asp Ser Arg Glu Asn

725 730 735

Asp Trp Lys Gln Phe Phe Ser Ser Glu Arg Glu Tyr Glu Lys Ile Met

740 745 750

Lys Gly Tyr Val Val Glu Glu Leu Tyr Gln Arg Glu Pro Tyr Arg Gln

755 760 765

Ser Asp Gly Lys Thr Pro Ile Leu Phe Arg Gly Val Glu Gln Ala Arg

770 775 780

Lys Tyr Gly Thr Glu Thr Val Ile Gln Arg Leu Phe Asp Ala Asn Pro

785 790 795 800

Glu Phe Lys Val Ser Lys Cys Asn Leu Ala Glu Trp Glu Arg Gln Lys

805 810 815

Glu Thr Ile Glu Glu Thr Ile Lys Arg Arg Lys Glu Leu His Asn Glu

820 825 830

Trp Ala Lys Asn Pro Lys Lys Pro Gln Asn Asn Ala Phe Phe Lys Glu

835 840 845

Tyr Lys Glu Cys Cys Asp Ala Ile Asp Ala Tyr Asn Trp His Lys Asn

850 855 860

Lys Thr Thr Leu Ala Tyr Val Asn Glu Leu His His Leu Leu Ile Glu

865 870 875 880

Ile Leu Gly Arg Tyr Val Gly Tyr Val Ala Ile Ala Asp Arg Asp Phe

885 890 895

Gln Cys Met Ala Asn Gln Tyr Phe Lys His Ser Gly Ile Thr Glu Arg

900 905 910

Val Glu Tyr Trp Gly Asp Asn Arg Leu Lys Ser Ile Lys Lys Leu Asp

915 920 925

Thr Phe Leu Lys Lys Glu Gly Leu Phe Val Ser Glu Lys Asn Ala Arg

930 935 940

Asn His Ile Ala His Leu Asn Tyr Leu Ser Leu Lys Ser Glu Cys Thr

945 950 955 960

Leu Leu Tyr Leu Ser Glu Arg Leu Arg Glu Ile Phe Lys Tyr Asp Arg

965 970 975

Lys Leu Lys Asn Ala Val Ser Lys Ser Leu Ile Asp Ile Leu Asp Arg

980 985 990

His Gly Met Ser Val Val Phe Ala Asn Leu Lys Glu Asn Lys His Arg

995 1000 1005

Leu Val Ile Lys Ser Leu Glu Pro Lys Lys Leu Arg His Leu Gly

1010 1015 1020

Gly Lys Lys Ile Asp Gly Gly Tyr Ile Glu Thr Asn Gln Val Ser

1025 1030 1035

Glu Glu Tyr Cys Gly Ile Val Lys Arg Leu Leu Glu Met

1040 1045 1050

<210> 584

<211> 1182

<212> PRT

<213> Leptotrichia wadei

<400> 584

Met Tyr Met Lys Ile Thr Lys Ile Asp Gly Val Ser His Tyr Lys Lys

1 5 10 15

Gln Asp Lys Gly Ile Leu Lys Lys Lys Trp Lys Asp Leu Asp Glu Arg

20 25 30

Lys Gln Arg Glu Lys Ile Glu Ala Arg Tyr Asn Lys Gln Ile Glu Ser

35 40 45

Lys Ile Tyr Lys Glu Phe Phe Arg Leu Lys Asn Lys Lys Arg Ile Glu

50 55 60

Lys Glu Glu Asp Gln Asn Ile Lys Ser Leu Tyr Phe Phe Ile Lys Glu

65 70 75 80

Leu Tyr Leu Asn Glu Lys Asn Glu Glu Trp Glu Leu Lys Asn Ile Asn

85 90 95

Leu Glu Ile Leu Asp Asp Lys Glu Arg Val Ile Lys Gly Tyr Lys Phe

100 105 110

Lys Glu Asp Val Tyr Phe Phe Lys Glu Gly Tyr Lys Glu Tyr Tyr Leu

115 120 125

Arg Ile Leu Phe Asn Asn Leu Ile Glu Lys Val Gln Asn Glu Asn Arg

130 135 140

Glu Lys Val Arg Lys Asn Lys Glu Phe Leu Asp Leu Lys Glu Ile Phe

145 150 155 160

Lys Lys Tyr Lys Asn Arg Lys Ile Asp Leu Leu Leu Lys Ser Ile Asn

165 170 175

Asn Asn Lys Ile Asn Leu Glu Tyr Lys Lys Glu Asn Val Asn Glu Glu

180 185 190

Ile Tyr Gly Ile Asn Pro Thr Asn Asp Arg Glu Met Thr Phe Tyr Glu

195 200 205

Leu Leu Lys Glu Ile Ile Glu Lys Lys Asp Glu Gln Lys Ser Ile Leu

210 215 220

Glu Glu Lys Leu Asp Asn Phe Asp Ile Thr Asn Phe Leu Glu Asn Ile

225 230 235 240

Glu Lys Ile Phe Asn Glu Glu Thr Glu Ile Asn Ile Ile Lys Gly Lys

245 250 255

Val Leu Asn Glu Leu Arg Glu Tyr Ile Lys Glu Lys Glu Glu Asn Asn

260 265 270

Ser Asp Asn Lys Leu Lys Gln Ile Tyr Asn Leu Glu Leu Lys Lys Tyr

275 280 285

Ile Glu Asn Asn Phe Ser Tyr Lys Lys Gln Lys Ser Lys Ser Lys Asn

290 295 300

Gly Lys Asn Asp Tyr Leu Tyr Leu Asn Phe Leu Lys Lys Ile Met Phe

305 310 315 320

Ile Glu Glu Val Asp Glu Lys Lys Glu Ile Asn Lys Glu Lys Phe Lys

325 330 335

Asn Lys Ile Asn Ser Asn Phe Lys Asn Leu Phe Val Gln His Ile Leu

340 345 350

Asp Tyr Gly Lys Leu Leu Tyr Tyr Lys Glu Asn Asp Glu Tyr Ile Lys

355 360 365

Asn Thr Gly Gln Leu Glu Thr Lys Asp Leu Glu Tyr Ile Lys Thr Lys

370 375 380

Glu Thr Leu Ile Arg Lys Met Ala Val Leu Val Ser Phe Ala Ala Asn

385 390 395 400

Ser Tyr Tyr Asn Leu Phe Gly Arg Val Ser Gly Asp Ile Leu Gly Thr

405 410 415

Glu Val Val Lys Ser Ser Lys Thr Asn Val Ile Lys Val Gly Ser His

420 425 430

Ile Phe Lys Glu Lys Met Leu Asn Tyr Phe Phe Asp Phe Glu Ile Phe

435 440 445

Asp Ala Asn Lys Ile Val Glu Ile Leu Glu Ser Ile Ser Tyr Ser Ile

450 455 460

Tyr Asn Val Arg Asn Gly Val Gly His Phe Asn Lys Leu Ile Leu Gly

465 470 475 480

Lys Tyr Lys Lys Lys Asp Ile Asn Thr Asn Lys Arg Ile Glu Glu Asp

485 490 495

Leu Asn Asn Asn Glu Glu Ile Lys Gly Tyr Phe Ile Lys Lys Arg Gly

500 505 510

Glu Ile Glu Arg Lys Val Lys Glu Lys Phe Leu Ser Asn Asn Leu Gln

515 520 525

Tyr Tyr Tyr Ser Lys Glu Lys Ile Glu Asn Tyr Phe Glu Val Tyr Glu

530 535 540

Phe Glu Ile Leu Lys Arg Lys Ile Pro Phe Ala Pro Asn Phe Lys Arg

545 550 555 560

Ile Ile Lys Lys Gly Glu Asp Leu Phe Asn Asn Lys Asn Asn Lys Lys

565 570 575

Tyr Glu Tyr Phe Lys Asn Phe Asp Lys Asn Ser Ala Glu Glu Glu Lys Lys

580 585 590

Glu Phe Leu Lys Thr Arg Asn Phe Leu Leu Lys Glu Leu Tyr Tyr Asn

595 600 605

Asn Phe Tyr Lys Glu Phe Leu Ser Lys Lys Glu Glu Phe Glu Lys Ile

610 615 620

Val Leu Glu Val Lys Glu Glu Lys Lys Ser Arg Gly Asn Ile Asn Asn

625 630 635 640

Lys Lys Ser Gly Val Ser Phe Gln Ser Ile Asp Asp Tyr Asp Thr Lys

645 650 655

Ile Asn Ile Ser Asp Tyr Ile Ala Ser Ile His Lys Lys Glu Met Glu

660 665 670

Arg Val Glu Lys Tyr Asn Glu Glu Lys Gln Lys Asp Thr Ala Lys Tyr

675 680 685

Ile Arg Asp Phe Val Glu Glu Ile Phe Leu Thr Gly Phe Ile Asn Tyr

690 695 700

Leu Glu Lys Asp Lys Arg Leu His Phe Leu Lys Glu Glu Phe Ser Ile

705 710 715 720

Leu Cys Asn Asn Asn Asn Asn Val Val Asp Phe Asn Ile Asn Ile Asn

725 730 735

Glu Glu Lys Ile Lys Glu Phe Leu Lys Glu Asn Asp Ser Lys Thr Leu

740 745 750

Asn Leu Tyr Leu Phe Phe Asn Met Ile Asp Ser Lys Arg Ile Ser Glu

755 760 765

Phe Arg Asn Glu Leu Val Lys Tyr Lys Gln Phe Thr Lys Lys Arg Leu

770 775 780

Asp Glu Glu Lys Glu Phe Leu Gly Ile Lys Ile Glu Leu Tyr Glu Thr

785 790 795 800

Leu Ile Glu Phe Val Ile Leu Thr Arg Glu Lys Leu Asp Thr Lys Lys

805 810 815

Ser Glu Glu Ile Asp Ala Trp Leu Val Asp Lys Leu Tyr Val Lys Asp

820 825 830

Ser Asn Glu Tyr Lys Glu Tyr Glu Glu Ile Leu Lys Leu Phe Val Asp

835 840 845

Glu Lys Ile Leu Ser Ser Lys Glu Ala Pro Tyr Tyr Ala Thr Asp Asn

850 855 860

Lys Thr Pro Ile Leu Leu Ser Asn Phe Glu Lys Thr Arg Lys Tyr Gly

865 870 875 880

Thr Gln Ser Phe Leu Ser Glu Ile Gln Ser Asn Tyr Lys Tyr Ser Lys

885 890 895

Val Glu Lys Glu Asn Ile Glu Asp Tyr Asn Lys Lys Glu Glu Ile Glu

900 905 910

Gln Lys Lys Lys Ser Asn Ile Glu Lys Leu Gln Asp Leu Lys Val Glu

915 920 925

Leu His Lys Lys Trp Glu Gln Asn Lys Ile Thr Glu Lys Glu Ile Glu

930 935 940

Lys Tyr Asn Asn Thr Thr Arg Lys Ile Asn Glu Tyr Asn Tyr Leu Lys

945 950 955 960

Asn Lys Glu Glu Leu Gln Asn Val Tyr Leu Leu His Glu Met Leu Ser

965 970 975

Asp Leu Leu Ala Arg Asn Val Ala Phe Phe Asn Lys Trp Glu Arg Asp

980 985 990

Phe Lys Phe Ile Val Ile Ala Ile Lys Gln Phe Leu Arg Glu Asn Asp

995 1000 1005

Lys Glu Lys Val Asn Glu Phe Leu Asn Pro Pro Asp Asn Ser Lys

1010 1015 1020

Gly Lys Lys Val Tyr Phe Ser Val Ser Lys Tyr Lys Asn Thr Val

1025 1030 1035

Glu Asn Ile Asp Gly Ile His Lys Asn Phe Met Asn Leu Ile Phe

1040 1045 1050

Leu Asn Asn Lys Phe Met Asn Arg Lys Ile Asp Lys Met Asn Cys

1055 1060 1065

Ala Ile Trp Val Tyr Phe Arg Asn Tyr Ile Ala His Phe Leu His

1070 1075 1080

Leu His Thr Lys Asn Glu Lys Ile Ser Leu Ile Ser Gln Met Asn

1085 1090 1095

Leu Leu Ile Lys Leu Phe Ser Tyr Asp Lys Lys Val Gln Asn His

1100 1105 1110

Ile Leu Lys Ser Thr Lys Thr Leu Leu Glu Lys Tyr Asn Ile Gln

1115 1120 1125

Ile Asn Phe Glu Ile Ser Asn Asp Lys Asn Glu Val Phe Lys Tyr

1130 1135 1140

Lys Ile Lys Asn Arg Leu Tyr Ser Lys Lys Gly Lys Met Leu Gly

1145 1150 1155

Lys Asn Asn Lys Phe Glu Ile Leu Glu Asn Glu Phe Leu Glu Asn

1160 1165 1170

Val Lys Ala Met Leu Glu Tyr Ser Glu

1175 1180

<210> 585

<211> 1180

<212> PRT

<213> Leptotrichia wadei

<400> 585

Met Lys Ile Thr Lys Ile Asp Gly Val Ser His Tyr Lys Lys Gln Asp

1 5 10 15

Lys Gly Ile Leu Lys Lys Lys Trp Lys Asp Leu Asp Glu Arg Lys Gln

20 25 30

Arg Glu Lys Ile Glu Ala Arg Tyr Asn Lys Gln Ile Glu Ser Lys Ile

35 40 45

Tyr Lys Glu Phe Phe Arg Leu Lys Asn Lys Lys Arg Ile Glu Lys Glu

50 55 60

Glu Asp Gln Asn Ile Lys Ser Leu Tyr Phe Phe Ile Lys Glu Leu Tyr

65 70 75 80

Leu Asn Glu Lys Asn Glu Glu Trp Glu Leu Lys Asn Ile Asn Leu Glu

85 90 95

Ile Leu Asp Asp Lys Glu Arg Val Ile Lys Gly Tyr Lys Phe Lys Glu

100 105 110

Asp Val Tyr Phe Phe Lys Glu Gly Tyr Lys Glu Tyr Tyr Leu Arg Ile

115 120 125

Leu Phe Asn Asn Leu Ile Glu Lys Val Gln Asn Glu Asn Arg Glu Lys

130 135 140

Val Arg Lys Asn Lys Glu Phe Leu Asp Leu Lys Glu Ile Phe Lys Lys

145 150 155 160

Tyr Lys Asn Arg Lys Ile Asp Leu Leu Leu Lys Ser Ile Asn Asn Asn

165 170 175

Lys Ile Asn Leu Glu Tyr Lys Lys Glu Asn Val Asn Glu Glu Ile Tyr

180 185 190

Gly Ile Asn Pro Thr Asn Asp Arg Glu Met Thr Phe Tyr Glu Leu Leu

195 200 205

Lys Glu Ile Ile Glu Lys Lys Asp Glu Gln Lys Ser Ile Leu Glu Glu

210 215 220

Lys Leu Asp Asn Phe Asp Ile Thr Asn Phe Leu Glu Asn Ile Glu Lys

225 230 235 240

Ile Phe Asn Glu Glu Thr Glu Ile Asn Ile Ile Lys Gly Lys Val Leu

245 250 255

Asn Glu Leu Arg Glu Tyr Ile Lys Glu Lys Glu Glu Asn Asn Ser Asp

260 265 270

Asn Lys Leu Lys Gln Ile Tyr Asn Leu Glu Leu Lys Lys Tyr Ile Glu

275 280 285

Asn Asn Phe Ser Tyr Lys Lys Gln Lys Ser Lys Ser Lys Asn Gly Lys

290 295 300

Asn Asp Tyr Leu Tyr Leu Asn Phe Leu Lys Lys Ile Met Phe Ile Glu

305 310 315 320

Glu Val Asp Glu Lys Lys Glu Ile Asn Lys Glu Lys Phe Lys Asn Lys

325 330 335

Ile Asn Ser Asn Phe Lys Asn Leu Phe Val Gln His Ile Leu Asp Tyr

340 345 350

Gly Lys Leu Leu Tyr Tyr Lys Glu Asn Asp Glu Tyr Ile Lys Asn Thr

355 360 365

Gly Gln Leu Glu Thr Lys Asp Leu Glu Tyr Ile Lys Thr Lys Glu Thr

370 375 380

Leu Ile Arg Lys Met Ala Val Leu Val Ser Phe Ala Ala Asn Ser Tyr

385 390 395 400

Tyr Asn Leu Phe Gly Arg Val Ser Gly Asp Ile Leu Gly Thr Glu Val

405 410 415

Val Lys Ser Ser Lys Thr Asn Val Ile Lys Val Gly Ser His Ile Phe

420 425 430

Lys Glu Lys Met Leu Asn Tyr Phe Phe Asp Phe Glu Ile Phe Asp Ala

435 440 445

Asn Lys Ile Val Glu Ile Leu Glu Ser Ile Ser Tyr Ser Ile Tyr Asn

450 455 460

Val Arg Asn Gly Val Gly His Phe Asn Lys Leu Ile Leu Gly Lys Tyr

465 470 475 480

Lys Lys Lys Asp Ile Asn Thr Asn Lys Arg Ile Glu Glu Asp Leu Asn

485 490 495

Asn Asn Glu Glu Ile Lys Gly Tyr Phe Ile Lys Lys Arg Gly Glu Ile

500 505 510

Glu Arg Lys Val Lys Glu Lys Phe Leu Ser Asn Asn Leu Gln Tyr Tyr

515 520 525

Tyr Ser Lys Glu Lys Ile Glu Asn Tyr Phe Glu Val Tyr Glu Phe Glu

530 535 540

Ile Leu Lys Arg Lys Ile Pro Phe Ala Pro Asn Phe Lys Arg Ile Ile

545 550 555 560

Lys Lys Gly Glu Asp Leu Phe Asn Asn Lys Asn Asn Lys Lys Tyr Glu

565 570 575

Tyr Phe Lys Asn Phe Asp Lys Asn Ser Ala Glu Glu Lys Lys Glu Phe

580 585 590

Leu Lys Thr Arg Asn Phe Leu Leu Lys Glu Leu Tyr Tyr Asn Asn Phe

595 600 605

Tyr Lys Glu Phe Leu Ser Lys Lys Glu Glu Phe Glu Lys Ile Val Leu

610 615 620

Glu Val Lys Glu Glu Lys Lys Ser Arg Gly Asn Ile Asn Asn Lys Lys

625 630 635 640

Ser Gly Val Ser Phe Gln Ser Ile Asp Asp Tyr Asp Thr Lys Ile Asn

645 650 655

Ile Ser Asp Tyr Ile Ala Ser Ile His Lys Lys Glu Met Glu Arg Val

660 665 670

Glu Lys Tyr Asn Glu Glu Lys Gln Lys Asp Thr Ala Lys Tyr Ile Arg

675 680 685

Asp Phe Val Glu Glu Ile Phe Leu Thr Gly Phe Ile Asn Tyr Leu Glu

690 695 700

Lys Asp Lys Arg Leu His Phe Leu Lys Glu Glu Phe Ser Ile Leu Cys

705 710 715 720

Asn Asn Asn Asn Asn Val Val Asp Phe Asn Ile Asn Ile Asn Glu Glu

725 730 735

Lys Ile Lys Glu Phe Leu Lys Glu Asn Asp Ser Lys Thr Leu Asn Leu

740 745 750

Tyr Leu Phe Phe Asn Met Ile Asp Ser Lys Arg Ile Ser Glu Phe Arg

755 760 765

Asn Glu Leu Val Lys Tyr Lys Gln Phe Thr Lys Lys Arg Leu Asp Glu

770 775 780

Glu Lys Glu Phe Leu Gly Ile Lys Ile Glu Leu Tyr Glu Thr Leu Ile

785 790 795 800

Glu Phe Val Ile Leu Thr Arg Glu Lys Leu Asp Thr Lys Lys Ser Glu

805 810 815

Glu Ile Asp Ala Trp Leu Val Asp Lys Leu Tyr Val Lys Asp Ser Asn

820 825 830

Glu Tyr Lys Glu Tyr Glu Glu Ile Leu Lys Leu Phe Val Asp Glu Lys

835 840 845

Ile Leu Ser Ser Lys Glu Ala Pro Tyr Tyr Ala Thr Asp Asn Lys Thr

850 855 860

Pro Ile Leu Leu Ser Asn Phe Glu Lys Thr Arg Lys Tyr Gly Thr Gln

865 870 875 880

Ser Phe Leu Ser Glu Ile Gln Ser Asn Tyr Lys Tyr Ser Lys Val Glu

885 890 895

Lys Glu Asn Ile Glu Asp Tyr Asn Lys Lys Glu Glu Ile Glu Gln Lys

900 905 910

Lys Lys Ser Asn Ile Glu Lys Leu Gln Asp Leu Lys Val Glu Leu His

915 920 925

Lys Lys Trp Glu Gln Asn Lys Ile Thr Glu Lys Glu Ile Glu Lys Tyr

930 935 940

Asn Asn Thr Thr Arg Lys Ile Asn Glu Tyr Asn Tyr Leu Lys Asn Lys

945 950 955 960

Glu Glu Leu Gln Asn Val Tyr Leu Leu His Glu Met Leu Ser Asp Leu

965 970 975

Leu Ala Arg Asn Val Ala Phe Phe Asn Lys Trp Glu Arg Asp Phe Lys

980 985 990

Phe Ile Val Ile Ala Ile Lys Gln Phe Leu Arg Glu Asn Asp Lys Glu

995 1000 1005

Lys Val Asn Glu Phe Leu Asn Pro Pro Asp Asn Ser Lys Gly Lys

1010 1015 1020

Lys Val Tyr Phe Ser Val Ser Lys Tyr Lys Asn Thr Val Glu Asn

1025 1030 1035

Ile Asp Gly Ile His Lys Asn Phe Met Asn Leu Ile Phe Leu Asn

1040 1045 1050

Asn Lys Phe Met Asn Arg Lys Ile Asp Lys Met Asn Cys Ala Ile

1055 1060 1065

Trp Val Tyr Phe Arg Asn Tyr Ile Ala His Phe Leu His Leu His

1070 1075 1080

Thr Lys Asn Glu Lys Ile Ser Leu Ile Ser Gln Met Asn Leu Leu

1085 1090 1095

Ile Lys Leu Phe Ser Tyr Asp Lys Lys Val Gln Asn His Ile Leu

1100 1105 1110

Lys Ser Thr Lys Thr Leu Leu Glu Lys Tyr Asn Ile Gln Ile Asn

1115 1120 1125

Phe Glu Ile Ser Asn Asp Lys Asn Glu Val Phe Lys Tyr Lys Ile

1130 1135 1140

Lys Asn Arg Leu Tyr Ser Lys Lys Gly Lys Met Leu Gly Lys Asn

1145 1150 1155

Asn Lys Phe Glu Ile Leu Glu Asn Glu Phe Leu Glu Asn Val Lys

1160 1165 1170

Ala Met Leu Glu Tyr Ser Glu

1175 1180

<210> 586

<211> 1197

<212> PRT

<213> Leptotrichia wadei

<400> 586

Met Lys Val Thr Lys Ile Asp Gly Leu Ser His Lys Lys Phe Glu Asp

1 5 10 15

Glu Gly Lys Leu Val Lys Phe Arg Asn Asn Lys Asn Ile Asn Glu Ile

20 25 30

Lys Glu Arg Leu Lys Lys Leu Lys Glu Leu Lys Leu Asp Asn Tyr Ile

35 40 45

Lys Asn Pro Glu Asn Val Lys Asn Lys Asp Lys Asp Ala Glu Lys Glu

50 55 60

Thr Lys Ile Arg Arg Thr Asn Leu Lys Lys Tyr Phe Ser Glu Ile Ile

65 70 75 80

Leu Arg Lys Glu Asp Glu Lys Tyr Ile Leu Lys Lys Thr Lys Lys Phe

85 90 95

Lys Asp Ile Asn Gln Glu Ile Asp Tyr Tyr Asp Val Lys Ser Lys Lys

100 105 110

Asn Gln Gln Glu Ile Phe Asp Val Leu Lys Glu Ile Leu Glu Leu Lys

115 120 125

Ile Lys Glu Thr Glu Lys Glu Glu Ile Ile Thr Phe Asp Ser Glu Lys

130 135 140

Leu Lys Lys Val Phe Gly Glu Asp Phe Val Lys Lys Glu Ala Lys Ile

145 150 155 160

Lys Ala Ile Glu Lys Ser Leu Lys Ile Asn Lys Ala Asn Tyr Lys Lys

165 170 175

Asp Ser Ile Lys Ile Gly Asp Asp Lys Tyr Ser Asn Val Lys Gly Glu

180 185 190

Asn Lys Arg Ser Arg Ile Tyr Glu Tyr Tyr Lys Lys Ser Glu Asn Leu

195 200 205

Lys Lys Phe Glu Glu Asn Ile Arg Glu Ala Phe Glu Lys Leu Tyr Thr

210 215 220

Glu Glu Asn Ile Lys Glu Leu Tyr Ser Lys Ile Glu Glu Ile Leu Lys

225 230 235 240

Lys Thr His Leu Lys Ser Ile Val Arg Glu Phe Tyr Gln Asn Glu Ile

245 250 255

Ile Gly Glu Ser Glu Phe Ser Lys Lys Asn Gly Asp Gly Ile Ser Ile

260 265 270

Leu Tyr Asn Gln Ile Lys Asp Ser Ile Lys Lys Glu Glu Asn Phe Ile

275 280 285

Glu Phe Ile Glu Asn Thr Gly Asn Leu Glu Leu Lys Glu Leu Thr Lys

290 295 300

Ser Gln Ile Phe Tyr Lys Tyr Phe Leu Glu Asn Glu Glu Glu Leu Asn Asp

305 310 315 320

Glu Asn Ile Lys Phe Ala Phe Cys Tyr Phe Val Glu Ile Glu Val Asn

325 330 335

Asn Leu Leu Lys Glu Asn Val Tyr Lys Ile Lys Arg Phe Asn Glu Ser

340 345 350

Asn Lys Lys Arg Ile Glu Asn Ile Phe Glu Tyr Gly Lys Leu Lys Lys

355 360 365

Leu Ile Val Tyr Lys Leu Glu Asn Lys Leu Asn Asn Tyr Val Arg Asn

370 375 380

Cys Gly Lys Tyr Asn Tyr His Met Glu Asn Gly Asp Ile Ala Thr Ser

385 390 395 400

Asp Ile Asn Met Arg Asn Arg Gln Thr Glu Ala Phe Leu Arg Ser Ile

405 410 415

Ile Gly Val Ser Ser Phe Gly Tyr Phe Ser Leu Arg Asn Ile Leu Gly

420 425 430

Val Asn Asp Asp Asp Phe Tyr Glu Thr Glu Glu Asp Leu Thr Lys Lys

435 440 445

Glu Arg Arg Asn Leu Glu Lys Ala Lys Glu Asp Ile Thr Ile Lys Asn

450 455 460

Thr Phe Asp Glu Val Val Val Lys Ser Phe Gln Lys Lys Gly Ile Tyr

465 470 475 480

Asn Ile Lys Glu Asn Leu Lys Met Phe Tyr Gly Asp Ser Phe Asp Asn

485 490 495

Ala Asp Lys Asp Glu Leu Lys Gln Phe Phe Val Asn Met Leu Asn Ala

500 505 510

Ile Thr Ser Ile Arg His Arg Val Val His Tyr Asn Met Asn Thr Asn

515 520 525

Ser Glu Asn Ile Phe Asn Phe Ser Gly Ile Glu Val Ser Lys Leu Leu

530 535 540

Lys Ser Ile Phe Glu Lys Glu Thr Asp Lys Arg Glu Leu Lys Leu Lys

545 550 555 560

Ile Phe Arg Gln Leu Asn Ser Ala Gly Val Phe Asp Tyr Trp Glu Asn

565 570 575

Arg Lys Ile Asp Lys Tyr Leu Glu Asn Ile Glu Phe Lys Phe Val Asn

580 585 590

Lys Asn Ile Pro Phe Val Pro Ser Phe Thr Lys Leu Tyr Asn Arg Ile

595 600 605

Asp Asn Leu Lys Gly Asn Asn Ala Leu Asn Leu Gly Tyr Ile Asn Ile

610 615 620

Pro Lys Arg Lys Glu Ala Arg Asp Ser Gln Ile Tyr Leu Leu Lys Asn

625 630 635 640

Ile Tyr Tyr Gly Glu Phe Val Glu Lys Phe Val Asn Asn Asn Asp Asn

645 650 655

Phe Glu Lys Ile Phe Arg Glu Ile Ile Glu Ile Asn Lys Lys Asp Gly

660 665 670

Thr Asn Thr Lys Thr Lys Phe Tyr Lys Leu Glu Lys Phe Glu Thr Leu

675 680 685

Lys Ala Asn Ala Pro Ile Glu Tyr Leu Glu Lys Leu Gln Ser Leu His

690 695 700

Gln Ile Asn Tyr Asn Arg Glu Lys Val Glu Glu Asp Lys Asp Ile Tyr

705 710 715 720

Val Asp Phe Val Gln Lys Ile Phe Leu Lys Gly Phe Ile Asn Tyr Leu

725 730 735

Gln Gly Ser Asp Leu Leu Lys Ser Leu Asn Leu Leu Asn Leu Lys Lys

740 745 750

Asp Glu Ala Ile Ala Asn Lys Lys Ser Phe Tyr Asp Glu Lys Leu Lys

755 760 765

Leu Trp Gln Asn Asn Gly Ser Asn Leu Ser Lys Met Pro Glu Glu Ile

770 775 780

Tyr Asp Tyr Ile Lys Lys Ile Lys Ile Asn Lys Ile Asn Tyr Ser Asp

785 790 795 800

Arg Met Ser Ile Phe Tyr Leu Leu Leu Lys Leu Ile Asp His Lys Glu

805 810 815

Leu Thr Asn Leu Arg Gly Asn Leu Glu Lys Tyr Val Ser Met Asn Lys

820 825 830

Asn Lys Ile Tyr Ser Glu Glu Leu Asn Ile Val Asn Leu Val Ser Leu

835 840 845

Asp Asn Asn Lys Val Arg Ala Asn Phe Asn Leu Lys Pro Glu Asp Ile

850 855 860

Gly Lys Phe Leu Lys Thr Glu Thr Ser Ile Arg Asn Ile Asn Gln Leu

865 870 875 880

Asn Asn Phe Ser Glu Ile Phe Ala Asp Gly Glu Asn Val Ile Lys His

885 890 895

Arg Ser Phe Tyr Asn Ile Lys Lys Tyr Gly Ile Leu Asp Leu Leu Glu

900 905 910

Lys Ile Val Asp Lys Ala Asp Leu Lys Ile Thr Lys Glu Glu Ile Lys

915 920 925

Lys Tyr Glu Asn Leu Gln Asn Glu Leu Lys Arg Asn Asp Phe Tyr Lys

930 935 940

Ile Gln Glu Arg Ile His Arg Asn Tyr Asn Gln Lys Pro Phe Leu Ile

945 950 955 960

Lys Asn Asn Glu Lys Asp Phe Asn Asp Tyr Lys Lys Ala Ile Glu Asn

965 970 975

Ile Gln Asn Tyr Thr Gln Leu Lys Asn Lys Ile Glu Phe Asn Asp Leu

980 985 990

Asn Leu Leu Gln Ser Leu Leu Phe Arg Ile Leu His Arg Leu Ala Gly

995 1000 1005

Tyr Thr Ser Leu Trp Glu Arg Asp Leu Gln Phe Lys Leu Lys Gly

1010 1015 1020

Glu Tyr Pro Glu Asn Lys Tyr Ile Asp Glu Ile Phe Asn Phe Asp

1025 1030 1035

Asn Ser Lys Asn Lys Ile Tyr Asn Glu Lys Asn Glu Arg Gly Gly

1040 1045 1050

Ser Val Val Ser Lys Tyr Gly Tyr Phe Leu Val Glu Lys Asp Gly

1055 1060 1065

Glu Ile Gln Arg Lys Asn Ala Arg Asp Lys Lys Lys Asn Lys Ile

1070 1075 1080

Ile Lys Lys Glu Gly Leu Glu Ile Arg Asn Tyr Ile Ala His Phe

1085 1090 1095

Asn Tyr Ile Pro Asp Ala Thr Lys Ser Ile Leu Glu Ile Leu Glu

1100 1105 1110

Glu Leu Arg Asn Leu Leu Lys Tyr Asp Arg Lys Leu Lys Asn Ala

1115 1120 1125

Val Met Lys Ser Ile Lys Asp Ile Phe Lys Glu Tyr Gly Leu Ile

1130 1135 1140

Ile Glu Phe Lys Ile Ser His Val Asn Asn Ser Glu Lys Ile Glu

1145 1150 1155

Val Leu Asn Val Asp Ser Glu Lys Ile Lys His Leu Lys Asn Asn

1160 1165 1170

Gly Leu Val Thr Thr Arg Asn Ser Glu Asp Leu Cys Glu Leu Ile

1175 1180 1185

Lys Met Met Leu Glu Tyr Lys Lys Ser

1190 1195

<210> 587

<211> 1152

<212> PRT

<213> Leptotrichia wadei

<400> 587

Met Lys Val Thr Lys Val Asp Gly Ile Ser His Lys Lys Tyr Ile Glu

1 5 10 15

Glu Gly Lys Leu Val Lys Ser Thr Ser Glu Glu Asn Arg Thr Ser Glu

20 25 30

Arg Leu Ser Glu Leu Leu Ser Ile Arg Leu Asp Ile Tyr Ile Lys Asn

35 40 45

Pro Asp Asn Ala Ser Glu Glu Glu Asn Arg Ile Arg Arg Glu Asn Leu

50 55 60

Lys Lys Phe Phe Ser Asn Lys Val Leu His Leu Lys Asp Ser Val Leu

65 70 75 80

Tyr Leu Lys Asn Arg Lys Glu Lys Asn Ala Val Gln Asp Lys Asn Tyr

85 90 95

Ser Glu Glu Asp Ile Ser Glu Tyr Asp Leu Lys Asn Lys Asn Ser Phe

100 105 110

Ser Val Leu Lys Lys Ile Leu Leu Asn Glu Asp Val Asn Ser Glu Glu

115 120 125

Leu Glu Ile Phe Arg Lys Asp Val Glu Ala Lys Leu Asn Lys Ile Asn

130 135 140

Ser Leu Lys Tyr Ser Phe Glu Glu Asn Lys Ala Asn Tyr Gln Lys Ile

145 150 155 160

Asn Glu Asn Asn Val Glu Lys Val Gly Gly Lys Ser Lys Arg Asn Ile

165 170 175

Ile Tyr Asp Tyr Tyr Arg Glu Ser Ala Lys Arg Asn Asp Tyr Ile Asn

180 185 190

Asn Val Gln Glu Ala Phe Asp Lys Leu Tyr Lys Lys Glu Asp Ile Glu

195 200 205

Lys Leu Phe Phe Leu Ile Glu Asn Ser Lys Lys His Glu Lys Tyr Lys

210 215 220

Ile Arg Glu Tyr Tyr His Lys Ile Ile Gly Arg Lys Asn Asp Lys Glu

225 230 235 240

Asn Phe Ala Lys Ile Ile Tyr Glu Glu Ile Gln Asn Val Asn Asn Ile

245 250 255

Lys Glu Leu Ile Glu Lys Ile Pro Asp Met Ser Glu Leu Lys Lys Ser

260 265 270

Gln Val Phe Tyr Lys Tyr Tyr Leu Asp Lys Glu Glu Leu Asn Asp Lys

275 280 285

Asn Ile Lys Tyr Ala Phe Cys His Phe Val Glu Ile Glu Met Ser Gln

290 295 300

Leu Leu Lys Asn Tyr Val Tyr Lys Arg Leu Ser Asn Ile Ser Asn Asp

305 310 315 320

Lys Ile Lys Arg Ile Phe Glu Tyr Gln Asn Leu Lys Lys Leu Ile Glu

325 330 335

Asn Lys Leu Leu Asn Lys Leu Asp Thr Tyr Val Arg Asn Cys Gly Lys

340 345 350

Tyr Asn Tyr Tyr Leu Gln Val Gly Glu Ile Ala Thr Ser Asp Phe Ile

355 360 365

Ala Arg Asn Arg Gln Asn Glu Ala Phe Leu Arg Asn Ile Ile Gly Val

370 375 380

Ser Ser Val Ala Tyr Phe Ser Leu Arg Asn Ile Leu Glu Thr Glu Asn

385 390 395 400

Glu Asn Asp Ile Thr Gly Arg Met Arg Gly Lys Thr Val Lys Asn Asn

405 410 415

Lys Gly Glu Glu Lys Tyr Val Ser Gly Glu Val Asp Lys Ile Tyr Asn

420 425 430

Glu Asn Lys Gln Asn Glu Val Lys Glu Asn Leu Lys Met Phe Tyr Ser

435 440 445

Tyr Asp Phe Asn Met Asp Asn Lys Asn Glu Ile Glu Asp Phe Phe Ala

450 455 460

Asn Ile Asp Glu Ala Ile Ser Ser Ile Arg His Gly Ile Val His Phe

465 470 475 480

Asn Leu Glu Leu Glu Gly Lys Asp Ile Phe Ala Phe Lys Asn Ile Ala

485 490 495

Pro Ser Glu Ile Ser Lys Lys Met Phe Gln Asn Glu Ile Asn Glu Lys

500 505 510

Lys Leu Lys Leu Lys Ile Phe Lys Gln Leu Asn Ser Ala Asn Val Phe

515 520 525

Asn Tyr Tyr Glu Lys Asp Val Ile Ile Lys Tyr Leu Lys Asn Thr Lys

530 535 540

Phe Asn Phe Val Asn Lys Asn Ile Pro Phe Val Pro Ser Phe Thr Lys

545 550 555 560

Leu Tyr Asn Lys Ile Glu Asp Leu Arg Asn Thr Leu Lys Phe Phe Trp

565 570 575

Ser Val Pro Lys Asp Lys Glu Glu Lys Asp Ala Gln Ile Tyr Leu Leu

580 585 590

Lys Asn Ile Tyr Tyr Gly Glu Phe Leu Asn Lys Phe Val Lys Asn Ser

595 600 605

Lys Val Phe Phe Lys Ile Thr Asn Glu Val Ile Lys Ile Asn Lys Gln

610 615 620

Arg Asn Gln Lys Thr Gly His Tyr Lys Tyr Gln Lys Phe Glu Asn Ile

625 630 635 640

Glu Lys Thr Val Pro Val Glu Tyr Leu Ala Ile Ile Gln Ser Arg Glu

645 650 655

Met Ile Asn Asn Gln Asp Lys Glu Glu Lys Asn Thr Tyr Ile Asp Phe

660 665 670

Ile Gln Gln Ile Phe Leu Lys Gly Phe Ile Asp Tyr Leu Asn Lys Asn

675 680 685

Asn Leu Lys Tyr Ile Glu Ser Asn Asn Asn Asn Asp Asn Asn Asp Ile

690 695 700

Phe Ser Lys Ile Lys Ile Lys Lys Asp Asn Lys Glu Lys Tyr Asp Lys

705 710 715 720

Ile Leu Lys Asn Tyr Glu Lys His Asn Arg Asn Lys Glu Ile Pro His

725 730 735

Glu Ile Asn Glu Phe Val Arg Glu Ile Lys Leu Gly Lys Ile Leu Lys

740 745 750

Tyr Thr Glu Asn Leu Asn Met Phe Tyr Leu Ile Leu Lys Leu Leu Asn

755 760 765

His Lys Glu Leu Thr Asn Leu Lys Gly Ser Leu Glu Lys Tyr Gln Ser

770 775 780

Ala Asn Lys Glu Glu Thr Phe Ser Asp Glu Leu Glu Leu Ile Asn Leu

785 790 795 800

Leu Asn Leu Asp Asn Asn Arg Val Thr Glu Asp Phe Glu Leu Glu Ala

805 810 815

Asn Glu Ile Gly Lys Phe Leu Asp Phe Asn Glu Asn Lys Ile Lys Asp

820 825 830

Arg Lys Glu Leu Lys Lys Phe Asp Thr Asn Lys Ile Tyr Phe Asp Gly

835 840 845

Glu Asn Ile Ile Lys His Arg Ala Phe Tyr Asn Ile Lys Lys Tyr Gly

850 855 860

Met Leu Asn Leu Leu Glu Lys Ile Ala Asp Lys Ala Lys Tyr Lys Ile

865 870 875 880

Ser Leu Lys Glu Leu Lys Glu Tyr Ser Asn Lys Lys Asn Glu Ile Glu

885 890 895

Lys Asn Tyr Thr Met Gln Gln Asn Leu His Arg Lys Tyr Ala Arg Pro

900 905 910

Lys Lys Asp Glu Lys Phe Asn Asp Glu Asp Tyr Lys Glu Tyr Glu Lys

915 920 925

Ala Ile Gly Asn Ile Gln Lys Tyr Thr His Leu Lys Asn Lys Val Glu

930 935 940

Phe Asn Glu Leu Asn Leu Leu Gln Gly Leu Leu Leu Lys Ile Leu His

945 950 955 960

Arg Leu Val Gly Tyr Thr Ser Ile Trp Glu Arg Asp Leu Arg Phe Arg

965 970 975

Leu Lys Gly Glu Phe Pro Glu Asn His Tyr Ile Glu Glu Ile Phe Asn

980 985 990

Phe Asp Asn Ser Lys Asn Val Lys Tyr Lys Ser Gly Gln Ile Val Glu

995 1000 1005

Lys Tyr Ile Asn Phe Tyr Lys Glu Leu Tyr Lys Asp Asn Val Glu

1010 1015 1020

Lys Arg Ser Ile Tyr Ser Asp Lys Lys Val Lys Lys Leu Lys Gln

1025 1030 1035

Glu Lys Lys Asp Leu Tyr Ile Arg Asn Tyr Ile Ala His Phe Asn

1040 1045 1050

Tyr Ile Pro His Ala Glu Ile Ser Leu Leu Glu Val Leu Glu Asn

1055 1060 1065

Leu Arg Lys Leu Leu Ser Tyr Asp Arg Lys Leu Lys Asn Ala Ile

1070 1075 1080

Met Lys Ser Ile Val Asp Ile Leu Lys Glu Tyr Gly Phe Val Ala

1085 1090 1095

Thr Phe Lys Ile Gly Ala Asp Lys Lys Ile Glu Ile Gln Thr Leu

1100 1105 1110

Glu Ser Glu Lys Ile Val His Leu Lys Asn Leu Lys Lys Lys Lys

1115 1120 1125

Leu Met Thr Asp Arg Asn Ser Glu Glu Leu Cys Glu Leu Val Lys

1130 1135 1140

Val Met Phe Glu Tyr Lys Ala Leu Glu

1145 1150

<210> 588

<211> 1159

<212> PRT

<213> Leptotrichia buccalis

<400> 588

Met Lys Val Thr Lys Val Gly Gly Ile Ser His Lys Lys Tyr Thr Ser

1 5 10 15

Glu Gly Arg Leu Val Lys Ser Glu Ser Glu Glu Asn Arg Thr Asp Glu

20 25 30

Arg Leu Ser Ala Leu Leu Asn Met Arg Leu Asp Met Tyr Ile Lys Asn

35 40 45

Pro Ser Ser Thr Glu Thr Lys Glu Asn Gln Lys Arg Ile Gly Lys Leu

50 55 60

Lys Lys Phe Phe Ser Asn Lys Met Val Tyr Leu Lys Asp Asn Thr Leu

65 70 75 80

Ser Leu Lys Asn Gly Lys Lys Glu Asn Ile Asp Arg Glu Tyr Ser Glu

85 90 95

Thr Asp Ile Leu Glu Ser Asp Val Arg Asp Lys Lys Asn Phe Ala Val

100 105 110

Leu Lys Lys Ile Tyr Leu Asn Glu Asn Val Asn Ser Glu Glu Leu Glu

115 120 125

Val Phe Arg Asn Asp Ile Lys Lys Lys Leu Asn Lys Ile Asn Ser Leu

130 135 140

Lys Tyr Ser Phe Glu Lys Asn Lys Ala Asn Tyr Gln Lys Ile Asn Glu

145 150 155 160

Asn Asn Ile Glu Lys Val Glu Gly Lys Ser Lys Arg Asn Ile Ile Tyr

165 170 175

Asp Tyr Tyr Arg Glu Ser Ala Lys Arg Asp Ala Tyr Val Ser Asn Val

180 185 190

Lys Glu Ala Phe Asp Lys Leu Tyr Lys Glu Glu Asp Ile Ala Lys Leu

195 200 205

Val Leu Glu Ile Glu Asn Leu Thr Lys Leu Glu Lys Tyr Lys Ile Arg

210 215 220

Glu Phe Tyr His Glu Ile Ile Gly Arg Lys Asn Asp Lys Glu Asn Phe

225 230 235 240

Ala Lys Ile Ile Tyr Glu Glu Ile Gln Asn Val Asn Asn Met Lys Glu

245 250 255

Leu Ile Glu Lys Val Pro Asp Met Ser Glu Leu Lys Lys Ser Gln Val

260 265 270

Phe Tyr Lys Tyr Tyr Leu Asp Lys Glu Glu Leu Asn Asp Lys Asn Ile

275 280 285

Lys Tyr Ala Phe Cys His Phe Val Glu Ile Glu Met Ser Gln Leu Leu

290 295 300

Lys Asn Tyr Val Tyr Lys Arg Leu Ser Asn Ile Ser Asn Asp Lys Ile

305 310 315 320

Lys Arg Ile Phe Glu Tyr Gln Asn Leu Lys Lys Leu Ile Glu Asn Lys

325 330 335

Leu Leu Asn Lys Leu Asp Thr Tyr Val Arg Asn Cys Gly Lys Tyr Asn

340 345 350

Tyr Tyr Leu Gln Asp Gly Glu Ile Ala Thr Ser Asp Phe Ile Ala Arg

355 360 365

Asn Arg Gln Asn Glu Ala Phe Leu Arg Asn Ile Ile Gly Val Ser Ser

370 375 380

Val Ala Tyr Phe Ser Leu Arg Asn Ile Leu Glu Thr Glu Asn Glu Asn

385 390 395 400

Asp Ile Thr Gly Arg Met Arg Gly Lys Thr Val Lys Asn Asn Lys Gly

405 410 415

Glu Glu Lys Tyr Val Ser Gly Glu Val Asp Lys Ile Tyr Asn Glu Asn

420 425 430

Lys Lys Asn Glu Val Lys Glu Asn Leu Lys Met Phe Tyr Ser Tyr Asp

435 440 445

Phe Asn Met Asp Asn Lys Asn Glu Ile Glu Asp Phe Phe Ala Asn Ile

450 455 460

Asp Glu Ala Ile Ser Ser Ile Arg His Gly Ile Val His Phe Asn Leu

465 470 475 480

Glu Leu Glu Gly Lys Asp Ile Phe Ala Phe Lys Asn Ile Ala Pro Ser

485 490 495

Glu Ile Ser Lys Lys Met Phe Gln Asn Glu Ile Asn Glu Lys Lys Leu

500 505 510

Lys Leu Lys Ile Phe Arg Gln Leu Asn Ser Ala Asn Val Phe Arg Tyr

515 520 525

Leu Glu Lys Tyr Lys Ile Leu Asn Tyr Leu Lys Arg Thr Arg Phe Glu

530 535 540

Phe Val Asn Lys Asn Ile Pro Phe Val Pro Ser Phe Thr Lys Leu Tyr

545 550 555 560

Ser Arg Ile Asp Asp Leu Lys Asn Ser Leu Gly Ile Tyr Trp Lys Thr

565 570 575

Pro Lys Thr Asn Asp Asp Asn Lys Thr Lys Glu Ile Ile Asp Ala Gln

580 585 590

Ile Tyr Leu Leu Lys Asn Ile Tyr Tyr Gly Glu Phe Leu Asn Tyr Phe

595 600 605

Met Ser Asn Asn Gly Asn Phe Phe Glu Ile Ser Lys Glu Ile Ile Glu

610 615 620

Leu Asn Lys Asn Asp Lys Arg Asn Leu Lys Thr Gly Phe Tyr Lys Leu

625 630 635 640

Gln Lys Phe Glu Asp Ile Gln Glu Lys Ile Pro Lys Glu Tyr Leu Ala

645 650 655

Asn Ile Gln Ser Leu Tyr Met Ile Asn Ala Gly Asn Gln Asp Glu Glu

660 665 670

Glu Lys Asp Thr Tyr Ile Asp Phe Ile Gln Lys Ile Phe Leu Lys Gly

675 680 685

Phe Met Thr Tyr Leu Ala Asn Asn Gly Arg Leu Ser Leu Ile Tyr Ile

690 695 700

Gly Ser Asp Glu Glu Thr Asn Thr Ser Leu Ala Glu Lys Lys Gln Glu

705 710 715 720

Phe Asp Lys Phe Leu Lys Lys Tyr Glu Gln Asn Asn Asn Ile Lys Ile

725 730 735

Pro Tyr Glu Ile Asn Glu Phe Leu Arg Glu Ile Lys Leu Gly Asn Ile

740 745 750

Leu Lys Tyr Thr Glu Arg Leu Asn Met Phe Tyr Leu Ile Leu Lys Leu

755 760 765

Leu Asn His Lys Glu Leu Thr Asn Leu Lys Gly Ser Leu Glu Lys Tyr

770 775 780

Gln Ser Ala Asn Lys Glu Glu Ala Phe Ser Asp Gln Leu Glu Leu Ile

785 790 795 800

Asn Leu Leu Asn Leu Asp Asn Asn Arg Val Thr Glu Asp Phe Glu Leu

805 810 815

Glu Ala Asp Glu Ile Gly Lys Phe Leu Asp Phe Asn Gly Asn Lys Val

820 825 830

Lys Asp Asn Lys Glu Leu Lys Lys Phe Asp Thr Asn Lys Ile Tyr Phe

835 840 845

Asp Gly Glu Asn Ile Ile Lys His Arg Ala Phe Tyr Asn Ile Lys Lys

850 855 860

Tyr Gly Met Leu Asn Leu Leu Glu Lys Ile Ala Asp Lys Ala Gly Tyr

865 870 875 880

Lys Ile Ser Ile Glu Glu Leu Lys Lys Tyr Ser Asn Lys Lys Asn Glu

885 890 895

Ile Glu Lys Asn His Lys Met Gln Glu Asn Leu His Arg Lys Tyr Ala

900 905 910

Arg Pro Arg Lys Asp Glu Lys Phe Thr Asp Glu Asp Tyr Glu Ser Tyr

915 920 925

Lys Gln Ala Ile Glu Asn Ile Glu Glu Tyr Thr His Leu Lys Asn Lys

930 935 940

Val Glu Phe Asn Glu Leu Asn Leu Leu Gln Gly Leu Leu Leu Arg Ile

945 950 955 960

Leu His Arg Leu Val Gly Tyr Thr Ser Ile Trp Glu Arg Asp Leu Arg

965 970 975

Phe Arg Leu Lys Gly Glu Phe Pro Glu Asn Gln Tyr Ile Glu Glu Ile

980 985 990

Phe Asn Phe Glu Asn Lys Lys Asn Val Lys Tyr Lys Gly Gly Gln Ile

995 1000 1005

Val Glu Lys Tyr Ile Lys Phe Tyr Lys Glu Leu His Gln Asn Asp

1010 1015 1020

Glu Val Lys Ile Asn Lys Tyr Ser Ser Ala Asn Ile Lys Val Leu

1025 1030 1035

Lys Gln Glu Lys Lys Asp Leu Tyr Ile Arg Asn Tyr Ile Ala His

1040 1045 1050

Phe Asn Tyr Ile Pro His Ala Glu Ile Ser Leu Leu Glu Val Leu

1055 1060 1065

Glu Asn Leu Arg Lys Leu Leu Ser Tyr Asp Arg Lys Leu Lys Asn

1070 1075 1080

Ala Val Met Lys Ser Val Val Asp Ile Leu Lys Glu Tyr Gly Phe

1085 1090 1095

Val Ala Thr Phe Lys Ile Gly Ala Asp Lys Lys Ile Gly Ile Gln

1100 1105 1110

Thr Leu Glu Ser Glu Lys Ile Val His Leu Lys Asn Leu Lys Lys

1115 1120 1125

Lys Lys Leu Met Thr Asp Arg Asn Ser Glu Glu Leu Cys Lys Leu

1130 1135 1140

Val Lys Ile Met Phe Glu Tyr Lys Met Glu Glu Lys Lys Ser Glu

1145 1150 1155

Asn

<210> 589

<211> 913

<212> PRT

<213> Leptotrichia sp.

<400> 589

Met Lys Glu Leu Ile Glu Lys Val Pro Asn Val Ser Glu Leu Lys Lys

1 5 10 15

Ser Gln Val Phe Tyr Lys Tyr Tyr Leu Asn Lys Glu Lys Leu Asn Asp

20 25 30

Glu Asn Ile Lys Tyr Val Phe Cys His Phe Val Glu Ile Glu Met Ser

35 40 45

Lys Leu Leu Lys Asn Tyr Val Tyr Lys Lys Pro Ser Asn Ile Ser Asn

50 55 60

Asp Lys Val Lys Arg Ile Phe Glu Tyr Gln Ser Leu Lys Lys Leu Ile

65 70 75 80

Glu Asn Lys Leu Leu Asn Lys Leu Asp Thr Tyr Ile Arg Asn Cys Gly

85 90 95

Lys Tyr Ser Phe Tyr Leu Gln Asp Gly Glu Ile Ala Thr Ser Asp Phe

100 105 110

Ile Val Gly Asn Arg Gln Asn Glu Ala Phe Leu Arg Asn Ile Ile Gly

115 120 125

Val Ser Ser Ala Ala Tyr Phe Ser Leu Arg Asn Ile Leu Glu Thr Glu

130 135 140

Asn Glu Asn Asp Ile Thr Gly Lys Met Arg Gly Lys Thr Val Lys Asn

145 150 155 160

Lys Lys Gly Glu Glu Lys Tyr Ile Ser Gly Glu Ile Asp Lys Leu Tyr

165 170 175

Asp Asn Asn Lys Gln Asn Glu Val Lys Lys Asn Leu Lys Met Phe Tyr

180 185 190

Ser Tyr Asp Phe Asn Met Asn Ser Lys Lys Glu Ile Glu Asp Phe Phe

195 200 205

Ser Asn Ile Asp Glu Ala Ile Ser Ser Ile Arg His Gly Ile Val His

210 215 220

Phe Asn Leu Glu Leu Glu Gly Lys Asp Ile Phe Thr Phe Lys Asn Ile

225 230 235 240

Val Pro Ser Gln Ile Ser Lys Lys Met Phe His Asp Glu Ile Asn Glu

245 250 255

Lys Lys Leu Lys Leu Lys Ile Phe Lys Gln Leu Asn Ser Ala Asn Val

260 265 270

Phe Arg Tyr Leu Glu Lys Tyr Lys Ile Leu Asn Tyr Leu Asn Arg Thr

275 280 285

Arg Phe Glu Phe Val Asn Lys Asn Ile Pro Phe Val Pro Ser Phe Thr

290 295 300

Lys Leu Tyr Ser Arg Ile Asp Asp Leu Lys Asn Ser Leu Cys Ile Tyr

305 310 315 320

Trp Lys Ile Pro Lys Ala Asn Asp Asn Asn Lys Thr Lys Glu Ile Thr

325 330 335

Asp Ala Gln Ile Tyr Leu Leu Lys Asn Ile Tyr Tyr Ser Glu Phe Leu

340 345 350

Asn Tyr Phe Met Ser Asn Asn Gly Asn Phe Phe Glu Ile Ile Lys Glu

355 360 365

Ile Ile Glu Leu Asn Lys Asn Asp Lys Arg Asn Leu Lys Thr Gly Phe

370 375 380

Tyr Lys Leu Gln Lys Phe Glu Asn Leu Gln Glu Lys Thr Pro Lys Glu

385 390 395 400

Tyr Leu Ala Asn Ile Gln Ser Phe Tyr Met Ile Asp Ala Gly Asn Lys

405 410 415

Asp Glu Glu Glu Lys Asp Ala Tyr Ile Asp Phe Ile Gln Lys Ile Phe

420 425 430

Leu Lys Gly Phe Met Thr Tyr Leu Ala Asn Asn Gly Arg Leu Ser Leu

435 440 445

Met Tyr Ile Gly Asn Asp Glu Gln Ile Asn Thr Ser Leu Ala Glu Lys

450 455 460

Lys Gln Glu Phe Asp Lys Phe Leu Lys Lys Tyr Glu Gln Asn Asn Asn

465 470 475 480

Ile Lys Ile Pro Tyr Glu Ile Asn Glu Phe Leu Arg Glu Ile Lys Leu

485 490 495

Gly Asn Ile Leu Lys Tyr Thr Glu Arg Leu Asn Met Phe Tyr Leu Ile

500 505 510

Leu Lys Leu Leu Asn His Lys Glu Leu Thr Asn Leu Lys Gly Ser Leu

515 520 525

Glu Lys Tyr Gln Ser Ala Asn Lys Glu Glu Glu Ala Phe Ser Asp Gln Leu

530 535 540

Glu Leu Ile Asn Leu Leu Asn Leu Asp Asn Asn Arg Val Thr Glu Asp

545 550 555 560

Phe Glu Leu Glu Ala Asp Glu Ile Gly Lys Phe Leu Asp Phe Asn Gly

565 570 575

Asn Lys Val Lys Asp Asn Lys Glu Leu Lys Lys Phe Asp Thr Asn Lys

580 585 590

Ile Tyr Phe Asp Gly Glu Asn Ile Ile Lys His Arg Ala Phe Tyr Asn

595 600 605

Ile Lys Lys Tyr Gly Met Leu Asn Leu Leu Glu Lys Ile Ser Asp Glu

610 615 620

Ala Lys Tyr Lys Ile Ser Ile Glu Glu Leu Lys Asn Tyr Ser Asn Lys

625 630 635 640

Lys Asn Glu Ile Glu Lys Asn His Thr Asn Gln Glu Asn Leu His Arg

645 650 655

Lys Tyr Ala Arg Pro Arg Lys Asp Glu Lys Phe Asn Asp Glu Asp Tyr

660 665 670

Lys Lys Tyr Glu Lys Ala Ile Arg Asn Ile Gln Gln Tyr Thr His Leu

675 680 685

Lys Asn Lys Val Glu Phe Asn Glu Leu Asn Leu Leu Gln Ser Leu Leu

690 695 700

Leu Arg Ile Leu His Arg Leu Val Gly Tyr Thr Ser Ile Trp Glu Arg

705 710 715 720

Asp Leu Arg Phe Arg Leu Lys Gly Glu Phe Pro Glu Asn Gln Tyr Ile

725 730 735

Glu Glu Ile Phe Asn Phe Asn Asn Ser Lys Asn Val Lys Tyr Lys Asn

740 745 750

Gly Gln Ile Val Glu Lys Tyr Ile Ser Phe Tyr Lys Glu Leu Tyr Lys

755 760 765

Asp Asp Thr Glu Lys Ile Ser Ile Tyr Ser Asp Lys Lys Val Lys Glu

770 775 780

Leu Lys Lys Glu Lys Lys Asp Leu Tyr Ile Arg Asn Tyr Ile Ala His

785 790 795 800

Phe Asn Tyr Ile Pro Asn Ala Glu Ile Ser Leu Leu Glu Val Leu Glu

805 810 815

Asn Leu Arg Lys Leu Leu Ser Tyr Asp Arg Lys Leu Lys Asn Ala Ile

820 825 830

Met Lys Ser Ile Val Asp Ile Leu Lys Glu Tyr Gly Phe Val Val Thr

835 840 845

Phe Lys Ile Glu Lys Asp Lys Lys Ile Arg Ile Glu Ser Leu Lys Ser

850 855 860

Glu Glu Val Val His Leu Lys Lys Leu Lys Leu Lys Asp Asn Asp Lys

865 870 875 880

Lys Lys Glu Pro Ile Lys Thr Tyr Arg Asn Ser Lys Glu Leu Cys Glu

885 890 895

Leu Val Lys Val Met Phe Glu Tyr Lys Met Lys Glu Lys Lys Ser Glu

900 905 910

Asn

<210> 590

<211> 1385

<212> PRT

<213> Leptotrichia sp.

<400> 590

Met Gly Asn Leu Phe Gly His Lys Arg Trp Tyr Glu Val Arg Asp Lys

1 5 10 15

Lys Asp Phe Lys Ile Lys Arg Lys Val Lys Val Lys Arg Asn Tyr Asp

20 25 30

Gly Asn Lys Tyr Ile Leu Asn Ile Asn Glu Asn Asn Asn Lys Glu Lys

35 40 45

Ile Asp Asn Asn Lys Phe Ile Gly Glu Phe Val Asn Tyr Lys Lys Asn

50 55 60

Asn Asn Val Leu Lys Glu Phe Lys Arg Lys Phe His Ala Gly Asn Ile

65 70 75 80

Leu Phe Lys Leu Lys Gly Lys Glu Glu Ile Ile Arg Ile Glu Asn Asn

85 90 95

Asp Asp Phe Leu Glu Thr Glu Glu Val Val Leu Tyr Ile Glu Val Tyr

100 105 110

Gly Lys Ser Glu Lys Leu Lys Ala Leu Glu Ile Thr Lys Lys Lys Ile

115 120 125

Ile Asp Glu Ala Ile Arg Gln Gly Ile Thr Lys Asp Asp Lys Lys Ile

130 135 140

Glu Ile Lys Arg Gln Glu Asn Glu Glu Glu Ile Glu Ile Asp Ile Arg

145 150 155 160

Asp Glu Tyr Thr Asn Lys Thr Leu Asn Asp Cys Ser Ile Ile Leu Arg

165 170 175

Ile Ile Glu Asn Asp Glu Leu Glu Thr Lys Lys Ser Ile Tyr Glu Ile

180 185 190

Phe Lys Asn Ile Asn Met Ser Leu Tyr Lys Ile Ile Glu Lys Ile Ile

195 200 205

Glu Asn Glu Thr Glu Lys Val Phe Glu Asn Arg Tyr Tyr Glu Glu His

210 215 220

Leu Arg Glu Lys Leu Leu Lys Asp Asn Lys Ile Asp Val Ile Leu Thr

225 230 235 240

Asn Phe Met Glu Ile Arg Glu Lys Ile Lys Ser Asn Leu Glu Ile Met

245 250 255

Gly Phe Val Lys Phe Tyr Leu Asn Val Ser Gly Asp Lys Lys Lys Ser

260 265 270

Glu Asn Lys Lys Met Phe Val Glu Lys Ile Leu Asn Thr Asn Val Asp

275 280 285

Leu Thr Val Glu Asp Ile Val Asp Phe Ile Val Lys Glu Leu Lys Phe

290 295 300

Trp Asn Ile Thr Lys Arg Ile Glu Lys Val Lys Lys Phe Asn Asn Glu

305 310 315 320

Phe Leu Glu Asn Arg Arg Asn Arg Thr Tyr Ile Lys Ser Tyr Val Leu

325 330 335

Leu Asp Lys His Glu Lys Phe Lys Ile Glu Arg Glu Asn Lys Lys Asp

340 345 350

Lys Ile Val Lys Phe Phe Val Glu Asn Ile Lys Asn Asn Ser Ile Lys

355 360 365

Glu Lys Ile Glu Lys Ile Leu Ala Glu Phe Lys Ile Asn Glu Leu Ile

370 375 380

Lys Lys Leu Glu Lys Glu Leu Lys Lys Gly Asn Cys Asp Thr Glu Ile

385 390 395 400

Phe Gly Ile Phe Lys Lys His Tyr Lys Val Asn Phe Asp Ser Lys Lys

405 410 415

Phe Ser Asn Lys Ser Asp Glu Glu Lys Glu Leu Tyr Lys Ile Ile Tyr

420 425 430

Arg Tyr Leu Lys Gly Arg Ile Glu Lys Ile Leu Val Asn Glu Gln Lys

435 440 445

Val Arg Leu Lys Lys Met Glu Lys Ile Glu Ile Glu Lys Ile Leu Asn

450 455 460

Glu Ser Ile Leu Ser Glu Lys Ile Leu Lys Arg Val Lys Gln Tyr Thr

465 470 475 480

Leu Glu His Ile Met Tyr Leu Gly Lys Leu Arg His Asn Asp Ile Val

485 490 495

Lys Met Thr Val Asn Thr Asp Asp Phe Ser Arg Leu His Ala Lys Glu

500 505 510

Glu Leu Asp Leu Glu Leu Ile Thr Phe Phe Ala Ser Thr Asn Met Glu

515 520 525

Leu Asn Lys Ile Phe Asn Gly Lys Glu Lys Val Thr Asp Phe Phe Gly

530 535 540

Phe Asn Leu Asn Gly Gln Lys Ile Thr Leu Lys Glu Lys Val Pro Ser

545 550 555 560

Phe Lys Leu Asn Ile Leu Lys Lys Leu Asn Phe Ile Asn Asn Glu Asn

565 570 575

Asn Ile Asp Glu Lys Leu Ser His Phe Tyr Ser Phe Gln Lys Glu Gly

580 585 590

Tyr Leu Leu Arg Asn Lys Ile Leu His Asn Ser Tyr Gly Asn Ile Gln

595 600 605

Glu Thr Lys Asn Leu Lys Gly Glu Tyr Glu Asn Val Glu Lys Leu Ile

610 615 620

Lys Glu Leu Lys Val Ser Asp Glu Glu Ile Ser Lys Ser Leu Ser Leu

625 630 635 640

Asp Val Ile Phe Glu Gly Lys Val Asp Ile Ile Asn Lys Ile Asn Ser

645 650 655

Leu Lys Ile Gly Glu Tyr Lys Asp Lys Lys Tyr Leu Pro Ser Phe Ser

660 665 670

Lys Ile Val Leu Glu Ile Thr Arg Lys Phe Arg Glu Ile Asn Lys Asp

675 680 685

Lys Leu Phe Asp Ile Glu Ser Glu Lys Ile Ile Leu Asn Ala Val Lys

690 695 700

Tyr Val Asn Lys Ile Leu Tyr Glu Lys Ile Thr Ser Asn Glu Glu Asn

705 710 715 720

Glu Phe Leu Lys Thr Leu Pro Asp Lys Leu Val Lys Lys Ser Asn Asn

725 730 735

Lys Lys Glu Asn Lys Asn Leu Leu Ser Ile Glu Glu Tyr Tyr Lys Asn

740 745 750

Ala Gln Val Ser Ser Ser Lys Gly Asp Lys Lys Ala Ile Lys Lys Tyr

755 760 765

Gln Asn Lys Val Thr Asn Ala Tyr Leu Glu Tyr Leu Glu Asn Thr Phe

770 775 780

Thr Glu Ile Ile Asp Phe Ser Lys Phe Asn Leu Asn Tyr Asp Glu Ile

785 790 795 800

Lys Thr Lys Ile Glu Glu Arg Lys Asp Asn Lys Ser Lys Ile Ile Ile

805 810 815

Asp Ser Ile Ser Thr Asn Ile Asn Ile Thr Asn Asp Ile Glu Tyr Ile

820 825 830

Ile Ser Ile Phe Ala Leu Leu Asn Ser Asn Thr Tyr Ile Asn Lys Ile

835 840 845

Arg Asn Arg Phe Phe Ala Thr Ser Val Trp Leu Glu Lys Gln Asn Gly

850 855 860

Thr Lys Glu Tyr Asp Tyr Glu Asn Ile Ile Ser Ile Leu Asp Glu Val

865 870 875 880

Leu Leu Ile Asn Leu Leu Arg Glu Asn Asn Ile Thr Asp Ile Leu Asp

885 890 895

Leu Lys Asn Ala Ile Ile Asp Ala Lys Ile Val Glu Asn Asp Glu Thr

900 905 910

Tyr Ile Lys Asn Tyr Ile Phe Glu Ser Asn Glu Glu Lys Leu Lys Lys

915 920 925

Arg Leu Phe Cys Glu Glu Leu Val Asp Lys Glu Asp Ile Arg Lys Ile

930 935 940

Phe Glu Asp Glu Asn Phe Lys Phe Lys Ser Phe Ile Lys Lys Asn Glu

945 950 955 960

Ile Gly Asn Phe Lys Ile Asn Phe Gly Ile Leu Ser Asn Leu Glu Cys

965 970 975

Asn Ser Glu Val Glu Ala Lys Lys Ile Ile Gly Lys Asn Ser Lys Lys

980 985 990

Leu Glu Ser Phe Ile Gln Asn Ile Ile Asp Glu Tyr Lys Ser Asn Ile

995 1000 1005

Arg Thr Leu Phe Ser Ser Glu Phe Leu Glu Lys Tyr Lys Glu Glu

1010 1015 1020

Ile Asp Asn Leu Val Glu Asp Thr Glu Ser Glu Asn Lys Asn Lys

1025 1030 1035

Phe Glu Lys Ile Tyr Tyr Pro Lys Glu His Lys Asn Glu Leu Tyr

1040 1045 1050

Ile Tyr Lys Lys Asn Leu Phe Leu Asn Ile Gly Asn Pro Asn Phe

1055 1060 1065

Asp Lys Ile Tyr Gly Leu Ile Ser Lys Asp Ile Lys Asn Val Asp

1070 1075 1080

Thr Lys Ile Leu Phe Asp Asp Asp Ile Lys Lys Asn Lys Ile Ser

1085 1090 1095

Glu Ile Asp Ala Ile Leu Lys Asn Leu Asn Asp Lys Leu Asn Gly

1100 1105 1110

Tyr Ser Asn Asp Tyr Lys Ala Lys Tyr Val Asn Lys Leu Lys Glu

1115 1120 1125

Asn Asp Asp Phe Phe Ala Lys Asn Ile Gln Asn Glu Asn Tyr Ser

1130 1135 1140

Ser Phe Gly Glu Phe Glu Lys Asp Tyr Asn Lys Val Ser Glu Tyr

1145 1150 1155

Lys Lys Ile Arg Asp Leu Val Glu Phe Asn Tyr Leu Asn Lys Ile

1160 1165 1170

Glu Ser Tyr Leu Ile Asp Ile Asn Trp Lys Leu Ala Ile Gln Met

1175 1180 1185

Ala Arg Phe Glu Arg Asp Met His Tyr Ile Val Asn Gly Leu Arg

1190 1195 1200

Glu Leu Gly Ile Ile Lys Leu Ser Gly Tyr Asn Thr Gly Ile Ser

1205 1210 1215

Arg Ala Tyr Pro Lys Arg Asn Gly Ser Asp Gly Phe Tyr Thr Thr

1220 1225 1230

Thr Ala Tyr Tyr Lys Phe Phe Asp Glu Glu Ser Tyr Lys Lys Phe

1235 1240 1245

Glu Lys Ile Cys Tyr Gly Phe Gly Ile Asp Leu Ser Glu Asn Ser

1250 1255 1260

Glu Ile Asn Lys Pro Glu Asn Glu Ser Ile Arg Asn Tyr Ile Ser

1265 1270 1275

His Phe Tyr Ile Val Arg Asn Pro Phe Ala Asp Tyr Ser Ile Ala

1280 1285 1290

Glu Gln Ile Asp Arg Val Ser Asn Leu Leu Ser Tyr Ser Thr Arg

1295 1300 1305

Tyr Asn Asn Ser Thr Tyr Ala Ser Val Phe Glu Val Phe Lys Lys

1310 1315 1320

Asp Val Asn Leu Asp Tyr Asp Glu Leu Lys Lys Lys Phe Arg Leu

1325 1330 1335

Ile Gly Asn Asn Asp Ile Leu Glu Arg Leu Met Lys Pro Lys Lys

1340 1345 1350

Val Ser Val Leu Glu Leu Glu Ser Tyr Asn Ser Asp Tyr Ile Lys

1355 1360 1365

Asn Leu Ile Ile Glu Leu Leu Thr Lys Ile Glu Asn Thr Asn Asp

1370 1375 1380

Thr Leu

1385

<210> 591

<211> 1389

<212> PRT

<213> Leptotrichia shahii

<400> 591

Met Gly Asn Leu Phe Gly His Lys Arg Trp Tyr Glu Val Arg Asp Lys

1 5 10 15

Lys Asp Phe Lys Ile Lys Arg Lys Val Lys Val Lys Arg Asn Tyr Asp

20 25 30

Gly Asn Lys Tyr Ile Leu Asn Ile Asn Glu Asn Asn Asn Lys Glu Lys

35 40 45

Ile Asp Asn Asn Lys Phe Ile Arg Lys Tyr Ile Asn Tyr Lys Lys Asn

50 55 60

Asp Asn Ile Leu Lys Glu Phe Thr Arg Lys Phe His Ala Gly Asn Ile

65 70 75 80

Leu Phe Lys Leu Lys Gly Lys Glu Gly Ile Ile Arg Ile Glu Asn Asn

85 90 95

Asp Asp Phe Leu Glu Thr Glu Glu Val Val Leu Tyr Ile Glu Ala Tyr

100 105 110

Gly Lys Ser Glu Lys Leu Lys Ala Leu Gly Ile Thr Lys Lys Lys Ile

115 120 125

Ile Asp Glu Ala Ile Arg Gln Gly Ile Thr Lys Asp Asp Lys Lys Ile

130 135 140

Glu Ile Lys Arg Gln Glu Asn Glu Glu Glu Ile Glu Ile Asp Ile Arg

145 150 155 160

Asp Glu Tyr Thr Asn Lys Thr Leu Asn Asp Cys Ser Ile Ile Leu Arg

165 170 175

Ile Ile Glu Asn Asp Glu Leu Glu Thr Lys Lys Ser Ile Tyr Glu Ile

180 185 190

Phe Lys Asn Ile Asn Met Ser Leu Tyr Lys Ile Ile Glu Lys Ile Ile

195 200 205

Glu Asn Glu Thr Glu Lys Val Phe Glu Asn Arg Tyr Tyr Glu Glu His

210 215 220

Leu Arg Glu Lys Leu Leu Lys Asp Asp Lys Ile Asp Val Ile Leu Thr

225 230 235 240

Asn Phe Met Glu Ile Arg Glu Lys Ile Lys Ser Asn Leu Glu Ile Leu

245 250 255

Gly Phe Val Lys Phe Tyr Leu Asn Val Gly Gly Asp Lys Lys Lys Ser

260 265 270

Lys Asn Lys Lys Met Leu Val Glu Lys Ile Leu Asn Ile Asn Val Asp

275 280 285

Leu Thr Val Glu Asp Ile Ala Asp Phe Val Ile Lys Glu Leu Glu Phe

290 295 300

Trp Asn Ile Thr Lys Arg Ile Glu Lys Val Lys Lys Val Asn Asn Glu

305 310 315 320

Phe Leu Glu Lys Arg Arg Asn Arg Thr Tyr Ile Lys Ser Tyr Val Leu

325 330 335

Leu Asp Lys His Glu Lys Phe Lys Ile Glu Arg Glu Asn Lys Lys Asp

340 345 350

Lys Ile Val Lys Phe Phe Val Glu Asn Ile Lys Asn Asn Ser Ile Lys

355 360 365

Glu Lys Ile Glu Lys Ile Leu Ala Glu Phe Lys Ile Asp Glu Leu Ile

370 375 380

Lys Lys Leu Glu Lys Glu Leu Lys Lys Gly Asn Cys Asp Thr Glu Ile

385 390 395 400

Phe Gly Ile Phe Lys Lys His Tyr Lys Val Asn Phe Asp Ser Lys Lys

405 410 415

Phe Ser Lys Lys Ser Asp Glu Glu Lys Glu Leu Tyr Lys Ile Ile Tyr

420 425 430

Arg Tyr Leu Lys Gly Arg Ile Glu Lys Ile Leu Val Asn Glu Gln Lys

435 440 445

Val Arg Leu Lys Lys Met Glu Lys Ile Glu Ile Glu Lys Ile Leu Asn

450 455 460

Glu Ser Ile Leu Ser Glu Lys Ile Leu Lys Arg Val Lys Gln Tyr Thr

465 470 475 480

Leu Glu His Ile Met Tyr Leu Gly Lys Leu Arg His Asn Asp Ile Asp

485 490 495

Met Thr Thr Val Asn Thr Asp Asp Phe Ser Arg Leu His Ala Lys Glu

500 505 510

Glu Leu Asp Leu Glu Leu Ile Thr Phe Phe Ala Ser Thr Asn Met Glu

515 520 525

Leu Asn Lys Ile Phe Ser Arg Glu Asn Ile Asn Asn Asp Glu Asn Ile

530 535 540

Asp Phe Phe Gly Gly Asp Arg Glu Lys Asn Tyr Val Leu Asp Lys Lys

545 550 555 560

Ile Leu Asn Ser Lys Ile Lys Ile Ile Arg Asp Leu Asp Phe Ile Asp

565 570 575

Asn Lys Asn Asn Ile Thr Asn Asn Phe Ile Arg Lys Phe Thr Lys Ile

580 585 590

Gly Thr Asn Glu Arg Asn Arg Ile Leu His Ala Ile Ser Lys Glu Arg

595 600 605

Asp Leu Gln Gly Thr Gln Asp Asp Tyr Asn Lys Val Ile Asn Ile Ile

610 615 620

Gln Asn Leu Lys Ile Ser Asp Glu Glu Val Ser Lys Ala Leu Asn Leu

625 630 635 640

Asp Val Val Phe Lys Asp Lys Lys Asn Ile Ile Thr Lys Ile Asn Asp

645 650 655

Ile Lys Ile Ser Glu Glu Asn Asn Asn Asp Ile Lys Tyr Leu Pro Ser

660 665 670

Phe Ser Lys Val Leu Pro Glu Ile Leu Asn Leu Tyr Arg Asn Asn Pro

675 680 685

Lys Asn Glu Pro Phe Asp Thr Ile Glu Thr Glu Lys Ile Val Leu Asn

690 695 700

Ala Leu Ile Tyr Val Asn Lys Glu Leu Tyr Lys Lys Leu Ile Leu Glu

705 710 715 720

Asp Asp Leu Glu Glu Asn Glu Ser Lys Asn Ile Phe Leu Gln Glu Leu

725 730 735

Lys Lys Thr Leu Gly Asn Ile Asp Glu Ile Asp Glu Asn Ile Ile Glu

740 745 750

Asn Tyr Tyr Lys Asn Ala Gln Ile Ser Ala Ser Lys Gly Asn Asn Lys

755 760 765

Ala Ile Lys Lys Tyr Gln Lys Lys Val Ile Glu Cys Tyr Ile Gly Tyr

770 775 780

Leu Arg Lys Asn Tyr Glu Glu Leu Phe Asp Phe Ser Asp Phe Lys Met

785 790 795 800

Asn Ile Gln Glu Ile Lys Lys Gln Ile Lys Asp Ile Asn Asp Asn Lys

805 810 815

Thr Tyr Glu Arg Ile Thr Val Lys Thr Ser Asp Lys Thr Ile Val Ile

820 825 830

Asn Asp Asp Phe Glu Tyr Ile Ile Ser Ile Phe Ala Leu Leu Asn Ser

835 840 845

Asn Ala Val Ile Asn Lys Ile Arg Asn Arg Phe Phe Ala Thr Ser Val

850 855 860

Trp Leu Asn Thr Ser Glu Tyr Gln Asn Ile Ile Asp Ile Leu Asp Glu

865 870 875 880

Ile Met Gln Leu Asn Thr Leu Arg Asn Glu Cys Ile Thr Glu Asn Trp

885 890 895

Asn Leu Asn Leu Glu Glu Phe Ile Gln Lys Met Lys Glu Ile Glu Lys

900 905 910

Asp Phe Asp Asp Phe Lys Ile Gln Thr Lys Lys Glu Ile Phe Asn Asn

915 920 925

Tyr Tyr Glu Asp Ile Lys Asn Asn Ile Leu Thr Glu Phe Lys Asp Asp

930 935 940

Ile Asn Gly Cys Asp Val Leu Glu Lys Lys Leu Glu Lys Ile Val Ile

945 950 955 960

Phe Asp Asp Glu Thr Lys Phe Glu Ile Asp Lys Lys Ser Asn Ile Leu

965 970 975

Gln Asp Glu Gln Arg Lys Leu Ser Asn Ile Asn Lys Lys Asp Leu Lys

980 985 990

Lys Lys Val Asp Gln Tyr Ile Lys Asp Lys Asp Gln Glu Ile Lys Ser

995 1000 1005

Lys Ile Leu Cys Arg Ile Ile Phe Asn Ser Asp Phe Leu Lys Lys

1010 1015 1020

Tyr Lys Lys Glu Ile Asp Asn Leu Ile Glu Asp Met Glu Ser Glu

1025 1030 1035

Asn Glu Asn Lys Phe Gln Glu Ile Tyr Tyr Pro Lys Glu Arg Lys

1040 1045 1050

Asn Glu Leu Tyr Ile Tyr Lys Lys Asn Leu Phe Leu Asn Ile Gly

1055 1060 1065

Asn Pro Asn Phe Asp Lys Ile Tyr Gly Leu Ile Ser Asn Asp Ile

1070 1075 1080

Lys Met Ala Asp Ala Lys Phe Leu Phe Asn Ile Asp Gly Lys Asn

1085 1090 1095

Ile Arg Lys Asn Lys Ile Ser Glu Ile Asp Ala Ile Leu Lys Asn

1100 1105 1110

Leu Asn Asp Lys Leu Asn Gly Tyr Ser Lys Glu Tyr Lys Glu Lys

1115 1120 1125

Tyr Ile Lys Lys Leu Lys Glu Asn Asp Asp Phe Phe Ala Lys Asn

1130 1135 1140

Ile Gln Asn Lys Asn Tyr Lys Ser Phe Glu Lys Asp Tyr Asn Arg

1145 1150 1155

Val Ser Glu Tyr Lys Lys Ile Arg Asp Leu Val Glu Phe Asn Tyr

1160 1165 1170

Leu Asn Lys Ile Glu Ser Tyr Leu Ile Asp Ile Asn Trp Lys Leu

1175 1180 1185

Ala Ile Gln Met Ala Arg Phe Glu Arg Asp Met His Tyr Ile Val

1190 1195 1200

Asn Gly Leu Arg Glu Leu Gly Ile Ile Lys Leu Ser Gly Tyr Asn

1205 1210 1215

Thr Gly Ile Ser Arg Ala Tyr Pro Lys Arg Asn Gly Ser Asp Gly

1220 1225 1230

Phe Tyr Thr Thr Thr Ala Tyr Tyr Lys Phe Phe Asp Glu Glu Ser

1235 1240 1245

Tyr Lys Lys Phe Glu Lys Ile Cys Tyr Gly Phe Gly Ile Asp Leu

1250 1255 1260

Ser Glu Asn Ser Glu Ile Asn Lys Pro Glu Asn Glu Ser Ile Arg

1265 1270 1275

Asn Tyr Ile Ser His Phe Tyr Ile Val Arg Asn Pro Phe Ala Asp

1280 1285 1290

Tyr Ser Ile Ala Glu Gln Ile Asp Arg Val Ser Asn Leu Leu Ser

1295 1300 1305

Tyr Ser Thr Arg Tyr Asn Asn Ser Thr Tyr Ala Ser Val Phe Glu

1310 1315 1320

Val Phe Lys Lys Asp Val Asn Leu Asp Tyr Asp Glu Leu Lys Lys

1325 1330 1335

Lys Phe Lys Leu Ile Gly Asn Asn Asp Ile Leu Glu Arg Leu Met

1340 1345 1350

Lys Pro Lys Lys Val Ser Val Leu Glu Leu Glu Ser Tyr Asn Ser

1355 1360 1365

Asp Tyr Ile Lys Asn Leu Ile Ile Glu Leu Leu Thr Lys Ile Glu

1370 1375 1380

Asn Thr Asn Asp Thr Leu

1385

<210> 592

<211> 10

<212> PRT

<213> Lactococcus lactis

<400> 592

Phe Leu Val Asn His Asn Tyr Tyr Ser Phe

1 5 10

<210> 593

<211> 16

<212> PRT

<213> Lactococcus lactis

<400> 593

Leu Gln Lys Phe Thr Gly Asp Ile Glu Asn Leu Val Lys Ala Ser Leu

1 5 10 15

<210> 594

<211> 9

<212> PRT

<213> Lactococcus lactis

<400> 594

Val Ile Val Pro Glu Leu Thr Phe Gly

fifteen

<210> 595

<211> 15

<212> PRT

<213> Lactococcus lactis

<400> 595

Trp Ile Arg Ala Gly Trp Phe Ile Arg Asn Arg Ser Ala His Tyr

1 5 10 15

<210> 596

<211> 13

<212> PRT

<213> Lactococcus lactis

<400> 596

Asn Lys Asp Leu Phe Ala Phe Met Leu Ser Ile Lys Gln

1 5 10

<210> 597

<211> 10

<212> PRT

<213> Lactococcus lactis

<400> 597

Phe Leu His Lys Asn Ser Tyr Phe Arg Phe

1 5 10

<210> 598

<211> 16

<212> PRT

<213> Lactococcus lactis

<400> 598

Leu Phe Ile Phe Ser Thr Arg Leu Glu Ile Phe Trp Lys Lys Lys Ile

1 5 10 15

<210> 599

<211> 9

<212> PRT

<213> Lactococcus lactis

<400> 599

Ala Leu Val Glu Glu Leu Thr Phe Gly

fifteen

<210> 600

<211> 15

<212> PRT

<213> Lactococcus lactis

<400> 600

Trp Met Asn Val Val Arg Leu Tyr Arg Asn Lys Ser Ala His Gly

1 5 10 15

<210> 601

<211> 13

<212> PRT

<213> Lactococcus lactis

<400> 601

Lys Ser Tyr Leu Tyr Gly Ala Leu Tyr Val Phe Lys His

1 5 10

<210> 602

<211> 10

<212> PRT

<213> Corynebacterium diphtheriae

<400> 602

Leu Leu Ala Gln Leu Asn Tyr Tyr Arg Leu

1 5 10

<210> 603

<211> 16

<212> PRT

<213> Corynebacterium diphtheriae

<400> 603

Val Phe Ile Glu Leu Asp Arg Val Glu Leu Ala Ile Gln Thr Arg Leu

1 5 10 15

<210> 604

<211> 9

<212> PRT

<213> Corynebacterium diphtheriae

<400> 604

Ala Ala Val Glu Val Met Asp Trp Gly

fifteen

<210> 605

<211> 15

<212> PRT

<213> Corynebacterium diphtheriae

<400> 605

Trp Leu Lys Ser Leu Asn Ile Leu Arg Asn Tyr Ala Ala His His

1 5 10 15

<210> 606

<211> 13

<212> PRT

<213> Corynebacterium diphtheriae

<400> 606

Gly Gln Leu Ser Met Ile Gln Tyr Leu His His Gln Leu

1 5 10

<210> 607

<211> 12

<212> PRT

<213> Shewanella baltica

<400> 607

Met Leu Ile Glu Asn Asp Leu Asp Gly Ile Glu Asn

1 5 10

<210> 608

<211> 16

<212> PRT

<213> Shewanella baltica

<400> 608

Asn Tyr Gln Leu Phe Tyr Phe Leu Glu Lys Thr Ile Arg Asn Gln Ile

1 5 10 15

<210> 609

<211> 15

<212> PRT

<213> Shewanella baltica

<400> 609

Val Met Phe Asn Leu Asn Thr Leu Arg Asn Pro Ile Ala His Cys

1 5 10 15

<210> 610

<211> 13

<212> PRT

<213> Shewanella baltica

<400> 610

Asp Glu Lys Leu Arg Leu Glu Ile Ser Leu Arg Asp Trp

1 5 10

<210> 611

<211> 12

<212> PRT

<213> Lactococcus lactis

<400> 611

Leu Arg Glu Ile Asn Ile Lys Ala Ser Lys Ser Arg

1 5 10

<210> 612

<211> 16

<212> PRT

<213> Lactococcus lactis

<400> 612

Leu Leu Pro Leu Leu His Lys Tyr Glu Trp Ser Leu Arg Lys Leu Ile

1 5 10 15

<210> 613

<211> 9

<212> PRT

<213> Lactococcus lactis

<400> 613

Tyr Asp Phe Glu Glu Tyr Leu Phe Gly

fifteen

<210> 614

<211> 15

<212> PRT

<213> Lactococcus lactis

<400> 614

Asp Met Arg Leu Ile Arg Asp Gly Arg Asn Ile Val Gly His Asn

1 5 10 15

<210> 615

<211> 13

<212> PRT

<213> Lactococcus lactis

<400> 615

Leu Ser Lys Gly Leu Lys Lys Tyr Ile Lys Lys Leu Asp

1 5 10

<210> 616

<211> 12

<212> PRT

<213> Geobacter bemidjiensis

<400> 616

Arg Leu Pro Leu Thr Ser His Ile Gln Lys Gln Asp

1 5 10

<210> 617

<211> 16

<212> PRT

<213> Geobacter bemidjiensis

<400> 617

Ile Tyr Pro Lys Leu Asn Arg Ile Glu Asn Arg Leu Arg His Tyr Leu

1 5 10 15

<210> 618

<211> 9

<212> PRT

<213> Geobacter bemidjiensis

<400> 618

Phe Glu Leu Gly Lys Ile Val Tyr Ala

fifteen

<210> 619

<211> 15

<212> PRT

<213> Geobacter bemidjiensis

<400> 619

Lys Trp Ile Arg Leu Glu Glu Ile Arg His Lys Val Ala His Asn

1 5 10 15

<210> 620

<211> 13

<212> PRT

<213> Geobacter bemidjiensis

<400> 620

Ala Asn Glu Tyr Ile Asp Ser Leu Gln Ser Ile Ile Asp

1 5 10

<210> 621

<211> 12

<212> PRT

<213> Salmonella enterica

<400> 621

Phe Val Thr Ser Leu Glu His Leu Arg Gln Gln Gln

1 5 10

<210> 622

<211> 16

<212> PRT

<213> Salmonella enterica

<400> 622

Ala Gln Arg Gln Leu Arg Ala Ile Glu Leu Thr Leu Lys Ala Leu Ile

1 5 10 15

<210> 623

<211> 9

<212> PRT

<213> Salmonella enterica

<400> 623

Asn His Tyr Leu Lys Gln His Phe Gly

fifteen

<210> 624

<211> 15

<212> PRT

<213> Salmonella enterica

<400> 624

Phe Leu Asp Asp Cys Arg Leu Ala Arg Asn Glu Val Ile Ala Arg

1 5 10 15

<210> 625

<211> 13

<212> PRT

<213> Salmonella enterica

<400> 625

Leu Met Leu Leu Asn Val Gln Tyr Gln Gln Ile Val Arg

1 5 10

<210> 626

<211> 12

<212> PRT

<213> Shigella flexneri

<400> 626

Phe Leu Trp Gln Leu Glu Tyr Leu Arg Glu Lys Gln

1 5 10

<210> 627

<211> 16

<212> PRT

<213> Shigella flexneri

<400> 627

Ser Leu Gln Gln Val Arg Ala Leu Glu Leu Thr Ile Arg Ser Leu Ile

1 5 10 15

<210> 628

<211> 9

<212> PRT

<213> Shigella flexneri

<400> 628

Leu Glu His Leu Asn Lys Leu Phe Gly

fifteen

<210> 629

<211> 15

<212> PRT

<213> Shigella flexneri

<400> 629

Phe Leu Asp Asp Ile Arg Val Ile Arg Asn Arg Leu Ala His His

1 5 10 15

<210> 630

<211> 13

<212> PRT

<213> Shigella flexneri

<400> 630

Thr Thr Leu Val Asn Tyr Tyr Tyr Arg Glu Ile Thr Glu

1 5 10

<210> 631

<211> 16

<212> PRT

<213> Streptomyces avermitilis

<400> 631

Ala Tyr Ile Trp Leu Asn Leu Val Glu Gln Arg Leu Arg Ala Val Val

1 5 10 15

<210> 632

<211> 9

<212> PRT

<213> Streptomyces avermitilis

<400> 632

Asn Val Leu Ser Phe Leu Thr Leu Pro

fifteen

<210> 633

<211> 11

<212> PRT

<213> Streptomyces avermitilis

<400> 633

Leu Glu Val Thr Arg Asn Val Val Ser Arg Asn

1 5 10

<210> 634

<211> 13

<212> PRT

<213> Streptomyces avermitilis

<400> 634

Arg Tyr Gly Asp Val Val Gly Val His Pro Asp Arg Val

1 5 10

<210> 635

<211> 10

<212> PRT

<213> Helicobacter pylori

<400> 635

Ser Ile Ser Val Leu His Tyr Asp Tyr Leu

1 5 10

<210> 636

<211> 16

<212> PRT

<213> Helicobacter pylori

<400> 636

Leu Phe Leu Trp Ile His Phe Phe Glu Thr Ala Leu Arg Ser Lys Met

1 5 10 15

<210> 637

<211> 9

<212> PRT

<213> Helicobacter pylori

<400> 637

Gln Ile Leu Asn Leu Phe Thr Leu Gly

fifteen

<210> 638

<211> 15

<212> PRT

<213> Helicobacter pylori

<400> 638

Thr Phe Ser Leu Ile Arg Lys Ala Arg Asn Asp Leu Phe His Asn

1 5 10 15

<210> 639

<211> 13

<212> PRT

<213> Helicobacter pylori

<400> 639

Thr Leu Lys Leu Glu Arg Ala Ile Phe Phe Lys Thr Ile

1 5 10

<210> 640

<211> 11

<212> PRT

<213> Methylomirabilis oxyfera

<400> 640

Gly Pro Pro Glu Tyr Tyr Tyr Arg Leu Cys Arg

1 5 10

<210> 641

<211> 16

<212> PRT

<213> Methylomirabilis oxyfera

<400> 641

Ala Asp Ser Lys Leu Lys Asp Thr Val Ser Glu Met Arg Lys Phe Ile

1 5 10 15

<210> 642

<211> 15

<212> PRT

<213> Methylomirabilis oxyfera

<400> 642

Trp Met Asn Arg Ile Asn Glu Leu Arg Arg Ile Pro Ala His Pro

1 5 10 15

<210> 643

<211> 13

<212> PRT

<213> Methylomirabilis oxyfera

<400> 643

Asp Phe Glu Tyr Ile Asp Phe Ile Tyr Asp Glu Leu Met

1 5 10

<210> 644

<211> 12

<212> PRT

<213> Novosphingobium aromaticivorans

<400> 644

Thr Ala Val Lys Gln Gln Ser Phe Gly Met Glu Ala

1 5 10

<210> 645

<211> 16

<212> PRT

<213> Novosphingobium aromaticivorans

<400> 645

Ala Ala Ala Lys Val Thr Gln Ile His Lys Lys Leu Phe Asn Tyr Val

1 5 10 15

<210> 646

<211> 15

<212> PRT

<213> Novosphingobium aromaticivorans

<400> 646

Trp Ile Lys Val Leu Asn Asp Ile Arg Gln Tyr Thr Ala His Pro

1 5 10 15

<210> 647

<211> 13

<212> PRT

<213> Novosphingobium aromaticivorans

<400> 647

Gln Val Ser Phe Val Asn Glu Val Tyr Glu Lys Val Glu

1 5 10

<210> 648

<211> 11

<212> PRT

<213> Elizabethkingia anophelis

<400> 648

Gly Glu Ile Lys Tyr Trp Arg Thr Phe Gln Lys

1 5 10

<210> 649

<211> 16

<212> PRT

<213> Elizabethkingia anophelis

<400> 649

Ala Ile Ala Tyr Ile Arg Asp Ile Glu Thr Glu Phe Lys Ser Asp Phe

1 5 10 15

<210> 650

<211> 15

<212> PRT

<213> Elizabethkingia anophelis

<400> 650

Trp Met Val Lys Leu Glu Arg Ile Arg Asn Gln Asn Phe His Ser

1 5 10 15

<210> 651

<211> 13

<212> PRT

<213> Elizabethkingia anophelis

<400> 651

Glu Leu Ser Phe Leu Glu Glu Leu His Asp Trp Ile Tyr

1 5 10

<210> 652

<211> 12

<212> PRT

<213> Escherichia coli

<400> 652

Phe Ser Ala Leu Pro Arg Ile Ile Glu Tyr Ala Tyr

1 5 10

<210> 653

<211> 16

<212> PRT

<213> Escherichia coli

<400> 653

Pro Phe Leu Leu Leu Ser Glu Ile Glu Asn His Ile Arg Lys Leu Ile

1 5 10 15

<210> 654

<211> 9

<212> PRT

<213> Escherichia coli

<400> 654

Glu Ser Val Ala Asp Leu Thr Phe Gly

fifteen

<210> 655

<211> 15

<212> PRT

<213> Escherichia coli

<400> 655

Glu Leu Asp Lys Val Arg Ile Ile Arg Asn Asp Val Met His Phe

1 5 10 15

<210> 656

<211> 13

<212> PRT

<213> Escherichia coli

<400> 656

Asn His Glu Leu Leu His Asn Phe Val Arg Phe Ile His

1 5 10

<210> 657

<211> 12

<212> PRT

<213> Haloarcula marismortui

<400> 657

Phe Glu Leu Phe Asp Thr Leu Ala Glu Asp Asp Tyr

1 5 10

<210> 658

<211> 16

<212> PRT

<213> Haloarcula marismortui

<400> 658

Pro Phe Leu Gln Ile Gly Glu Ile Glu Glu Ser Leu Arg His Leu Phe

1 5 10 15

<210> 659

<211> 9

<212> PRT

<213> Haloarcula marismortui

<400> 659

Asp Arg Pro Glu Asp Phe Ser Phe Asp

fifteen

<210> 660

<211> 15

<212> PRT

<213> Haloarcula marismortui

<400> 660

Leu Leu Glu Asp Ile Arg Glu Thr Arg Asn Ala Leu Leu His Phe

1 5 10 15

<210> 661

<211> 13

<212> PRT

<213> Haloarcula marismortui

<400> 661

Asp Arg Asp Gln Leu Asp Met Ala His Gly Tyr Phe Thr

1 5 10

<210> 662

<211> 12

<212> PRT

<213> Nostoc sp.

<400> 662

Met Lys Leu Leu Pro Ile Leu Gln Gln Asn Pro Arg

1 5 10

<210> 663

<211> 15

<212> PRT

<213> Nostoc sp.

<400> 663

Phe Gly Leu Val Thr Leu Leu Glu Met Asn Leu Leu Arg Leu Val

1 5 10 15

<210> 664

<211> 9

<212> PRT

<213> Nostoc sp.

<400> 664

Asp Leu Leu Asp Tyr Leu Gln Phe Cys

fifteen

<210> 665

<211> 15

<212> PRT

<213> Nostoc sp.

<400> 665

Phe Leu Lys Ser Ala Glu Gln Leu Arg Asn Arg Leu Ala His Ala

1 5 10 15

<210> 666

<211> 13

<212> PRT

<213> Nostoc sp.

<400> 666

Ser Trp Asn Asp Leu Ile Ser Leu Ala Glu Ala Met Glu

1 5 10

<210> 667

<211> 10

<212> PRT

<213> Xanthobacter autotrophicus

<400> 667

Val Phe Glu Gly Met Glu Leu Leu Pro Ala

1 5 10

<210> 668

<211> 14

<212> PRT

<213> Xanthobacter autotrophicus

<400> 668

Ala Leu Ile Pro Phe Val Glu Lys Arg Leu Glu Thr Ser Leu

1 5 10

<210> 669

<211> 8

<212> PRT

<213> Xanthobacter autotrophicus

<400> 669

Glu Ala Phe Lys Ala Val Leu Gly

fifteen

<210> 670

<211> 15

<212> PRT

<213> Xanthobacter autotrophicus

<400> 670

Leu Val Asn Glu Leu Gly Asp Val Arg Asn Lys Leu Ser His Asn

1 5 10 15

<210> 671

<211> 13

<212> PRT

<213> Xanthobacter autotrophicus

<400> 671

Tyr Asp Asp Ala Glu Arg Ala Leu Asp Thr Met Arg Arg

1 5 10

<210> 672

<211> 10

<212> PRT

<213> Methanospirillum hungatei

<400> 672

Val Gly Arg Ala Met Asp Gln Leu Lys Thr

1 5 10

<210> 673

<211> 14

<212> PRT

<213> Methanospirillum hungatei

<400> 673

Gly Leu Met Arg Phe Val Glu Arg Glu Met Lys Ser Ala Tyr

1 5 10

<210> 674

<211> 8

<212> PRT

<213> Methanospirillum hungatei

<400> 674

Lys Val Phe Ser Gln Ile Leu Gly

fifteen

<210> 675

<211> 15

<212> PRT

<213> Methanospirillum hungatei

<400> 675

Leu Val Ser Glu Leu Arg Glu Thr Arg Asn Gln Trp Ala His Gln

1 5 10 15

<210> 676

<211> 13

<212> PRT

<213> Methanospirillum hungatei

<400> 676

Thr Asn Asp Thr Leu Arg Ala Leu Asp Ser Thr Ala Arg

1 5 10

<210> 677

<211> 10

<212> PRT

<213> Roseiflexus sp.

<400> 677

Ile Gly Lys Ala Leu Asp Leu Leu Arg Gln

1 5 10

<210> 678

<211> 14

<212> PRT

<213> Roseiflexus sp.

<400> 678

Gly Leu Gln Pro Phe Ile Glu Arg Glu Leu Gln Asn His Tyr

1 5 10

<210> 679

<211> 8

<212> PRT

<213> Roseiflexus sp.

<400> 679

Asp Val Phe Arg Lys Thr Leu Gly

fifteen

<210> 680

<211> 15

<212> PRT

<213> Roseiflexus sp.

<400> 680

Leu Val Ser Glu Leu Arg Glu Trp Arg Asn Lys Trp Ala His Gln

1 5 10 15

<210> 681

<211> 13

<212> PRT

<213> Roseiflexus sp.

<400> 681

Thr Asp Asp Thr Tyr Arg Val Leu Asp Ser Ala Ala Arg

1 5 10

<210> 682

<211> 10

<212> PRT

<213> Plasmodium yoelii

<400> 682

Ile Leu Asn Ile Phe His Ile Leu Ser Ala

1 5 10

<210> 683

<211> 14

<212> PRT

<213> Plasmodium yoelii

<400> 683

His Leu Ser Pro Ile Ile Glu Gln Ile Met Glu Met Glu Tyr

1 5 10

<210> 684

<211> 7

<212> PRT

<213> Plasmodium yoelii

<400> 684

Asp Ile Phe Glu Asn Arg Ile

fifteen

<210> 685

<211> 15

<212> PRT

<213> Plasmodium yoelii

<400> 685

Ile Leu Glu Asn Leu Gln Lys Ala Ser Ile Phe Trp Ala Asn Gln

1 5 10 15

<210> 686

<211> 13

<212> PRT

<213> Plasmodium yoelii

<400> 686

Glu Phe Phe Leu Ser Asn Leu Val Ser Ser Tyr Phe Phe

1 5 10

<210> 687

<211> 10

<212> PRT

<213> Theileria parva

<400> 687

Val Val Met Ile Phe Gln Cys Val Cys Asp

1 5 10

<210> 688

<211> 14

<212> PRT

<213> Theileria parva

<400> 688

Ala Phe Gln Pro Phe Ile Ser Lys Cys Met Leu Lys Lys Phe

1 5 10

<210> 689

<211> 7

<212> PRT

<213> Theileria parva

<400> 689

Asp Ile Phe Glu Gln Val Leu

fifteen

<210> 690

<211> 15

<212> PRT

<213> Theileria parva

<400> 690

His Leu Asn Thr Ile Gln Thr Ala Ser Ile Tyr Trp Ala Asn Gln

1 5 10 15

<210> 691

<211> 8

<212> PRT

<213> Theileria parva

<400> 691

Asn Tyr Gly Lys Cys Arg Lys Ile

fifteen

<210> 692

<211> 10

<212> PRT

<213> Daphnia pulex

<400> 692

Ser Ser Lys Glu Ser Ala Ala Ile Ala Ile

1 5 10

<210> 693

<211> 14

<212> PRT

<213> Daphnia pulex

<400> 693

Gly His Ile Val Phe Asp Thr Phe Leu Glu Asp Val Ala Pro

1 5 10

<210> 694

<211> 8

<212> PRT

<213> Daphnia pulex

<400> 694

Asp Cys Phe Ile Ile Pro Pro Gly

fifteen

<210> 695

<211> 15

<212> PRT

<213> Daphnia pulex

<400> 695

Ile Leu Glu Arg Ala Met Asp Gly Arg His Ala Val Ser His His

1 5 10 15

<210> 696

<211> 13

<212> PRT

<213> Daphnia pulex

<400> 696

Trp Glu Gln His Leu Lys Asp Tyr Val Tyr Ile Leu Thr

1 5 10

<210> 697

<211> 10

<212> PRT

<213> Homo sapiens

<400> 697

Ala Gly His Cys Leu Leu Leu Leu Arg Ser

1 5 10

<210> 698

<211> 16

<212> PRT

<213> Homo sapiens

<400> 698

Cys Leu Gln Gly Phe Val Gly Arg Glu Val Leu Ser Phe His Arg Gly

1 5 10 15

<210> 699

<211> 15

<212> PRT

<213> Homo sapiens

<400> 699

Lys Val Thr Glu Val Ile Lys Cys Arg Asn Glu Ile Met His Ser

1 5 10 15

<210> 700

<211> 13

<212> PRT

<213> Homo sapiens

<400> 700

Ser Ser Thr Trp Leu Arg Asp Phe Gln Met Lys Ile Gln

1 5 10

<210> 701

<211> 10

<212> PRT

<213> Branchiostoma floridae

<400> 701

Val Gly Ile Ala Leu Leu Thr Thr Arg Asp

1 5 10

<210> 702

<211> 16

<212> PRT

<213> Branchiostoma floridae

<400> 702

Gly Leu Thr Asn Val Thr Glu Gln Ala Ala Lys Glu Leu Gln Ala Glu

1 5 10 15

<210> 703

<211> 15

<212> PRT

<213> Branchiostoma floridae

<400> 703

Pro Leu Lys Asn Val Ile Glu Val Arg Asn Lys Thr Met His Ser

1 5 10 15

<210> 704

<211> 13

<212> PRT

<213> Branchiostoma floridae

<400> 704

Asp Arg Gln Thr Phe Asn Glu Tyr Met Asp Lys Met Glu

1 5 10

<210> 705

<211> 10

<212> PRT

<213> Homo sapiens

<400> 705

Val Ser Asp Leu Glu Lys Ser Leu Gly Thr

1 5 10

<210> 706

<211> 14

<212> PRT

<213> Homo sapiens

<400> 706

Gly Leu Ser Ser Ile Leu Glu Thr Glu Met Lys Ile Ala Phe

1 5 10

<210> 707

<211> 8

<212> PRT

<213> Homo sapiens

<400> 707

Lys His Trp Leu Ala Val Phe Gly

fifteen

<210> 708

<211> 16

<212> PRT

<213> Homo sapiens

<400> 708

Thr Ile Glu Ser Leu Tyr Lys Asn Leu Arg Lys Ala Asn Lys Ala Val

1 5 10 15

<210> 709

<211> 13

<212> PRT

<213> Homo sapiens

<400> 709

Ser Arg Ser Leu Leu His Ala Phe Ser Thr Arg Ser Asn

1 5 10

<210> 710

<211> 10

<212> PRT

<213> Ostreococcus lucimarinus

<400> 710

Met Glu Arg Leu Met Met Val Leu Asp His

1 5 10

<210> 711

<211> 14

<212> PRT

<213> Ostreococcus lucimarinus

<400> 711

Val Leu Ala Ile Val Leu Glu Gly Gly Leu Arg Ala Glu Phe

1 5 10

<210> 712

<211> 8

<212> PRT

<213> Ostreococcus lucimarinus

<400> 712

Ala Asn Trp Gly Ser Leu Phe Ser

fifteen

<210> 713

<211> 14

<212> PRT

<213> Ostreococcus lucimarinus

<400> 713

Glu Ile Glu Val Leu Leu Asp Ala Ala Ile Arg Gln Arg Lys

1 5 10

<210> 714

<211> 13

<212> PRT

<213> Ostreococcus lucimarinus

<400> 714

Ala Arg Asp Val Ser Ser Ala Ala Val Ala Leu Leu Asn

1 5 10

<210> 715

<211> 10

<212> PRT

<213> Branchiostoma floridae

<400> 715

Leu Cys Gly Met Lys Thr Leu Leu Lys Ala

1 5 10

<210> 716

<211> 14

<212> PRT

<213> Branchiostoma floridae

<400> 716

Val Leu Ala Val Val Leu Glu Thr Glu Met Lys Ala Val Phe

1 5 10

<210> 717

<211> 8

<212> PRT

<213> Branchiostoma floridae

<400> 717

Lys His Trp Ile Ala Val Phe Gly

fifteen

<210> 718

<211> 17

<212> PRT

<213> Branchiostoma floridae

<400> 718

His Leu Asp Ser Leu Val Lys His Phe Thr Arg Gly Arg Ser Tyr Gly

1 5 10 15

Val

<210> 719

<211> 13

<212> PRT

<213> Branchiostoma floridae

<400> 719

Ala Leu Gln Leu Val Arg Gln Leu His Asn His Ser Thr

1 5 10

<210> 720

<211> 10

<212> PRT

<213> Microcystis aeruginosa

<400> 720

Leu Asn Trp Leu Asp Gln Leu His Asp Asp

1 5 10

<210> 721

<211> 16

<212> PRT

<213> Microcystis aeruginosa

<400> 721

Leu Ile Glu Leu Cys Gly Trp Ile Glu Glu Thr Met Asp Asp Ile Val

1 5 10 15

<210> 722

<211> 9

<212> PRT

<213> Microcystis aeruginosa

<400> 722

Phe Arg Lys Met Leu Met Met Val Ile

fifteen

<210> 723

<211> 15

<212> PRT

<213> Microcystis aeruginosa

<400> 723

Tyr Leu Gly Asn Leu Lys Asp Ser Arg Asn Arg Ala Ala His Thr

1 5 10 15

<210> 724

<211> 13

<212> PRT

<213> Microcystis aeruginosa

<400> 724

Phe Asp Lys Ile Tyr Gly Leu Leu Lys Glu Leu Asp Ala

1 5 10

<210> 725

<211> 12

<212> PRT

<213> Lactococcus lactis

<400> 725

Leu Ser Glu Leu His Glu Phe Ile Lys Lys Leu Asn

1 5 10

<210> 726

<211> 16

<212> PRT

<213> Lactococcus lactis

<400> 726

Val Ile Arg Ser Cys Gly Ile Ile Glu Gln Leu Thr Lys Thr Leu Ile

1 5 10 15

<210> 727

<211> 9

<212> PRT

<213> Lactococcus lactis

<400> 727

Ile Asn Gly Leu Ile Asp Thr Phe Asp

fifteen

<210> 728

<211> 15

<212> PRT

<213> Lactococcus lactis

<400> 728

His Ile Asp Ser Leu Arg Gln Leu Arg Asn Ser Ile Ala His Gly

1 5 10 15

<210> 729

<211> 13

<212> PRT

<213> Lactococcus lactis

<400> 729

Met Gly Tyr Phe Asp Ser Cys Ile Ile Leu Met Phe Arg

1 5 10

<210> 730

<211> 12

<212> PRT

<213> Frankia sp.

<400> 730

Leu Ser Glu Leu Ala Ala Leu Val Gln Asp Gln Ala

1 5 10

<210> 731

<211> 16

<212> PRT

<213> Frankia sp.

<400> 731

Val Ile Arg Ser Cys Gly Tyr Leu Glu Gln Thr Val Ala Gly Thr Phe

1 5 10 15

<210> 732

<211> 9

<212> PRT

<213> Frankia sp.

<400> 732

Leu Glu Thr Leu Ala Gly Arg Phe Asp

fifteen

<210> 733

<211> 15

<212> PRT

<213> Frankia sp.

<400> 733

Glu Leu Ala Thr Leu Val Asp Arg Arg Asn Arg Ile Ala His Gly

1 5 10 15

<210> 734

<211> 13

<212> PRT

<213> Frankia sp.

<400> 734

Leu Glu Leu His Arg Val Ala Cys Glu Ala Ala Asp Trp

1 5 10

<210> 735

<211> 10

<212> PRT

<213> Neisseria meningitidis

<400> 735

Cys Cys Ser Ile Phe Ser Asp Phe Arg Met

1 5 10

<210> 736

<211> 16

<212> PRT

<213> Neisseria meningitidis

<400> 736

Leu Phe His Val Val Ser Ile Phe Glu Ile Val Leu Arg Asn Lys Ile

1 5 10 15

<210> 737

<211> 9

<212> PRT

<213> Neisseria meningitidis

<400> 737

Gln Leu Val Ala Gly Leu Gly Phe Gly

fifteen

<210> 738

<211> 15

<212> PRT

<213> Neisseria meningitidis

<400> 738

Glu Leu Ser Asn Ile Asn Lys Phe Arg Asn Arg Leu Ala His His

1 5 10 15

<210> 739

<211> 13

<212> PRT

<213> Neisseria meningitidis

<400> 739

Asp Val Asp Thr Ala Ser Val Phe Ser His Phe Ser Asp

1 5 10

<210> 740

<211> 10

<212> PRT

<213> Pseudomonas syringae

<400> 740

Leu Glu Lys His Phe Ser Ser Ala Arg Leu

1 5 10

<210> 741

<211> 16

<212> PRT

<213> Pseudomonas syringae

<400> 741

Met Met Pro Met Leu Ser Val Leu Glu Ile Ala Leu Lys Asn Gly Ile

1 5 10 15

<210> 742

<211> 9

<212> PRT

<213> Pseudomonas syringae

<400> 742

Lys Ile Val Ala Glu Leu Ala Phe Gly

fifteen

<210> 743

<211> 15

<212> PRT

<213> Pseudomonas syringae

<400> 743

Ala Leu Asn Leu Ile Arg Asn Leu Arg Asn Arg Val Phe His His

1 5 10 15

<210> 744

<211> 13

<212> PRT

<213> Pseudomonas syringae

<400> 744

Asp Pro Gln Leu Val Pro Trp Leu Ala Gln Tyr Asp Arg

1 5 10

<210> 745

<211> 10

<212> PRT

<213> Geobacter uraniireducens

<400> 745

Leu Arg Arg Ala Ile Ser His Glu Arg Leu

1 5 10

<210> 746

<211> 16

<212> PRT

<213> Geobacter uraniireducens

<400> 746

Leu Tyr Thr Pro Leu Gln Cys Leu Glu Val Cys Leu Arg Asn Ser Ile

1 5 10 15

<210> 747

<211> 9

<212> PRT

<213> Geobacter uraniireducens

<400> 747

Arg Ile Ile Pro Glu Leu Thr Phe Gly

fifteen

<210> 748

<211> 15

<212> PRT

<213> Geobacter uraniireducens

<400> 748

Arg Phe Asn His Ile Arg Thr Leu Arg Asn Arg Ile Phe His His

1 5 10 15

<210> 749

<211> 13

<212> PRT

<213> Geobacter uraniireducens

<400> 749

Asn Pro Ala Met Met Thr Phe Val Glu Pro Phe Asp Ser

1 5 10

<210> 750

<211> 16

<212> PRT

<213> Sulfuricurvum kujiense

<400> 750

Glu Glu Lys Ser Glu Phe Ile Arg Glu Phe Phe Lys Arg Thr Leu His

1 5 10 15

<210> 751

<211> 9

<212> PRT

<213> Sulfuricurvum kujiense

<400> 751

Thr Gln Thr Ile Asn Ser Phe Leu Gly

fifteen

<210> 752

<211> 14

<212> PRT

<213> Sulfuricurvum kujiense

<400> 752

Phe Arg Asn Tyr Leu Lys Arg Leu Arg Asn Ala Val Ser His

1 5 10

<210> 753

<211> 13

<212> PRT

<213> Sulfuricurvum kujiense

<400> 753

Val Asn Leu Leu Ile Thr Leu Leu Ser Arg Asn Ile Leu

1 5 10

<210> 754

<211> 13

<212> PRT

<213> Dethiobacter alkaliphilus

<400> 754

Gln Val Val Glu Lys Asp Phe Val Ala Arg Thr Met His

1 5 10

<210> 755

<211> 9

<212> PRT

<213> Dethiobacter alkaliphilus

<400> 755

Thr Leu Leu Ile Asn Cys Leu Leu Gly

fifteen

<210> 756

<211> 14

<212> PRT

<213> Dethiobacter alkaliphilus

<400> 756

Ala Ser Arg Phe Leu Gln Cys Met Arg Asn Ser Val Ala His

1 5 10

<210> 757

<211> 10

<212> PRT

<213> Dethiobacter alkaliphilus

<400> 757

Leu Ala Thr Lys Leu Ala Gln Tyr Val Gln

1 5 10

<210> 758

<211> 13

<212> PRT

<213> Klebsiella pneumoniae

<400> 758

Ser Asp Phe Glu Thr Asp Phe Val Gln Arg Thr Leu Ala

1 5 10

<210> 759

<211> 9

<212> PRT

<213> Klebsiella pneumoniae

<400> 759

Thr Leu Thr Leu Asn Cys Leu Leu Gly

fifteen

<210> 760

<211> 14

<212> PRT

<213> Klebsiella pneumoniae

<400> 760

Leu Arg Gln Leu Ile His Lys Met Arg Asn Ser Val Ala His

1 5 10

<210> 761

<211> 13

<212> PRT

<213> Klebsiella pneumoniae

<400> 761

Leu Leu Pro Phe Leu Lys Tyr Tyr Ala Thr Leu Leu Leu

1 5 10

<210> 762

<211> 10

<212> PRT

<213> Lactobacillus casei

<400> 762

Lys Ile Asp Arg Glu Met Phe Trp Arg Arg

1 5 10

<210> 763

<211> 16

<212> PRT

<213> Lactobacillus casei

<400> 763

Tyr Leu Leu Leu Tyr Ser Ser Trp Glu Gly Phe Ile Arg Ser Ile Ala

1 5 10 15

<210> 764

<211> 9

<212> PRT

<213> Lactobacillus casei

<400> 764

Leu Ala Arg Ile Val Ser Val Leu Asp

fifteen

<210> 765

<211> 14

<212> PRT

<213> Lactobacillus casei

<400> 765

Asp Arg Asp Leu Leu Lys Val Arg Asn Glu Ile Ala His Gly

1 5 10

<210> 766

<211> 13

<212> PRT

<213> Lactobacillus casei

<400> 766

Thr Val Ser His Val Leu Glu Met Met Asp Leu Phe Ser

1 5 10

<210> 767

<211> 10

<212> PRT

<213> Caulobacter sp.

<400> 767

Asp Leu Asp Ala Ala Arg Leu Arg Arg Ala

1 5 10

<210> 768

<211> 16

<212> PRT

<213> Caulobacter sp.

<400> 768

Ile Val Leu Ala Tyr Ser His Trp Glu Gly Phe Tyr Asn Glu Cys Ile

1 5 10 15

<210> 769

<211> 9

<212> PRT

<213> Caulobacter sp.

<400> 769

Leu Lys Glu Asn Phe Arg Ile Leu Gly

fifteen

<210> 770

<211> 14

<212> PRT

<213> Caulobacter sp.

<400> 770

Asn Lys Glu Leu Val Gly Trp Arg His Ser Ile Ala His Gly

1 5 10

<210> 771

<211> 13

<212> PRT

<213> Caulobacter sp.

<400> 771

His Ile Ile Leu Thr Asn Ser Leu Leu Leu Thr Leu Ser

1 5 10

<210> 772

<211> 10

<212> PRT

<213> Microcystis aeruginosa

<400> 772

Asn Leu Asp Glu Asp Met Ala Trp Arg Ile

1 5 10

<210> 773

<211> 16

<212> PRT

<213> Microcystis aeruginosa

<400> 773

Ile Thr Thr Leu Tyr Ala His Trp Glu Gly Phe Ile Lys Tyr Ala Ala

1 5 10 15

<210> 774

<211> 9

<212> PRT

<213> Microcystis aeruginosa

<400> 774

Phe Thr Asp Ile Cys Thr Ile Leu Gly

fifteen

<210> 775

<211> 14

<212> PRT

<213> Microcystis aeruginosa

<400> 775

Asp Glu Gln Leu Leu Thr Gln Arg Asn Lys Ile Ala His Gly

1 5 10

<210> 776

<211> 13

<212> PRT

<213> Microcystis aeruginosa

<400> 776

Thr Tyr Asn Leu Val Ile Lys Leu Ile Arg Asp Phe Lys

1 5 10

<210> 777

<211> 11

<212> PRT

<213> Arabidopsis thaliana

<400> 777

Pro Trp Leu Ser Trp Glu Glu Trp Asp Ser Val

1 5 10

<210> 778

<211> 15

<212> PRT

<213> Arabidopsis thaliana

<400> 778

Gly Ser Leu Pro Ala Pro Val Asp Val Thr Cys Ser Leu Ile Glu

1 5 10 15

<210> 779

<211> 9

<212> PRT

<213> Arabidopsis thaliana

<400> 779

Ile Ala Asp Ala Ala Arg Ala Ile Gly

fifteen

<210> 780

<211> 15

<212> PRT

<213> Arabidopsis thaliana

<400> 780

Ile Pro Arg Lys Leu Ile Asp Leu Arg His Glu Gly Ser His Arg

1 5 10 15

<210> 781

<211> 13

<212> PRT

<213> Arabidopsis thaliana

<400> 781

Ala Ala Asp Glu Ala Leu Glu Trp Leu Lys Ser Tyr Tyr

1 5 10

<210> 782

<211> 11

<212> PRT

<213> Homo sapiens

<400> 782

Ala Trp Leu Ser Arg Ala Glu Trp Asp Gln Val

1 5 10

<210> 783

<211> 16

<212> PRT

<213> Homo sapiens

<400> 783

Gly Asn Glu Leu Pro Leu Ala Val Ala Ser Thr Ala Asp Leu Ile Arg

1 5 10 15

<210> 784

<211> 9

<212> PRT

<213> Homo sapiens

<400> 784

Leu Lys Cys Leu Ala Gln Glu Val Asn

fifteen

<210> 785

<211> 15

<212> PRT

<213> Homo sapiens

<400> 785

Ile Pro Asp Trp Ile Val Asp Leu Arg His Glu Leu Thr His Lys

1 5 10 15

<210> 786

<211> 13

<212> PRT

<213> Homo sapiens

<400> 786

Gly Cys Tyr Phe Val Leu Asp Trp Leu Gln Lys Thr Tyr

1 5 10

<210> 787

<211> 11

<212> PRT

<213> Saccharomyces cerevisiae

<400> 787

Pro Trp Arg Asp Phe Ala Glu Leu Glu Glu Leu

1 5 10

<210> 788

<211> 16

<212> PRT

<213> Saccharomyces cerevisiae

<400> 788

Ser Gln Tyr Leu Pro His Val Val Asp Ser Thr Ala Gln Ile Thr Cys

1 5 10 15

<210> 789

<211> 9

<212> PRT

<213> Saccharomyces cerevisiae

<400> 789

Leu His Thr Leu Ala Ala Lys Ile Gly

fifteen

<210> 790

<211> 15

<212> PRT

<213> Saccharomyces cerevisiae

<400> 790

Leu Pro Ser Trp Phe Val Asp Leu Arg His Trp Gly Thr His Glu

1 5 10 15

<210> 791

<211> 13

<212> PRT

<213> Saccharomyces cerevisiae

<400> 791

Ala Ala Asn Glu Ala Leu Ser Trp Leu Tyr Asp His Tyr

1 5 10

<210> 792

<211> 11

<212> PRT

<213> Streptococcus pneumoniae

<400> 792

Ser Lys Pro Cys Ile Glu Ala Glu Asn Met Ile

1 5 10

<210> 793

<211> 16

<212> PRT

<213> Streptococcus pneumoniae

<400> 793

Ala Phe Met Ala Arg Arg Ala Leu Glu Gln Ala Val His Trp Ile Tyr

1 5 10 15

<210> 794

<211> 8

<212> PRT

<213> Streptococcus pneumoniae

<400> 794

Ser Ser Leu Val Trp Asp Asp Asp

fifteen

<210> 795

<211> 15

<212> PRT

<213> Streptococcus pneumoniae

<400> 795

Gln Ile Val Leu Leu Ile Arg Trp Gly Asn His Ala Ala His Gly

1 5 10 15

<210> 796

<211> 13

<212> PRT

<213> Streptococcus pneumoniae

<400> 796

Ala Leu His His Leu Tyr Gln Phe Val Asn Phe Ile Asp

1 5 10

<210> 797

<211> 11

<212> PRT

<213> Microcystis aeruginosa

<400> 797

Tyr Asp His Ala Ser Gln Ala Glu Gly Leu Val

1 5 10

<210> 798

<211> 16

<212> PRT

<213> Microcystis aeruginosa

<400> 798

Cys Phe Tyr Thr Arg Phe Val Leu Glu Gln Met Val Cys Trp Leu Tyr

1 5 10 15

<210> 799

<211> 8

<212> PRT

<213> Microcystis aeruginosa

<400> 799

Gly Ala Leu Ile His Glu Gln Thr

fifteen

<210> 800

<211> 15

<212> PRT

<213> Microcystis aeruginosa

<400> 800

Lys Ile Arg Thr Ile His Lys Val Gly Asn Asn Ala Ala His Asp

1 5 10 15

<210> 801

<211> 13

<212> PRT

<213> Microcystis aeruginosa

<400> 801

Leu Ile Glu Glu Leu Phe His Leu Thr Tyr Trp Leu Val

1 5 10

<210> 802

<211> 11

<212> PRT

<213> Escherichia coli

<400> 802

Tyr Ala Ile Ala Cys Ala Ala Glu Asn Asn Tyr

1 5 10

<210> 803

<211> 16

<212> PRT

<213> Escherichia coli

<400> 803

Leu Ile Lys Met Arg Met Phe Gly Glu Ala Thr Ala Lys His Leu Gly

1 5 10 15

<210> 804

<211> 8

<212> PRT

<213> Escherichia coli

<400> 804

His Asp Leu Leu Arg Glu Leu Gly

fifteen

<210> 805

<211> 15

<212> PRT

<213> Escherichia coli

<400> 805

Val Phe His Lys Leu Arg Arg Ile Gly Asn Gln Ala Val His Glu

1 5 10 15

<210> 806

<211> 13

<212> PRT

<213> Escherichia coli

<400> 806

Cys Leu Arg Leu Gly Phe Arg Leu Ala Val Trp Tyr Tyr

1 5 10

<210> 807

<211> 11

<212> PRT

<213> Bradyrhizobium japonicum

<400> 807

Val Gln Lys Leu Ile Lys Ala Ser Gln Leu Ala

1 5 10

<210> 808

<211> 16

<212> PRT

<213> Bradyrhizobium japonicum

<400> 808

Leu Thr Glu Val Arg Arg Ala Met Lys Ala Ala Ala Asp Leu Phe Trp

1 5 10 15

<210> 809

<211> 10

<212> PRT

<213> Bradyrhizobium japonicum

<400> 809

Leu Asn Arg Leu Gln Glu Phe Ala Arg Val

1 5 10

<210> 810

<211> 13

<212> PRT

<213> Bradyrhizobium japonicum

<400> 810

Arg Arg Leu Asn Asp Leu Ala Ser Lys Gly Val His Ala

1 5 10

<210> 811

<211> 13

<212> PRT

<213> Bradyrhizobium japonicum

<400> 811

Ala Glu Ala Arg Gln Gly Leu Val Gly Leu Tyr Phe Phe

1 5 10

<210> 812

<211> 11

<212> PRT

<213> Leptospira meyeri

<400> 812

Leu Pro Lys Phe Ser Ala Ile Tyr Ser Asn Leu

1 5 10

<210> 813

<211> 16

<212> PRT

<213> Leptospira meyeri

<400> 813

Val His Ser Cys Arg Arg Leu Leu Gln Ser Val Ala Asp Lys Leu Met

1 5 10 15

<210> 814

<211> 10

<212> PRT

<213> Leptospira meyeri

<400> 814

Ile Asn Arg Leu Ile Tyr Tyr Ile Glu Thr

1 5 10

<210> 815

<211> 13

<212> PRT

<213> Leptospira meyeri

<400> 815

Asp Ser Val Phe Gln Ala Ser Gln Lys Gly Ser His Ser

1 5 10

<210> 816

<211> 13

<212> PRT

<213> Leptospira meyeri

<400> 816

Gln Glu Ala Asp Arg Tyr Val Ile His Thr Phe Leu Leu

1 5 10

<210> 817

<211> 11

<212> PRT

<213> Bacteroides coprosuis

<400> 817

Val Val Asp Asp Arg Asp Phe Ser Leu Leu Ala

1 5 10

<210> 818

<211> 16

<212> PRT

<213> Bacteroides coprosuis

<400> 818

Leu Asp Arg Leu His Thr Tyr Val Ile Lys Phe Ile Arg Gln Leu Cys

1 5 10 15

<210> 819

<211> 9

<212> PRT

<213> Bacteroides coprosuis

<400> 819

Phe Gly Lys Tyr Val Lys Phe Ile Val

fifteen

<210> 820

<211> 16

<212> PRT

<213> Bacteroides coprosuis

<400> 820

Ile Glu Ala Phe Asn Asp Ile Arg Asn Asn Lys Ser Phe Ala His Asp

1 5 10 15

<210> 821

<211> 13

<212> PRT

<213> Bacteroides coprosuis

<400> 821

Tyr Ala Glu Ser Val Leu Ile Phe Asn Asn Val Thr Asn

1 5 10

<210> 822

<211> 10

<212> PRT

<213> Escherichia coli

<400> 822

Asn Val Asn Glu Asn Ile Tyr Gln Ala Leu

1 5 10

<210> 823

<211> 16

<212> PRT

<213> Escherichia coli

<400> 823

Tyr Asp Arg Val His Thr Ala Leu His Ala Ser Leu Arg Gln Met Cys

1 5 10 15

<210> 824

<211> 9

<212> PRT

<213> Escherichia coli

<400> 824

Leu Ser Leu Ile Thr Ala His Leu Lys

fifteen

<210> 825

<211> 16

<212> PRT

<213> Escherichia coli

<400> 825

Leu His Gly Ile Asn Asn Leu Arg Asn Asn Tyr Ser Met Ala His Pro

1 5 10 15

<210> 826

<211> 13

<212> PRT

<213> Escherichia coli

<400> 826

Glu Ala Asp Ala Arg Phe Ala Ile Asn Leu Val Arg Ser

1 5 10

<210> 827

<211> 11

<212> PRT

<213> Lactococcus lactis

<400> 827

Ile Met Asn Ile Gly Tyr Val Glu Lys Ile Leu

1 5 10

<210> 828

<211> 16

<212> PRT

<213> Lactococcus lactis

<400> 828

Val Thr Lys Ser Arg Thr Ile Ile Glu Thr Val Phe Ile Ala Ile Leu

1 5 10 15

<210> 829

<211> 9

<212> PRT

<213> Lactococcus lactis

<400> 829

Arg Ser Leu Val Asn Lys Thr Leu Gly

fifteen

<210> 830

<211> 16

<212> PRT

<213> Lactococcus lactis

<400> 830

Val Asp Ser Ile Thr Thr Met Arg Asn Ile Asn Ser Asp Ser His Gly

1 5 10 15

<210> 831

<211> 13

<212> PRT

<213> Lactococcus lactis

<400> 831

Glu Ala Glu Ala Glu Leu Ile Leu Asn Ser Ala Val Asn

1 5 10

<210> 832

<211> 11

<212> PRT

<213> Peptoniphilus indolicus

<400> 832

Phe Leu Tyr Leu Lys Thr Leu Lys Asn Lys Glu

1 5 10

<210> 833

<211> 16

<212> PRT

<213> Peptoniphilus indolicus

<400> 833

Arg Gly Ile Thr Pro Leu Val Thr Glu Leu Phe Ile Leu Ile Ile Asp

1 5 10 15

<210> 834

<211> 9

<212> PRT

<213> Peptoniphilus indolicus

<400> 834

Leu Ile Glu Ile Ile Lys Asn Glu Arg

fifteen

<210> 835

<211> 15

<212> PRT

<213> Peptoniphilus indolicus

<400> 835

Ile Arg Asp Val Glu Gly Lys Leu Arg Asn Arg Ala Ala His Glu

1 5 10 15

<210> 836

<211> 13

<212> PRT

<213> Peptoniphilus indolicus

<400> 836

Gly Asn Asn His Tyr Asp Ser Tyr Asp Leu Met Asn Lys

1 5 10

<210> 837

<211> 11

<212> PRT

<213> Mycobacterium tuberculosis

<400> 837

Ile Ser Ala Leu Ala Leu Leu Ala Lys Arg Glu

1 5 10

<210> 838

<211> 16

<212> PRT

<213> Mycobacterium tuberculosis

<400> 838

Arg Ser Ala Thr Pro Ala Ile Thr Ile Val Leu Arg Ala Ala Val Ala

1 5 10 15

<210> 839

<211> 9

<212> PRT

<213> Mycobacterium tuberculosis

<400> 839

Trp Leu Ala Leu Leu Arg Gln Phe Ala

fifteen

<210> 840

<211> 15

<212> PRT

<213> Mycobacterium tuberculosis

<400> 840

Leu Gly Arg Phe Glu Ser Arg Val Arg Asn Thr Ala Ala His Glu

1 5 10 15

<210> 841

<211> 11

<212> PRT

<213> Mycobacterium tuberculosis

<400> 841

Ala Asp Leu Thr Leu Tyr Asp Arg Leu Asn Asp

1 5 10

<210> 842

<211> 12

<212> PRT

<213> Streptococcus thermophilus

<400> 842

Tyr Leu Met Ile Asp Val Leu Lys Glu Arg Glu His

1 5 10

<210> 843

<211> 16

<212> PRT

<213> Streptococcus thermophilus

<400> 843

Ile Glu Glu Ile Ile Lys Lys Asp His Glu Gly Leu Ile Val Phe Asp

1 5 10 15

<210> 844

<211> 9

<212> PRT

<213> Streptococcus thermophilus

<400> 844

Tyr Leu Asn Ile Leu Glu Phe Tyr Glu

fifteen

<210> 845

<211> 14

<212> PRT

<213> Streptococcus thermophilus

<400> 845

Ile Leu Ser Leu Asn Gly Glu Arg Asn Lys Val Ala His Gly

1 5 10

<210> 846

<211> 13

<212> PRT

<213> Streptococcus thermophilus

<400> 846

Asp Ser Ser Tyr Phe Asn Tyr Tyr Asp Lys Gln Asn Lys

1 5 10

<210> 847

<211> 10

<212> PRT

<213> Synechocystis sp.

<400> 847

Leu Ile Ser Val Val Ala Phe Arg Leu Gly

1 5 10

<210> 848

<211> 16

<212> PRT

<213> Synechocystis sp.

<400> 848

Ile Leu Asp His Arg Lys Gln Ile Asn Phe Ala Leu Asn Asn Gly Gly

1 5 10 15

<210> 849

<211> 10

<212> PRT

<213> Synechocystis sp.

<400> 849

Thr Glu Ile Arg Asn Asp Leu Ala His Cys

1 5 10

<210> 850

<211> 12

<212> PRT

<213> Synechocystis sp.

<400> 850

Asn Lys Ile Phe Pro Gln Leu Glu Glu Ile Ala Asn

1 5 10

<210> 851

<211> 5

<212> PRT

<213> Methanocaldococcus jannaschii

<400> 851

Lys Asn Thr Leu Phe

fifteen

<210> 852

<211> 14

<212> PRT

<213> Methanocaldococcus jannaschii

<400> 852

Lys Glu Asn Pro Asn Ser Gln Tyr Ile Lys Asn Glu Ile Ser

1 5 10

<210> 853

<211> 15

<212> PRT

<213> Methanocaldococcus jannaschii

<400> 853

Glu Asn Ile Asp Lys Phe Lys Ile Arg Asn Phe Leu Ala His Ala

1 5 10 15

<210> 854

<211> 13

<212> PRT

<213> Methanocaldococcus jannaschii

<400> 854

Ser Glu Lys Thr Ser Leu Arg Tyr Asn Lys Asn Tyr Ile

1 5 10

<210> 855

<211> 11

<212> PRT

<213> Pyrococcus furiosus

<400> 855

Ser Lys Ile Phe Glu Ser Leu Pro Arg Ile Gly

1 5 10

<210> 856

<211> 15

<212> PRT

<213> Pyrococcus furiosus

<400> 856

Arg Gln Val Glu Trp Leu Arg Asn Leu Val Tyr Gly Arg Leu Trp

1 5 10 15

<210> 857

<211> 15

<212> PRT

<213> Pyrococcus furiosus

<400> 857

Thr Ile Glu Ser Pro Asn Val Val Arg Asn Phe Ile Ala His Ser

1 5 10 15

<210> 858

<211> 13

<212> PRT

<213> Pyrococcus furiosus

<400> 858

Asp Lys Glu Lys Ala Ala Asn Leu Ala Tyr Glu Ala Leu

1 5 10

<210> 859

<211> 5

<212> PRT

<213> Sulfolobus solfataricus

<400> 859

Ala Glu Thr Tyr Ala

fifteen

<210> 860

<211> 13

<212> PRT

<213> Sulfolobus solfataricus

<400> 860

Asp Lys Val Thr Arg Ala Ile Ile Glu Asn Glu Val Asp

1 5 10

<210> 861

<211> 13

<212> PRT

<213> Sulfolobus solfataricus

<400> 861

Gly Lys Gly Phe Asp Lys Arg Ile Leu Tyr Ala His Gly

1 5 10

<210> 862

<211> 10

<212> PRT

<213> Sulfolobus solfataricus

<400> 862

Asp Lys Ile Asp Glu Ile Glu Arg Gln Ile

1 5 10

<210> 863

<211> 12

<212> PRT

<213> Desulfococcus oleovorans

<400> 863

Phe Ala Asn Ala Glu Arg Arg Phe Asp Glu Gly Lys

1 5 10

<210> 864

<211> 16

<212> PRT

<213> Desulfococcus oleovorans

<400> 864

Val Leu Arg Leu Tyr Arg Ile Val Glu Met Ala Gly Gln Gln Arg Leu

1 5 10 15

<210> 865

<211> 9

<212> PRT

<213> Desulfococcus oleovorans

<400> 865

Gly Tyr Ser Leu Leu Lys Glu Met Gly

fifteen

<210> 866

<211> 17

<212> PRT

<213> Desulfococcus oleovorans

<400> 866

Ser Phe Leu Lys Ile Gln Asp Ser Arg Asn His Ser Phe Leu Ala His

1 5 10 15

gly

<210> 867

<211> 13

<212> PRT

<213> Desulfococcus oleovorans

<400> 867

Tyr Met Ser Leu Arg Asp Phe Ile Val Ser Leu Asn Ile

1 5 10

<210> 868

<211> 12

<212> PRT

<213> Oscillochloris trichoides

<400> 868

Leu Arg Asn Ala Glu Arg Arg Ala Ala Arg Ala Arg

1 5 10

<210> 869

<211> 16

<212> PRT

<213> Oscillochloris trichoides

<400> 869

Val Ala Arg Leu Tyr Arg Ala Thr Glu Leu Phe Ala Gln Ile Arg Leu

1 5 10 15

<210> 870

<211> 9

<212> PRT

<213> Oscillochloris trichoides

<400> 870

Ser Tyr Ala Leu Leu Gly Lys Leu Asp

fifteen

<210> 871

<211> 17

<212> PRT

<213> Oscillochloris trichoides

<400> 871

Pro Leu Asn Asn Ala Leu Thr Arg Arg Asn Gln Ser Ile Leu Ala His

1 5 10 15

gly

<210> 872

<211> 13

<212> PRT

<213> Oscillochloris trichoides

<400> 872

Tyr His Asp Leu Ala Ser His Leu Tyr Thr Leu Ile Asn

1 5 10

<210> 873

<211> 12

<212> PRT

<213> Homo sapiens

<400> 873

Phe Pro Glu Ile Phe Asp Ala Leu Glu Ser Leu Gln

1 5 10

<210> 874

<211> 14

<212> PRT

<213> Homo sapiens

<400> 874

Lys Leu Thr Ser Cys Leu Glu Arg Ala Leu Gly Asp Val Phe

1 5 10

<210> 875

<211> 9

<212> PRT

<213> Homo sapiens

<400> 875

Ser Glu Glu Leu Ala Gln Val Phe Ser

fifteen

<210> 876

<211> 15

<212> PRT

<213> Homo sapiens

<400> 876

Gly Ser Pro Cys Gly Leu Asn Leu Arg Asn Val Leu Trp His Gly

1 5 10 15

<210> 877

<211> 13

<212> PRT

<213> Homo sapiens

<400> 877

Tyr Cys Ser Met Met Ile Leu Leu Thr Ala Gly Leu Gly

1 5 10

<210> 878

<211> 12

<212> PRT

<213> Entamoeba histolytica

<400> 878

Trp Phe Glu Ser Phe Gln Glu Ile Ile Gln Thr Pro

1 5 10

<210> 879

<211> 14

<212> PRT

<213> Entamoeba histolytica

<400> 879

Leu Leu Ser Val Gln Phe Asn Val His Leu Lys Asp Asn Ile

1 5 10

<210> 880

<211> 9

<212> PRT

<213> Entamoeba histolytica

<400> 880

Lys Met Tyr Glu Glu His Thr Val Pro

fifteen

<210> 881

<211> 15

<212> PRT

<213> Entamoeba histolytica

<400> 881

Gly Pro Pro Thr Gly Leu Asn Leu Arg Asn Leu Leu Trp His Gly

1 5 10 15

<210> 882

<211> 13

<212> PRT

<213> Entamoeba histolytica

<400> 882

His Ile Cys Leu Leu Ile Ile Leu Tyr Gln Thr Ile Gln

1 5 10

<210> 883

<211> 12

<212> PRT

<213> Staphylococcus aureus

<400> 883

Ile Glu His Gly Ile Ser Arg Phe Leu Glu Lys Asp

1 5 10

<210> 884

<211> 14

<212> PRT

<213> Staphylococcus aureus

<400> 884

Ile Leu Val Pro Gln Phe Glu Ser Thr Val Arg Arg Met Phe

1 5 10

<210> 885

<211> 9

<212> PRT

<213> Staphylococcus aureus

<400> 885

Arg Asp Asp Val Lys Ser Thr Leu Gly

fifteen

<210> 886

<211> 15

<212> PRT

<213> Staphylococcus aureus

<400> 886

Val Glu Gln Ser Gly Leu Asn Leu Arg Asn Glu Ile Ala His Gly

1 5 10 15

<210> 887

<211> 12

<212> PRT

<213> Staphylococcus aureus

<400> 887

Lys Cys Ile Leu Val Ile Tyr Leu Phe Leu Ile Leu

1 5 10

<210> 888

<211> 12

<212> PRT

<213> Cyanothece sp.

<400> 888

Leu Leu Lys Gly Ile Gln Ala Tyr Leu Glu Glu Asp

1 5 10

<210> 889

<211> 14

<212> PRT

<213> Cyanothece sp.

<400> 889

Leu Leu Ile Pro Gln Ile Glu Ala Ala Ile Arg Asn Leu Val

1 5 10

<210> 890

<211> 9

<212> PRT

<213> Cyanothece sp.

<400> 890

Ser Glu Gln Val Lys Gln Ser Leu Gly

fifteen

<210> 891

<211> 15

<212> PRT

<213> Cyanothece sp.

<400> 891

Thr Asp Gln Arg Gly Trp Asn Val Arg Asn Asn Val Cys His Gly

1 5 10 15

<210> 892

<211> 12

<212> PRT

<213> Cyanothece sp.

<400> 892

Leu Thr Glu Arg Leu Ile His Ile Leu Leu Ile Leu

1 5 10

<210> 893

<211> 9

<212> PRT

<213> Sulfolobus solfataricus

<400> 893

Ile Ser Thr Ser Ala Glu Val Tyr Tyr

fifteen

<210> 894

<211> 16

<212> PRT

<213> Sulfolobus solfataricus

<400> 894

Cys Glu Lys Tyr Tyr Lys Ala Ala Glu Glu Ala Ile Lys Leu Leu Val

1 5 10 15

<210> 895

<211> 8

<212> PRT

<213> Sulfolobus solfataricus

<400> 895

Lys Leu Leu Arg Ser Asn Asn Thr

fifteen

<210> 896

<211> 15

<212> PRT

<213> Sulfolobus solfataricus

<400> 896

Leu Trp Lys Ser Ala Trp Thr Leu His Val Glu Gly Phe His Glu

1 5 10 15

<210> 897

<211> 13

<212> PRT

<213> Sulfolobus solfataricus

<400> 897

Leu Lys Glu Asp Val Arg Lys Leu Val Ile Phe Ala Val

1 5 10

<210> 898

<211> 9

<212> PRT

<213> Pyrobaculum aerophilum

<400> 898

Tyr Ala Glu Ala Ala Arg Glu Leu Leu

fifteen

<210> 899

<211> 16

<212> PRT

<213> Pyrobaculum aerophilum

<400> 899

Ser Glu Lys Ala Trp Gly Ala Ala Ala Leu Ala Val Lys Ala Tyr Ala

1 5 10 15

<210> 900

<211> 7

<212> PRT

<213> Pyrobaculum aerophilum

<400> 900

Lys Ile Ala Gly Glu Leu Gly

fifteen

<210> 901

<211> 14

<212> PRT

<213> Pyrobaculum aerophilum

<400> 901

Ala Trp Ala Gln Ala Asn Ala Met His Ile Asn Phe Tyr Glu

1 5 10

<210> 902

<211> 13

<212> PRT

<213> Pyrobaculum aerophilum

<400> 902

Ala Leu Lys Lys Val Ser Arg Leu Val Glu Glu Leu Thr

1 5 10

<210> 903

<211> 11

<212> PRT

<213> Homo sapiens

<400> 903

Arg Arg Trp Leu Arg Gln Ala Arg Ala Asn Phe

1 5 10

<210> 904

<211> 16

<212> PRT

<213> Homo sapiens

<400> 904

Asn Glu Trp Val Cys Phe Lys Cys Tyr Leu Ser Thr Lys Leu Ala Leu

1 5 10 15

<210> 905

<211> 8

<212> PRT

<213> Homo sapiens

<400> 905

Ala Gln Lys Ile Glu Glu Tyr Ser

fifteen

<210> 906

<211> 15

<212> PRT

<213> Homo sapiens

<400> 906

Val His Thr Leu Glu Ala Tyr Gly Val Asp Ser Leu Lys Thr Arg

1 5 10 15

<210> 907

<211> 13

<212> PRT

<213> Homo sapiens

<220>

<221> MOD_RES

<222> (2)..(2)

<223> Any amino acids

<400> 907

Val Xaa Glu Cys Thr Ala Cys Ile Ile Ile Lys Leu Glu

1 5 10

<210> 908

<211> 11

<212> PRT

<213> Heamophilus influenzae

<400> 908

Lys Leu Asn Leu Asn Val Leu Asp Ala Ala Phe

1 5 10

<210> 909

<211> 16

<212> PRT

<213> Heamophilus influenzae

<220>

<221> MOD_RES

<222> (14)..(15)

<223> Any amino acids

<400> 909

Ile Gln Lys Phe Glu Phe Val Tyr Glu Leu Ser Leu Lys Xaa Xaa Lys

1 5 10 15

<210> 910

<211> 8

<212> PRT

<213> Heamophilus influenzae

<400> 910

Leu Arg Glu Ala Leu Arg Phe Gly

fifteen

<210> 911

<211> 15

<212> PRT

<213> Heamophilus influenzae

<220>

<221> MOD_RES

<222>(8)..(8)

<223> Any amino acids

<400> 911

Lys Trp Val Ala Tyr Arg Asp Xaa Arg Asn Ile Thr Ser His Thr

1 5 10 15

<210> 912

<211> 13

<212> PRT

<213> Heamophilus influenzae

<400> 912

Asp Phe Leu Ile Glu Ser Ser Phe Leu Leu Glu Gln Leu

1 5 10

<210> 913

<211> 6

<212> PRT

<213> Thermus thermophilus

<220>

<221> MOD_RES

<222> (1)..(1)

<223> Any amino acids

<400> 913

Xaa Ala Glu Lys Ala Leu

fifteen

<210> 914

<211> 16

<212> PRT

<213> Thermus thermophilus

<400> 914

Ile Gln Arg Phe Glu Tyr Thr Phe Glu Ala Phe Trp Lys Ala Leu Gln

1 5 10 15

<210> 915

<211> 8

<212> PRT

<213> Thermus thermophilus

<400> 915

Ile Arg Leu Ala Arg Glu Val Gly

fifteen

<210> 916

<211> 15

<212> PRT

<213> Thermus thermophilus

<220>

<221> MOD_RES

<222> (5)..(5)

<223> Any amino acids

<400> 916

Leu Ala Leu Gly Xaa Val Asp Asp Arg Ser Leu Thr Val His Thr

1 5 10 15

<210> 917

<211> 13

<212> PRT

<213> Thermus thermophilus

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Any amino acids

<400> 917

Ile Phe Arg Arg Leu Pro Asp Tyr Ala Arg Leu Xaa Glu

1 5 10

<210> 918

<211> 11

<212> PRT

<213> Rhodococcus equi

<400> 918

Val Asn Leu Leu Arg Arg Ala Asp Gly Leu Leu

1 5 10

<210> 919

<211> 16

<212> PRT

<213> Rhodococcus equi

<400> 919

Phe Cys Ala Ala Tyr Val Gly Ala Leu Arg Gly Ala Ala Ala Val Leu

1 5 10 15

<210> 920

<211> 8

<212> PRT

<213> Rhodococcus equi

<400> 920

Trp Val Leu Met Ala Arg Ala Glu

fifteen

<210> 921

<211> 15

<212> PRT

<213> Rhodococcus equi

<400> 921

Tyr Phe Ala Gly Tyr Ser Gly Leu Arg Ala Asp Leu Glu Ala Gly

1 5 10 15

<210> 922

<211> 13

<212> PRT

<213> Rhodococcus equi

<400> 922

Asp Ala Glu Glu Val Asp Gly Phe Tyr Ala Glu Val Gly

1 5 10

<210> 923

<211> 11

<212> PRT

<213> Streptomyces avermitilis

<400> 923

Leu Asp Leu Leu Ala Gln Ala Arg Ala Gly Leu

1 5 10

<210> 924

<211> 16

<212> PRT

<213> Streptomyces avermitilis

<400> 924

Tyr Ala Thr Ala His Leu Ala Ala Leu Arg Thr Ala Ala Ala Val Leu

1 5 10 15

<210> 925

<211> 8

<212> PRT

<213> Streptomyces avermitilis

<400> 925

Trp Glu Val Leu Pro Glu Ile Ala

fifteen

<210> 926

<211> 15

<212> PRT

<213> Streptomyces avermitilis

<400> 926

Leu Phe Ala Ser Gly Ala Gly Arg Arg Ala Arg Ala Glu Ala Gly

1 5 10 15

<210> 927

<211> 13

<212> PRT

<213> Streptomyces avermitilis

<400> 927

Ser Asn Arg Asp Ala Asp Asp Leu Ile Arg Asp Val Ala

1 5 10

<210> 928

<211> 11

<212> PRT

<213> Staphylococcus aureus

<400> 928

Ala Leu Ile Val Glu Glu Leu Phe Glu Tyr Ala

1 5 10

<210> 929

<211> 16

<212> PRT

<213> Staphylococcus aureus

<400> 929

Pro Ser Leu Thr Val Gln Val Ala Met Ala Gly Ala Met Leu Ile Gly

1 5 10 15

<210> 930

<211> 8

<212> PRT

<213> Staphylococcus aureus

<400> 930

Thr Glu Ala Val Lys Gln Ser Asp

fifteen

<210> 931

<211> 10

<212> PRT

<213> Staphylococcus aureus

<400> 931

His Leu Cys Gln Phe Val Met Ser Gly Gln

1 5 10

<210> 932

<211> 13

<212> PRT

<213> Staphylococcus aureus

<400> 932

Ser Glu Lys Leu Leu Glu Ser Leu Glu Asn Phe Trp Asn

1 5 10

<210> 933

<211> 11

<212> PRT

<213> Enterococcus faecium

<400> 933

Asn Phe Leu Leu Cys Asn Phe Ser Asn Leu Trp

1 5 10

<210> 934

<211> 16

<212> PRT

<213> Enterococcus faecium

<400> 934

Leu Glu Leu Leu Ser Gln Leu Gln Lys Asn Thr Leu Gln Leu Ile Arg

1 5 10 15

<210> 935

<211> 10

<212> PRT

<213> Enterococcus faecium

<400> 935

Lys Lys Phe Ala Lys Thr Thr Ala Arg Leu

1 5 10

<210> 936

<211> 13

<212> PRT

<213> Enterococcus faecium

<400> 936

Lys Val Glu Leu Phe Glu Ala Tyr Lys Asn Ser Leu Leu

1 5 10

<210> 937

<211> 9

<212> PRT

<213> Escherichia coli

<400> 937

Gly Val Tyr Ala Asn Glu Leu Arg Ala

fifteen

<210> 938

<211> 16

<212> PRT

<213> Escherichia coli

<400> 938

Gly Gly Ile Arg Glu Ile Glu Phe Ile Val Gln Val Phe Gln Leu Ile

1 5 10 15

<210> 939

<211> 8

<212> PRT

<213> Escherichia coli

<400> 939

Thr Leu Ser Ala Ile Ala Glu Leu

fifteen

<210> 940

<211> 15

<212> PRT

<213> Escherichia coli

<400> 940

Glu Gln Leu Arg Val Ala Tyr Leu Phe Leu Arg Arg Leu Glu Asn

1 5 10 15

<210> 941

<211> 13

<212> PRT

<213> Escherichia coli

<400> 941

Leu Thr Gly His Met Thr Asn Val Arg Arg Val Phe Asn

1 5 10

<210> 942

<211> 11

<212> PRT

<213> Sebaldella termitidis

<400> 942

Ser Arg Cys Met Lys Ile Ala Gln Ser Gly Gln

1 5 10

<210> 943

<211> 16

<212> PRT

<213> Sebaldella termitidis

<400> 943

Ile Ala Glu Ala Glu Phe Ile Asn Glu Ser Ile Tyr Met Ile Tyr Leu

1 5 10 15

<210> 944

<211> 8

<212> PRT

<213> Sebaldella termitidis

<400> 944

Lys Asp Met Gln Phe Leu Pro Ile

fifteen

<210> 945

<211> 14

<212> PRT

<213> Sebaldella termitidis

<400> 945

Asn Leu Leu Asn Asn Leu Ile Ser Ile Gln Asn Ser Glu Lys

1 5 10

<210> 946

<211> 13

<212> PRT

<213> Sebaldella termitidis

<400> 946

Ala Glu Lys Ile Cys Gly Leu Ile Ile Asn Glu Leu Lys

1 5 10

<210> 947

<211> 10

<212> PRT

<213> Streptomyces coelicolor

<400> 947

Ala Arg Leu Asp Ala Tyr Ala Asn Ser His

1 5 10

<210> 948

<211> 16

<212> PRT

<213> Streptomyces coelicolor

<400> 948

Leu Asp Ala Ala Asp Ser Ile Gly Phe Leu Leu Glu Leu Leu Phe Ala

1 5 10 15

<210> 949

<211> 8

<212> PRT

<213> Streptomyces coelicolor

<400> 949

Trp Glu Leu Asp Arg Phe Pro Leu

fifteen

<210> 950

<211> 14

<212> PRT

<213> Streptomyces coelicolor

<400> 950

Glu Leu Leu Ala Thr Leu Gly Arg Ile Thr Gly Ala Gly Gly

1 5 10

<210> 951

<211> 13

<212> PRT

<213> Streptomyces coelicolor

<400> 951

Gln Arg Glu Leu Phe Gly Arg Val Glu Ala Ala Ala Arg

1 5 10

<210> 952

<211> 11

<212> PRT

<213> Flavobacterium psychrophilum

<400> 952

Tyr Ser Ile Tyr Lys Asn Ala Arg Gln Leu Arg

1 5 10

<210> 953

<211> 16

<212> PRT

<213> Flavobacterium psychrophilum

<400> 953

Thr Ser Leu Leu Ile Leu Ser Glu Glu Val Ile Lys Ser Ile Leu

1 5 10 15

<210> 954

<211> 8

<212> PRT

<213> Flavobacterium psychrophilum

<400> 954

Gln Leu Ile Glu Leu Ser Ile Gly

fifteen

<210> 955

<211> 15

<212> PRT

<213> Flavobacterium psychrophilum

<400> 955

Lys Leu Thr Glu Phe Asp Asp Lys Lys Asn Gln Gly Phe Tyr Val

1 5 10 15

<210> 956

<211> 13

<212> PRT

<213> Flavobacterium psychrophilum

<400> 956

Lys Thr Glu Phe Thr Glu Thr Lys Val Val Val Asp Arg

1 5 10

<210> 957

<211> 10

<212> PRT

<213> Lactococcus lactis

<400> 957

Lys Cys Ile Asp His Ile Ser Val Leu Ile

1 5 10

<210> 958

<211> 16

<212> PRT

<213> Lactococcus lactis

<400> 958

Thr Phe Ile Ser Ile Thr Ile Ile Glu Glu Val Gly Lys Thr His Ile

1 5 10 15

<210> 959

<211> 8

<212> PRT

<213> Lactococcus lactis

<400> 959

Ser Leu Pro Thr Ile Lys Met Gly

fifteen

<210> 960

<211> 14

<212> PRT

<213> Lactococcus lactis

<400> 960

Thr Gly Glu Leu Ile Ser Ile Arg Glu Ser Ser Leu Tyr Ala

1 5 10

<210> 961

<211> 13

<212> PRT

<213> Lactococcus lactis

<400> 961

Lys Glu Gln Ser Arg Ala Leu Leu Leu Tyr Ala Ile Glu

1 5 10

<210> 962

<211> 11

<212> PRT

<213> Pseudomonas putida

<400> 962

Asp Ala Leu Leu Thr Asn Ala Ala Ser Leu Ile

1 5 10

<210> 963

<211> 16

<212> PRT

<213> Pseudomonas putida

<400> 963

Phe Ala Leu Ala His Leu Ala Arg Glu Glu Ile Ala Lys Thr Leu Met

1 5 10 15

<210> 964

<211> 8

<212> PRT

<213> Pseudomonas putida

<400> 964

Thr Ile Asn Ser Ile Val Phe Cys

fifteen

<210> 965

<211> 12

<212> PRT

<213> Pseudomonas putida

<400> 965

Phe Arg Asn Asp Leu Lys Asn Asn Ser Leu Tyr Val

1 5 10

<210> 966

<211> 13

<212> PRT

<213> Pseudomonas putida

<400> 966

Ala Glu Arg Ala Leu Arg Thr Ile Thr Leu Ala Trp Asp

1 5 10

<210> 967

<211> 11

<212> PRT

<213> Selenomonas sputigena

<400> 967

Gln Ile Ala Tyr Tyr Leu Tyr Phe Met Tyr Leu

1 5 10

<210> 968

<211> 16

<212> PRT

<213> Selenomonas sputigena

<400> 968

Met Thr Ser Phe Ala Tyr Tyr Lys Ser Tyr Phe Asp Arg Val Thr Ala

1 5 10 15

<210> 969

<211> 10

<212> PRT

<213> Selenomonas sputigena

<400> 969

Arg Leu Cys Glu Phe Tyr Glu Glu Phe Asp

1 5 10

<210> 970

<211> 17

<212> PRT

<213> Selenomonas sputigena

<400> 970

Ile Ile Asp Lys Ala Gln Ala Leu Arg Tyr Ala Asn Pro Leu Thr His

1 5 10 15

Ser

<210> 971

<211> 13

<212> PRT

<213> Selenomonas sputigena

<400> 971

Ile Arg Glu Leu Ser Thr Leu Leu Asp Arg Tyr Ile Ala

1 5 10

<210> 972

<211> 11

<212> PRT

<213> Lactobacillus helveticus

<400> 972

Trp Ile Ser Tyr Tyr Leu Tyr Phe Glu Ser Ile

1 5 10

<210> 973

<211> 16

<212> PRT

<213> Lactobacillus helveticus

<400> 973

Leu Thr Ser Tyr Ala Phe Phe Lys Asn Tyr Phe Asp Arg Thr Thr Ala

1 5 10 15

<210> 974

<211> 10

<212> PRT

<213> Lactobacillus helveticus

<400> 974

Gln Leu Gln Lys Val Tyr Arg Ile Leu Asn

1 5 10

<210> 975

<211> 17

<212> PRT

<213> Lactobacillus helveticus

<400> 975

Ile Ile Ser Lys Ala Asn Asp Leu Arg Asn Asn Asn Pro Leu Ser His

1 5 10 15

Ala

<210> 976

<211> 13

<212> PRT

<213> Lactobacillus helveticus

<400> 976

Ile Ala Thr Met Arg Ser Leu Phe Lys Leu Leu Val Glu

1 5 10

<210> 977

<211> 11

<212> PRT

<213> Lactococcus lactis

<400> 977

Lys Ile Leu Asn Phe Ile Tyr Phe Arg Ala Lys

1 5 10

<210> 978

<211> 16

<212> PRT

<213> Lactococcus lactis

<400> 978

Leu Glu Ser Phe Ala Tyr Tyr Lys Asn Tyr Phe Asp Arg Phe Val Ala

1 5 10 15

<210> 979

<211> 10

<212> PRT

<213> Lactococcus lactis

<400> 979

Lys Leu Ile Asp Gly Leu Lys Gln Leu Asn

1 5 10

<210> 980

<211> 17

<212> PRT

<213> Lactococcus lactis

<400> 980

Ile Ile Asn Glu Ala His Lys Ile Arg Asn Ser Asn Pro Val Ser His

1 5 10 15

Ser

<210> 981

<211> 13

<212> PRT

<213> Lactococcus lactis

<400> 981

Leu Asn Asp Leu Lys Ile Ile Ile Glu Gln Leu Ser Thr

1 5 10

<210> 982

<211> 11

<212> PRT

<213> Pseudomonas sp.

<400> 982

Lys Trp Leu Phe Ile Asp Gln Met Val Asp Leu

1 5 10

<210> 983

<211> 12

<212> PRT

<213> Pseudomonas sp.

<400> 983

Phe Lys Phe Arg Glu Ile Arg Ile Glu Tyr Ser Gln

1 5 10

<210> 984

<211> 9

<212> PRT

<213> Pseudomonas sp.

<400> 984

Tyr Glu Tyr Ala Gln Glu Ile Arg Ser

fifteen

<210> 985

<211> 14

<212> PRT

<213> Pseudomonas sp.

<400> 985

Arg Lys Ile Pro Asp Phe Arg Gly Lys Tyr Ala Ala His Ile

1 5 10

<210> 986

<211> 13

<212> PRT

<213> Pseudomonas sp.

<400> 986

Lys Ala Leu Glu Phe Tyr Asn Trp Ile His Ser Asn Glu

1 5 10

<210> 987

<211> 11

<212> PRT

<213> Vibrio paracholerae

<400> 987

Glu Glu Ile Leu Ser Gly Leu Ile Gly Asp Leu

1 5 10

<210> 988

<211> 12

<212> PRT

<213> Vibrio paracholerae

<400> 988

Arg Lys Tyr Val Glu Leu Asn Gln Lys Tyr Gly Lys

1 5 10

<210> 989

<211> 10

<212> PRT

<213> Vibrio paracholerae

<400> 989

Gly Val Tyr Asn Asn Glu Ile Asn Lys Asn

1 5 10

<210> 990

<211> 14

<212> PRT

<213> Vibrio paracholerae

<400> 990

Thr Ala Ile Lys Lys Leu Arg Asn His Cys Val Ala His Val

1 5 10

<210> 991

<211> 13

<212> PRT

<213> Vibrio paracholerae

<400> 991

Phe Ala Asp Glu Phe Leu Asp Trp Ile Cys Pro Asp Asn

1 5 10

<210> 992

<211> 11

<212> PRT

<213> Escherichia coli

<400> 992

Thr Met Ala Asp His Met Val Asn Glu Ala Trp

1 5 10

<210> 993

<211> 16

<212> PRT

<213> Escherichia coli

<400> 993

Phe Asn Leu Ile Leu Gln Ser Ile Glu Phe Arg Leu Lys Gly Leu Ile

1 5 10 15

<210> 994

<211> 10

<212> PRT

<213> Escherichia coli

<400> 994

Lys Val Tyr Asn Thr Phe Ala Ser Lys Ser

1 5 10

<210> 995

<211> 14

<212> PRT

<213> Escherichia coli

<400> 995

Trp Phe Asn Ser Met Arg Ile Leu Arg Asn Arg Phe Met His

1 5 10

<210> 996

<211> 13

<212> PRT

<213> Escherichia coli

<400> 996

Asp Ile Met Pro Glu Leu Ile Phe Thr Ser Val Val Arg

1 5 10

<210> 997

<211> 11

<212> PRT

<213> Geobacter sulfurreducens

<400> 997

Leu Asn Tyr Glu Ala Leu Tyr Val Lys Ser Lys

1 5 10

<210> 998

<211> 16

<212> PRT

<213> Geobacter sulfurreducens

<400> 998

Gln Leu Trp Ala Ser Met Ala Leu Glu Leu Leu Ala Lys Ser Ser Leu

1 5 10 15

<210> 999

<211> 9

<212> PRT

<213> Geobacter sulfurreducens

<400> 999

Gln Arg Leu Gly His Ile Ser Lys Leu

fifteen

<210> 1000

<211> 15

<212> PRT

<213> Geobacter sulfurreducens

<400> 1000

Phe Cys Glu Gln Leu Ser Leu Arg Arg Asn Ser Glu Ile His Ser

1 5 10 15

<210> 1001

<211> 13

<212> PRT

<213> Geobacter sulfurreducens

<400> 1001

Asp Ala Trp Glu Val Lys Tyr Trp Tyr Ala Ile Glu Val

1 5 10

<210> 1002

<211> 11

<212> PRT

<213> Streptomyces coelicolor

<400> 1002

Asp Val Ser Tyr Thr Pro Val Ser Asn Gly Met

1 5 10

<210> 1003

<211> 16

<212> PRT

<213> Streptomyces coelicolor

<400> 1003

Val Leu His Leu Gln Ala Ala Thr Glu Val Leu Leu Lys Ala Arg Leu

1 5 10 15

<210> 1004

<211> 10

<212> PRT

<213> Streptomyces coelicolor

<400> 1004

Asp Arg Leu Arg Asp Ile Ala Arg Leu Asp

1 5 10

<210> 1005

<211> 15

<212> PRT

<213> Streptomyces coelicolor

<400> 1005

Arg Ile Lys Glu Pro Gly Glu Ser Arg Asn Ala Leu Gln His Tyr

1 5 10 15

<210> 1006

<211> 13

<212> PRT

<213> Streptomyces coelicolor

<400> 1006

Tyr Ala Ile Glu Ser Arg Ala Ala Arg Val Leu Asp Phe

1 5 10

<210> 1007

<211> 10

<212> PRT

<213> Leptospira interrogans

<400> 1007

Cys Thr Arg Leu Tyr Asn Gln Ile Leu Glu

1 5 10

<210> 1008

<211> 16

<212> PRT

<213> Leptospira interrogans

<400> 1008

Tyr Thr Lys Leu Phe Asn Ile Leu Asp Lys Val Ala Ala Ile Val Tyr

1 5 10 15

<210> 1009

<211> 6

<212> PRT

<213> Leptospira interrogans

<400> 1009

Phe Pro Ser Thr Phe Gly

fifteen

<210> 1010

<211> 13

<212> PRT

<213> Leptospira interrogans

<400> 1010

His His Leu Arg Val Arg Arg Asn Asn Ile Val His Trp

1 5 10

<210> 1011

<211> 13

<212> PRT

<213> Leptospira interrogans

<400> 1011

Glu Glu Asp Val Gln Arg Leu Phe Leu Ile Ser Lys Ala

1 5 10

<210> 1012

<211> 10

<212> PRT

<213> Shigella boydii

<400> 1012

Met Glu Met Val Leu Asn Arg Leu Lys Ser

1 5 10

<210> 1013

<211> 16

<212> PRT

<213> Shigella boydii

<400> 1013

Phe Arg Leu Cys Phe Gly Ile Leu Asp Lys Ile Ala Val Ala Ile Cys

1 5 10 15

<210> 1014

<211> 8

<212> PRT

<213> Shigella boydii

<400> 1014

Pro Gln Lys Asn Ile Tyr Phe Gln

fifteen

<210> 1015

<211> 15

<212> PRT

<213> Shigella boydii

<400> 1015

Glu Leu Ala Phe Tyr Lys Glu Trp Arg Asn Gly Leu Glu His Lys

1 5 10 15

<210> 1016

<211> 13

<212> PRT

<213> Shigella boydii

<400> 1016

Ile His His Phe Glu His Leu Leu Gln Ile Thr Arg Ser

1 5 10

<210> 1017

<211> 10

<212> PRT

<213> Enterococcus faecalis

<400> 1017

Phe Tyr Ser Leu Phe Asn Gln Ile Lys Gln

1 5 10

<210> 1018

<211> 16

<212> PRT

<213> Enterococcus faecalis

<400> 1018

Tyr Arg Ser Val Tyr Ser Ile Phe Asp Lys Ile Ala Tyr Phe Leu Asn

1 5 10 15

<210> 1019

<211> 8

<212> PRT

<213> Enterococcus faecalis

<400> 1019

Pro Lys Asn Leu Ile Thr Phe His

fifteen

<210> 1020

<211> 15

<212> PRT

<213> Enterococcus faecalis

<400> 1020

Asn Leu Glu Lys Ile Ala Glu Ile Arg Asn Ala Met Glu His Lys

1 5 10 15

<210> 1021

<211> 13

<212> PRT

<213> Enterococcus faecalis

<400> 1021

Glu Lys Ile Thr Leu Glu Leu Phe Lys Leu Thr Arg Glu

1 5 10

<210> 1022

<211> 10

<212> PRT

<213> Clostridium thermocellum

<400> 1022

Phe Asn Asn Arg Ala Phe Asp Leu Ile Val

1 5 10

<210> 1023

<211> 16

<212> PRT

<213> Clostridium thermocellum

<400> 1023

Tyr Thr Arg Phe Glu Gly Leu Ile Asp Thr Ile Tyr His Ile Ile Asn

1 5 10 15

<210> 1024

<211> 7

<212> PRT

<213> Clostridium thermocellum

<400> 1024

Lys Pro Ser Ser Glu Phe Arg

fifteen

<210> 1025

<211> 15

<212> PRT

<213> Clostridium thermocellum

<400> 1025

Val Tyr Lys Lys Ile Asn Lys Phe Arg Asn Asn Ile Val His Asn

1 5 10 15

<210> 1026

<211> 13

<212> PRT

<213> Clostridium thermocellum

<400> 1026

Tyr Thr Thr Ser Thr Glu Phe Leu Asn Asn Ile Lys Asp

1 5 10

<210> 1027

<211> 10

<212> PRT

<213> Bacillus cereus

<400> 1027

Leu Asn Asn Arg Ile Phe Gln Leu Asp Leu

1 5 10

<210> 1028

<211> 16

<212> PRT

<213> Bacillus cereus

<400> 1028

Phe Pro Lys Ala Phe Thr Ala Leu Asp Leu Leu Ala His Leu Leu Phe

1 5 10 15

<210> 1029

<211> 7

<212> PRT

<213> Bacillus cereus

<400> 1029

Lys Thr Glu Lys Lys Ile Lys

fifteen

<210> 1030

<211> 15

<212> PRT

<213> Bacillus cereus

<400> 1030

Glu Phe Gln Lys Ala Ser Lys Val Arg Asn Asp Ile Ile His Asn

1 5 10 15

<210> 1031

<211> 13

<212> PRT

<213> Bacillus cereus

<400> 1031

Tyr Thr Pro Ser Lys Glu Ile Leu Asn Ile Ala Arg Gly

1 5 10

<210> 1032

<211> 10

<212> PRT

<213> Pseudomonas syringae

<400> 1032

Glu Tyr Leu Arg Cys Lys Asp Ala Phe Glu

1 5 10

<210> 1033

<211> 16

<212> PRT

<213> Pseudomonas syringae

<400> 1033

Ser Ser Phe Ile His His Leu Tyr Glu Leu Tyr Met Ala Leu Phe Ala

1 5 10 15

<210> 1034

<211> 8

<212> PRT

<213> Pseudomonas syringae

<400> 1034

Ser Ile Asp Arg Gly Ala Val Ser

fifteen

<210> 1035

<211> 16

<212> PRT

<213> Pseudomonas syringae

<400> 1035

Phe Gly Pro Ala Phe Arg Ser Met Arg Asn Lys Ile Ala Gly His Val

1 5 10 15

<210> 1036

<211> 13

<212> PRT

<213> Pseudomonas syringae

<400> 1036

Val Lys Leu Thr Glu Phe Phe Gln Lys Tyr His Pro Tyr

1 5 10

<210> 1037

<211> 10

<212> PRT

<213> Burkholderia xenovorans

<400> 1037

Glu Tyr Leu Arg Cys Asp Asp Ala Leu His

1 5 10

<210> 1038

<211> 16

<212> PRT

<213> Burkholderia xenovorans

<400> 1038

Ala Arg Phe Ile His His Leu Tyr Glu Phe Asn Ile Ala Cys Ala Gln

1 5 10 15

<210> 1039

<211> 8

<212> PRT

<213> Burkholderia xenovorans

<400> 1039

Arg Val Arg Arg Gln Ala Tyr Asn

fifteen

<210> 1040

<211> 16

<212> PRT

<213> Burkholderia xenovorans

<400> 1040

Phe Ala Lys Ala Phe Arg Thr Ala Arg Asn Thr Thr Asn Gly His Ala

1 5 10 15

<210> 1041

<211> 13

<212> PRT

<213> Burkholderia xenovorans

<400> 1041

Leu Asn Leu Ser Asp Phe Phe Thr Arg Tyr His Arg Phe

1 5 10

<210> 1042

<211> 10

<212> PRT

<213> Microcystis aeruginosa

<400> 1042

Glu His Leu Asp Cys Glu Leu Trp Glu Arg

1 5 10

<210> 1043

<211> 16

<212> PRT

<213> Microcystis aeruginosa

<400> 1043

Ile Arg Asn Ala Thr Val Ile Leu Glu Asp Arg Met Arg Lys Leu Gly

1 5 10 15

<210> 1044

<211> 8

<212> PRT

<213> Microcystis aeruginosa

<400> 1044

Gly Ile Val Asn Leu Ile Phe Gly

fifteen

<210> 1045

<211> 15

<212> PRT

<213> Microcystis aeruginosa

<400> 1045

Tyr Ser Gly Thr Met Lys Ile Phe Arg Asn Arg Tyr Ala His Arg

1 5 10 15

<210> 1046

<211> 13

<212> PRT

<213> Microcystis aeruginosa

<400> 1046

Ile Ile Val Phe Ile Asp Leu Leu Leu Lys Met Leu Asp

1 5 10

<210> 1047

<211> 11

<212> PRT

<213> Vibrio parahaemolyticus

<400> 1047

Ser Arg Asn Val His Pro Asp Val Leu Lys Tyr

1 5 10

<210> 1048

<211> 16

<212> PRT

<213> Vibrio parahaemolyticus

<400> 1048

Val Phe Glu Ala Thr Lys Ser Val Ala Asp Lys Ile Arg Asn Lys Thr

1 5 10 15

<210> 1049

<211> 8

<212> PRT

<213> Vibrio parahaemolyticus

<400> 1049

Val Leu Val Asp Glu Ala Phe Ser

fifteen

<210> 1050

<211> 15

<212> PRT

<213> Vibrio parahaemolyticus

<400> 1050

Leu Lys Gly Leu Phe Gly Thr Phe Arg Asn Thr Thr Ala His Ala

1 5 10 15

<210> 1051

<211> 13

<212> PRT

<213> Vibrio parahaemolyticus

<400> 1051

Ile Leu Ser Met Val Ser Leu Val His Arg Arg Leu Asp

1 5 10

<210> 1052

<211> 11

<212> PRT

<213> Lactococcus lactis

<400> 1052

Ala Leu Glu Leu His Ser Glu Val Thr Lys Tyr

1 5 10

<210> 1053

<211> 16

<212> PRT

<213> Lactococcus lactis

<400> 1053

Val Phe Glu Ser Cys Lys Gly Leu Phe Asp Arg Ile Arg Leu Ile Ser

1 5 10 15

<210> 1054

<211> 8

<212> PRT

<213> Lactococcus lactis

<400> 1054

Thr Leu Ile Asn Gln Ala Phe Asn

fifteen

<210> 1055

<211> 15

<212> PRT

<213> Lactococcus lactis

<400> 1055

Ile Lys Thr Cys Leu Tyr Leu Tyr Arg Asn His Gln Ala His Val

1 5 10 15

<210> 1056

<211> 13

<212> PRT

<213> Lactococcus lactis

<400> 1056

Gly Leu Met Ser Ile Ser Leu Ala His Glu Leu Leu Asp

1 5 10

<210> 1057

<211> 11

<212> PRT

<213> Nematostella vectensis

<400> 1057

Ser Thr Thr Leu Thr Thr Phe Leu Asn Leu His

1 5 10

<210> 1058

<211> 16

<212> PRT

<213> Nematostella vectensis

<400> 1058

Glu Asp Tyr Asp Ile Thr Leu Leu Thr Cys Leu Leu Arg Asn Ile Cys

1 5 10 15

<210> 1059

<211> 8

<212> PRT

<213> Nematostella vectensis

<400> 1059

Asp Lys Leu Pro Pro Ala Tyr Asp

fifteen

<210> 1060

<211> 15

<212> PRT

<213> Nematostella vectensis

<400> 1060

Val Val Arg Leu Arg His Tyr Arg Asn Asp Leu Tyr Ala His Ile

1 5 10 15

<210> 1061

<211> 13

<212> PRT

<213> Nematostella vectensis

<400> 1061

Trp Ala Asp Ile Ser Ala Ala Leu Leu Ser Leu Gly Gly

1 5 10

<210> 1062

<211> 11

<212> PRT

<213> Branchiostoma floridae

<400> 1062

Pro Pro Ser Leu Pro Ala Gln Leu Lys Lys His

1 5 10

<210> 1063

<211> 16

<212> PRT

<213> Branchiostoma floridae

<400> 1063

Glu Glu Phe Asp Ile Ser Leu Leu Leu Leu Leu Leu Lys Glu Leu Val

1 5 10 15

<210> 1064

<211> 8

<212> PRT

<213> Branchiostoma floridae

<400> 1064

Gly Arg Asp Ala Pro Tyr Ser Asp

fifteen

<210> 1065

<211> 13

<212> PRT

<213> Branchiostoma floridae

<400> 1065

Lys Leu Gly Gln Phe Arg Asn Lys Asn Tyr Gly His Ile

1 5 10

<210> 1066

<211> 13

<212> PRT

<213> Branchiostoma floridae

<400> 1066

Trp Asp Glu Leu Thr Glu Ile Leu Val Asp Leu Gly Gly

1 5 10

<210> 1067

<211> 11

<212> PRT

<213> Homo sapiens

<400> 1067

Pro Pro Leu Leu Lys Lys Glu Leu Leu Ile His

1 5 10

<210> 1068

<211> 16

<212> PRT

<213> Homo sapiens

<400> 1068

Lys Gln Phe Asp Leu Cys Leu Leu Leu Ala Leu Ile Lys His Leu Asn

1 5 10 15

<210> 1069

<211> 8

<212> PRT

<213> Homo sapiens

<400> 1069

Asn Met Glu Pro Pro Ser Ser Asp

fifteen

<210> 1070

<211> 15

<212> PRT

<213> Homo sapiens

<400> 1070

Ile Leu Arg Leu Cys Lys Tyr Arg Asp Ile Leu Leu Ser Glu Ile

1 5 10 15

<210> 1071

<211> 13

<212> PRT

<213> Homo sapiens

<400> 1071

Trp Lys Lys Val Ser Asp Ile Leu Leu Arg Leu Gly Met

1 5 10

<210> 1072

<211> 11

<212> PRT

<213> Escherichia coli

<400> 1072

Val Thr Ala Glu Lys Leu Leu Val Ser Gly Leu

1 5 10

<210> 1073

<211> 16

<212> PRT

<213> Escherichia coli

<400> 1073

Leu Tyr Pro Glu Leu Arg Thr Ile Glu Gly Val Leu Lys Ser Lys Met

1 5 10 15

<210> 1074

<211> 8

<212> PRT

<213> Escherichia coli

<400> 1074

Tyr Ile Leu Lys Pro Gln Phe Ala

fifteen

<210> 1075

<211> 14

<212> PRT

<213> Escherichia coli

<400> 1075

Ala Tyr Thr Phe Phe Asn Val Glu Arg His Ser Leu Phe His

1 5 10

<210> 1076

<211> 13

<212> PRT

<213> Escherichia coli

<400> 1076

Met Ile Ser Asp Met Ala Arg Leu Met Gly Lys Ala Thr

1 5 10

<210> 1077

<211> 11

<212> PRT

<213> Photobacterium profundum

<400> 1077

Asp Thr Tyr Arg Ser Leu Leu Ser Ser Ser Ser Tyr

1 5 10

<210> 1078

<211> 16

<212> PRT

<213> Photobacterium profundum

<400> 1078

Ile Tyr Pro Asp Leu Arg Val Leu Glu Gly Val Ile Lys Glu Ala Met

1 5 10 15

<210> 1079

<211> 8

<212> PRT

<213> Photobacterium profundum

<400> 1079

Thr Glu Leu Lys Thr Glu Tyr Asn

fifteen

<210> 1080

<211> 14

<212> PRT

<213> Photobacterium profundum

<400> 1080

Cys Tyr Ala Tyr Phe Lys Ala His Arg His Ser Leu Phe His

1 5 10

<210> 1081

<211> 13

<212> PRT

<213> Photobacterium profundum

<400> 1081

Thr Thr Asp Thr Ile Gly Glu Val Met Gln Met Ser Glu

1 5 10

<210> 1082

<211> 11

<212> PRT

<213> Geobacillus thermoglucosidasius

<400> 1082

Leu Tyr Asp Arg Asp Arg Ile Glu Ala Ser Glu

1 5 10

<210> 1083

<211> 16

<212> PRT

<213> Geobacillus thermoglucosidasius

<400> 1083

Val Ser Gly Thr Leu Arg Ala Phe Glu Gly Phe Phe Lys Lys Leu Leu

1 5 10 15

<210> 1084

<211> 8

<212> PRT

<213> Geobacillus thermoglucosidasius

<400> 1084

Asp Ile Ser Glu Lys Val Phe Asn

fifteen

<210> 1085

<211> 14

<212> PRT

<213> Geobacillus thermoglucosidasius

<400> 1085

Met Leu Asn His Met Ser Gln Asp Arg Asn Pro Tyr Ser His

1 5 10

<210> 1086

<211> 13

<212> PRT

<213> Geobacillus thermoglucosidasius

<400> 1086

Pro Leu Arg Thr Leu Asn Gln Ala Ile Ser Leu His Asn

1 5 10

<210> 1087

<211> 12

<212> PRT

<213> Teredinibacter turnerae

<400> 1087

Cys Arg Ser Ile Arg Lys Leu Leu Asn Met Asn Ala

1 5 10

<210> 1088

<211> 16

<212> PRT

<213> Teredinibacter turnerae

<400> 1088

Ser Tyr Pro Leu Ile Tyr Glu Ile Glu Asn Leu Val Arg Lys Leu Ile

1 5 10 15

<210> 1089

<211> 9

<212> PRT

<213> Teredinibacter turnerae

<400> 1089

Ile Gln Leu Ser Asn Phe Leu Phe Asp

fifteen

<210> 1090

<211> 15

<212> PRT

<213> Teredinibacter turnerae

<400> 1090

Arg Trp Gly Lys Leu Tyr Lys Leu Arg Cys Lys Ile Ala His Asn

1 5 10 15

<210> 1091

<211> 13

<212> PRT

<213> Teredinibacter turnerae

<400> 1091

Thr Thr Lys Leu Val Glu Glu Val Lys Leu Lys Ile Leu

1 5 10

<210> 1092

<211> 5

<212> PRT

<213> Methanococcus maripaludis

<400> 1092

Phe Arg Leu Met Tyr

fifteen

<210> 1093

<211> 16

<212> PRT

<213> Methanococcus maripaludis

<400> 1093

Phe Leu Asp Ser Val Leu Ala Leu Glu Ile Tyr His Thr Leu Lys Phe

1 5 10 15

<210> 1094

<211> 10

<212> PRT

<213> Methanococcus maripaludis

<400> 1094

Phe Ile Asn Lys Met Lys Asp Val Phe Asn

1 5 10

<210> 1095

<211> 15

<212> PRT

<213> Methanococcus maripaludis

<400> 1095

Ile Cys Arg Ile Ile Arg Asp Thr Arg Asn Lys Leu Val His Asp

1 5 10 15

<210> 1096

<211> 13

<212> PRT

<213> Methanococcus maripaludis

<400> 1096

Pro Tyr Phe Leu Ile Glu Leu Leu Lys Asn Ile Phe Lys

1 5 10

<210> 1097

<211> 11

<212> PRT

<213> Novosphingobium pentaromativorans

<400> 1097

Val His Arg Ala Leu Ser Trp Leu Arg Arg Ala

1 5 10

<210> 1098

<211> 14

<212> PRT

<213> Novosphingobium pentaromativorans

<400> 1098

Phe Ile Leu Leu Trp Ile Gly Phe Asn Ala Ala Tyr Ala Gly

1 5 10

<210> 1099

<211> 10

<212> PRT

<213> Novosphingobium pentaromativorans

<400> 1099

Glu Arg Ser Arg Thr Ala Ile Asn Tyr Ala

1 5 10

<210> 1100

<211> 15

<212> PRT

<213> Novosphingobium pentaromativorans

<400> 1100

Leu Phe Asp Arg Leu Tyr Val Leu Arg Asn Gln Leu Val His Gly

1 5 10 15

<210> 1101

<211> 13

<212> PRT

<213> Novosphingobium pentaromativorans

<400> 1101

Arg Asp Gln Val Arg Asp Gly Ala Ser Leu Leu Gly Cys

1 5 10

<210> 1102

<211> 12

<212> PRT

<213> Chlorobium chlorochromatii

<400> 1102

Ile Met Glu Gln Arg Lys Ala Ile Leu Glu Pro Leu

1 5 10

<210> 1103

<211> 16

<212> PRT

<213> Chlorobium chlorochromatii

<400> 1103

Ala Val Ala Tyr Asn His Phe Val Pro Leu Leu Ala Gln Asp Leu Ile

1 5 10 15

<210> 1104

<211> 6

<212> PRT

<213> Chlorobium chlorochromatii

<400> 1104

Lys Ile Ser Asn Lys Lys

fifteen

<210> 1105

<211> 15

<212> PRT

<213> Chlorobium chlorochromatii

<400> 1105

Ser Glu Lys Leu Lys Thr Phe Arg Asp Lys Tyr Tyr Ala His Leu

1 5 10 15

<210> 1106

<211> 13

<212> PRT

<213> Chlorobium chlorochromatii

<400> 1106

Phe Leu Gly Ile His Arg Lys Ser Ala Asn Glu Met Trp

1 5 10

<210> 1107

<211> 12

<212> PRT

<213> Lactococcus lactis

<400> 1107

Asp Ala Tyr Asn Lys Leu Ile Leu Leu Lys Gln Tyr

1 5 10

<210> 1108

<211> 15

<212> PRT

<213> Lactococcus lactis

<400> 1108

Phe Phe Tyr Asn Asn Leu Leu Asp Ser Leu Val Ile Ala Ile Phe

1 5 10 15

<210> 1109

<211> 6

<212> PRT

<213> Lactococcus lactis

<400> 1109

Asn Tyr Thr Asn Phe Pro

fifteen

<210> 1110

<211> 15

<212> PRT

<213> Lactococcus lactis

<400> 1110

Leu Glu Tyr Leu Tyr Ala Gln Arg Asn Lys Ile Tyr Val His Asn

1 5 10 15

<210> 1111

<211> 13

<212> PRT

<213> Lactococcus lactis

<400> 1111

Asn Tyr Ala Trp Glu Pro Thr Asn Ile Asn Asp Trp Glu

1 5 10

<210> 1112

<211> 16

<212> PRT

<213> Escherichia coli

<400> 1112

Glu Ser Val Ile Ala His Met Asn Glu Leu Leu Ile Ala Leu Ser Asp

1 5 10 15

<210> 1113

<211> 15

<212> PRT

<213> Escherichia coli

<400> 1113

Arg Tyr Thr Gln Gln Gln Arg Leu Arg Thr Ala Ile Ala His His

1 5 10 15

<210> 1114

<211> 12

<212> PRT

<213> Escherichia coli

<400> 1114

Glu Ala Arg His Glu Gln Leu Thr Lys Gly Gly Thr

1 5 10

<210> 1115

<211> 16

<212> PRT

<213> Cronobacter sakazakii

<400> 1115

Gln His Val Ile Ala Pro Met Asn Glu Leu Leu Ile Ala Leu Ser Asp

1 5 10 15

<210> 1116

<211> 15

<212> PRT

<213> Cronobacter sakazakii

<400> 1116

Arg Tyr Asp Leu Gln Gln Gln Leu Arg Thr Ala Ile Ala His His

1 5 10 15

<210> 1117

<211> 13

<212> PRT

<213> Cronobacter sakazakii

<400> 1117

Ala Ala Glu Arg Leu Ala Glu Leu Thr Arg Gly Gly Thr

1 5 10

<210> 1118

<211> 16

<212> PRT

<213> Homo sapiens

<400> 1118

Glu Ser Arg Tyr Arg Thr Leu Arg Asn Val Gly Asn Glu Ser Asp Ile

1 5 10 15

<210> 1119

<211> 10

<212> PRT

<213> Homo sapiens

<400> 1119

Leu Gln Pro Gly Pro Ser Glu His Ser Lys

1 5 10

<210> 1120

<211> 14

<212> PRT

<213> Homo sapiens

<400> 1120

Val Gly Asp Leu Leu Lys Phe Ile Arg Asn Leu Gly Glu His

1 5 10

<210> 1121

<211> 13

<212> PRT

<213> Homo sapiens

<400> 1121

Ile Gly Asp Pro Ser Leu Tyr Phe Gln Lys Thr Phe Pro

1 5 10

<210> 1122

<211> 16

<212> PRT

<213> Arabidopsis thaliana

<400> 1122

Glu Met Arg Leu Ser Phe Leu Arg Asp Ala Ser Asp Arg Val Glu Leu

1 5 10 15

<210> 1123

<211> 10

<212> PRT

<213> Arabidopsis thaliana

<400> 1123

Met Glu Ser Thr Ala Pro Val Ala Ile Gly

1 5 10

<210> 1124

<211> 15

<212> PRT

<213> Arabidopsis thaliana

<400> 1124

Ile Arg Asp Leu Leu Arg Val Ile Arg Asn Lys Leu Asn His His

1 5 10 15

<210> 1125

<211> 13

<212> PRT

<213> Arabidopsis thaliana

<400> 1125

Pro Glu Gly Phe Asp Glu Tyr Phe Ala Val Arg Phe Pro

1 5 10

<210> 1126

<211> 12

<212> PRT

<213> Helicobacter pylori

<400> 1126

Tyr Glu Leu Leu Trp Gln Glu Val Ile Arg Ala Lys

1 5 10

<210> 1127

<211> 15

<212> PRT

<213> Helicobacter pylori

<400> 1127

Trp Val Ser Leu Gln Asn Val Met Arg Arg Ile Ile Glu Tyr Tyr

1 5 10 15

<210> 1128

<211> 5

<212> PRT

<213> Helicobacter pylori

<400> 1128

Phe Arg Ile Leu Gly

fifteen

<210> 1129

<211> 17

<212> PRT

<213> Helicobacter pylori

<400> 1129

Lys Gln Val Phe Ser Ser Phe Ile Ser Trp Phe Asn Asp Gly Ser His

1 5 10 15

gly

<210> 1130

<211> 13

<212> PRT

<213> Helicobacter pylori

<400> 1130

Ile Glu Thr Tyr Leu Lys Val Phe Glu Asn Ile Phe Lys

1 5 10

<210> 1131

<211> 12

<212> PRT

<213> Streptococcus mutans

<400> 1131

His Leu Met Leu Val Asp Glu Leu Lys Lys Ala Ile

1 5 10

<210> 1132

<211> 15

<212> PRT

<213> Streptococcus mutans

<400> 1132

Glu Lys Tyr His Phe Asn Leu Leu Arg Asn Leu Leu Glu Lys Thr

1 5 10 15

<210> 1133

<211> 5

<212> PRT

<213> Streptococcus mutans

<400> 1133

Ala Thr Phe Leu Gly

fifteen

<210> 1134

<211> 14

<212> PRT

<213> Streptococcus mutans

<400> 1134

Pro Ala Pro Tyr Ile Arg Arg Ile Asn Leu His Ser His Ser

1 5 10

<210> 1135

<211> 13

<212> PRT

<213> Streptococcus mutans

<400> 1135

Lys Lys Val Leu Glu Arg Val Phe Asn Gln Phe Leu Gln

1 5 10

<210> 1136

<211> 12

<212> PRT

<213> Escherichia coli

<400> 1136

His Leu His Leu Lys Gln Thr Ile Glu Gln Ala Ile

1 5 10

<210> 1137

<211> 15

<212> PRT

<213> Escherichia coli

<400> 1137

Glu Arg Tyr His Phe Thr Leu Leu Arg Asn Leu Tyr Glu Lys Thr

1 5 10 15

<210> 1138

<211> 5

<212> PRT

<213> Escherichia coli

<400> 1138

Ala Ser Phe Leu Gly

fifteen

<210> 1139

<211> 13

<212> PRT

<213> Escherichia coli

<400> 1139

Leu Tyr Leu Ser Arg Ile Ile Asn Phe Thr Ser His Ser

1 5 10

<210> 1140

<211> 13

<212> PRT

<213> Escherichia coli

<400> 1140

Lys Ala Thr Val Lys Leu Leu Leu Asp His Leu Lys Asn

1 5 10

<210> 1141

<211> 12

<212> PRT

<213> Bacteroides fragilis

<400> 1141

Lys Glu Ile Glu Glu Glu Arg Thr Val Gln Asn Ile

1 5 10

<210> 1142

<211> 16

<212> PRT

<213> Bacteroides fragilis

<400> 1142

Thr Ser Phe Gly Glu Val Thr Glu Glu Tyr His Asp Glu Leu Tyr Ser

1 5 10 15

<210> 1143

<211> 8

<212> PRT

<213> Bacteroides fragilis

<400> 1143

Tyr Ile Lys Glu Leu Ser Asn Gly

fifteen

<210> 1144

<211> 15

<212> PRT

<213> Bacteroides fragilis

<400> 1144

Gln Lys Thr Leu Thr Glu Lys Ile Arg His Gln Ile His His Pro

1 5 10 15

<210> 1145

<211> 13

<212> PRT

<213> Bacteroides fragilis

<400> 1145

Glu Thr Glu Ile Arg Gln Ser Ile Glu Asp Met Arg Ala

1 5 10

<210> 1146

<211> 12

<212> PRT

<213> Methylobacillus flagellates

<400> 1146

Ser Asn Gln Ile Pro Thr Arg Val Ser Pro Val Leu

1 5 10

<210> 1147

<211> 16

<212> PRT

<213> Methylobacillus flagellates

<400> 1147

Ser Ala Phe Gly Glu Ala Ser Tyr Glu Tyr His Asn Glu Leu Tyr Gly

1 5 10 15

<210> 1148

<211> 8

<212> PRT

<213> Methylobacillus flagellates

<400> 1148

Tyr Asn Arg Leu Arg Arg Asp Gly

fifteen

<210> 1149

<211> 15

<212> PRT

<213> Methylobacillus flagellates

<400> 1149

Gln Val Ile Leu Thr Glu Tyr Ile Arg His Gln Ile His His Pro

1 5 10 15

<210> 1150

<211> 13

<212> PRT

<213> Methylobacillus flagellates

<400> 1150

Thr Ala Glu Leu Thr Glu Ser Ile Glu Thr Met Arg Leu

1 5 10

<210> 1151

<211> 12

<212> PRT

<213> Campylobacter hominis

<400> 1151

Lys Asp Gly Glu Gln Lys Lys Glu Val Lys Asn Val

1 5 10

<210> 1152

<211> 16

<212> PRT

<213> Campylobacter hominis

<400> 1152

Met Ala Phe Gly Glu Ile Thr Glu Glu Tyr His Asn Glu Leu Tyr Gly

1 5 10 15

<210> 1153

<211> 8

<212> PRT

<213> Campylobacter hominis

<400> 1153

Tyr Lys Lys Leu Lys Lys Asp Gly

fifteen

<210> 1154

<211> 15

<212> PRT

<213> Campylobacter hominis

<400> 1154

Lys Leu Thr Leu Thr Glu Tyr Ile Arg His Gln Ile His His Pro

1 5 10 15

<210> 1155

<211> 13

<212> PRT

<213> Campylobacter hominis

<400> 1155

Leu Ser Glu Leu Lys Asp Ser Ile Glu Met Met Arg Asn

1 5 10

<210> 1156

<211> 15

<212> PRT

<213> Homo sapiens

<400> 1156

Ile Pro Asp Trp Ile Val Asp Leu Arg His Glu Leu Thr His Lys

1 5 10 15

<210> 1157

<211> 15

<212> PRT

<213> Mycobacterium tuberculosis

<400> 1157

Leu Gly Arg Phe Glu Ser Arg Val Arg Asn Thr Ala Ala His Glu

1 5 10 15

<210> 1158

<211> 15

<212> PRT

<213> Lactococcus lactis

<400> 1158

Trp Ile Arg Ala Gly Trp Phe Ile Arg Asn Arg Ser Ala His Tyr

1 5 10 15

<210> 1159

<211> 15

<212> PRT

<213> Thermus thermophilus

<220>

<221> MOD_RES

<222> (5)..(5)

<223> Any amino acids

<400> 1159

Leu Ala Leu Gly Xaa Val Asp Asp Arg Ser Leu Thr Val His Thr

1 5 10 15

<210> 1160

<211> 22

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1160

Leu Lys Ser Met Leu Tyr Ser Met Arg Asn Ser Ser Phe His Phe Ser

1 5 10 15

Thr Glu Asn Val Asp Asn

twenty

<210> 1161

<211> 22

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1161

Leu Lys Asp Val Ile Tyr Ser Met Arg Asn Asp Ser Phe His Tyr Ala

1 5 10 15

Thr Glu Asn His Asn Asn

twenty

<210> 1162

<211> 22

<212> PRT

<213> Clostridium aminophilum

<400> 1162

Leu Arg Lys Ala Ile Tyr Ser Leu Arg Asn Glu Thr Phe His Phe Thr

1 5 10 15

Thr Leu Asn Lys Gly Ser

twenty

<210> 1163

<211> 22

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1163

Ile Ile Gln Ile Ile Tyr Ser Leu Arg Asn Lys Ser Phe His Phe Lys

1 5 10 15

Thr Tyr Asp His Gly Asp

twenty

<210> 1164

<211> 22

<212> PRT

<213> Carnobacterium gallinarum

<400> 1164

Leu Arg Gly Ser Val Gln Gln Ile Arg Asn Glu Ile Phe His Ser Phe

1 5 10 15

Asp Lys Asn Gln Lys Phe

twenty

<210> 1165

<211> 21

<212> PRT

<213> Carnobacterium gallinarum

<400> 1165

Ile Arg Gly Ala Val Gln Arg Val Arg Asn Gln Ile Phe His Gln Gln

1 5 10 15

Ile Asn Lys Arg His

twenty

<210> 1166

<211> 20

<212> PRT

<213> Paludibacter propionicigenes

<400> 1166

Ile Arg Gly Ala Val Gln Gln Ile Arg Asn Asn Val Asn His Tyr Lys

1 5 10 15

Lys Asp Ala Leu

twenty

<210> 1167

<211> 20

<212> PRT

<213> Listeria seeligeri

<400> 1167

Leu Arg Gly Ala Ile Ala Pro Ile Arg Asn Glu Ile Ile His Leu Lys

1 5 10 15

Lys His Ser Trp

twenty

<210> 1168

<211> 20

<212> PRT

<213> Listeria weihenstephanensis

<400> 1168

Ile Arg Gly Ser Ile Gln Gln Ile Arg Asn Glu Val Tyr His Cys Lys

1 5 10 15

Lys His Ser Trp

twenty

<210> 1169

<211> 20

<212> PRT

<213> Listeria newyorkensis

<400> 1169

Ile Arg Gly Ser Ile Gln Gln Ile Arg Asn Glu Val Tyr His Cys Lys

1 5 10 15

Lys His Ser Trp

twenty

<210> 1170

<211> 21

<212> PRT

<213> Leptotrichia wadei

<400> 1170

Ile Ser Tyr Ser Ile Tyr Asn Val Arg Asn Gly Val Gly His Phe Asn

1 5 10 15

Lys Leu Ile Leu Gly

twenty

<210> 1171

<211> 21

<212> PRT

<213> Leptotrichia wadei

<400> 1171

Met Leu Asn Ala Ile Thr Ser Ile Arg His Arg Val Val His Tyr Asn

1 5 10 15

Met Asn Thr Asn Ser

twenty

<210> 1172

<211> 21

<212> PRT

<213> Leptotrichia wadei

<400> 1172

Ile Asp Glu Ala Ile Ser Ser Ile Arg His Gly Ile Val His Phe Asn

1 5 10 15

Leu Glu Leu Glu Gly

twenty

<210> 1173

<211> 22

<212> PRT

<213> Rhodobacter capsulatus

<400> 1173

Leu Leu Arg Tyr Leu Arg Gly Cys Arg Asn Gln Thr Phe His Leu Gly

1 5 10 15

Ala Arg Ala Gly Phe Leu

twenty

<210> 1174

<211> 21

<212> PRT

<213> Leptotrichia buccalis

<400> 1174

Ile Asp Glu Ala Ile Ser Ser Ile Arg His Gly Ile Val His Phe Asn

1 5 10 15

Leu Glu Leu Glu Gly

twenty

<210> 1175

<211> 21

<212> PRT

<213> Leptotrichia sp.

<400> 1175

Ile Asp Glu Ala Ile Ser Ser Ile Arg His Gly Ile Val His Phe Asn

1 5 10 15

Leu Glu Leu Glu Gly

twenty

<210> 1176

<211> 16

<212> PRT

<213> Leptotrichia sp.

<400> 1176

Phe Gln Lys Glu Gly Tyr Leu Leu Arg Asn Lys Ile Leu His Asn Ser

1 5 10 15

<210> 1177

<211> 15

<212> PRT

<213> Leptotrichia shahii

<400> 1177

Phe Thr Lys Ile Gly Thr Asn Glu Arg Asn Arg Ile Leu His Ala

1 5 10 15

<210> 1178

<211> 14

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1178

Phe Arg Asn Glu Ile Asp His Phe His Tyr Phe Tyr Asp Arg

1 5 10

<210> 1179

<211> 14

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1179

Leu Arg Asn Tyr Ile Glu His Phe Arg Tyr Tyr Ser Ser Phe

1 5 10

<210> 1180

<211> 14

<212> PRT

<213> Clostridium aminophilum

<400> 1180

Val Arg Lys Tyr Val Asp His Phe Lys Tyr Tyr Ala Thr Ser

1 5 10

<210> 1181

<211> 14

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1181

Leu Arg Lys Tyr Val Asp His Phe Lys Tyr Tyr Ala Tyr Gly

1 5 10

<210> 1182

<211> 14

<212> PRT

<213> Carnobacterium gallinarum

<400> 1182

Ile Arg Asn Gln Thr Ala His Leu Ser Val Leu Gln Leu Glu

1 5 10

<210> 1183

<211> 14

<212> PRT

<213> Carnobacterium gallinarum

<400> 1183

Ile Arg Asn Asn Ile Ala His Leu His Val Leu Arg Asn Asp

1 5 10

<210> 1184

<211> 14

<212> PRT

<213> Paludibacter propionicigenes

<400> 1184

Ile Arg Asn His Ile Ala His Phe Asn Tyr Leu Thr Lys Asp

1 5 10

<210> 1185

<211> 14

<212> PRT

<213> Listeria seeligeri

<400> 1185

Lys Arg Asn Asn Ile Ser His Phe Asn Tyr Leu Asn Gly Gln

1 5 10

<210> 1186

<211> 14

<212> PRT

<213> Listeria weihenstephanensis

<400> 1186

Ala Arg Asn His Ile Ala His Leu Asn Tyr Leu Ser Leu Lys

1 5 10

<210> 1187

<211> 14

<212> PRT

<213> Listeria newyorkensis

<400> 1187

Ala Arg Asn His Ile Ala His Leu Asn Tyr Leu Ser Leu Lys

1 5 10

<210> 1188

<211> 14

<212> PRT

<213> Leptotrichia wadei

<400> 1188

Phe Arg Asn Tyr Ile Ala His Phe Leu His Leu His Thr Lys

1 5 10

<210> 1189

<211> 14

<212> PRT

<213> Leptotrichia wadei

<400> 1189

Ile Arg Asn Tyr Ile Ala His Phe Asn Tyr Ile Pro Asp Ala

1 5 10

<210> 1190

<211> 14

<212> PRT

<213> Leptotrichia wadei

<400> 1190

Ile Arg Asn Tyr Ile Ala His Phe Asn Tyr Ile Pro His Ala

1 5 10

<210> 1191

<211> 14

<212> PRT

<213> Rhodobacter capsulatus

<400> 1191

Thr Arg Lys Asp Leu Ala His Phe Asn Val Leu Asp Arg Ala

1 5 10

<210> 1192

<211> 14

<212> PRT

<213> Leptotrichia buccalis

<400> 1192

Ile Arg Asn Tyr Ile Ala His Phe Asn Tyr Ile Pro His Ala

1 5 10

<210> 1193

<211> 14

<212> PRT

<213> Leptotrichia sp.

<400> 1193

Ile Arg Asn Tyr Ile Ala His Phe Asn Tyr Ile Pro Asn Ala

1 5 10

<210> 1194

<211> 14

<212> PRT

<213> Leptotrichia sp.

<400> 1194

Ile Arg Asn Tyr Ile Ser His Phe Tyr Ile Val Arg Asn Pro

1 5 10

<210> 1195

<211> 14

<212> PRT

<213> Leptotrichia shahii

<400> 1195

Ile Arg Asn Tyr Ile Ser His Phe Tyr Ile Val Arg Asn Pro

1 5 10

<210> 1196

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1196

gugccacuuc ucagaucgcu cgcucaguga uccgac 36

<210> 1197

<211> 105

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1197

gucagaacac ugagcgagcg uucuuuuuga gaagcucaac gggcuuugcc accuggaaag 60

uggccauugg cacacccguu gaaaaaauuc uguccucuag acaga 105

<210> 1198

<211> 105

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1198

gucagaacac ugagcgagcg uucuuuuuga gaagcucaac gggcuuugcc accuggaaag 60

uggccauugg cacacccguu gaaaaaauuc uguccucuag acaga 105

<210> 1199

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1199

gugccacuuc ucagaucgcu cgcucaguga uccgac 36

<210> 1200

<211> 37

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1200

gugccaauca cccaacacug accaagcuug cggagac 37

<210> 1201

<211> 64

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1201

cuuggggaaa gcuaggcaag uuuuggauga uaagaaauaa ucaugucaca aggagggagu 60

uuu 64

<210> 1202

<211> 64

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1202

cuuggggaaa gcuaggcaag uuuuggauga uaagaaauaa ucaugucaca aggagggagu 60

uuu 64

<210> 1203

<211> 37

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1203

gugccaauca cccaacacug accaagcuug cggagac 37

<210> 1204

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1204

gccgcagcga augccguuuc acgaaucguc aggcgg 36

<210> 1205

<211> 75

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1205

gcuggagacg uuuuuugaaa cggcgagugc ugcggauagc gaguuucucu uggggaggcg 60

cucgcggcca cuuuu 75

<210> 1206

<211> 75

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1206

gcuggagacg uuuuuugaaa cggcgagugc ugcggauagc gaguuucucu uggggaggcg 60

cucgcggcca cuuuu 75

<210> 1207

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1207

gccgcagcga augccguuuc acgaaucguc aggcgg 36

<210> 1208

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1208

guccaagaaa aaagaaauga uacgaggcau uagcac 36

<210> 1209

<211> 107

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1209

cuggacgaug ucucuuuuau uucuuuuuuc uuggaucuga guacgagcac ccacauugga

cauuucgcau ggugggugcu cguacuauag guaaaacaaa ccuuuuu 107

<210> 1210

<211> 107

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1210

cuggacgaug ucucuuuuau uucuuuuuuc uuggaucuga guacgagcac ccacauugga

cauuucgcau ggugggugcu cguacuauag guaaaacaaa ccuuuuu 107

<210> 1211

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1211

guccaagaaa aaagaaauga uacgaggcau uagcac 36

<210> 1212

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1212

guucgaaagc uuaguggaaa gcuucguccu uagcac 36

<210> 1213

<211> 69

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1213

cacggauaau cacgacuuuc cacuaagcuu ucgaauuuua ugaugcgagc auccucucag 60

69

<210> 1214

<211> 69

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1214

cacggauaau cacgacuuuc cacuaagcuu ucgaauuuua ugaugcgagc auccucucag 60

69

<210> 1215

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1215

guucgaaagc uuaguggaaa gcuucguggu uagcac 36

<210> 1216

<211> 35

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1216

guauugagaa aagccagaua uaguuggcaa uagac 35

<210> 1217

<211> 62

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1217

auauuuugau ucccauuuau gguuauuuac cauaaauggg aaucaacuaa aaaauauuuuu

uu 62

<210> 1218

<211> 35

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1218

guauugagaa aagccagaua uaguuggcaa uagac 35

<210> 1219

<211> 62

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1219

auauuuugau ucccauuuau gguuauuuac cauaaauggg aaucaacuaa aaaauauuuuu

uu 62

<210> 1220

<211> 35

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1220

guugaugaga agagcccaag auagagggca auaac 35

<210> 1221

<211> 35

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1221

gcuggagaag auagcccaag aaagagggca auaac 35

<210> 1222

<211> 78

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1222

auuauuacca uuuugguugg aaugcuauua uaaaggauca uucgauuauu accucuaccu 60

cccuucccac gauuucuu 78

<210> 1223

<211> 78

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1223

attatattacca ttttggttgg aatgctatta taaaggatca ttcgattatt acctctacct 60

cccttcccac gatttctt 78

<210> 1224

<211> 35

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1224

guugaugaga agagcccaag auagagggca auaac 35

<210> 1225

<211> 78

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1225

attatattacca ttttggttgg aatgctatta taaaggatca ttcgattatt acctctacct 60

cccttcccac gatttctt 78

<210> 1226

<211> 35

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1226

gcuggagaag auagcccaag aaagagggca auaac 35

<210> 1227

<211> 35

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1227

guuuggagaa cagcccgaua uagagggcaa uagac 35

<210> 1228

<211> 81

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1228

gucuuacgac cucaguauua ggaagauuuc aaccaagaaa acuuaguuuc aggcuuaaug 60

aucgagucau gcagccaaag u 81

<210> 1229

<211> 81

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1229

gucuuacgac cucaguauua ggaagauuuc aaccaagaaa acuuaguuuc aggcuuaaug 60

aucgagucau gcagccaaag u 81

<210> 1230

<211> 35

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1230

guuuggagaa cagcccgaua uagagggcaa uagac 35

<210> 1231

<211> 35

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1231

guuuugagaa uagcccgaca uagaggggcaa uagac 35

<210> 1232

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1232

guuaugaaaa cagcccgaca uagaggggcaa uagaca 36

<210> 1233

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1233

guuauagucc ucuuacauuu agagguaguc uuuaau 36

<210> 1234

<211> 98

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1234

ucuuaagaac uucucuaccu gaaguuggau uauaaaugac ucuugcucuc auagauaucc 60

uccuuugaaa auauacacug ccgauuaauu accguuuu 98

<210> 1235

<211> 98

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1235

ucuuaagaac uucucuaccu gaaguuggau uauaaaugac ucuugcucuc auagauaucc 60

uccuuugaaa auauacacug ccgauuaauu accguuuu 98

<210> 1236

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1236

guuauagucc ucuuacauuu agagguaguc uuuaau 36

<210> 1237

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1237

guuauagucc ucuuacauuu agagguaguu uauauu 36

<210> 1238

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1238

guuauagucc ccuuacauuu agggguaguc uuuaau 36

<210> 1239

<211> 102

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1239

aauauaaauu cucccuaaau auaagagaau aauaacucaa ucucuucauu cguauuuugu

cuaguuaaga uaaguaccac caaauacaau caauccaaaa aa 102

<210> 1240

<211> 102

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1240

aauauaaauu cucccuaaau auaagagaau aauaacucaa ucucuucauu cguauuuugu

cuaguuaaga uaaguaccac caaauacaau caauccaaaa aa 102

<210> 1241

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1241

guuauagucc ucuuacauuu agagguaguu uauauu 36

<210> 1242

<211> 102

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1242

aauauaaauu cucccuaaau auaagagaau aauaacucaa ucucuucauu cguauuuugu

cuaguuaaga uaaguaccac caaauacaau caauccaaaa aa 102

<210> 1243

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1243

guuauagucc ccuuacauuu agggguaguc uuuaau 36

<210> 1244

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1244

guuguaguuc ccuucaauuu gggauaauc cacaag 36

<210> 1245

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1245

guuuuagucc ucuuucauau agagguaguc ucuuac 36

<210> 1246

<211> 99

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1246

augaaaagag gacuaaaacu gaaagaggac uaaaacacca gauguggaua acuauauuag 60

uggcuauuaa aaauucgucg auauuagaga ggaaacuuu 99

<210> 1247

<211> 99

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1247

augaaaagag gacuaaaacu gaaagaggac uaaaacacca gauguggaua acuauauuag 60

uggcuauuaa aaauucgucg auauuagaga ggaaacuuu 99

<210> 1248

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1248

guuuuagucc ucuuucauau agagguaguc ucuuac 36

<210> 1249

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1249

guuuuagacc ucuucuauuu ugagguacuc uaaauc 36

<210> 1250

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1250

guuuuagucc ucuuuuguuu ugagguacuc uaaauc 36

<210> 1251

<211> 147

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1251

60

uugccacaca gccaacauua uaagcguuaa aaccagcacc augaguacau uucacccaac 120

aaucagaauc cccguuucuc cguuuuu 147

<210> 1252

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1252

guuuuagucc ucuuuuguuu ugagguacuc uaaauc 36

<210> 1253

<211> 147

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1253

60

uugccacaca gccaacauua uaagcguuaa aaccagcacc augaguacau uucacccaac 120

aaucagaauc cccguuucuc cguuuuu 147

<210> 1254

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1254

guuuuagauc ccuucguuuu ugggguuauc uauauc 36

<210> 1255

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1255

guuuuagucc ccuucguuuu ugggguaguc uaaauc 36

<210> 1256

<211> 113

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1256

gauuuagagc accccaaaag uaaugaaaau uugcaauuaa auaaggaaua uuaaaaaaau60

gugauuuuaa aaaaauugaa gaaauuaaau gaaaaauugu ccaaguaaaa aaa 113

<210> 1257

<211> 70

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1257

auuuagauua ccccuuuaau uuauuuuacc auauuuuucu cauaaugcaa acuaauauuc 60

caaaauuuuu 70

<210> 1258

<211> 113

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1258

gauuuagagc accccaaaag uaaugaaaau uugcaauuaa auaaggaaua uuaaaaaaau

gugauuuuaa aaaaauugaa gaaauuaaau gaaaaauugu ccaaguaaaa aaa 113

<210> 1259

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1259

guuuuagucc ccuucguuuu ugggguaguc uaaauc 36

<210> 1260

<211> 70

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1260

auuuagauua ccccuuuaau uuauuuuacc auauuuuucu cauaaugcaa acuaauauuc 60

caaaauuuuu 70

<210> 1261

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1261

guuuuagucc ccuucguuuu ugggguaguc uaaauc 36

<210> 1262

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1262

guuuuagucc ccuucgauau uggggugguc uauauc 36

<210> 1263

<211> 95

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1263

60

uuuaauuuaa aaauauuuua uuagauuaaa guaga 95

<210> 1264

<211> 95

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1264

60

uuuaauuuaa aaauauuuua uuagauuaaa guaga 95

<210> 1265

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1265

guuuuagucc ccuucgauau uggggugguc uauauc 36

<210> 1266

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1266

guucaguccg ccgucgucuu ggcggugaug ugaggc 36

<210> 1267

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1267

guucaguccg ccgucauuuu ggcggugaug ugcucc 36

<210> 1268

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1268

guucaguccg ccgucgucuu ggcggugaug ugaggc 36

<210> 1269

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1269

guucaguccg ccgucauuuu ggcggugaug ugcucc 36

<210> 1270

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1270

guucaguccg ccgucgucuu ggcggugaug ugaggc 36

<210> 1271

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1271

guucaguccg ccgucauuuu ggcggugaug ugcucc 36

<210> 1272

<211> 81

<212> DNA

<213> Alicyclobacillus acidoterrestris

<400> 1272

agcgcccgct gtcggatcac tgagcgagcg atctgagaag tggcactgtt tggtaaaggt 60

aaaaagacga atgatgcatc c 81

<210> 1273

<211> 83

<212> DNA

<213> Alicyclobacillus acidoterrestris

<400> 1273

tgatgcatcc gtcggatcac tgagcgagcg atctgagaag tggcaccctt tataaaaagg 60

ggcgtccttt agtaccgtgt act 83

<210> 1274

<211> 84

<212> DNA

<213> Alicyclobacillus acidoterrestris

<400> 1274

accgtgtact gtcggatcac tgagcgagcg atctgagaag tggcacaagc cttgagtaat 60

tcgccgtggg attccccgcc gtat 84

<210> 1275

<211> 80

<212> DNA

<213> Alicyclobacillus acidoterrestris

<400> 1275

cccgccgtat gtcggatcac tgagcgagcg atctgagaag tggcactaat gaagttaaag 60

gagatgagac aatgaaagaa 80

<210> 1276

<211> 83

<212> DNA

<213> Alicyclobacillus acidoterrestris

<400> 1276

aatgaaagaa gtcggatcac tgagcgagcg atctgagaag tggcactgca atgcgttgga 60

ttatgacgat gcaggccaag gaa 83

<210> 1277

<211> 56

<212> DNA

<213> Alicyclobacillus acidoterrestris

<400> 1277

ggccaaggaa gtcggatcac tgagcgagcg atctgagaag tggcactgat gtcagc 56

<210> 1278

<211> 36

<212> DNA

<213> Alicyclobacillus acidoterrestris

<400> 1278

gtcggatcac tgagcgagcg atctgagaag tggcac 36

<210> 1279

<211> 36

<212> DNA

<213> Alicyclobacillus acidoterrestris

<400> 1279

gtcggatcac tgagcgagcg atctgagaag tggcac 36

<210> 1280

<211> 84

<212> DNA

<213> Desulfonatronum thiodismutans

<400> 1280

ccggctcgag gtctcggcaa gcttggtcag tgttgggtga ttggcacatc caggtggttg 60

gatgcgggac ataccttccg cctt 84

<210> 1281

<211> 84

<212> DNA

<213> Desulfonatronum thiodismutans

<400> 1281

cttccgcctt gtctcggcaa gcttggtcag tgttgggtga ttggcactgc ttcccggcga 60

acggcgagct gacctcctag atgt 84

<210> 1282

<211> 83

<212> DNA

<213> Desulfonatronum thiodismutans

<400> 1282

tcctagatgt gtctcggcaa gcttggtcag tgttgggtga ttggcaccgt ctgctcggtc 60

tcggacttca ccaccacgtc cac 83

<210> 1283

<211> 83

<212> DNA

<213> Desulfonatronum thiodismutans

<400> 1283

ccacgtccac gtctcggcaa gcttggtcag tgttgggtga ttggcacgggg tgcggcattt 60

gcgggtgttg ggggagtggc agg 83

<210> 1284

<211> 37

<212> DNA

<213> Desulfonatronum thiodismutans

<400> 1284

gtctcggcaa gcttggtcag tgttgggtga ttggcac 37

<210> 1285

<211> 15

<212> DNA

<213> Desulfonatronum thiodismutans

<400> 1285

gtctcggcaa gcttg 15

<210> 1286

<211> 22

<212> DNA

<213> Desulfonatronum thiodismutans

<400> 1286

gtcagtgttg ggtgattggc ac 22

<210> 1287

<211> 80

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1287

acatccactg ccgcctgacg attcgtgaaa cggcattcgc tgcggcaata gtctctggaa 60

atgttatagt agctcctaca 80

<210> 1288

<211> 81

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1288

agctcctaca ccgcctgacg attcgtgaaa cggcattcgc tgcggcaaga aaaagagtcg 60

tggtgttggc gcgggtcaga c 81

<210> 1289

<211> 80

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1289

cgggtcagac ccgcctgacg attcgtgaaa cggcattcgc tgcggcaatg taacgcctgg 60

agcatggctt gacccgaacc 80

<210> 1290

<211> 82

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1290

gacccgaacc ccgcctgacg attcgtgaaa cggcattcgc tgcggcaata tacgtctgat 60

taaaggtatg ggattccctg tt 82

<210> 1291

<211> 80

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1291

attccctgtt ccgcctgacg attcgtgaaa cggcattcgc tgcggctctc agtcaattcg 60

aatatgatgc ggggtactgg 80

<210> 1292

<211> 80

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1292

ggggtactgg ccgcctgacg attcgtgaaa cggcattcgc tgcggcgctc cacaaaagcg 60

attatcattt cccggttata 80

<210> 1293

<211> 82

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1293

cccggttata ccgcctgacg attcgtgaaa cggcattcgc tgcggcgtgc ccggccatgc 60

ggttatcggt ctcgatggcc tt 82

<210> 1294

<211> 79

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1294

cgatggcctt ccgcctgacg attcgtgaaa cggcattcgc tgcggctcgc ggggaaacga 60

gtgcgtagtc gatcgtcac 79

<210> 1295

<211> 79

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1295

cgatcgtcac ccgcctgacg attcgtgaaa cggcattcgc tgcggcgtag ctgtcgccgt 60

ctttcttgta ttcttttttt 79

<210> 1296

<211> 82

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1296

attctttttt ccgcctgacg attcgtgaaa cggcattcgc tgcggcgatc ggacaatcac 60

gccagacatt gccggtcatg at 82

<210> 1297

<211> 83

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1297

cggtcatgat ccgcctgacg attcgtgaaa cggcattcgc tgcggcttgg cgaccttcag 60

gcgagcgtta ttggcggcat aga 83

<210> 1298

<211> 81

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1298

gcggcataga ccgcctgacg attcgtgaaa cggcattcgc tgcggcaaaa atggcgaaac 60

cgaagccgcc gacgcgatac a 81

<210> 1299

<211> 80

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1299

acgcgataca ccgcctgacg attcgtgaaa cggcattcgc tgcggcatgt caattttggt 60

aacacttcgc cttggcacca 80

<210> 1300

<211> 80

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1300

cttggcacca ccgcctgacg attcgtgaaa cggcattcgc tgcggcatgt caattttggt 60

aacacttcgc cttggcacca 80

<210> 1301

<211> 80

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1301

cttggcacca ccgcctgacg attcgtgaaa cggcattcgc tgcggcagca cgtggggttt 60

ttgctctcac aaagtaaatt 80

<210> 1302

<211> 79

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1302

aaagtaaatt ccgcctgacg attcgtgaaa cggcattcgc tgcggcatag catcggcgag 60

tgtgtctgac agtcctact 79

<210> 1303

<211> 83

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1303

cagtcctact ccgcctgacg attcgtgaaa cggcattcgc tgcggcggcg aaccctcgtc 60

aaccgcgacc tcaagatggc aca 83

<210> 1304

<211> 81

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1304

agatggcaca ccgcctgacg attcgtgaaa cggcattcgc tgcggcaacg catcacgcgc 60

ctccggctcc atttccttgc c 81

<210> 1305

<211> 80

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1305

tttccttgcc ccgcctgacg attcgtgaaa cggcattcgc tgcggctccg ctcacagggg 60

caatctacgc tcaggagatg 80

<210> 1306

<211> 81

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1306

tcaggagatg ccgcctgacg attcgtgaaa cggcattcgc tgcggcgaaa acgccccgaa 60

acacagttcc cgaattcaaa t 81

<210> 1307

<211> 79

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1307

gaattcaaat ccgcctgacg attcgtgaaa cggcattcgc tgcggcagca caattccgag 60

aaacggtttc aggatcata 79

<210> 1308

<211> 81

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1308

caggatcata ccgcctgacg attcgtgaaa cggcattcgc tgcggcgcca gcgttccctg 60

cgatgctgtt gcgaaatccc c 81

<210> 1309

<211> 80

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1309

cgaaatcccc ccgcctgacg attcgtgaaa cggcattcgc tgcggcttgc actgcgtggt 60

gaaaggttc aaagcgtcca 80

<210> 1310

<211> 56

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1310

aaagcgtcca ccgcctgacg attcgtgaaa cggcattcgc tgcggcccgt gctcgc 56

<210> 1311

<211> 36

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1311

ccgcctgacg attcgtgaaa cggcattcgc tgcggc 36

<210> 1312

<211> 36

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1312

ccgcctgacg attcgtgaaa cggcattcgc tgcggc 36

<210> 1313

<211> 10

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1313

ccgcctgacg 10

<210> 1314

<211> 12

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1314

attcgtgaaa cg 12

<210> 1315

<211> 14

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1315

gcattcgctg cggc 14

<210> 1316

<211> 10

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1316

ccgcctgacg 10

<210> 1317

<211> 12

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1317

attcgtgaaa cg 12

<210> 1318

<211> 14

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1318

gcattcgctg cggc 14

<210> 1319

<211> 36

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1319

ccgcctgacg attcgtgaaa cggcattcgc tgcggc 36

<210> 1320

<211> 10

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1320

ccgcctgacg 10

<210> 1321

<211> 12

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1321

attcgtgaaa cg 12

<210> 1322

<211> 14

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1322

gcattcgctg cggc 14

<210> 1323

<211> 36

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1323

ccgcctgacg attcgtgaaa cggcattcgc tgcggc 36

<210> 1324

<211> 10

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1324

ccgcctgacg 10

<210> 1325

<211> 12

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1325

attcgtgaaa cg 12

<210> 1326

<211> 14

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1326

gcattcgctg cggc 14

<210> 1327

<211> 81

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1327

accccctgcg gtccaagaaa aaagaaatga tacgaggcat tagcaccatg caaacggatt 60

gttatataaa tcttcttgaa c 81

<210> 1328

<211> 82

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1328

cttcttgaac gtccaagaaa aaagaaatga tacgaggcat tagcacattg ttccggcggc 60

taatttgtct gcggtaatcg aa 82

<210> 1329

<211> 87

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1329

ggtaatcgaa gtccaagaaa aaagaaatga tacgaggcat tagcaccttc gccatcctca 60

tcccttatca gttgattgcc tagcgtt 87

<210> 1330

<211> 83

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1330

gcctagcgtt gtccaagaaa aaagaaatga tacgaggcat tagcacacac agaaaccaaa 60

tgggaacacg ttttcgttaa taa 83

<210> 1331

<211> 84

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1331

tcgttaataa gtccaagaaa aaagaaatga tacgaggcat tagcacgaca ttaaaaaatt 60

ccaaccaagc gagttattga gtgg 84

<210> 1332

<211> 80

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1332

tattgagtgg gtccaagaaa aaagaaatga tacgaggcat tagcacttat gagcttaaaa 60

gcttgttagc gacattaaac 80

<210> 1333

<211> 83

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1333

gacattaaac gtccaagaaa aaagaaatga tacgaggcat tagcacgttt tgttacagct 60

ttattcctta cttgatcgac tct 83

<210> 1334

<211> 83

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1334

gatcgactct gtccaagaaa aaagaaatga tacgaggcat tagcaccaga acctatctca 60

agaggatgca ttctggaaag gaa 83

<210> 1335

<211> 86

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1335

tggaaaggaa gtccaagaaa aaagaaatga tacgaggcat tagcacctta taacaataat 60

ttaaaagcaa tttatgactg tataga 86

<210> 1336

<211> 82

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1336

actgtataga gtccaagaaa aaagaaatga tacgaggcat tagcactttt aagggacatc 60

agaaacacta taagctcact tg 82

<210> 1337

<211> 81

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1337

agctcacttg gtccaagaaa aaagaaatga tacgaggcat tagcacataa tcgactttgc 60

atttctatag tgtcgttcat c 81

<210> 1338

<211> 85

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1338

gtcgttcatc gtccaagaaa aaagaaatga tacgaggcat tagcacaaaa tggaacaagg 60

aacaatagac gtttataagt atgga 85

<210> 1339

<211> 83

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1339

taagtatgga gtccaagaaa aaagaaatga tacgaggcat tagcactttc aataaagcat 60

caaactcttt ttgcattttt tca 83

<210> 1340

<211> 83

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1340

cattttttca gtccaagaaa aaagaaatga tacgaggcat tagcacaccg agtaaggaat 60

cgtttaatca acaaacttga aac 83

<210> 1341

<211> 84

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1341

aacttgaaac gtccaagaaa aaagaaatga tacgaggcat tagcacggta gtcggcggct 60

aagtgtcgca gggttaacac cgat 84

<210> 1342

<211> 85

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1342

taacaccgat gtccaagaaa aaagaaatga tacgaggcat tagcacacgc tgaacaaaac 60

tcacgaaacc aaaagtttat aaaat 85

<210> 1343

<211> 81

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1343

tttataaaat gtccaagaaa aaagaaatga tacgaggcat tagcacggaa aggatttact 60

agatctcgca agaaaggtaa c 81

<210> 1344

<211> 82

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1344

gaaaggtaac gtccaagaaa aaagaaatga tacgaggcat tagcacattt agtatattgt 60

tgttttcatt tgcttttttc gc 82

<210> 1345

<211> 83

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1345

cttttttcgc gtccaagaaa aaagaaatga tacgaggcat tagcacttat accgtaaaaa 60

attttggatt tgatgtcacc gtc 83

<210> 1346

<211> 82

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1346

tgtcaccgtc gtccaagaaa aaagaaatga tacgaggcat tagcacagaa cacaaaaagc 60

ggaaaaattg cacttatttt cg 82

<210> 1347

<211> 82

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1347

cttattttcg gtccaagaaa aaagaaatga tacgaggcat tagcacaata ctcgtctaca 60

aactttttct gctttttctgt ta 82

<210> 1348

<211> 84

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1348

ttttctgtta gtccaagaaa aaagaaatga tacgaggcat tagcaccata caggacactt 60

aaactctact ttacgatttt caaa 84

<210> 1349

<211> 82

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1349

gattttcaaa gtccaagaaa aaagaaatga tatgaggcat tagcacgatt taaaacttct 60

tctggcatcc agtacataga tt 82

<210> 1350

<211> 82

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1350

tacatagatt gtccaagaaa aaagaaatga tatgaggcat tagcacgaaa aaactgtttc 60

cttatcacac ctatagcaat aa 82

<210> 1351

<211> 84

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1351

atagcaataa gtccaagaaa aaagaaatga tatgaggcat tagcactcta gatatgttca 60

agaaaatatg cagtctatcg gtca 84

<210> 1352

<211> 85

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1352

ctatcggtca gtccaagaaa aaagaaatga tatgaggcat tagcacgaat caattaatcc 60

aatttgttgc aacttaggta gagag 85

<210> 1353

<211> 84

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1353

aggtagagag gtccaagaaa aaagaaatga tatgaggcat tagcacccga attagcatcg 60

taccaattac atccatatga tgct 84

<210> 1354

<211> 82

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1354

atatgatgct gtccaagaaa aaagaaatga tatgaggcat tagcactttt ctatttttat 60

attactgttt gtttggtgat aa 82

<210> 1355

<211> 84

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1355

ttggtgataa gtccaagaaa aaagaaatga tatgaggcat tagcactgaa tctaaccaag 60

gaagaaataa aagatattta taag 84

<210> 1356

<211> 81

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1356

tatttataag gtccaagaaa aaagaaatga tatgaggcat tagcacaaaa aggagtgttt 60

caaaatggca aatgaaaaaa g 81

<210> 1357

<211> 82

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1357

atgaaaaaag gtccaagaaa aaagaaatga tatgaggcat tagcacagct tggattgaat 60

caaacaatgg tggtcgaggt tt 82

<210> 1358

<211> 82

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1358

gtcgaggttt gtccaagaaa aaagaaatga tatgaggcat tagcacttga gttgtaatta 60

atcaccgttc tacctgaaaa ct 82

<210> 1359

<211> 80

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1359

cctgaaaact gtccaagaaa aaagaaatga tatgaggcat tagcactaga tgttgaaaac 60

ggcggttttg tctattatcc 80

<210> 1360

<211> 84

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1360

tctattatcc gtccaagaaa aaagaaatga tatgaggcat tagcacaaac ccgcaaaaaa 60

taagctgttg acgagtctag ttag 84

<210> 1361

<211> 85

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1361

gtctagttag gtccaagaaa aaagaaatga tacgaggcat tagcacttgt caatcctcga 60

aaatgccggg cggacggggct tgcca 85

<210> 1362

<211> 81

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1362

gggcttgcca gtccaagaaa aaagaaatga aacgaggcat tagcacccac caccgacggc 60

gtccacgcca attgcctgct t 81

<210> 1363

<211> 72

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1363

ttgcctgctt gtccaagaaa aaagaaatga tacgaggcat tagcacaaca atataaacga 60

ctactttacc gt 72

<210> 1364

<211> 56

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1364

actttaccgt gttcaagaaa aaagaaatga tatgaggcat tagcacgatg ggatgg 56

<210> 1365

<211> 36

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1365

gtccaagaaa aaagaaatga tacgaggcat tagcac 36

<210> 1366

<211> 36

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1366

gtccaagaaa aaagaaatga tacgaggcat tagcac 36

<210> 1367

<211> 36

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1367

gtccaagaaa aaagaaatga tacgaggcat tagcac 36

<210> 1368

<211> 83

<212> DNA

<213> Bacillus sp.

<400> 1368

ggactttagc gtgctaacca cgaagctttc cactaagctt tcgaacaccg gattggtgga 60

aagacgccgc tgccgaccga aaa 83

<210> 1369

<211> 83

<212> DNA

<213> Bacillus sp.

<400> 1369

cgaccgaaaa gtgctaacca cgaagctttc cactaagctt tcgaacaccg gattggtgga 60

aagacgccgc tgccgaccga aaa 83

<210> 1370

<211> 87

<212> DNA

<213> Bacillus sp.

<400> 1370

cgaccgaaaa gtgctaacca cgaagctttc cactaagctt tcgaacggga aaacgtggggc 60

gcaactttgc tattctggca tgtttgc 87

<210> 1371

<211> 83

<212> DNA

<213> Bacillus sp.

<400> 1371

gcatgtttgc gtgctaacca cgaagctttc cactaagctt tcgaacaagt ttggaagggg 60

gcgatagcga tgggttttaa gat 83

<210> 1372

<211> 81

<212> DNA

<213> Bacillus sp.

<400> 1372

gttttaagat gtgctaacca cgaagctttc cactaagctt tcgaacccga tcaccgtcaa 60

aaccgtagta gtaagactcg c 81

<210> 1373

<211> 81

<212> DNA

<213> Bacillus sp.

<400> 1373

taagactcgc gtgctaacca cgaagctttc cactaagctt tcgaacaagg cggcctaaat 60

cacttgggcc gcccttaaga t 81

<210> 1374

<211> 86

<212> DNA

<213> Bacillus sp.

<400> 1374

cccttaagat gtgctaacca cgaagctttc cactaagctt tcgaactgct tttatttcca 60

tgctaccatt ggctgaatcg ggactg 86

<210> 1375

<211> 82

<212> DNA

<213> Bacillus sp.

<400> 1375

atcgggactg gtgctaacca cgaagctttc cactaagctt tcgaacaaac aggtatctgc 60

ttatcaacgt gcagcacagg cg 82

<210> 1376

<211> 82

<212> DNA

<213> Bacillus sp.

<400> 1376

agcacaggcg gtgctaacca cgaagctttc cactaagctt tcgaacctat atgagtagaa 60

cgtctctcaa taagcgtaga at 82

<210> 1377

<211> 81

<212> DNA

<213> Bacillus sp.

<400> 1377

agcgtagaat gtgctaacca cgaagctttc cactaagctt tcgaacgcgc cttgttgcta 60

tcattagggt cgcgatcaac a 81

<210> 1378

<211> 88

<212> DNA

<213> Bacillus sp.

<400> 1378

gcgatcaaca gtgctaacca cgaagctttc cactaagctt tcgaacgtat aacttttaga 60

taacaaatgt tatacaaaat gcttgacg 88

<210> 1379

<211> 81

<212> DNA

<213> Bacillus sp.

<400> 1379

atgcttgacg gtgctaacca cgaagctttc cactaagctt tcgaacagcc caagctttac 60

atacacctat gcgtatgctt t 81

<210> 1380

<211> 56

<212> DNA

<213> Bacillus sp.

<400> 1380

cgtatgcttt gtgctaacca cgaagctttc cactaagctt tcgaactcct tcccac 56

<210> 1381

<211> 36

<212> DNA

<213> Bacillus sp.

<400> 1381

gtgctaacca cgaagctttc cactaagctt tcgaac 36

<210> 1382

<211> 84

<212> DNA

<213> Bacillus sp.

<400> 1382

tggacaagct gtgctaacca cgaagctttc cactaagctt tcgaacctct ggtgtaaggc 60

ggtttcttgc ttgtaagtgc gcgg 84

<210> 1383

<211> 84

<212> DNA

<213> Bacillus sp.

<400> 1383

aagtgcgcgg gtgctaatcc cgaagctttc cactaagctt tcgaacaaaa gagtataagt 60

accactctta gccgagtagt tcaa 84

<210> 1384

<211> 80

<212> DNA

<213> Bacillus sp.

<400> 1384

agtagttcaa gtgctaatcc cgaagctttc cactaagctt tcgaacactg gagaaccttc 60

caagtgtgcg atagcgtatc 80

<210> 1385

<211> 80

<212> DNA

<213> Bacillus sp.

<400> 1385

atagcgtatc gtgctaatcc cgaagctttc cactaagctt tcgaactttt ttgttccgcc 60

aaggtaaaac gtaccgagtt 80

<210> 1386

<211> 83

<212> DNA

<213> Bacillus sp.

<400> 1386

gtaccgagtt gtgctaatcc cgaagctttc cactaagctt tcgaactttc cgtaaaactt 60

tttgagaaaa caggagggcg act 83

<210> 1387

<211> 86

<212> DNA

<213> Bacillus sp.

<400> 1387

gagggcgact gtgctaatcc cgaagctttc cactaagctt tcgaaccagg ctttttgtgc 60

ataggtcatg cagtaaccta atccgc 86

<210> 1388

<211> 85

<212> DNA

<213> Bacillus sp.

<400> 1388

cctaatccgc gtgctaatcc cgaagctttc cactaagctt tcgaacgacc atgatctcga 60

aaagaaaatt acgcgtgcat cgcaa 85

<210> 1389

<211> 84

<212> DNA

<213> Bacillus sp.

<400> 1389

tgcatcgcaa gtgctaatcc cgaagctttc cactaagctt tcgaactggg ccgctccgat 60

tcgataaaga cgggtatctg gaga 84

<210> 1390

<211> 56

<212> DNA

<213> Bacillus sp.

<400> 1390

tatctggaga gtgctaatcc cgaagctttc cactaagctt tcgaaccagg gtaccc 56

<210> 1391

<211> 36

<212> DNA

<213> Bacillus sp.

<400> 1391

gtgctaatcc cgaagctttc cactaagctt tcgaac 36

<210> 1392

<211> 82

<212> DNA

<213> Bacillus sp.

<400> 1392

gctacctctg gtacatcccc ttcaatttcc actaagcttt cgaacgttcg gcgtagtagt 60

gtacacgctc ttgaatatga tg 82

<210> 1393

<211> 81

<212> DNA

<213> Bacillus sp.

<400> 1393

gaatatgatg gtacatcccc ttcaatttcc actaagcttt cgaacgccgc aacgtgctcc 60

gggagaggcg cacgttcaga g 81

<210> 1394

<211> 80

<212> DNA

<213> Bacillus sp.

<400> 1394

acgttcagag gtacatcccc ttcaatttcc actaagcttt cgaacgcgac cagtccgagc 60

agggcaatcc cgacaatgcg 80

<210> 1395

<211> 81

<212> DNA

<213> Bacillus sp.

<400> 1395

cgacaatgcg gtacatcccc ttcaatttcc actaagcttt cgaacaaccc gaaggactac 60

accatttccc gtgataaagc g 81

<210> 1396

<211> 81

<212> DNA

<213> Bacillus sp.

<400> 1396

tgataaagcg gtacatcccc ttcaatttcc actaagcttt cgaacaaaag cttggggagg 60

gcttcgagtc gggtatacag g 81

<210> 1397

<211> 79

<212> DNA

<213> Bacillus sp.

<400> 1397

ggtatacagg gtacatcccc ttcaatttcc actaagcttt cgaacccttg tccgcgataa 60

atctgtacgt cgaatccgt 79

<210> 1398

<211> 81

<212> DNA

<213> Bacillus sp.

<400> 1398

tcgaatccgt gtacatcccc ttcaatttcc actaagcttt cgaactgttt ggttgctgcg 60

cggccgaagt tgtctgacag t 81

<210> 1399

<211> 84

<212> DNA

<213> Bacillus sp.

<400> 1399

gtctgacagt gtacatcccc ttcaatttcc actaagcttt cgaacccgtt attaagttac 60

ctctttctag cataacctga gtgt 84

<210> 1400

<211> 88

<212> DNA

<213> Bacillus sp.

<400> 1400

acctgagtgt gtacatcccc ttcaatttcc actaagcttt cgaacatgta catcctgcat 60

cagttcgaag gaagcgggat cagctagt 88

<210> 1401

<211> 86

<212> DNA

<213> Bacillus sp.

<400> 1401

atcagctagt gtacatcccc ttcaatttcc actaagcttt cgaaccaaga gacgtgttga 60

cgatcatctt ttttgagatc tgccgt 86

<210> 1402

<211> 87

<212> DNA

<213> Bacillus sp.

<400> 1402

gatctgccgt gtacatcccc ttcaatttcc actaagcttt cgaacgcgca actcttgcaa 60

agttttttgc agcaacattt ttttccgt 87

<210> 1403

<211> 82

<212> DNA

<213> Bacillus sp.

<400> 1403

ttttttccgt gtacatcccc ttcaatttcc actaagcttt cgaactcaac catacttatt 60

tattttagca ctgccccaaa gt 82

<210> 1404

<211> 56

<212> DNA

<213> Bacillus sp.

<400> 1404

gccccaaagt gtacatcccc ttcaatttcc actaagcttt cgaacaacct ctgctg 56

<210> 1405

<211> 35

<212> DNA

<213> Bacillus sp.

<400> 1405

gtacatcccc ttcaatttcc actaagcttt cgaac 35

<210> 1406

<211> 36

<212> DNA

<213> Bacillus sp.

<400> 1406

gtgctaacca cgaagctttc cactaagctt tcgaac 36

<210> 1407

<211> 83

<212> DNA

<213> Desulfatirhabdium butyrativorans

<400> 1407

cccatccatt tgtccgtcga tcaagctgct ttcaccatcg gaaccccggc caccgaggca 60

gccaacacca cccaggccga gct 83

<210> 1408

<211> 83

<212> DNA

<213> Desulfatirhabdium butyrativorans

<400> 1408

aggccgagct gtgtcagtcg atcaagctgt tttcaccatc ggaacccctt gaattgagac 60

ggttccggag cgcaatatgg tga 83

<210> 1409

<211> 59

<212> DNA

<213> Desulfatirhabdium butyrativorans

<400> 1409

aatatggtga gtgtcagtcg atcaagctgt tttcaccatc ggaacccccc ccggccatc 59

<210> 1410

<211> 38

<212> DNA

<213> Desulfatirhabdium butyrativorans

<400> 1410

gtgtcagtcg atcaagctgt tttcaccatc ggaacccc 38

<210> 1411

<211> 38

<212> DNA

<213> Desulfatirhabdium butyrativorans

<400> 1411

gtgtcagtcg atcaagctgt tttcaccatc ggaacccc 38

<210> 1412

<211> 19

<212> DNA

<213> Desulfatirhabdium butyrativorans

<400> 1412

gtgtcagtcg atcaagctg 19

<210> 1413

<211> 19

<212> DNA

<213> Desulfatirhabdium butyrativorans

<400> 1413

ttttcaccat cggaaccc 19

<210> 1414

<211> 82

<212> DNA

<213> Brevibacillus agri

<400> 1414

cgaggatttt gtgctaacca cgaagctttc cactaagctt tcgaaccata ccgccgttta 60

ctttacctgg tacggcgact at 82

<210> 1415

<211> 81

<212> DNA

<213> Brevibacillus agri

<400> 1415

cggcgactat gtgctaacca cgaagctttc cactaagctt tcgaactctg caagtgcgcg 60

tctatgaaaa gaactgggaa a 81

<210> 1416

<211> 86

<212> DNA

<213> Brevibacillus agri

<400> 1416

aactgggaaa gtgctaacca cgaagctttc cactaagctt tcgaacgttc aaccttcttc 60

gtgagaaaat tgatgatatt ttgaag 86

<210> 1417

<211> 85

<212> DNA

<213> Brevibacillus agri

<400> 1417

tattttgaag gtgctaacca cgaagctttc cactaagctt tcgaaccgga tcactctcaa 60

cagctccatt acaacgatac ttatt 85

<210> 1418

<211> 83

<212> DNA

<213> Brevibacillus agri

<400> 1418

gatacttatt gtgctaacca cgaagctttc cactaagctt tcgaacgaag ttatgaagaa 60

agtctagcat gtattcccga tgt 83

<210> 1419

<211> 84

<212> DNA

<213> Brevibacillus agri

<400> 1419

ttcccgatgt gtgctaacca cgaagctttc cactaagctt tcgaaccatc aacagcaaca 60

tcaacacgag atcgaggttg aggc 84

<210> 1420

<211> 81

<212> DNA

<213> Brevibacillus agri

<400> 1420

aggttgaggc gtgctaacca cgaagctttc cactaagctt tcgaacctct gtttgcagca 60

gcatacttca aaaagtcttc a 81

<210> 1421

<211> 85

<212> DNA

<213> Brevibacillus agri

<400> 1421

aaagtcttca gtgctaacca cgaagctttc cactaagctt tcgaacataa tccgtttgct 60

gtacggatat aaaaatcttg tatag 85

<210> 1422

<211> 80

<212> DNA

<213> Brevibacillus agri

<400> 1422

tcttgtatag gtgctaacca cgaagctttc cactaagctt tcgaaccgta tgcgaagtct 60

gttatacacc gcatttttg 80

<210> 1423

<211> 57

<212> DNA

<213> Brevibacillus agri

<400> 1423

gcattttatg gtgctaacca cgaagctttc cactaagctt tcgaactcct tcccact 57

<210> 1424

<211> 36

<212> DNA

<213> Brevibacillus agri

<400> 1424

gtgctaacca cgaagctttc cactaagctt tcgaac 36

<210> 1425

<211> 87

<212> DNA

<213> Brevibacillus agri

<400> 1425

tggacaagct gtgctaacca cgaagctttc cactaagctt tcgaacttct taacacgcga 60

cgactctttc cccacacgat ttcgatg 87

<210> 1426

<211> 84

<212> DNA

<213> Brevibacillus agri

<400> 1426

gatttcgatg gtgctaatcc cgaagctttc cactaagctt tcgaacaact ataacataac 60

ccaagcaacc gcttatagtt tctg 84

<210> 1427

<211> 86

<212> DNA

<213> Brevibacillus agri

<400> 1427

atagtttctg gtgctaatcc cgaagctttc cactaagctt tcgaacacca gcgccttgtc 60

tgccatgtat tgctgtgcca aagcca 86

<210> 1428

<211> 81

<212> DNA

<213> Brevibacillus agri

<400> 1428

gccaaagcca gtgctaatcc cgaagctttc cactaagctt tcgaactaac cgtaagcctg 60

taacgaagcg ttcgctccaa c 81

<210> 1429

<211> 81

<212> DNA

<213> Brevibacillus agri

<400> 1429

tcgctccaac gtgctaatcc cgaagctttc cactaagctt tcgaactggg acggtgcaca 60

gcagaccgta aaagatgcgt t 81

<210> 1430

<211> 82

<212> DNA

<213> Brevibacillus agri

<400> 1430

aagatgcgtt gtgctaatcc cgaagctttc cactaagctt tcgaacatct agtcacccca 60

aggataattg tcgtcgtagt cc 82

<210> 1431

<211> 84

<212> DNA

<213> Brevibacillus agri

<400> 1431

gtcgtagtcc gtgctaatcc cgaagctttc cactaagctt tcgaacctct agcttgctgt 60

cgaagttagc cctactgttg atga 84

<210> 1432

<211> 84

<212> DNA

<213> Brevibacillus agri

<400> 1432

ctgttgatga gtgctaatcc cgaagctttc cactaagctt tcgaacatct gcatcacgggg 60

tagcaacgcc cgcagtcagg tctt 84

<210> 1433

<211> 82

<212> DNA

<213> Brevibacillus agri

<400> 1433

gtcaggtctt gtgctaatcc cgaagctttc cactaagctt tcgaacgagg atcgtgtctg 60

tcaacgggta ttgcacgctt cc 82

<210> 1434

<211> 84

<212> DNA

<213> Brevibacillus agri

<400> 1434

gcacgcttcc gtgctaatcc cgaagctttc cactaagctt tcgaacacgg agtggatttt 60

agagaggggt ggtttttgtt tcta 84

<210> 1435

<211> 81

<212> DNA

<213> Brevibacillus agri

<400> 1435

tttgtttcta gtgctaatcc cgaagctttc cactaagctt tcgaacattt tcgttgccca 60

gacgggagag aggaagagag t 81

<210> 1436

<211> 82

<212> DNA

<213> Brevibacillus agri

<400> 1436

ggaagagagt gtgctaatcc cgaagctttc cactaagctt tcgaactctc ctgatacgcc 60

ccatcagact gcttttctag ca 82

<210> 1437

<211> 83

<212> DNA

<213> Brevibacillus agri

<400> 1437

ttttctagca gtgctaatcc cgaagctttc cactaagctt tcgaactcat atttatcctg 60

taagcttccc tcactcattttta 83

<210> 1438

<211> 82

<212> DNA

<213> Brevibacillus agri

<400> 1438

ctcattttta gtgctaatcc cgaagctttc cactaagctt tcgaacttga ttgttcggct 60

aaggcgaagg acagagctaa gg 82

<210> 1439

<211> 82

<212> DNA

<213> Brevibacillus agri

<400> 1439

agagctaagg gtgctaatcc cgaagctttc cactaagctt tcgaacgtcc tgacataccg 60

atgttcggcg ctcggtcgaa gt 82

<210> 1440

<211> 86

<212> DNA

<213> Brevibacillus agri

<400> 1440

cggtcgaagt gtgctaatcc cgaagctttc cactaagctt tcgaacgcca ttcgccgagc 60

ctgcggacgt cttctttttcc ggatac 86

<210> 1441

<211> 81

<212> DNA

<213> Brevibacillus agri

<400> 1441

ttccggatac gtgctaatcc cgaagctttc cactaagctt tcgaacccca ggaaacttat 60

atcggcaata gtagctgtct g 81

<210> 1442

<211> 82

<212> DNA

<213> Brevibacillus agri

<400> 1442

tagctgtctg gtgctaatcc cgaagctttc cactaagctt tcgaacgtgc tgcgagtacg 60

agatcaagca ccgcaaggct aa 82

<210> 1443

<211> 85

<212> DNA

<213> Brevibacillus agri

<400> 1443

gcaaggctaa gtgctaatcc cgaagctttc cactaagctt tcgaacttgc cacatttctc 60

tacacggggc tgcgggtcag cgagc 85

<210> 1444

<211> 86

<212> DNA

<213> Brevibacillus agri

<400> 1444

gtcagcgagc gtgctaatcc cgaagctttc cactaagctt tcgaacgtaa cttgcttact 60

caaagtaagc gtgtagtcac ccgccg 86

<210> 1445

<211> 85

<212> DNA

<213> Brevibacillus agri

<400> 1445

tcacccgccg gtgctaatcc cgaagctttc cactaagctt tcgaacggca cgaattcgac 60

ttcgtccgta tgggcgacta tatga 85

<210> 1446

<211> 84

<212> DNA

<213> Brevibacillus agri

<400> 1446

gactatatga gtgctaatcc cgaagctttc cactaagctt tcgaacacta tggcaggatt 60

ggcagaactt tcgccgtttt ttca 84

<210> 1447

<211> 83

<212> DNA

<213> Brevibacillus agri

<400> 1447

cgttttttca gtgctaatcc cgaagctttc cactaagctt tcgaaccccc tttttgacca 60

tgccaagagg aaaagatagc gga 83

<210> 1448

<211> 85

<212> DNA

<213> Brevibacillus agri

<400> 1448

agatagcgga gtgctaatcc cgaagctttc cactaagctt tcgaacttat tttgggtctc 60

gtgagtcgat gcagttgcgt atcta 85

<210> 1449

<211> 82

<212> DNA

<213> Brevibacillus agri

<400> 1449

tgcgtatcta gtgctaatcc cgaagctttc cactaagctt tcgaacccac gtggcgaaag 60

caagttttcc atatcgtcat aa 82

<210> 1450

<211> 81

<212> DNA

<213> Brevibacillus agri

<400> 1450

atcgtcataa gtgctaatcc cgaagctttc cactaagctt tcgaaccttc tattttggga 60

gttggtgctg tgatggtggc g 81

<210> 1451

<211> 82

<212> DNA

<213> Brevibacillus agri

<400> 1451

gatggtggcg gtgctaatcc cgaagctttc cactaagctt tcgaacttct ttggtattca 60

ctccccatcc cgtctcatgg ct 82

<210> 1452

<211> 83

<212> DNA

<213> Brevibacillus agri

<400> 1452

tctcatggct gtgctaatcc cgaagctttc cactaagctt tcgaacgcta gccgtggaca 60

aggaagaaat aaaggaactc att 83

<210> 1453

<211> 82

<212> DNA

<213> Brevibacillus agri

<400> 1453

ggaactcatt gtgctaatcc cgaagctttc cactaagctt tcgaactacc actacctcaa 60

cgctcccgtc gaaaccatca tg 82

<210> 1454

<211> 80

<212> DNA

<213> Brevibacillus agri

<400> 1454

aaccatcatg gtgctaatcc cgaagctttc cactaagctt tcgaactata caggcccgct 60

tttttgatgt cgatgctgga 80

<210> 1455

<211> 82

<212> DNA

<213> Brevibacillus agri

<400> 1455

cgatgctgga gtgctaatcc cgaagctttc cactaagctt tcgaacggtt tcttgatgaa 60

acgcagcgcg tccgtaaagg at 82

<210> 1456

<211> 56

<212> DNA

<213> Brevibacillus agri

<400> 1456

cgtaaaggat gtgctaatcc cgaagctttc cactaagctt tcgaacccct gtcttc 56

<210> 1457

<211> 36

<212> DNA

<213> Brevibacillus agri

<400> 1457

gtgctaatcc cgaagctttc cactaagctt tcgaac 36

<210> 1458

<211> 36

<212> DNA

<213> Brevibacillus agri

<400> 1458

gtgctaacca cgaagctttc cactaagctt tcgaac 36

<210> 1459

<211> 397

<212> DNA

<213> Brevibacillus sp.

<400> 1459

gtgctaacca cgaagctttc cactaagctt tcgaacgctt acagcttccg gtcaaccccg 60

catccatccg catagtgcta atcccgaagc ttttcactaa gctttcgaac tcaagatcga 120

ggcactgtac ccgggcacga aagggtgcta atcccgaagc ttttcactaa gctttcgaac 180

ttaatatctt ggttgtagtt gcgcaaacct tttatgtgct aatcccgaag ctttcacta 240

agctttcgaa ccttactcag tttgcgtcct ccgaagaagt cgtccataag tgctaatccc 300

gaagctttcc actaagcttt cgaacgcgac acgaccgtat tcgtggacga gccggtcttt 360

gtgtgctaat cccgaagctt tcactaagc tttcgaa 397

<210> 1460

<211> 36

<212> DNA

<213> Brevibacillus sp.

<400> 1460

gtgctaacca cgaagctttc cactaagctt tcgaac 36

<210> 1461

<211> 80

<212> DNA

<213> Methylobacterium nodulans

<400> 1461

aacagatcgt gtgccaacgc gatcaggatc tggcgcccac tgcgaccccc cgtgtccaca 60

tcgacggccg cgatgcacca 80

<210> 1462

<211> 81

<212> DNA

<213> Methylobacterium nodulans

<400> 1462

cgatgcacca gtgccaacgc gctcaggatc tggcgcccac tgcgacttcc ggcgaccctg 60

gatctgcacg gctacacaac t 81

<210> 1463

<211> 81

<212> DNA

<213> Methylobacterium nodulans

<400> 1463

ctacacaact gtgccaacgc gctcaggatc tggcgcccac tgcgacttgg ccccgccggc 60

accgggtagg agacccgcca t 81

<210> 1464

<211> 79

<212> DNA

<213> Methylobacterium nodulans

<400> 1464

gacccgccat gtgccaacgc gctcaggatc tggcgcccac tgcgacatag ttctggctgt 60

tggaggccag attgttctg 79

<210> 1465

<211> 81

<212> DNA

<213> Methylobacterium nodulans

<400> 1465

gattgttctg gtgccaacgc gctcaggatc tggcgcccac tgcgacctga ccttcacgcg 60

tgtctactcg ggcgtggcca a 81

<210> 1466

<211> 81

<212> DNA

<213> Methylobacterium nodulans

<400> 1466

gcgtggccaa gtgccaacgc gctcaggatc tggcgcccac tgcgacgtcc aggccgagtt 60

gggcaaggtc cgccggatcc t 81

<210> 1467

<211> 56

<212> DNA

<213> Methylobacterium nodulans

<400> 1467

gccggatcct gtgccaacgc gctcaggatc tggcgcccac tgcgacggcg ggcacg 56

<210> 1468

<211> 36

<212> DNA

<213> Methylobacterium nodulans

<400> 1468

gtgccaacgc gctcaggatc tggcgcccac tgcgac 36

<210> 1469

<211> 36

<212> DNA

<213> Methylobacterium nodulans

<400> 1469

gtgccaacgc gctcaggatc tggcgcccac tgcgac 36

<210> 1470

<211> 12

<212> DNA

<213> Methylobacterium nodulans

<400> 1470

gtgccaacgc gc 12

<210> 1471

<211> 24

<212> DNA

<213> Methylobacterium nodulans

<400> 1471

tcaggatctg gcgcccactg cgac 24

<210> 1472

<211> 36

<212> DNA

<213> Alicyclobacillus acidoterrestris

<400> 1472

gtcggatcac tgagcgagcg atctgagaag tggcac 36

<210> 1473

<211> 37

<212> DNA

<213> Desulfonatronum thiodismutans

<400> 1473

gtctcggcaa gcttggtcag tgttgggtga ttggcac 37

<210> 1474

<211> 36

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1474

ccgcctgacg attcgtgaaa cggcattcgc tgcggc 36

<210> 1475

<211> 36

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1475

gtccaagaaa aaagaaatga tacgaggcat tagcac 36

<210> 1476

<211> 36

<212> DNA

<213> Bacillus sp.

<400> 1476

gtgctaacca cgaagctttc cactaagctt tcgaac 36

<210> 1477

<211> 38

<212> DNA

<213> Desulfatirhabdium butyrativorans

<400> 1477

gtgtcagtcg atcaagctgt tttcaccatc ggaacccc 38

<210> 1478

<211> 36

<212> DNA

<213> Brevibacillus agri

<400> 1478

gtgctaacca cgaagctttc cactaagctt tcgaac 36

<210> 1479

<211> 36

<212> DNA

<213> Brevibacillus sp.

<400> 1479

gtgctaacca cgaagctttc cactaagctt tcgaac 36

<210> 1480

<211> 36

<212> DNA

<213> Methylobacterium nodulans

<400> 1480

gtgccaacgc gctcaggatc tggcgcccac tgcgac 36

<210> 1481

<211> 36

<212> DNA

<213> Alicyclobacillus acidoterrestris

<400> 1481

gtcggatcac tgagcgagcg atctgagaag tggcac 36

<210> 1482

<211> 37

<212> DNA

<213> Desulfonatronum thiodismutans

<400> 1482

gtctcggcaa gcttggtcag tgttgggtga ttggcac 37

<210> 1483

<211> 36

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Opitutaceae bacterium sequence"

<400> 1483

ccgcctgacg attcgtgaaa cggcattcgc tgcggc 36

<210> 1484

<211> 36

<212> DNA

<213> Bacillus thermoamylovorans

<400> 1484

gtccaagaaa aaagaaatga tacgaggcat tagcac 36

<210> 1485

<211> 36

<212> DNA

<213> Bacillus sp.

<400> 1485

gtgctaacca cgaagctttc cactaagctt tcgaac 36

<210> 1486

<211> 38

<212> DNA

<213> Desulfatirhabdium butyrativorans

<400> 1486

gtgtcagtcg atcaagctgt tttcaccatc ggaacccc 38

<210> 1487

<211> 36

<212> DNA

<213> Brevibacillus agri

<400> 1487

gtgctaacca cgaagctttc cactaagctt tcgaac 36

<210> 1488

<211> 36

<212> DNA

<213> Brevibacillus sp.

<400> 1488

gtgctaacca cgaagctttc cactaagctt tcgaac 36

<210> 1489

<211> 36

<212> DNA

<213> Methylobacterium nodulans

<400> 1489

gtcgcagtgg gcgccagatc ctgagcgcgt tggcac 36

<210> 1490

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1490

gtcggatcac tgagcgagcg atctgagaag tggcac 36

<210> 1491

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1491

gtctcggcaa gcttggtcag tgttgggtga ttggcac 37

<210> 1492

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1492

gccgcagcga atgccgtttc acgaatcgtc aggcgg 36

<210> 1493

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1493

gtccaagaaa aaagaaatga tacgaggcat tagcac 36

<210> 1494

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1494

gttcgaaagc ttagtggaaa gcttcgtggt tagcac 36

<210> 1495

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1495

gttcgaaagc ttagtggaaa ttgaagggga tgtacca 37

<210> 1496

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1496

ggggttccga tggtgaaaac agcttgatcg actgac 36

<210> 1497

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1497

gttcgaaagc ttagtggaaa gcttcgtggt tagcac 36

<210> 1498

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1498

gttcgaaagc ttagtggaaa gcttcgtggt tagcac 36

<210> 1499

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1499

gtctcaacgg gcgccagttc ctgagctcgt tggcac 36

<210> 1500

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1500

tgatgtcagc gaaacgacca ccatcggggt tataac 36

<210> 1501

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1501

gggtgcggca tttgcgggtg ttgggggagt ggcagg 36

<210> 1502

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1502

cagtggatgt ttttccatga ggcgaagaat ttcatc 36

<210> 1503

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1503

aacaatataa acgactactt taccgt 26

<210> 1504

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1504

agcttgtcca acttgatgct ccttttcatc 30

<210> 1505

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1505

atcatattca agagcgtgta cactactacg ccgaac 36

<210> 1506

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1506

acagctcggc ctgggtggtg ttggctgcct cggtggcc 38

<210> 1507

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1507

agcttgtcca acttgatgct ccttttcatc 30

<210> 1508

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1508

agcttgtcca acttgatgct ccttttcatc 30

<210> 1509

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1509

tgcatgcgct cctcgaggcg gaggatccga tcgc 34

<210> 1510

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1510

gtcggatcac tgagtgatct aacctatcaa atgcccaaac cacctcctgc ggggttcgaa 60

tcccttcgag tgcgccacaa acataagtaa aacgccggat tcagtttatc gagtccagcg 120

tttttctttt ctactgcagt gttctacgca tcttactatg ccgttgtaat acgaagattt 180

gcgaacatcttggaatacag 200

<210> 1511

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1511

gtctcggcaa gcttggtcag tgatgggtga ttgacatcag tgatgggtga ttgacactag 60

aagcgcacgg cctaggttac gttcttgggg aaagctagggc aagttttggga tgataagaaa 120

taatcatgtc acaaggaggg agtttttgtg gcgagaatca ccatgtatcg aattcaaatc 180

aatacagaag ctataagaaa 200

<210> 1512

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1512

gccgcagtga atgccgtttc accattgatg aagaatgcga ggtgaaaaca gagaaattgg 60

gtcaactcta tcactcttat tcagccatcg tttcaagaaa ggatacctcg tattggatac 120

aacacagctc gttcgttctc tctacctccc tcgacaatct caaggactat ggcgatgcca 180

gcgatggaac agccgaggca 200

<210> 1513

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1513

gttcaagaaa aaagaaatga tatgaggcat tagcacgatg ggatgggaga gagaggacag 60

ttctactctt gctgtatcca gcttctttta ctttatccgg tatcatttct tcacttcttt 120

ctgcacataa aaaagcacct aactatttgg ataagttaag tgcttttatt tccgtttgaa 180

gttgtctatt gcttttttct 200

<210> 1514

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1514

gttcgaaaac ttggtgaaat accttgaaaa ttaacacatc aaagatgccc ctgctttacg 60

ttaggggcat ctttctatta aataacctat agtgaacttc aaaatcattg atctcttgat 120

aataatatat accaaattca atagtcatag tgagaggttt acaggtgctc gatacgtttt 180

cttgcaaaaa agaagcagtc 200

<210> 1515

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1515

gttcgaaagc ttagtggaaa ttgaagggga tgtaccagag gtagcactta gcaagttttt 60

tgagaaacaa gtatctggcc ccaatagcta ggcttatgtg aaatgtagca tgagtggtgt 120

gcaatgaaaa aatagggagg atattagcac tatgaaaaag cagcttaaaa aaacagttaa 180

atgcgtgatg gcgtctatat 200

<210> 1516

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1516

ggggttccga tggtgaaagc agcttgatcg acggacaaat ggatgggcat gcgtagggggc 60

ggttcgcgaa ccgcccctac aactacaccg aaagataccg cgactgaatc tcggtgttgg 120

ccagaagctc ctgcctggtg ccttcgtagc ggatgaggcc tttgtcgatg acatagcccc 180

gatcggagat ggagaggggcg 200

<210> 1517

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1517

gttcgaaaac ttggtgaaat accttgaaaa ttaacacatc aaagatgccc ctgctttacg 60

ttaggggcat ctttctatta aataacctat agtgaacttc aaaatcattg atctcttgat 120

aataatatat accaaattca atagtcatag tgagaggttt acaggtgctc gatacgtttt 180

cttgcaaaaa agaagcagtc 200

<210> 1518

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1518

gttcgaaaac ttggtgaaat accttgaaaa ttaacacatc aaagatgccc ctgctttacg 60

ttaggggcat ctttctatta aataacctat agtgaacttc aaaatcattg atctcttgat 120

180

cttgcaaaaa agaagcagtc 200

<210> 1519

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1519

gtcgcaacgg atccgatc ctgatctcgc tggcaggtgc cctggtagat gacgactcgc 60

gctcgttgca gggaggctcg cgcagacgag cgggaaactg cttgtcaggc aatcgcctgt 120

gctgggtccg ctggattctg tgttcgttgg cacgggggag ccgtgggaat gcctactgcc 180

tggatggggt cgcaccaagt 200

<210> 1520

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1520

gttcgaaagc ttagtggaaa ttgaagggga tgtaccc 37

<210> 1521

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1521

gucggaucac ugagcgagcg aucugagaag uggcac 36

<210> 1522

<211> 158

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1522

ucuagaggac agaauuuuuc aacgggugug ccaauggcca cuuuccaggu ggcaaagccc 60

guugagcuuc ucaaaaagaa cgcucgcuca guguucugac cuuucgagcg ccuguucagg 120

gcgaaaaccc ugggaggcgc ucgaaucaua ggugggac 158

<210> 1523

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1523

gccgcagcga augccguuuc acgaaucguc aggcgg 36

<210> 1524

<211> 75

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1524

gcuggagacg uuuuuugaaa cggcgagugc ugcggauagc gaguuucucu uggggaggcg 60

cucgcggcca cuuuu 75

<210> 1525

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1525

guccaagaaa aaagaaauga uacgaggcau uagcac 36

<210> 1526

<211> 107

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1526

cuggacgaug ucucuuuuau uucuuuuuuc uuggaucuga guacgagcac ccacauugga

cauuucgcau ggugggugcu cguacuauag guaaaacaaa ccuuuuu 107

<210> 1527

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1527

guucgaaagc uuaguggaaa gcuucguccu uagcac 36

<210> 1528

<211> 69

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1528

cacggauaau cacgacuuuc cacuaagcuu ucgaauuuua ugaugcgagc auccucucag 60

69

<210> 1529

<211> 79

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1529

gaagaaatga ctattgccaa ctatatctgg cttttctcaa tacccttagc gcgaaaatac 60

ccctcgcca taaccaacc 79

<210> 1530

<211> 84

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1530

ataaccaacc gtctattgcc atctttatct ggcttttctc aatactatca aggtacagca 60

aatatgctca tcagtgctat gaag 84

<210> 1531

<211> 82

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1531

tgctatgaag gtctattgcc atctttatct ggcttttctc aatacagtag aaatgattat 60

acctgacaac gttaaagaga gt 82

<210> 1532

<211> 79

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1532

taaagagagt gtctattgcc atctttatct ggcttttctc aatacaaaag aaaccaaaaa 60

attcattgcg taacaccat 79

<210> 1533

<211> 80

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1533

gtaacaccat gtctattgcc aactatatct ggcttttctc aatacaaaat acgcacagcc 60

tcattaccac tcagtttatt 80

<210> 1534

<211> 80

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1534

tcagtttatt gtctattgcc aactatatct ggcttttctc aatactaaaa tggatgattg 60

gtggctcgat gacactgtcc 80

<210> 1535

<211> 82

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1535

gacactgtcc gtctattgcc aactatatct ggcttttctc aatacagctt ctagtggtat 60

gccttcggga ccatcaaagg aa 82

<210> 1536

<211> 81

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1536

atcaaaggaa gtctattgcc aactatatct ggcttttctc aatacgcata tcttgaggat 60

tgggattctg aactcttaga g 81

<210> 1537

<211> 82

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1537

actcttagag gtctattgcc aactatatct ggcttttctc aatacctcac ttattcacct 60

gggttaactg catgaactca ac 82

<210> 1538

<211> 55

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1538

tgaactcaac gtctattgcc aactttatct ggcttttctc aataccctac cttct 55

<210> 1539

<211> 35

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1539

gtctattgcc aactatatct ggcttttctc aatac 35

<210> 1540

<211> 35

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1540

gtctattgcc aactatatct ggcttttctc aatac 35

<210> 1541

<211> 78

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1541

accttccaga gttattgccc tctatcttgg actcttctca tcaactcata ataccaagaa 60

gaaatcaagc atattatc 78

<210> 1542

<211> 80

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1542

gcatattatc gttattgccc tctatcttgg gctcttctca tcaacacgag ataggcaaac 60

cgaagaacat gatatatata 80

<210> 1543

<211> 78

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1543

gatatatata gttattgccc tctatcttgg gctcttctca tcaacgaata cgatacaact 60

ggcgactggt ttgattaa 78

<210> 1544

<211> 55

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1544

gtttgattaa gttattgccc tctatcttgg gctcttctca tcaacaccgt cctct 55

<210> 1545

<211> 35

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1545

gttattgccc tctatcttgg gctcttctca tcaac 35

<210> 1546

<211> 35

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1546

gttattgccc tctatcttgg gctcttctca tcaac 35

<210> 1547

<211> 80

<212> DNA

<213> Clostridium aminophilum

<400> 1547

aaagagcagg gtttggagaa cagcccgata tagagggcaa tagacgcctt ttgtcgcaag 60

tgttgccgta ccggataacg 80

<210> 1548

<211> 80

<212> DNA

<213> Clostridium aminophilum

<400> 1548

ccggataacg gtttggagaa cagcccgata tagagggcaa tagacgcaac atatgcttca 60

tcggggaaga gttctttccc 80

<210> 1549

<211> 80

<212> DNA

<213> Clostridium aminophilum

<400> 1549

60

ccaatggcct gtccaagcca 80

<210> 1550

<211> 79

<212> DNA

<213> Clostridium aminophilum

<400> 1550

gtccaagcca gtttggagaa cagcccgata tagagggcaa tagacttaat tgccggatac 60

ttgagatgga acttgtcac 79

<210> 1551

<211> 78

<212> DNA

<213> Clostridium aminophilum

<400> 1551

aacttgtcac gtttggagaa cagcccgata tagagggcaa tagacatatc atcatcaaga 60

gtctgccttg catcacaa 78

<210> 1552

<211> 79

<212> DNA

<213> Clostridium aminophilum

<400> 1552

tgcatcacaa gtttggagaa cagcccgata tagagggcaa tagacgttta tgtaaactct 60

taacgagtaa ccatcttaa 79

<210> 1553

<211> 81

<212> DNA

<213> Clostridium aminophilum

<400> 1553

accatcttaa gtttggagaa cagcccgata aagagggcaa tagaccagac catacctttt 60

cagacgggat ctttaccatc t 81

<210> 1554

<211> 79

<212> DNA

<213> Clostridium aminophilum

<400> 1554

tttaccatct gtttggagaa cagcccgata aagagggcaa tagacctgat gctgcgaatc 60

tgccagaact cttgcgacc 79

<210> 1555

<211> 56

<212> DNA

<213> Clostridium aminophilum

<400> 1555

tcttgcgacc gtttggagaa cagcccgata tagagggcaa tagacggtgt acttgt 56

<210> 1556

<211> 35

<212> DNA

<213> Clostridium aminophilum

<400> 1556

gtttggagaa cagcccgata tagagggcaa tagac 35

<210> 1557

<211> 35

<212> DNA

<213> Clostridium aminophilum

<400> 1557

gtttggagaa cagcccgata tagagggcaa tagac 35

<210> 1558

<211> 81

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1558

agaagacagg gttttgagaa tagcccgaca tagagggcaa tagacaacgg ttttaccatt 60

gtcaaactcg cctgctgtcg t 81

<210> 1559

<211> 84

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1559

ctgctgtcgt gttttgagaa tagcccgaca tagagggcaa tagacttttg cttcgtcacg 60

gatggacttc acaatggcaa caac 84

<210> 1560

<211> 79

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1560

tggcaacaac gttttgagaa tagcccgaca tagagggcaa tagaccttca taaatccaag 60

atacggatgc atgattacg 79

<210> 1561

<211> 77

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1561

catgattacg gttttgagaa tagcccgaca tagagggcaa tagactttta aagggatgat 60

aaggaagccg tatactc 77

<210> 1562

<211> 82

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1562

60

catgttgaca ccttgcctag tt 82

<210> 1563

<211> 81

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1563

ttgcctagtt gttttgagaa tagcccgaca tagagggcaa tagacttctt ctcttgtcat 60

tcttctacct ctaaaatctc a 81

<210> 1564

<211> 35

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1564

gttttgagaa tagcccgaca tagagggcaa tagac 35

<210> 1565

<211> 35

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1565

gttttgagaa tagcccgaca tagagggcaa tagac 35

<210> 1566

<211> 35

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1566

gttttgagaa tagcccgaca tagagggcaa tagac 35

<210> 1567

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1567

aaaatggaaa gttatagtcc tcttacattt agaggtagtc tttaattaaa agattacaat 60

gaaggttatc cacttc 76

<210> 1568

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1568

tatccacttc gttatagtcc tcttacattt agaggtagtc tttaatttaa ctcatgggat 60

atgaattatc atttag 76

<210> 1569

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1569

tatcatttag gttatagtcc tcttacattt agaggtagtc tttaatgtat taggaacaat 60

aaacgactctttttta 76

<210> 1570

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1570

ctctttttta gttatagtcc tcttacattt agaggtagtc tttaattcaa attaatcgtt 60

ctctttatat ctggga 76

<210> 1571

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1571

atatctggga gttatagtcc tcttacattt agaggtagtc tttaattgat tatatcgaaa 60

atcaaataaa tgcgct 76

<210> 1572

<211> 37

<212> DNA

<213> Carnobacterium gallinarum

<400> 1572

gttatagtcc tcttacattt agaggtagtc tttaatt 37

<210> 1573

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1573

ccaataaacg gttatagtcc tcttacattt agaggtagtc tttaattctc ctttttcatg 60

aatggccgtt aaccct 76

<210> 1574

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1574

cgttaaccct gttatagtcc tcttacattt agaggtagtc tttaattgcg acaatcagta 60

gattacgat gctgac 76

<210> 1575

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1575

cgatgctgac gttatagtcc tcttacattt agaggtagtc tttaattttt acataaaaaa 60

aggagggtat aatcat 76

<210> 1576

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1576

gtataatcat gttatagtcc tcttacattt agaggtagtc tttaatttac gccaagaatt 60

attaatgttg atgcaa 76

<210> 1577

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1577

gttgatgcaa gttatagtcc tcttacattt agaggtagtc tttaatcttt atccatgaat 60

taactcatgc gattgc 76

<210> 1578

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1578

atgcgattgc gttatagtcc tcttacattt agaggtagtc tttaatagaa atatattaat 60

agcgacttat attaca 76

<210> 1579

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1579

ttatattaca gttatagtcc tcttacattt agaggtagtc tttaatatat tggagaaaaa 60

agaaaaagat tagtca 76

<210> 1580

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1580

agattagtca gttatagtcc tcttacattt agaggtagtc tttaattcct ctattgtact 60

taacatcatc tactga 76

<210> 1581

<211> 56

<212> DNA

<213> Carnobacterium gallinarum

<400> 1581

catctactga gttatagtcc tcttacattt agaggtagtc tttaataccc ttctat 56

<210> 1582

<211> 36

<212> DNA

<213> Carnobacterium gallinarum

<400> 1582

gttatagtcc tcttacattt agaggtagtc tttaat 36

<210> 1583

<211> 37

<212> DNA

<213> Carnobacterium gallinarum

<400> 1583

gttatagtcc tcttacattt agaggtagtc tttaatt 37

<210> 1584

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1584

tctagttctg attaaagact acccctaaat gtaaggggac tataactcct caatgctttt 60

attttgagcg ttcgct 76

<210> 1585

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1585

agcgttcgct attaaagact acccctaaat gtaaggggac tataactacc gactagagaa 60

ctctttagtc acttga 76

<210> 1586

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1586

agtcacttga attaaagact acccctaaat gtaaggggac tataactgct tttgttaaat 60

tgattattgg gtcttc 76

<210> 1587

<211> 37

<212> DNA

<213> Carnobacterium gallinarum

<400> 1587

attaaagact acccctaaat gtaaggggac tataact 37

<210> 1588

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1588

gttgaggggt aatataaact acctctaaat gtaagaggac tataacttta ttagttgttc 60

atctgtttttttttt 76

<210> 1589

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1589

ttttttttaa aatataaact acctctaaat gtaagaggac tataacctac aaaaacgact 60

aaaaaagcgt ttaaag 76

<210> 1590

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1590

gcgtttaaag aatataaact acctctaaat gtaagaggac tataactttg gttgtacccc 60

tttttgaaca tatagg 76

<210> 1591

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1591

aacatatagg aatataaact acctctaaat gtaagaggac tataactgcg taatgatgag 60

gtggcagaat caatgc 76

<210> 1592

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1592

gaatcaatgc aatataaact acctctaaat gtaagaggac tataaccata aaaccttgat 60

tttcataaga tatatc 76

<210> 1593

<211> 76

<212> DNA

<213> Carnobacterium gallinarum

<400> 1593

aagatatatc aatataaact acctctaaat gtaagaggac tataacccaa tagtcaattc 60

tgttagccag taggga 76

<210> 1594

<211> 57

<212> DNA

<213> Carnobacterium gallinarum

<400> 1594

ccagtaggga aatataaact acctctaaat gtaagaggac tataactgct atataaa 57

<210> 1595

<211> 36

<212> DNA

<213> Carnobacterium gallinarum

<400> 1595

aatataaact acctctaaat gtaagaggac tataac 36

<210> 1596

<211> 37

<212> DNA

<213> Carnobacterium gallinarum

<400> 1596

attaaagact acccctaaat gtaaggggac tataact 37

<210> 1597

<211> 75

<212> DNA

<213> Paludibacter propionicigenes

<400> 1597

gcatgtcctt gttttagttc ccttcaattt tgggataatc cacaagtatt actgctgcaa 60

tgcagattgg cgtat 75

<210> 1598

<211> 75

<212> DNA

<213> Paludibacter propionicigenes

<400> 1598

attggcgtat gttgtagttc ccttcaattt tgggataatc cacaagtatt actgctgcaa 60

tgcagattgg cgtat 75

<210> 1599

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1599

attggcgtat gttgtagttc ccttcaattt tgggataatc cacaaggaaa acgaaaatta 60

taacttcaat aaattc 76

<210> 1600

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1600

caataaattc gttgtagttc cctttaattt tgggataatc cacaagagta aaaccgtaca 60

atggagaaga taacgt 76

<210> 1601

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1601

aagataacgt gatgtagttc ccttcaattt tgggatagtc cacaagagcg gtgcggattt 60

gagcgatgcg aatttg 76

<210> 1602

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1602

tgcgaatttg gttgtagttc ccttcaatat tgggataatc cacaaggaat taggattatg 60

agtttatata gtttga 76

<210> 1603

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1603

60

ataaacggaa gttcga 76

<210> 1604

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1604

ggaagttcga gttgtagttc ccttcaattt tgggataatc cataagaaag caaaaatgaa 60

cacaaaacaa tttgaa 76

<210> 1605

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1605

acaatttgaa gttttagttc ccttcgattt tgggataatc cacaagtaca gcagtaagcg 60

gaaacactca ggcaat 76

<210> 1606

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1606

ctcaggcaat gttgtagttc ccttcaattt tgggataatc cacaaggccg taaaaaatcc 60

acacaacgat aagatg 76

<210> 1607

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1607

cgataagatg gttttagttc ccttcaattt tgggataatc cataagctct tactggctgg 60

tgccgggctt gctgca 76

<210> 1608

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1608

gcttgctgca gttgtagttc ccttcaattt tgggattatc cacaagagaa agagtgatga 60

acgacgagca ggatat 76

<210> 1609

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1609

agcaggatat gttgtagttc ccttcaattt tgggattatc cacaagttac acctgatcaa 60

tggaaattgc ttagtg 76

<210> 1610

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1610

ttgcttagtg gttgtagttc ccttcaattt tgggataatc cacaagtgtg attaatgata 60

ttaccgtagc taacgg 76

<210> 1611

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1611

tagctaacgg gttgtagttc ccttcattat tgggatagtc cacaagaaaa caacctgcat 60

ggataaatta tatgac 76

<210> 1612

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1612

attatatgac gttctagctc ccttcaattt tgggataatc cacaagtaaa aacatatttg 60

ctgaagacta tggaag 76

<210> 1613

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1613

actatggaag gttgtagttc ccttcaatat tgggataatc cacaagtgct attcggaata 60

gatttttacc ccacca 76

<210> 1614

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1614

taccccacca gttgtagttc ccttcaattt tgggataatt cacaagtgggg attatgaaat 60

aaccgattat aaacta 76

<210> 1615

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1615

ttataaacta gttgtagttc ccttcaattt tgggataatt cacaagagaa atcaaaaatt 60

gattcttcag tagctt 76

<210> 1616

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1616

tcagtagctt gttgtagttc ccttcaattt tgggataatt cacaagcgaa tcctaatggt 60

tatattactg cttcat 76

<210> 1617

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1617

actgcttcat gttgtagttc ccttcaattt tgggataatt cacaagtaaa ttgaaaattt 60

ctggacaagc tccagt 76

<210> 1618

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1618

aagctccagt gttgtagttc ccttcaatat tgggataatc cacaagaaac gaatcacaga 60

acatgtgaac ctggta 76

<210> 1619

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1619

gaacctggta gttgtagttc ccttcaattt tgggataatc cacaagagat gtaacgtag 60

gcaacatccc tgataa 76

<210> 1620

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1620

tccctgataa gttttagttc ccttcaattt tgggataatc cacaagcgtt attcaggaga 60

gagtttcaaa gagtat 76

<210> 1621

<211> 76

<212> DNA

<213> Paludibacter propionicigenes

<400> 1621

caaagagtat gttttagttc ccttcaatat tgggataatc cacaagtaaa acattggcta 60

ataacagaac tagggc 76

<210> 1622

<211> 56

<212> DNA

<213> Paludibacter propionicigenes

<400> 1622

gaactagggc gttttagttc ccttcaatat tgggataatc cacaagatag tatctg 56

<210> 1623

<211> 36

<212> DNA

<213> Paludibacter propionicigenes

<400> 1623

gttgtagttc ccttcaattt tgggataatc cacaag 36

<210> 1624

<211> 36

<212> DNA

<213> Paludibacter propionicigenes

<400> 1624

gttgtagttc ccttcaattt tgggataatc cacaag 36

<210> 1625

<211> 36

<212> DNA

<213> Paludibacter propionicigenes

<400> 1625

gttgtagttc ccttcaattt tgggataatc cacaag 36

<210> 1626

<211> 36

<212> DNA

<213> Paludibacter propionicigenes

<400> 1626

gttgtagttc ccttcaattt tgggataatc cacaag 36

<210> 1627

<211> 76

<212> DNA

<213> Listeria seeligeri

<400> 1627

tgttattgca gtaagagact acctctatat gaaagaggac taaaaccata tttccaaact 60

ccactttgac tacacc 76

<210> 1628

<211> 76

<212> DNA

<213> Listeria seeligeri

<400> 1628

tgactacacc gtaagagact acctctatat gaaagaggac taaaacggtc ccactacttg 60

aggtacgaac atatca 76

<210> 1629

<211> 76

<212> DNA

<213> Listeria seeligeri

<400> 1629

gaacatatca gtaagagact acctctatat gaaagaggac taaaacttag tcaacccctc 60

gctgcatttt cacatt 76

<210> 1630

<211> 76

<212> DNA

<213> Listeria seeligeri

<400> 1630

ttttcacatt gtaaagagact acctctatat gaaagaggac taaaacgatg gataataggg 60

atagatcatt agtccg 76

<210> 1631

<211> 56

<212> DNA

<213> Listeria seeligeri

<400> 1631

cattagtccg gtaagagact acctctatat gaaagaggac taaaacgtct aatgtg 56

<210> 1632

<211> 36

<212> DNA

<213> Listeria seeligeri

<400> 1632

gtaagagact acctctatat gaaagaggac taaaac 36

<210> 1633

<211> 36

<212> DNA

<213> Listeria seeligeri

<400> 1633

gtaagagact acctctatat gaaagaggac taaaac 36

<210> 1634

<211> 76

<212> DNA

<213> Listeria weihenstephanensis

<400> 1634

tattttcata gatttagagt acctcaaaac agaagaggac taaaacgcac tctccgacaa 60

taatctcgtc catttt 76

<210> 1635

<211> 76

<212> DNA

<213> Listeria weihenstephanensis

<400> 1635

cgtccatttt gatttagagt acctcaaaac aaaagaggac taaaacaact ctgtacttgt 60

gaagtacgtt aaatcc 76

<210> 1636

<211> 56

<212> DNA

<213> Listeria weihenstephanensis

<400> 1636

cgttaaatcc gatttagagt acctcaaaac aaaagaggac taaaacctct tttgtg 56

<210> 1637

<211> 36

<212> DNA

<213> Listeria weihenstephanensis

<400> 1637

gatttagagt acctcaaaac aaaagaggac taaaac 36

<210> 1638

<211> 76

<212> DNA

<213> Listeria weihenstephanensis

<400> 1638

gtctacaagt gatttagagt agctcaaaaa agaagaggtc taaaacagag atactgatac 60

tattgagccc agtcat 76

<210> 1639

<211> 76

<212> DNA

<213> Listeria weihenstephanensis

<400> 1639

gcccagtcat gatttagagt acctcaaaat agaagaggtc taaaactgtt ccaaggcttg 60

ctaactcacc tttttc 76

<210> 1640

<211> 76

<212> DNA

<213> Listeria weihenstephanensis

<400> 1640

cacctttttc gatttagagt acctcaaaat agaagaggtc taaaacatct tgtttatatc 60

cgtatcggca cccaaa 76

<210> 1641

<211> 76

<212> DNA

<213> Listeria weihenstephanensis

<400> 1641

ggcacccaaa gatttagagt gcctcaaaat agaagaggtc taaaactcac tctccatcca 60

ctcaatcaga tcattt 76

<210> 1642

<211> 76

<212> DNA

<213> Listeria weihenstephanensis

<400> 1642

cagatcattt gatttagagt acctcaaaat agaagaggtc taaaactatc aatttccttt 60

tgatttctta aatcag 76

<210> 1643

<211> 76

<212> DNA

<213> Listeria weihenstephanensis

<400> 1643

cttaaatcag gatttagagt acctcaaaat agaagaggtc taaaacgttc caatatggtt 60

ttttccacat cttccg 76

<210> 1644

<211> 57

<212> DNA

<213> Listeria weihenstephanensis

<400> 1644

acatcttccg gatttagagt acctcaaaat agaagaggtc taaaacctca aattgaa 57

<210> 1645

<211> 36

<212> DNA

<213> Listeria weihenstephanensis

<400> 1645

gatttagagt acctcaaaat agaagaggtc taaaac 36

<210> 1646

<211> 36

<212> DNA

<213> Listeria weihenstephanensis

<400> 1646

gatttagagt acctcaaaac aaaagaggac taaaac 36

<210> 1647

<211> 36

<212> DNA

<213> Listeria weihenstephanensis

<400> 1647

gatttagagt acctcaaaac aaaagaggac taaaac 36

<210> 1648

<211> 75

<212> DNA

<213> Listeria newyorkensis

<400> 1648

cgtaatgctt gatttagagt acctcaaaac aaaagaggac taaaactact tgtcgatatg 60

gtatagcttttttca 75

<210> 1649

<211> 76

<212> DNA

<213> Listeria newyorkensis

<400> 1649

gcttttttca gatttagagt acctcaaaac aaaagaggac taaaactaaa gcttctaaat 60

ggtggcgcgt tacgcc 76

<210> 1650

<211> 76

<212> DNA

<213> Listeria newyorkensis

<400> 1650

gcgttacgcc gatttagaat acctcaaaac aaaagaggac taaaacctag caagccggtc 60

gccgcgctca aagtaa 76

<210> 1651

<211> 57

<212> DNA

<213> Listeria newyorkensis

<400> 1651

ctcaaagtaa gatttagagt acctcaaaac aaaagaggac taaaacctct tttgtgg 57

<210> 1652

<211> 36

<212> DNA

<213> Listeria newyorkensis

<400> 1652

gatttagagt acctcaaaac aaaagaggac taaaac 36

<210> 1653

<211> 36

<212> DNA

<213> Listeria newyorkensis

<400> 1653

gatttagagt acctcaaaac aaaagaggac taaaac 36

<210> 1654

<211> 36

<212> DNA

<213> Listeria newyorkensis

<400> 1654

gatttagagt acctcaaaac aaaagaggac taaaac 36

<210> 1655

<211> 78

<212> DNA

<213> Leptotrichia wadei

<400> 1655

atgatgtatg taagttttag tccccttcgt ttttggggta gtctaaatcc tggtaaacca 60

atccacatcg aagaaaag 78

<210> 1656

<211> 76

<212> DNA

<213> Leptotrichia wadei

<400> 1656

cgaagaaaag aaagttttag tccccttcgt ttttggggta gtctaaatca tgtagaagaa 60

gttattgtat ctattt 76

<210> 1657

<211> 59

<212> DNA

<213> Leptotrichia wadei

<400> 1657

gtatctattt tacgttttag tccccttcgt ttttggggta gtctaaatct ttatatcaa 59

<210> 1658

<211> 39

<212> DNA

<213> Leptotrichia wadei

<400> 1658

taagttttag tccccttcgt ttttggggta gtctaaatc 39

<210> 1659

<211> 39

<212> DNA

<213> Leptotrichia wadei

<400> 1659

taagttttag tccccttcgt ttttggggta gtctaaatc 39

<210> 1660

<211> 39

<212> DNA

<213> Leptotrichia wadei

<400> 1660

taagttttag tccccttcgt ttttggggta gtctaaatc 39

<210> 1661

<211> 78

<212> DNA

<213> Leptotrichia shahii

<400> 1661

attctttaga gttttagtcc ccttcgatat tggggtggtc tatatcgaaa aagaagagtt 60

tattcagata gatttgtc 78

<210> 1662

<211> 77

<212> DNA

<213> Leptotrichia shahii

<400> 1662

tagatttgtc gttttagtcc ccttcgatat tggggtggtc tatatcaata tggattactt 60

ggtagaacag caatcta 77

<210> 1663

<211> 57

<212> DNA

<213> Leptotrichia shahii

<400> 1663

cagcaatcta gttttagtcc ccttcgatat tggggtggtc tatatcccat cctaatt 57

<210> 1664

<211> 36

<212> DNA

<213> Leptotrichia shahii

<400> 1664

gttttagtcc ccttcgatat tggggtggtc tatatc 36

<210> 1665

<211> 36

<212> DNA

<213> Leptotrichia shahii

<400> 1665

gttttagtcc ccttcgatat tggggtggtc tatatc 36

<210> 1666

<211> 80

<212> DNA

<213> Rhodobacter capsulatus

<400> 1666

gaacatcatg ggttcagtcc gccgtcgtct tggcggtgat gtgaggctct cccagcatac 60

caaaccgctg gcgaccatca 80

<210> 1667

<211> 78

<212> DNA

<213> Rhodobacter capsulatus

<400> 1667

gcgaccatca ggttcagtcc gccgtcgtct tggcggtgat gtgatgcaca ggagagaccg 60

atgaaaatca cggccttc 78

<210> 1668

<211> 79

<212> DNA

<213> Rhodobacter capsulatus

<400> 1668

cacggccttc ggttcagtcc gccgtcgtct tggcggtgac gtgaggcacc taaacaagag 60

gttctacgat gccgaaagg 79

<210> 1669

<211> 58

<212> DNA

<213> Rhodobacter capsulatus

<400> 1669

tgccgaaagg ggttcagtcc gccgtcgtct tggcggtgat gtgaggctca ggtcccgc 58

<210> 1670

<211> 37

<212> DNA

<213> Rhodobacter capsulatus

<400> 1670

ggttcagtcc gccgtcgtct tggcggtgat gtgaggc 37

<210> 1671

<211> 37

<212> DNA

<213> Rhodobacter capsulatus

<400> 1671

ggttcagtcc gccgtcgtct tggcggtgat gtgaggc 37

<210> 1672

<211> 37

<212> DNA

<213> Rhodobacter capsulatus

<400> 1672

ggttcagtcc gccgtcgtct tggcggtgat gtgaggc 37

<210> 1673

<211> 37

<212> DNA

<213> Rhodobacter capsulatus

<400> 1673

ggttcagtcc gccgtcgtct tggcggtgat gtgaggc 37

<210> 1674

<211> 80

<212> DNA

<213> Rhodobacter capsulatus

<400> 1674

gaacatcatg ggttcagtcc gccgtcgtct tggcggtgat gtgaggctct cccagcatac 60

caaaccgctg gcgaccatca 80

<210> 1675

<211> 78

<212> DNA

<213> Rhodobacter capsulatus

<400> 1675

gcgaccatca ggttcagtcc gccgtcgtct tggcggtgat gtgatgcaca ggagagaccg 60

atgaaaatca acggcttc 78

<210> 1676

<211> 79

<212> DNA

<213> Rhodobacter capsulatus

<400> 1676

caacggcttc ggttcagtcc gccgtcgtct tggcggtgac gtgaggcacc taaacaagag 60

gttctacgat gccgaaagg 79

<210> 1677

<211> 58

<212> DNA

<213> Rhodobacter capsulatus

<400> 1677

tgccgaaagg ggttcagtcc gccgtcgtct tggcggtgat gtgaggctca ggtcccgc 58

<210> 1678

<211> 37

<212> DNA

<213> Rhodobacter capsulatus

<400> 1678

ggttcagtcc gccgtcgtct tggcggtgat gtgaggc 37

<210> 1679

<211> 37

<212> DNA

<213> Rhodobacter capsulatus

<400> 1679

ggttcagtcc gccgtcgtct tggcggtgat gtgaggc 37

<210> 1680

<211> 37

<212> DNA

<213> Rhodobacter capsulatus

<400> 1680

ggttcagtcc gccgtcgtct tggcggtgat gtgaggc 37

<210> 1681

<211> 37

<212> DNA

<213> Rhodobacter capsulatus

<400> 1681

ggttcagtcc gccgtcgtct tggcggtgat gtgaggc 37

<210> 1682

<211> 80

<212> DNA

<213> Rhodobacter capsulatus

<400> 1682

gaacatcatg ggttcagtcc gccgtcgtct tggcggtgat gtgaggctct cccagcatac 60

caaaccgctg gcgaccatca 80

<210> 1683

<211> 78

<212> DNA

<213> Rhodobacter capsulatus

<400> 1683

gcgaccatca ggttcagtcc gccgtcgtct tggcggtgat gtgatgcaca ggagagaccg 60

atgaaaatca acggcttc 78

<210> 1684

<211> 79

<212> DNA

<213> Rhodobacter capsulatus

<400> 1684

caacggcttc ggttcagtcc gccgtcgtct tggcggtgac gtgaggcacc taaacaagag 60

gttctacgat gccgaaagg 79

<210> 1685

<211> 58

<212> DNA

<213> Rhodobacter capsulatus

<400> 1685

tgccgaaagg ggttcagtcc gccgtcgtct tggcggtgat gtgaggctca ggtcccgc 58

<210> 1686

<211> 37

<212> DNA

<213> Rhodobacter capsulatus

<400> 1686

ggttcagtcc gccgtcgtct tggcggtgat gtgaggc 37

<210> 1687

<211> 37

<212> DNA

<213> Rhodobacter capsulatus

<400> 1687

ggttcagtcc gccgtcgtct tggcggtgat gtgaggc 37

<210> 1688

<211> 37

<212> DNA

<213> Rhodobacter capsulatus

<400> 1688

ggttcagtcc gccgtcgtct tggcggtgat gtgaggc 37

<210> 1689

<211> 37

<212> DNA

<213> Rhodobacter capsulatus

<400> 1689

ggttcagtcc gccgtcgtct tggcggtgat gtgaggc 37

<210> 1690

<211> 35

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1690

gtctattgcc aactatatct ggcttttctc aatac 35

<210> 1691

<211> 35

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1691

gttattgccc tctatcttgg gctcttctca tcaac 35

<210> 1692

<211> 35

<212> DNA

<213> Clostridium aminophilum

<400> 1692

gtttggagaa cagcccgata tagagggcaa tagac 35

<210> 1693

<211> 35

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1693

gttttgagaa tagcccgaca tagagggcaa tagac 35

<210> 1694

<211> 37

<212> DNA

<213> Carnobacterium gallinarum

<400> 1694

gttatagtcc tcttacattt agaggtagtc tttaatt 37

<210> 1695

<211> 36

<212> DNA

<213> Carnobacterium gallinarum

<400> 1695

aatataaact acctctaaat gtaagaggac tataac 36

<210> 1696

<211> 36

<212> DNA

<213> Paludibacter propionicigenes

<400> 1696

gttgtagttc ccttcaattt tgggataatc cacaag 36

<210> 1697

<211> 36

<212> DNA

<213> Listeria seeligeri

<400> 1697

gtaagagact acctctatat gaaagaggac taaaac 36

<210> 1698

<211> 36

<212> DNA

<213> Listeria weihenstephanensis

<400> 1698

gatttagagt acctcaaaac aaaagaggac taaaac 36

<210> 1699

<211> 36

<212> DNA

<213> Listeria newyorkensis

<400> 1699

gatttagagt acctcaaaac aaaagaggac taaaac 36

<210> 1700

<211> 39

<212> DNA

<213> Leptotrichia wadei

<400> 1700

taagttttag tccccttcgt ttttggggta gtctaaatc 39

<210> 1701

<211> 36

<212> DNA

<213> Leptotrichia shahii

<400> 1701

gttttagtcc ccttcgatat tggggtggtc tatatc 36

<210> 1702

<211> 37

<212> DNA

<213> Rhodobacter capsulatus

<400> 1702

ggttcagtcc gccgtcgtct tggcggtgat gtgaggc 37

<210> 1703

<211> 37

<212> DNA

<213> Rhodobacter capsulatus

<400> 1703

ggttcagtcc gccgtcgtct tggcggtgat gtgaggc 37

<210> 1704

<211> 37

<212> DNA

<213> Rhodobacter capsulatus

<400> 1704

ggttcagtcc gccgtcgtct tggcggtgat gtgaggc 37

<210> 1705

<211> 35

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1705

gtctattgcc aactatatct ggcttttctc aatac 35

<210> 1706

<211> 35

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1706

gttattgccc tctatcttgg gctcttctca tcaac 35

<210> 1707

<211> 35

<212> DNA

<213> Clostridium aminophilum

<400> 1707

gtttggagaa cagcccgata tagagggcaa tagac 35

<210> 1708

<211> 35

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Lachnospiraceae bacterium sequence"

<400> 1708

gttttgagaa tagcccgaca tagagggcaa tagac 35

<210> 1709

<211> 37

<212> DNA

<213> Carnobacterium gallinarum

<400> 1709

gttatagtcc tcttacattt agaggtagtc tttaatt 37

<210> 1710

<211> 36

<212> DNA

<213> Carnobacterium gallinarum

<400> 1710

aatataaact acctctaaat gtaagaggac tataac 36

<210> 1711

<211> 36

<212> DNA

<213> Paludibacter propionicigenes

<400> 1711

gttgtagttc ccttcaattt tgggataatc cacaag 36

<210> 1712

<211> 36

<212> DNA

<213> Listeria seeligeri

<400> 1712

gtaagagact acctctatat gaaagaggac taaaac 36

<210> 1713

<211> 36

<212> DNA

<213> Listeria weihenstephanensis

<400> 1713

gatttagagt acctcaaaac aaaagaggac taaaac 36

<210> 1714

<211> 36

<212> DNA

<213> Listeria newyorkensis

<400> 1714

gatttagagt acctcaaaac aaaagaggac taaaac 36

<210> 1715

<211> 39

<212> DNA

<213> Leptotrichia wadei

<400> 1715

taagttttag tccccttcgt ttttggggta gtctaaatc 39

<210> 1716

<211> 36

<212> DNA

<213> Leptotrichia shahii

<400> 1716

gttttagtcc ccttcgatat tggggtggtc tatatc 36

<210> 1717

<211> 37

<212> DNA

<213> Rhodobacter capsulatus

<400> 1717

ggttcagtcc gccgtcgtct tggcggtgat gtgaggc 37

<210> 1718

<211> 37

<212> DNA

<213> Rhodobacter capsulatus

<400> 1718

ggttcagtcc gccgtcgtct tggcggtgat gtgaggc 37

<210> 1719

<211> 37

<212> DNA

<213> Rhodobacter capsulatus

<400> 1719

ggttcagtcc gccgtcgtct tggcggtgat gtgaggc 37

<210> 1720

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1720

gtattgagaa aagccagata tagttggcaa tagtc 35

<210> 1721

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1721

attaaagact acctctaaat gtaagaggac tataac 36

<210> 1722

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1722

gttatagtcc tcttacattt agaggtagtc tttaat 36

<210> 1723

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1723

attaaagact acctctaaat gtaagaggac tataac 36

<210> 1724

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1724

gttttagtcc tcttctgttt tgaggtactc taaatc 36

<210> 1725

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1725

gttttagccc tcttttgttc tgaggtactc taaatc 36

<210> 1726

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1726

gttttgagaa tagcccgaca tagagggcaa tagac 35

<210> 1727

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1727

gtttggagaa cagcccgata tagagggcaa tagac 35

<210> 1728

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1728

tcacatcacc gccaagacga cggcggactg aacc 34

<210> 1729

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1729

tcacatcacc gccaagacga cggcggactg aacc 34

<210> 1730

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1730

tcacatcacc gccaagacga cggcggactg aacc 34

<210> 1731

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1731

gctggagaag atagcccaag aaaggggca ataac 35

<210> 1732

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1732

gttttagtcc tcttttgttt tgaggtactc taaatc 36

<210> 1733

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1733

gatatagacc accccaatat cgaaggggac taaaac 36

<210> 1734

<211> 28

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1734

tggattatcc caaaattgaa gggaacta 28

<210> 1735

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1735

gttttagtcc tctttcatat agaggtagtc tcttac 36

<210> 1736

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1736

atttcttctc ttgtcattct tctacctttc gcacaa 36

<210> 1737

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1737

tttccatttt cccactactt tctaaaaaga 30

<210> 1738

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1738

tgattatatc gaaaatcaaa taaatgcgct 30

<210> 1739

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1739

cgtttattgg tcagagtaaa ctcaactccg 30

<210> 1740

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1740

tatgaaaata atgtaacacc aatcgtttgg 30

<210> 1741

<211> 29

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1741

atattttttg taacggcttg caatcattt 29

<210> 1742

<211> 36

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1742

ttcttctctt gtcattcttc tacctctaaa atctca 36

<210> 1743

<211> 35

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1743

ggtgtacttg ttccactcaa tccacttatc atctt 35

<210> 1744

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1744

catgatgttc ctttcttgggg tatggggtaa gcc 33

<210> 1745

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1745

catgatgttc ctttcttgggg tatggggtaa gcc 33

<210> 1746

<211> 33

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1746

catgatgttc ctttcttgggg tatggggtaa gcc 33

<210> 1747

<211> 34

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1747

gctaacatct ccggtgttat taccactcca ttct 34

<210> 1748

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1748

aagcattacg gcgtatcacg ccaccaatta 30

<210> 1749

<211> 32

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1749

tctaaagaat tatctattct gtcttttaaa tt 32

<210> 1750

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1750

aaacaaggac atgcacatac ccacatgttt ttctcttg 38

<210> 1751

<211> 30

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1751

tgcaataaca ttttctgata cttttggcgg 30

<210> 1752

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1752

gtattgagaa aagccagata tagcatgaac acttcggtgt ttgtgctttt ttagtatgac 60

gggcatgccg tcagtctgtg gtgaaagtcc acaaggggcg tagttgccaa cgaaccccaa 120

agcaactccc aaggtttaca ccgtgaggtg taggcgagaa gaagggatag caaaatcgta 180

gcctgacgaa cagaaacctg 200

<210> 1753

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1753

attaaagact acctctaaat gtaagaaaaa taaaaaaata aaagagttac atatagttaa 60

gaaaaagaag taggaatatt tattcctact tctttttcgt tgtatttaat ttatttatat 120

gaaaatatga taagatagat atatagtatt agaatggagg gtattgttaa gatgcgtata 180

acaaaagtga aaataaaatt 200

<210> 1754

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1754

gttatagtcc tcttacattt agaggttgga atggcaacag ttttttttgac aaattttata 60

aggtgcagaa cttctttccg tatgctattc cgattgtcct tgacaatgag cctcctcgga 120

tgtgacgatc ttcggcagga gctaacagtt aaagttagac tgcctcgcta gcgttagcac 180

atccgagctcattatcaagg 200

<210> 1755

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1755

attaaagact acctggatag gctacaatta actagacaga aaaattaagg tgtgtagact 60

agaagaaaac ataccaggag gaatttttat gtctaagaga acacgaagaa ctttttcaca 120

agaattcaag caacaaatcg tcaatcttta cttagctgga aagccacgtg tagaaatcat 180

tcgagaatat gaactaacgg 200

<210> 1756

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1756

gttttagtcc tcttctgttt tgaggtaata tatccgccta ttcgcttgca acaacataac 60

tataccagat tattaatgaa tctgcagctt

ataaattaag agacagctca gcagccatct cttaaattca accaattatc ttctgctaac 180

aaaatacctt ccttcttgaa 200

<210> 1757

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1757

gttttagtcc tcttctgttt tgaggtattt tttaatagca aaatgaaatt gcattctccc 60

atccaatttc attttgaaat taactgcaac attctatatc aaatttctaa tagtctcttt 120

actataccac aatactcttc tgaaacttga tttgtttcta tataaccatt atcgattttt 180

ttctcaccta gatgtctcaa 200

<210> 1758

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1758

gttttgagaa tagcccgaca tagttataga gatgtataaa tataaccgat aaacattgac 60

taatttgttg aagtcagtgt ttatcggttt tttgtgtaaa tataggagtt gttagaatga 120

tactttttgc ctaattttgg aactttatga ggatataaga tagacttgat aaaaaggtaa 180

aagaaaggtt aaagagcatg 200

<210> 1759

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1759

gtttgtagaa cagcctgata tagagggcga taggactttg gctgcatgac tcgatcatta 60

agcctgaaac taagttttct tgtgttgaaa tcttcctaat actgaggtcg taagaccatc 120

ttgattattc acgaatctgt gactctgttc tgagaacaat ctcatactat aaggacaatg 180

ttttttgaaa tggaggattt 200

<210> 1760

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1760

tcacatcacc gccaagatga cggcgggaac ccaatgcaaa cggaggtgcg gcaacagcaa 60

ggttgcaccg gctggacttc ggcggcagtc tggcgcaacg gggcgcagga cacggaagat 120

gtggcggggg caagatggac ctgtttttca aggccacgga atatgagacc ctgcaggcct 180

catggctcaa ggtccagcaa 200

<210> 1761

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1761

tcacatcacc gccaagatga cggcgggaac ccaatgcaaa cggaggtgcg gcaacagcaa 60

ggttgcaccg gctggacttc ggcggcagtc tggcgcaacg gggcgcagga cacggaagat 120

gtggcggggg caagatggac ctgtttttca aggccacgga atatgagacc ctgcaggcct 180

catggctcaa ggtccagcaa 200

<210> 1762

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1762

tcacatcacc gccaagatga cggcgggaac ccaatgcaaa cggaggtgcg gcaacagcaa 60

ggttgcaccg gctggacttc ggcggcagtc tggcgcaacg gggcgcagga cacggaagat 120

gtggcggggg caagatggac ctgtttttca aggccacgga atatgagacc ctgcaggcct 180

catggctcaa ggtccagcaa 200

<210> 1763

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1763

gttggagaag agagcccaag atagaggaga ttgacattta ttacaagcgg agattaaaac 60

gataactgag aaaaaatgaa taacgctgat gaaaacggcc ggattcttgg ccgttttttt 120

gtctatttgc taagtgcaca aagattgtga aataacatct gctactatgt atttatcgag 180

gtacgtaaat ctaggtggtg 200

<210> 1764

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1764

gttttagtcc tcttttattt tgaggtaata tattcacttg caacaacata actataccag 60

attattttt tcataaatta 120

agagacaact cagaatacag aattgcctct acatgtctaa ttttcccact gtcaattcct 180

ctgcgcataa tatctccacc 200

<210> 1765

<211> 200

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1765

gatatagacc accccaatat cgaaaagtga tatttaataa aaataacttc tgagtgagaa 60

taaaatttca attcttgctc attttttatt gttttttgaa tatggttgct tggttgttct 120

180

tataataaat aattatgcga 200

<210> 1766

<211> 250

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1766

tggattatcc caaaattgaa gggtaacact acagctgaca tcaaagcaca aacaaccccg 60

aatgaaattc atcattcggg gttgttttta taaaggttag cttagctaat tgcagtccta 120

cagcaaatca ctacttcttc aaacgcaata tctccggatt ttctgcaata aatttattgg 180

cagcttcgta gccctgatga tagatagtga agtaagagtc gtagctaaac tcatgaaagg 240

tttcgatggc 250

<210> 1767

<211> 250

<212> DNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1767

gttttagtcc tctttcattt agaggtatat cgtattccta cttaataata gtaattaaaa 60

120

attcataaat atcatatcat ttataagctc tattttccat tttctaaggc taataaataa 180

aactgctgta cctatggatc taaggaagac ttatgcacac agtacagcaa cttttcagca 240

tgatttgtgt 250

<210> 1768

<211> 78

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1768

auuauuacca uuuugguugg aaugcuauua uaaaggauca uucgauuauu accucuaccu 60

cccuucccac gauuucuu 78

<210> 1769

<211> 35

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1769

gcuggagaag auagcccaag aaagagggca auaac 35

<210> 1770

<211> 81

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1770

gucuuacgac cucaguauua ggaagauuuc aaccaagaaa acuuaguuuc aggcuuaaug 60

aucgagucau gcagccaaag u 81

<210> 1771

<211> 35

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1771

guuuggagaa cagcccgaua uagagggcaa uagac 35

<210> 1772

<211> 99

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1772

augaaaagag gacuaaaacu gaaagaggac uaaaacacca gauguggaua acuauauuag 60

uggcuauuaa aaauucgucg auauuagaga ggaaacuuu 99

<210> 1773

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1773

guuuuagucc ucuuucauau agagguaguc ucuuac 36

<210> 1774

<211> 106

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

polynucleotide"

<400> 1774

uuaguauauacc acaucaauau uaaaucucaa aaaaauaagg agccgucaaa cauagcuccc 60

uacuucuuuu uacucauaau ccccaucuau ccuuacuuuu cguaaa 106

<210> 1775

<211> 36

<212>RNA

<213> Artificial Sequence

<220>

<221> source

<223> /note="Description of Artificial Sequence: Synthetic

oligonucleotide"

<400> 1775

guuuuagucc ccuucgauau uggggugguc uauauc 36

<210> 1776

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 1776

cccataattg ataggatcta tgaggt 26

<210> 1777

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 1777

ctcccgaaaa gccttgt 17

<210> 1778

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 1778

cccatgattg ataggatcta tgaggt 26

<210> 1779

<211> 18

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 1779

tttccccga caggcgta 18

<210> 1780

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 1780

cccataattg ataggatcta tgaggt 26

<210> 1781

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 1781

tccatatgaa tggcgcg 17

<210> 1782

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 1782

cccataattg ataggatcta tgaggt 26

<210> 1783

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 1783

tgccgccgtc ctgcatg 17

<210> 1784

<211> 26

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 1784

cccataattg ataggatcta tgaggt 26

<210> 1785

<211> 17

<212> DNA

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 1785

gcccggacca catgcac 17

<210> 1786

<211> 51

<212> PRT

<213> Unknown

<220>

<221> source

<223> /note="Description of Unknown:

Gut metagenome sequence"

<400> 1786

Met Ser Lys Asp Thr Thr Gln Ser Arg Tyr Phe Cys Val Phe Lys Asn

1 5 10 15

Cys Pro Cys His Asn Lys Glu Gln His Ala Asp Val Asn Ala Ala Ile

20 25 30

Asn Ile Gly Arg Arg Phe Leu Lys Asp Cys Ile Leu Asp Asp Asn Lys

35 40 45

Glu Lys Asp

fifty

<---

Claims (78)

1. A method for modifying a target locus of interest, comprising delivering to said locus a non-naturally occurring or engineered composition comprising a single molecule CRISPR-Cas effector protein containing three RuvC nuclease catalytic domains, but not containing an HNH domain, and one or more components, being nucleic acids, comprising a heterologous guide sequence and a tracr-RNA sequence, where the effector protein forms a complex with one or more components that are nucleic acids, and upon binding of the specified complex to the target locus of interest, the effector protein induces modification of the target locus of interest, and wherein the method of modification does not include modifying the genetic integrity of a human germline cell. 2. The method of claim 1 wherein the target locus of interest is DNA. 3. The method of claim 1 or 2, wherein the modification of the target locus of interest is a chain break. 4. The method according to any one of the preceding claims, wherein the target locus of interest is located on the DNA molecule in vitro. 5. The method of any one of the preceding claims, wherein the target locus of interest is in the DNA in the cell. 6. The method of claim 5, wherein the cell is a prokaryotic cell. 7. The method of claim 5, wherein the cell is a eukaryotic cell. 8. The method of any one of the preceding claims, wherein the target locus of interest is a genomic locus of interest. 9. The method of any one of the preceding claims, wherein, in complex with the effector protein, the nucleic acid component(s) is or is capable of performing or performing sequence-specific binding of the complex to the sequence of the target locus of interest. 10. The method according to any one of the preceding claims, wherein the nucleic acid component(s) is a CRISPR RNA sequence (cr-RNA) and/or a CRISPR transactivating RNA sequence (tracr-RNA). 11. The method according to any one of paragraphs. 3-10, where the chain break is a single strand break. 12. The method according to any one of paragraphs. 3-10, where the chain break is a double-strand break. 13. The method according to any one of the preceding claims, wherein the effector protein and nucleic acid component(s) are provided by one or more polynucleotide molecules encoding the polypeptide and/or nucleic acid component(s), and wherein the one or more polynucleotide molecules are functionally organized in such a way that the expression of polypeptides and/or component(s) that is a nucleic acid occurs. 14. The method according to claim 13, where one or more polynucleotide molecules contain one or more regulatory elements, functionally organized in such a way that the expression of polypeptides and / or component (s), which is a nucleic acid, in which optionally one or more regulatory elements include inducible promoters. 15. The method according to claim 13 or 14, where one or more polynucleotide molecules are in one or more vectors. 16. The method according to any one of paragraphs. 13-15, where one or more polynucleotide molecules are in the delivery system. 17. The method of claim 15 wherein the one or more vectors are in the delivery system. 18. The method of any one of the preceding claims, wherein the non-naturally occurring or engineered composition is delivered via a delivery vehicle comprising liposome(s), particle(s), exosome(s), microvesicle(s), gene gun, or one or more viral vectors. 19. A non-naturally occurring or engineered composition for modifying a target locus of interest, comprising a single molecule CRISPR-Cas effector protein containing three RuvC nuclease catalytic domains but no HNH domain, and one or more nucleic acid components comprising a heterologous guide sequence and tracr-RNA sequence, wherein the effector protein forms a complex with one or more nucleic acid components, and upon binding said complex to the target locus of interest, the effector protein induces modification of the target locus of interest, nor does the modification include modification of the genetic integrity of a human germline cell. 20. The composition of claim 19, wherein the target locus of interest is DNA. 21. The composition of claim 19 or 20, wherein the modification of the target locus of interest is a chain break. 22. The composition according to any one of paragraphs. 19-21, where the target locus of interest is in the DNA molecule in vitro. 23. The composition according to any one of paragraphs. 19-21 where the target locus of interest is in the DNA in the cell. 24. The composition of claim 23, wherein the cell is a prokaryotic cell. 25. The composition of claim 23, wherein the cell is a eukaryotic cell. 26. The composition according to any one of paragraphs. 19-25, where the target locus of interest is the genomic locus of interest. 27. The composition according to any one of paragraphs. 19-26, wherein, in complex with the effector protein, the nucleic acid component(s) is or is capable of performing sequence-specific binding of the complex to the sequence of the target locus of interest. 28. The composition according to any one of paragraphs. 19-27, where the nucleic acid component(s) is a CRISPR RNA sequence (cr-RNA) and/or a CRISPR transactivating RNA sequence (tracr-RNA). 29. The composition according to any one of paragraphs. 21-28 where the chain break is a single strand break. 30. The composition according to any one of paragraphs. 21-28, where the chain break is a double-strand break. 31. The composition according to any one of paragraphs. 19-30, wherein the effector protein and nucleic acid component(s) are provided by one or more polynucleotide molecules encoding the polypeptide and/or nucleic acid component(s), and wherein the one or more polynucleotide molecules are operatively organized in such a manner to express the polypeptides and/or the nucleic acid component(s). 32. The composition according to claim 31, where one or more polynucleotide molecules contain one or more regulatory elements, functionally organized in such a way that the expression of polypeptides and / or component (s), which is a nucleic acid, in which optionally one or more regulatory elements include inducible promoters. 33. The composition according to claim 31 or 32, where one or more polynucleotide molecules are in one or more vectors. 34. The composition according to any one of paragraphs. 31, 32, where one or more polynucleotide molecules are in the delivery system. 35. The composition of claim 33 wherein the one or more vectors are in the delivery system. 36. The composition according to any one of paragraphs. 19-35, wherein the non-naturally occurring or engineered composition is delivered via a delivery vehicle comprising liposome(s), particle(s), exosome(s), microvesicle(s), gene gun, or one or more viral vectors. 37. A vector system for modifying a target locus of interest, comprising one or more vectors, the one or more vectors comprising one or more polynucleotide molecules encoding components of a non-naturally occurring or engineered composition according to any one of paragraphs. 19-36, where one or more vectors contain: a) a first regulatory element operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas polynucleotide sequences, including a guide RNA that contains a guide sequence connected to a direct repeat sequence where the guide sequence is capable of hybridizing to the target locus; b) a second regulatory element operably linked to a nucleotide sequence encoding a single molecule CRISPR-Cas effector protein containing three RuvC nuclease catalytic domains but not containing an HNH domain; where components (a) and (b) are located on the same or different vectors of the system, where during transcription one or more guide sequences hybridize with the specified target locus, the target locus is located on the 3' side of the motif adjacent to the protospacer ( PAM) and said guide RNA forms a complex with said effector protein. 38. A delivery system for modifying a target sequence of interest, comprising one or more vectors or one or more polynucleotide molecules, wherein the one or more vectors or polynucleotide molecules comprise one or more polynucleotide molecules encoding components of a non-naturally occurring or engineered composition that is a composition according to any one of paragraphs. 19-36, wherein one or more vectors comprise: (a) a first regulatory element operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas polynucleotide sequences comprising a guide RNA that contains a guide sequence fused to a direct repeat, where the guide sequence is able to hybridize with the target locus; (b) a second regulatory element operably linked to a nucleotide sequence encoding a single molecule CRISPR-Cas effector protein containing three RuvC nuclease catalytic domains but no HNH domain; where components (a) and (b) are located on the same or different vectors of the system, where during transcription one or more guide sequences hybridize with the specified target locus, the target locus is located on the 3' side of the motif adjacent to the protospacer ( PAM) and said guide RNA forms a complex with said effector protein. 39. Non-naturally occurring or engineered composition according to any one of paragraphs. 19-36, the vector system of claim 37, or the delivery system of claim 38 for use in a therapeutic method of treatment. 40. Non-naturally occurring or engineered composition according to any one of paragraphs. 19-36 for use in a therapeutic treatment, wherein said therapeutic treatment comprises gene or genome editing or gene therapy. 41. Non-naturally occurring or engineered composition according to any one of paragraphs. 19-36, the vector system of claim 37, or the delivery system of claim 38 for use in: - site-specific gene knockout; - site-specific genome editing; - sequence-specific DNA interference; or - multiplex modification of the genome by engineering methods. 42. A cell that is not a human embryonic cell modified by engineering methods so that it contains or expresses a non-naturally occurring or engineered composition according to any one of paragraphs. 19-36 for modifying a target locus of interest comprising a single molecule CRISPR-Cas effector protein containing three RuvC nuclease catalytic domains but no HNH domain and one or more nucleic acid components comprising a heterologous targeting sequence and a tracr- RNA, wherein the effector protein forms a complex with one or more nucleic acid components, and upon binding of said complex to the target locus of interest, the effector protein induces modification of the target locus of interest. 43. A cell line consisting of cells according to claim 42, modified by engineering methods so as to contain or express a non-naturally occurring or engineered composition according to any one of paragraphs. 19-36 to modify the target locus of interest. 44. An engineered, non-naturally occurring CRISPR-Cas system for modifying a target locus of interest, comprising: a. one or more CRISPR-Cas polynucleotide sequences comprising a guide RNA (gRNA) that contains a guide sequence connected to a direct repeat sequence, where the guide sequence is capable of hybridizing to a target sequence, or one or more nucleotide sequences encoding one or more polynucleotide sequences CRISPR Cas; and b. a CRISPR-Cas single molecule effector protein containing three RuvC nuclease catalytic domains but no HNH domain, or one or more nucleotide sequences encoding said single molecule effector protein; wherein one or more guide sequences hybridize to said target locus, said target locus is 3' to a protospacer adjacent motif (PAM) and said guide RNA forms a complex with said effector protein. 45. An engineered, non-naturally occurring CRISPR-Cas vector system for modifying a target locus of interest, comprising one or more vectors containing: a. a first regulatory element operably linked to one or more nucleotide sequences encoding one or more CRISPR-Cas polynucleotide sequences comprising a guide RNA that contains a guide sequence linked to a direct repeat sequence, where the guide sequence is capable of hybridizing to a target locus; b. a second regulatory element operably linked to a nucleotide sequence encoding a single molecule CRISPR-Cas effector protein containing three RuvC nuclease catalytic domains but no HNH domain; where components (a) and (b) are located on the same or different vectors of the system, where during transcription one or more guide sequences hybridize with the specified target locus, the target locus is located on the 3' side of the motif adjacent to the protospacer ( PAM) and said guide RNA forms a complex with said effector protein. 46. The system according to claim 44 or 45, where the target locus is located in the cell. 47. The system of claim 45 wherein the cell is a eukaryotic cell. 48. The system according to claim 44 or 45, wherein during transcription, one or more guide sequences hybridize with the target locus and the guide RNA forms a complex with a single-molecule CRISPR-Cas effector protein containing three RuvC nuclease catalytic domains, but not containing an HNH domain, which causes cleavage distal to the target locus. 49. The system of claim 48 wherein said cleavage forms a stepped double strand break with a 4 or 5 nucleotide 5' overhang. 50. The system according to claim 44 or 45, where the PAM contains a 5'-thymine-rich motif selected from TTN, TTTV or ATTN, where N is A, C, G or T and V is A, C or G . 51. The system according to claim 44 or 45, wherein the effector protein is an effector protein derived from bacterial species selected from the group consisting of Alicyclobacillus acidoterrestris, Alicyclobacillus contaminans, Desulfovibrio inopinaius, Desulfonatronum ihiodismutans, bacteria Opitutaceae TAV5, Tuberibacillus calidus, Bacillus thermoamyiovorans , Brevibacillus sp.CF112, Bacillus sp.NSP2.1, Desulfatirhabdium butyrativorans, Alicyclobacillus herbarius, Citrobacier freundii, Brevibacillus agri, Methylobacterium nodularis. 52. The system according to claim 51, in which the PAM sequence is TTN, where N is A, C, G, or T, or where the PAM sequence is TTTV, where V is A, C, or G, and the effector protein obtained from the bacteria Alicyclobacillus acidoterrestris. 53. The system of claim 44 or 45, wherein the effector protein contains one or more nuclear localization signals. 54. The system of claim 44 or 45, wherein the nucleic acid sequences encoding the effector protein are codon-optimized for expression in a eukaryotic cell. 55. The system according to claim 44 or 45, where components (a) and (b) or nucleotide sequences are located on the same vector. 56. A method for producing a plant having a modified trait of interest encoded by a gene of interest, said method comprising bringing the plant cell into contact with the system of claim 44 or 45 or exposing the plant cell to the method of claim 1, thereby either modifying or by introducing said gene of interest and regenerating the plant from said plant cell. 57. Particle for delivering the system into the cell, containing the system according to claim 44 or 45. 58. The particle of claim 57, wherein the particle contains an effector protein in combination with a guide RNA. 59. The system of claim 44 or 45, or the method of claim 1, wherein the complex, guide RNA or protein is conjugated to at least one sugar moiety, optionally N-acetylgalactosamine (GalNAc), in particular a three-antennary GalNAc. 60. An engineered, non-naturally occurring composition for modifying a target locus of interest, comprising the CRISPR-Cas system, said system comprising a functional single molecular effector protein of the CRISPR-Cas system and a guide RNA (gRNA), where gRNA contains a non-functional guide sequence; where gRNA is able to hybridize with the target locus; where the CRISPR-Cas system is directed with reduced insertion-deletion activity to the target locus, and where the functional single-molecule effector protein of the CRISPR-Cas system is a protein containing three RuvC nuclease catalytic domains, but not containing an HNH domain. 61. A method for inhibiting cell growth, the method comprising delivering to a cell a non-naturally occurring or engineered composition containing a functional single molecular effector protein of the CRISPR-Cas system and a guide RNA (gRNA), where the gRNA is capable of hybridizing with the target DNA sequence of the cell; where the CRISPR-Cas system is directed with reduced insertion-deletion activity to the target DNA sequence; and where the functional single-molecule effector protein of the CRISPR-Cas system is a protein containing three RuvC nuclease catalytic domains, but not containing an HNH domain.

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