RU2845909C2 - Crispr enzyme mutations that reduce off-target effects - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, in particular to an engineered Cas9 protein comprising at least one modification as compared to a wild-type Cas9 protein, where said engineered Cas9 protein has reduced activity with respect to non-target sites compared to the wild-type Cas9 protein, as well as to a composition and system containing same. Also disclosed is a nucleic acid molecule encoding said protein, as well as a vector system and a cell containing same.

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

29 cl, 37 dwg, 7 tbl, 19 ex

Description

Cross-referencing/inclusion by reference

Reference is made to U.S. Provisional Patent Application Serial No. 62/181,453, filed June 18, 2015, U.S. Provisional Patent Application Serial No. 62/207,312, filed August 19, 2015, U.S. Provisional Patent Application Serial No. 62/237,360, filed October 5, 2015, U.S. Provisional Patent Application Serial No. 62/255,256, filed November 13, 2015, and U.S. Provisional Patent Application Serial No. 62/269,876, filed December 18, 2015.

The above-mentioned application(s) and all documents cited or incorporated by reference herein (the "documents cited herein"), and all documents cited or incorporated by reference in documents cited herein, together with any manufacturer's instructions, descriptions, product specifications, and process sheets for any products mentioned herein or in any document incorporated herein by reference, are hereby incorporated herein by reference and may be used in the practice of the present invention. More specifically, all documents incorporated by reference are hereby incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

Statement Regarding Federally Funded Research

This invention was made with support from the Government under Grant Numbers MH100706 and MH110049 from the National Institutes of Health. The Government has certain rights in this invention.

Field of invention

The present invention generally relates to clustered regularly interspaced short palindromic repeats (CRISPR), a CRISPR enzyme (e.g., Cas or Cas9), a CRISPR-Cas or CRISPR system or CRISPR-Cas complex, components thereof, nucleic acid molecules, e.g., vectors, comprising them, and uses of any of the above, among other aspects.

Background of the invention

The first publication with sufficient disclosure of how to generate and use the CRISPR-Cas system in eukaryotic cells is Cong et al., Science 2013; 339:819–823 (published online January 3, 2013). The first patent application filing with a sufficient disclosure of how to make and use the CRISPR-Cas system in eukaryotic cells is Zhang et al., U.S. Provisional Patent Application Ser. No. 61/736,527, filed December 12, 2012, from which many patent applications claim priority, including those that have transitioned into fundamental U.S. Patent Nos. 8999641, 8993233, 8945839, 8932814, 8906616, 8895308, 8889418, 8889356, 8871445, 8865406, 8795965, 8771945, and 8697359.

Brief description of the invention

Given the breakthrough advances that have enabled the use of the CRISPR-Cas system in eukaryotic cells, the Zhang et al. lab at the Broad Institute recognized that there remains a need for improved CRISPR enzymes for use in making modifications at target loci that reduce or eliminate activity at off-target loci. There is a pressing need for alternative and robust systems and methods for reducing off-target activity of CRISPR enzymes complexed with guide RNAs. There is also a pressing need for alternative and robust systems and methods for enhancing activity of CRISPR enzymes complexed with guide RNAs.

Several strategies have been developed to enhance the specificity of Cas9, including reducing the amount of Cas9 in the cell, using mutant Cas9 nicks to create a pair of superimposable single-stranded DNA nicks, truncating the guide sequence at the 5' end, and using a pair of catalytically inactive Cas9 nucleases, each fused to the FokI nuclease domain.

The inventors of the present invention have surprisingly found that modifications can be made to CRISPR enzymes that provide reduced off-target activity compared to unmodified CRISPR enzymes and/or increased on-target activity compared to unmodified CRISPR enzymes. Thus, provided herein are improved CRISPR enzymes that can be used in a variety of gene modification applications. Also provided herein are CRISPR complexes, compositions, and systems, as well as methods and uses, all of which involve the modified CRISPR enzymes disclosed herein. Preferred is CRISPR-Cas9, including but not limited to SaCas9, SpCas9, and orthologs.

In one aspect, an engineered CRISPR protein is provided, wherein the protein is complexed with a nucleic acid molecule comprising RNA to form a CRISPR complex, wherein the nucleic acid molecule, when in the CRISPR complex, targets one or more target polynucleotide loci, wherein the protein comprises at least one modification compared to an unmodified CRISPR, and wherein the CRISPR complex comprising the modified protein is characterized by altered activity compared to a complex comprising an unmodified CRISPR protein. Preferred is CRISPR-Cas9, including but not limited to SaCas9, SpCas9 and orthologs. CRISPR proteins include proteins with enzymatic activity, such as nuclease activity.

In one aspect, the altered activity of the engineered CRISPR protein comprises an altered binding property with respect to the nucleic acid molecule providing RNA or target polynucleotide loci, altered binding kinetics with respect to the nucleic acid molecule providing RNA or target polynucleotide loci, or altered binding specificity with respect to the nucleic acid molecule providing RNA or target polynucleotide loci compared to non-target polynucleotide loci.

In certain embodiments, the altered activity of the engineered CRISPR protein provides for increased targeting efficiency or decreased off-target binding. In certain embodiments, the altered activity of the engineered CRISPR protein provides for modified cleavage activity.

In certain embodiments, the altered activity comprises increased cleavage activity at target polynucleotide loci. In certain embodiments, the altered activity comprises decreased cleavage activity at target polynucleotide loci. In certain embodiments, the altered activity comprises decreased cleavage activity at non-target polynucleotide loci. In certain embodiments, the altered activity comprises increased cleavage activity at non-target polynucleotide loci. Accordingly, in certain embodiments, there is increased specificity at target polynucleotide loci compared to non-target polynucleotide loci. In other embodiments, there is decreased specificity at target polynucleotide loci compared to non-target polynucleotide loci.

In one aspect of the present invention, the altered activity of the engineered CRISPR protein comprises altered kinetic properties of the helicase.

In one aspect of the present invention, the engineered CRISPR protein comprises a modification that alters the binding of the protein to a nucleic acid molecule comprising RNA, or a strand comprising target polynucleotide loci, or a strand comprising non-target polynucleotide loci. In one aspect of the present invention, the engineered CRISPR protein comprises a modification that alters the formation of a CRISPR complex.

The present invention provides:

a non-naturally occurring CRISPR enzyme where:

the enzyme combines with the guide RNA to form the CRISPR complex,

while in the CRISPR complex, the guide RNA targets one or more target polynucleotide loci, and the enzyme alters the polynucleotide loci, and

the enzyme contains at least one modification,

whereby the enzyme in the CRISPR complex has a reduced ability to modify one or more off-target loci compared to an unmodified enzyme, and/or whereby the enzyme in the CRISPR complex has an increased ability to modify one or more target loci compared to an unmodified enzyme.

In any such non-naturally occurring CRISPR enzyme, the modification may involve modification of one or more amino acid residues of the enzyme.

In any such non-naturally occurring CRISPR enzyme, the modification may involve modification of one or more amino acid residues located in a region that contains residues that are positively charged in the unmodified enzyme.

In any such non-naturally occurring CRISPR enzyme, the modification may involve modification of one or more amino acid residues that are positively charged in the unmodified enzyme.

In any such non-naturally occurring CRISPR enzyme, the modification may involve modification of one or more amino acid residues that are not positively charged in the unmodified enzyme.

The modification may involve modification of one or more amino acid residues that are uncharged in the unmodified enzyme.

The modification may involve modification of one or more amino acid residues that are negatively charged in the unmodified enzyme.

The modification may involve modification of one or more amino acid residues that are hydrophobic in the unmodified enzyme.

The modification may involve modification of one or more amino acid residues that are polar in the unmodified enzyme.

In the case of any of the above non-naturally occurring CRISPR enzymes, the enzyme may include a type II CRISPR enzyme. The enzyme may include a Cas9 enzyme.

For some of the non-naturally occurring CRISPR enzymes described above, the modification may involve modification of one or more residues located in the region between the RuvC domain and the HNH domain. The RuvC domain may involve a RuvCII domain or a RuvCIII domain. The modification may involve modification of one or more residues located in the groove.

In the case of some of the non-naturally occurring CRISPR enzymes described above, the modification may involve modification of one or more residues located outside the region between the RuvC domain and the HNH domain, or outside the groove.

In the case of some of the non-naturally occurring CRISPR enzymes described above, modification may involve modification of one or more residues in a region that includes:

residues R63-K1325 or K775-K1325 of Streptococcus pyogenes Cas9 (SpCas9) or the corresponding region in another Cas9 ortholog; or

residues K37-K736 of Staphylococcus aureus Cas9 (SaCas9) or the corresponding region in another Cas9 ortholog.

In the case of some of the non-naturally occurring CRISPR enzymes described above, the modification involves modifying one or more residues, where one or more residues include arginine, histidine, or lysine.

In the case of any of the above-described non-naturally occurring CRISPR enzymes, the enzyme may be modified by mutating said one or more residues.

In the case of some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by a mutation of said one or more residues, wherein the mutation involves replacing the residue in the unmodified enzyme with an alanine residue.

In the case of some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by a mutation of said one or more residues, and the mutation involves replacing the residue in the unmodified enzyme with aspartic acid or glutamic acid.

In the case of some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by a mutation of the specified one or more residues, and the mutation involves replacing the residue in the unmodified enzyme with serine, threonine, asparagine, or glutamine.

In the case of some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by a mutation of said one or more residues, wherein the mutation involves replacing the residue in the unmodified enzyme with alanine, glycine, isoleucine, leucine, methionine, phenylalanine, tryptophan, tyrosine, or valine.

In the case of some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by a mutation of said one or more residues, wherein the mutation involves replacing the residue in the unmodified enzyme with a polar amino acid residue.

In the case of some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by a mutation of said one or more residues, and the mutation involves replacing the residue in the unmodified enzyme with an amino acid residue that is not a polar amino acid residue.

In the case of some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by a mutation of said one or more residues, wherein the mutation involves replacing the residue in the unmodified enzyme with a negatively charged amino acid residue.

In the case of some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by a mutation of said one or more residues, and the mutation involves replacing the residue in the unmodified enzyme with an amino acid residue that is not a negatively charged amino acid residue.

In the case of some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutating said one or more residues, and the mutation involves replacing the residue in the unmodified enzyme with an uncharged amino acid residue.

In the case of some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by a mutation of said one or more residues, and the mutation involves replacing the residue in the unmodified enzyme with an amino acid residue that is not an uncharged amino acid residue.

In the case of some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by a mutation of said one or more residues, and the mutation involves replacing the residue in the unmodified enzyme with a hydrophobic amino acid residue.

In the case of some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by a mutation of said one or more residues, and the mutation involves replacing the residue in the unmodified enzyme with an amino acid residue that is not a hydrophobic amino acid residue.

The non-naturally occurring CRISPR enzyme may be SpCas9 or an ortholog of SpCas9, and:

the enzyme is modified by or comprises a modification, such as comprises, consists essentially of, or consists of a modification by mutation of any of the SpCas9 or SaCas9 residues listed in any of Tables 1-7 or the corresponding residue in a Cas9 ortholog; or

the enzyme comprises, consists essentially of, or consists of a modification at any one (single), two (double), three (triple), four (quadruple) or more positions as described herein throughout the present application, including but not limited to this summary and/or the summary of the drawings and/or the detailed description and/or any of the examples and/or any of the figures, or at a corresponding residue or position in a Cas9 ortholog, for example, the enzyme comprises, consists essentially of, or consists of a modification at any of the Cas9 residues mentioned in any of this summary and/or the summary of the drawings and/or the detailed description and/or any of the examples and/or any of the figures or elsewhere herein, or at a corresponding residue or position in a Cas9 ortholog. In such an enzyme, each residue may be modified by substitution with an alanine residue.

In the case of some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutation of one or more residues including, but not limited to, positions 12, 13, 63, 415, 610, 775, 779, 780, 810, 832, 848, 855, 861, 862, 866, 961, 968, 974, 976, 982, 983, 1000, 1003, 1014, 1047, 1060, 1107, 1108, 1109, 1114, 1129, 1240, 1289, 1296, 1297, 1300, 1311, and 1325 as numbered amino acid positions of SpCas9.

In the case of some of the above-described non-naturally occurring CRISPR enzymes, the enzyme is modified by mutation and contains one or more substitutions to alanine at residues including, but not limited to, positions 63, 415, 775, 779, 780, 810, 832, 848, 855, 861, 862, 866, 961, 968, 974, 976, 982, 983, 1000, 1003, 1014, 1047, 1060, 1107, 1108, 1109, 1114, 1129, 1240, 1289, 1296, 1297, 1300, 1311, or 1325 according to amino acid numbering provisions of SpCas9.

In the case of some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutation and contains one or more of the substitutions K775A, E779L, Q807A, R780A, K810A, R832A, K848A, K855A, K862A, K866A, K961A, K968A, K974A, R976A, H982A, H983A, K1000A, K1014A, K1047A, K1060A, K1003A, K1107A, S1109A, H1240A, K1289A, K1296A, H1297A, K1300A, H1311A, or K1325A.

In the case of some of the above-described non-naturally occurring CRISPR enzymes, the enzyme is modified by mutation and comprises two or more substitutions, wherein the two or more substitutions include, but are not limited to, R783A and A1322T, or R780A and K810A, or ER780A and K855A, or R780A and R976A, or K848A and R976A, or K855A and R976A, and R780A and K848A, or K810A and K848A, or K848A and K855A, or K810A and K855A, or H982A and R1060A, or H982A and R1003A, or K1003A and R1060A, or R780A and H982A, or K810A and H982A, or K848A and H982A, or K855A and H982A, or R780A and K1003A, or K810A and R1003A, or K848A and K1003A, or K848A and K1007A, or R780A and R1060A, or K810A and R1060A, or K848A and R1060A, or R780A and R1114A, or K848A and R1114A, or R63A and K855A, or R63A and H982A, or H415A and R780A, or H415A and K848A, or K848A and E1108A, or K810A and K1003A, or R780A and R1060A, K810A and R1060A, or K848A and R1060A.

In the case of some of the above-described non-naturally occurring CRISPR enzymes, the enzyme is modified by mutation and contains three or more substitutions, wherein the three or more substitutions include, but are not limited to, H982A, K1003A, and K1129E, or R780A, K1003A, and R1060A, or K810A, K1003A, and R1060A, or K848A, K1003A, and R1060A, or K855A, K1003A, and R1060A, or H982A, K1003A, and R1060A, or R63A, K848A, and R1060A, or T13I, R63A, and K810A, or G12D, R63A, and R1060A.

In the case of some of the non-naturally occurring CRISPR enzymes described above, the enzyme is modified by mutation and contains four or more substitutions, wherein the four or more substitutions include, but are not limited to, R63A, E610G, K855A, and R1060A, or R63A, K855A, R1060A, and E610G.

In one preferred embodiment, the mutation in the non-naturally occurring CRISPR enzyme is not a mutation listed in Table B. In a further preferred embodiment, the mutation in the non-naturally occurring CRISPR enzyme is not R63A, K866A, H982A, H983A, K1107A, K1107A, KES1107-1109AG, or KES1107-1109GG according to the amino acid position numbering of SpCas9. In a further preferred embodiment, the non-naturally occurring CRISPR enzyme is not an enzyme modified with a single mutation selected from R63A, K866A, H982A, H983A, K1107A and K1107A, or an enzyme modified with a mutation selected from KES1107-1109AG and KES1107-1109GG according to the amino acid position numbering of SpCas9.

In a preferred embodiment, the above-described non-naturally occurring CRISPR enzyme is modified by mutation of one or more residues including, but not limited to, positions 12, 13, 415, 610, 775, 779, 780, 810, 832, 848, 855, 861, 862, 961, 968, 974, 976, 1000, 1003, 1014, 1047, 1060, 1114, 1129, 1240, 1289, 1296, 1297, 1300, 1311, and 1325, according to the amino acid position numbering of SpCas9.

In a further preferred embodiment, the above-described non-naturally occurring CRISPR enzyme is modified by mutation and comprises one or more substitutions to alanine at residues including, but not limited to, positions 415, 775, 779, 780, 810, 832, 848, 855, 861, 862, 961, 968, 974, 976, 1000, 1003, 1014, 1047, 1060, 1114, 1129, 1240, 1289, 1296, 1297, 1300, 1311 or 1325 according to the amino acid position numbering of SpCas9.

In a further preferred embodiment, the above-described non-naturally occurring CRISPR enzyme is modified by mutation and comprises one or more of the substitutions K775A, E779L, Q807A, R780A, K810A, R832A, K848A, K855A, K862A, K961A, K968A, K974A, R976A, K1000A, K1014A, K1047A, K1060A, K1003A, S1109A, H1240A, K1289A, K1296A, H1297A, K1300A, H1311A or K1325A.

In any of the non-naturally occurring CRISPR enzymes:

a single mismatch may be between the target and the corresponding sequence of one or more non-target loci; and/or

two, three, or four or more mismatches may be between the target and the corresponding sequence of one or more non-target loci, and/or

where (ii) said two, three or four or more mismatches are adjacent.

For any of the non-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, and the enzyme in the CRISPR complex may have an increased ability to modify said target loci compared to the unmodified enzyme.

For any of the non-naturally occurring CRISPR enzymes, when it is in a CRISPR complex, the relative difference in the modifying ability of the enzyme with respect to the target and at least one non-target locus may be increased compared to the relative difference for the unmodified enzyme.

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

In the case of such non-naturally occurring CRISPR enzymes, the CRISPR enzyme may have reduced or abolished nuclease activity compared to an enzyme that lacks the specified one or more additional mutations.

In the case of some of these non-naturally occurring CRISPR enzymes, the CRISPR enzyme does not direct the cleavage of one or the other strand of DNA at a specific position in the target sequence.

For some of these unnaturally occurring CRISPR enzymes, one or more additional mutations involve mutation of D10 of SpCas9, E762 of SpCas9, H840 of SpCas9, N854 of SpCas9, N863 of SpCas9, and/or D986 of SpCas9 or corresponding residues from other Cas9 orthologs.

For some of these unnatural CRISPR enzymes, one or more additional mutations involve D10A, E762A, H840A, N854A, N863A, and/or D986A from SpCas9 or corresponding residues from other Cas9 orthologs.

For some of these unnatural CRISPR enzymes, one or more additional mutations result in two additional mutations. The two additional mutations may result in D10A of SpCas9 and H840A of SpCas9 or the corresponding residues from another Cas9 ortholog. For some of these unnatural CRISPR enzymes, the CRISPR enzyme may not direct the cleavage of one of the DNA strands at a particular position in the target sequence.

If the CRISPR enzyme contains one or more additional mutations in one or more catalytically active domains, the one or more additional mutations may be in a catalytically active domain of the CRISPR enzyme that contains RuvCI, RuvCII, or RuvCIII.

Without being limited by theory, one aspect of the present invention provides methods and mutations for improving the conformational rearrangement of Cas9 domains into positions that allow cleavage at target sites and avoiding such conformational states at off-target sites. Cas9 cleaves target DNA through a series of coordinated steps. Initially, the PAM-interacting domain recognizes a PAM sequence at the 5' end of the target DNA. Upon PAM binding, the first 10-12 nucleotides of the target sequence (the seed sequence) are tested for sgRNA:DNA complementarity, a process driven by separation of the DNA duplex. If the nucleotides of the seed sequence are complementary to the sgRNA, the remainder of the DNA is unwound and the entire length of the sgRNA hybridizes to the target DNA strand. The nt-groove between the RuvC and HNH domains stabilizes the non-targetable DNA strand and facilitates unwinding through non-specific interactions with the positive charges of the DNA phosphate backbone. RNA:cDNA and Cas9:ncDNA interactions drive DNA unwinding in competition with cDNA:ncDNA rehybridization. Other Cas9 domains also influence the conformation of the nuclease domains, such as the linkers connecting HNH to RuvCII and RuvCIII. Accordingly, the provided methods and mutations include, but are not limited to, the RuvCI, RuvCIII, RuvCIII, and HNH domains and linkers. Conformational changes in Cas9 induced by binding of target DNA, including interactions with the primer sequence and interactions with the target and non-targetable DNA strand, determine whether the domains are positioned to trigger nuclease activity. Thus, the mutations and methods provided herein demonstrate and provide modifications that extend beyond PAM recognition and RNA-DNA base pairing.

In one aspect, the present invention provides Cas9 nucleases that provide an improved equilibrium biased toward conformations associated with cleavage activity when engaged in on-target interactions and/or an improved equilibrium biased away from conformations associated with cleavage activity when engaged in off-target interactions. In one aspect, the present invention provides Cas9 nucleases with improved editing function, i.e., a Cas9 nuclease that adopts a conformation that provides nuclease activity at a target site, and wherein such conformation has an increased disfavor at an off-target site. Sternberg et al., Nature 527(7576):110-3, doi: 10.1038/nature15544, published online October 28, 2015. In Epub 2015 Oct 28, Forster resonance energy transfer (FRET) experiments were used to detect the relative orientation of the Cas9 catalytic domains when associated with target and non-target DNA.

The present invention also provides methods and mutations for modulating nuclease activity and/or specificity using modified guide RNAs. As discussed, the target nuclease activity can be increased or decreased. Also, the off-target nuclease activity can be increased or decreased. Additionally, the specificity for the target activity can be increased or decreased compared to the off-target activity. Modified guide RNAs include, but are not limited to, truncated guide RNAs, non-functional guide RNAs, chemically modified guide RNAs, guide RNAs associated with functional domains, modified guide RNAs containing functional domains, modified guide RNAs containing aptamers, modified guide RNAs containing adaptor proteins, and guide RNAs containing added or modified loops.

In one aspect, the present invention also provides methods and mutations for modulating the binding activity and/or binding specificity of Cas9. In certain embodiments, Cas9 proteins that lack nuclease activity are used. In certain embodiments, modified guide RNAs are used that promote binding but not the nuclease activity of the Cas9 nuclease. In such embodiments, target binding may be increased or decreased. Also, in such embodiments, off-target binding may be increased or decreased. Moreover, the specificity for on-target binding relative to off-target binding may be increased or decreased.

Methods and mutations that can be used in various combinations to increase or decrease the activity and/or specificity of a target versus an off-target activity, or to increase or decrease the binding and/or specificity of a target versus an off-target binding, can be used to compensate for or enhance the effect of mutations or modifications made to promote other effects. Such mutations or modifications made to promote other effects include mutations or a modification in Cas9 and/or a mutation or modification made in a guide RNA. In certain embodiments, the methods and mutations are used with chemically modified guide RNAs. Examples of chemical modifications of guide RNAs include, but are not limited to, the introduction of 2′-O-methyl (M), 2′-O-methyl-3′-phosphorothioate (MS), or 2′-O-methyl-3′-thio-PACE (MSP) at one or more terminal nucleotides. Such chemically modified guide RNAs may provide increased stability and increased activity compared to unmodified guide RNAs, although on-target versus off-target specificity is not predictable. (See, Hendel, 2015, Nat Biotechnol. 33(9):985-9, doi: 10.1038/nbt.3290, published online June 29, 2015.) Chemically modified guide RNAs also include, but are not limited to, phosphorothioate-linked RNAs and locked nucleic acid (LNA) nucleotides containing a methylene bridge between the 2′ and 4′ carbons of the ribose ring. The methods and mutations of the present invention are used to modulate the nuclease activity and/or binding of Cas9 to chemically modified guide RNAs.

In one aspect, the present invention provides methods and mutations for modulating the binding and/or binding specificity of Cas9 proteins comprising functional domains such as nucleases, transcriptional activators, transcriptional repressors, and the like. For example, a Cas9 protein can be made to lack nuclease activity by introducing mutations such as D10A, D839A, H840A, and N863A into the RuvC and HNH nuclease domains. Cas9 proteins lacking nuclease activity are suitable for RNA-directed, target sequence-dependent delivery of functional domains. The present invention provides methods and mutations for modulating the binding of Cas9 proteins. In one embodiment, the functional domain provides VP64, which provides an RNA-directed transcription factor. In another embodiment, the functional domain provides Fok I, which provides RNA-directed nuclease activity. Reference should be made herein to U.S. Patent Application Publication 2014/0356959, U.S. Patent Application Publication 2014/0342456, U.S. Patent Application Publication 2015/0031132, and Mali, P. et al., 2013, Science 339(6121):823-6, doi: 10.1126/science.1232033, published online January 3, 2013, and in the teachings herein, the present invention contemplates the methods and materials of these documents used in conjunction with the teachings herein. In certain embodiments, on-target binding is increased. In certain embodiments, off-target binding is decreased. In certain embodiments, on-target binding is decreased. In certain embodiments, off-target binding is increased. Accordingly, the present invention also provides for increasing or decreasing the specificity of target binding compared to off-target binding in functionalized Cas9 binding proteins.

The use of Cas9 as an RNA-guided binding protein is not limited to Cas9 lacking nuclease activity. Cas9 enzymes that provide nuclease activity can also function as RNA-guided binding proteins when used with certain guide RNAs. For example, short guide RNAs and guide RNAs containing nucleotides that are mismatched to the target can promote RNA-guided binding of Cas9 to a target sequence with little or no cleavage of the target sequence. (See, e.g., Dahlman, 2015, Nat Biotechnol. 33(11):1159-1161, doi: 10.1038/nbt.3390, published online October 5, 2015.) In one aspect, the present invention provides methods and mutations for modulating the binding of Cas9 proteins that provide nuclease activity. In certain embodiments, the target binding is increased. In certain embodiments, the off-target binding is decreased. In certain embodiments, the target binding is decreased. In certain embodiments, the off-target binding is increased. In certain embodiments, there is increased or decreased specificity of the target binding compared to the off-target binding. In certain embodiments, the nuclease activity of the Cas9 guide RNA enzyme is also modulated.

For cleavage activity and specificity, it is important to form an RNA-DNA heteroduplex throughout the target region, not just the region of the seed sequence closest to the PAM. Thus, truncated guide RNAs exhibit reduced cleavage activity and specificity. In one aspect of the present invention, a method and mutations are provided for increasing cleavage activity and specificity using altered guide RNAs.

The present invention also demonstrates that modifications to the specificity of the Cas9 nuclease can be made in combination with modifications to the targeting range. Cas9 mutants can be developed that have increased target specificity and also have modifications that allow recognition of PAMs, for example, by selecting mutations that alter specificity for PAMs and combining these mutations with nt groove mutations that increase (or, where appropriate, decrease) specificity for target sequences compared to non-target sequences. In one such embodiment, the PI domain residue is mutated to allow recognition of the desired PAM sequence, and one or more nt groove amino acids are mutated to alter target specificity. Kleinstiver reported SpCas9 and SaCas9 nucleases in which certain residues of the PI domain are mutated and recognize alternative PAM sequences (see Kleinstiver et al., Nature 523(7561):481-5 doi: 10.1038/nature14592, published online June 22, 2015; Kleinstiver et al., Nature Biotechnology, doi: 10.1038/nbt.3404, published online November 2, 2015). The methods and modifications of Cas9 described herein can be used to counteract a loss of specificity resulting from an alteration in PAM recognition, enhance a gain in specificity resulting from an alteration in PAM recognition, counteract a gain in specificity resulting from an alteration in PAM recognition, or enhance a loss of specificity resulting from an alteration in PAM recognition.

The methods and mutations can be applied to any Cas9 enzyme with altered PAM recognition. Non-limiting examples of PAMs include NGG, NNGRRT, NN[A/C/T]RRT, NGAN, NGCG, NGAG, NGNG, NGC, and NGA.

In additional embodiments of the methods and mutations, modified proteins are used.

For any of the non-naturally occurring CRISPR enzymes, the CRISPR enzyme may contain one or more heterologous functional domains.

One or more heterologous functional domains may provide one or more domains that represent a nuclear localization signal (NLS). One or more heterologous functional domains may provide at least two or more NLS.

In certain embodiments of the present invention, at least one nuclear localization signal (NLS) is attached to the nucleic acid sequences encoding the Cas9 effector proteins. In preferred embodiments, at least one or more C-terminal or N-terminal NLSs are attached (and thus the nucleic acid molecule(s) encoding the Cas9 effector protein may provide for encoding an NLS, such that the expressed product has the NLS(s) attached or tethered). In a preferred embodiment, the C-terminal NLS is attached for optimal expression and targeting to the nucleus in eukaryotic cells, preferably human cells. In a preferred embodiment, the codon-optimized effector protein is SpCas9 or SaCas9 and the guide RNA spacer length is from 15 to 35 nucleotides. In certain embodiments, the length of the guide RNA spacer is at least 16 nucleotides, such as at least 17 nucleotides. In certain embodiments, the length of the spacer is from 15 to 17 nucleotides, from 17 to 20 nucleotides, from 20 to 24 nucleotides, such as 20, 21, 22, 23 or 24 nucleotides, from 23 to 25 nucleotides, such as 23, 24 or 25 nucleotides, from 24 to 27 nucleotides, 27-30 nucleotides, 30-35 nucleotides, or 35 nucleotides or more. In certain embodiments of the present invention, the codon-optimized effector protein is SpCas9 or SaCas9, and the length of the direct repeat of the guide RNA is at least 16 nucleotides. In certain embodiments, the codon-optimized effector protein is FnCpf1p and the direct repeat length of the guide RNA is from 16 to 20 nucleotides, such as 16, 17, 18, 19, or 20 nucleotides. In certain preferred embodiments, the direct repeat length of the guide RNA is 19 nucleotides.

One or more heterologous functional domains provide one or more transcriptional activation domains. A transcriptional activation domain may provide VP64.

One or more heterologous functional domains provide one or more transcriptional repression domains. A transcriptional repression domain may provide a KRAB domain or a SID domain.

One or more heterologous functional domains may provide one or more nuclease domains. One or more nuclease domains may provide Fok1.

One or more heterologous functional domains may be characterized by one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcript release factor 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.

At least one or more heterologous functional domains may be located at or near the amino terminus of the enzyme and/or at or near the carboxy terminus of the enzyme.

One or more heterologous functional domains may be fused to a CRISPR enzyme, or linked to a CRISPR enzyme, or attached to a CRISPR enzyme via a linker moiety.

For any of the non-naturally occurring CRISPR enzymes, the CRISPR enzyme may include a CRISPR enzyme from an organism of the genus Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, or Corynebacter.

In the case of any of the non-naturally occurring CRISPR enzymes, the CRISPR enzyme may provide a chimeric Cas9 enzyme comprising a first fragment from a first Cas9 ortholog and a second fragment from a second Cas9 ortholog, and wherein the first and second Cas9 orthologs are different. At least one of the first and second Cas9 orthologs may provide a Cas9 from an organism comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, or Corynebacter.

For any of the non-naturally occurring CRISPR enzymes, the nucleotide sequence encoding the CRISPR enzyme can be codon optimized for expression in a eukaryotic organism.

In the case of 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 wherein the enzyme in the CRISPR complex has a reduced ability to modify one or more off-target loci in the cell compared to an unmodified enzyme, and/or wherein the enzyme in the CRISPR complex has an enhanced ability to modify one or more target loci compared to an unmodified enzyme.

The present invention also provides a non-naturally occurring, engineered composition comprising a CRISPR-Cas complex comprising any non-naturally occurring CRISPR enzyme described above.

The present invention also provides a non-naturally occurring, engineered composition comprising:

a delivery system operatively configured to deliver components of a CRISPR-Cas complex or one or more polynucleotide sequences comprising or encoding said components into a cell, and wherein said CRISPR-Cas complex is functional in the cell,

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

(I) a non-naturally occurring CRISPR enzyme according to any of the preceding paragraphs;

(II) CRISPR-Cas complex RNA containing:

guiding sequence,

paired tracr sequence, and

tracr-sequence,

Where

in a cage

the paired tracr sequence hybridizes with the tracr sequence;

the CRISPR complex is formed;

the guide RNA targets the target polynucleotide loci and the enzyme alters the polynucleotide loci, and

an enzyme in a CRISPR complex has a reduced ability to modify one or more off-target loci compared to an unmodified enzyme, and/or resulting in an enzyme in a CRISPR complex having an increased ability to modify one or more target loci compared to an unmodified enzyme.

In the case of any such composition, the delivery system may comprise a yeast system, a lipofection-based system, a microinjection-based system, a biolistics-based system, virosomes, liposomes, immunoliposomes, polycations, lipid:nucleic acid conjugates, or artificial virions.

In the case of any such compositions, the delivery system may provide a vector system comprising one or more vectors, and where component (II) comprises a first regulatory element operably linked to a polynucleotide sequence that comprises a guide sequence, a tracr mate sequence and a tracr sequence, and where component (I) comprises a second regulatory element operably linked to a polynucleotide sequence encoding a CRISPR enzyme. In the case of such compositions, the guide RNA or RNA of the CRISPR-Cas complex may provide a chimeric RNA.

In the case of any such compositions, the delivery system may provide a vector system comprising one or more vectors, and where component (II) comprises a first regulatory element operably linked to a guide sequence and a tracr pair sequence, and a third regulatory element operably linked to a tracr sequence, and where component (I) comprises a second regulatory element operably linked to a polynucleotide sequence encoding a CRISPR enzyme.

In the case of any such compositions, the composition may comprise more than one guide RNA, and each guide RNA has its own target, resulting in multiplexing.

In the case of any such compositions, the polynucleotide sequence(s) may be located on a single vector.

The present invention also provides a vector system based on an engineered, non-naturally occurring clustered regularly interspaced short palindromic repeats (CRISPR)-associated CRISPR (Cas) enzyme (CRISPR-Cas), comprising one or more vectors comprising:

(a) a first regulatory element operably linked to a nucleotide sequence encoding a non-naturally occurring CRISPR enzyme of any of the constructs of the present invention set forth herein; and

b) a second regulatory element operably linked to one or more nucleotide sequences encoding one or more guide RNAs, wherein the guide RNA comprises a guide sequence, a tracr sequence and a tracr mate sequence,

Where

components (a) and (b) are in the same or different vectors,

the paired tracr sequence hybridizes with the tracr sequence;

the CRISPR complex is formed;

the guide RNA targets the target polynucleotide loci and the enzyme modifies the polynucleotide loci, and

an enzyme in a CRISPR complex has a reduced ability to modify one or more off-target loci compared to an unmodified enzyme, and/or resulting in an enzyme in a CRISPR complex having an increased ability to modify one or more target loci compared to an unmodified enzyme.

In the case of such a system, component (II) may comprise a first regulatory element operably linked to a polynucleotide sequence that comprises a guide sequence, a tracr pair sequence and a tracr sequence, and where component (II) may comprise a second regulatory element operably linked to a polynucleotide sequence encoding a CRISPR enzyme. In the case of such a system, the guide RNA may comprise a chimeric RNA.

In the case of such a system, component (I) may comprise a first regulatory element operably linked to a guide sequence and a paired tracr sequence, and a third regulatory element operably linked to a tracr sequence, and where component (II) may comprise a second regulatory element operably linked to a polynucleotide sequence encoding a CRISPR enzyme. Such a system may comprise more than one guide RNA, and each guide RNA has its own target, resulting in multiplexing. Components (a) and (b) may be located on the same vector.

In the case of any of such vector-containing systems, the one or more vectors may comprise one or more viral vectors, such as one or more retroviral, lentiviral, adenoviral, adeno-associated virus, or herpes simplex virus vectors.

In the case of any of such systems containing regulatory elements, at least one of said regulatory elements may provide a tissue-specific promoter. The tissue-specific promoter may direct expression in a mammalian blood cell, in a mammalian liver cell, or in a mammalian eye.

In any of the above compositions or systems, the tracr sequence may provide one or more RNA aptamers that interact with the protein. The one or more aptamers may be in a tetraloop and/or a stem-loop 2 structure of the tracr sequence. The one or more aptamers may be capable of binding the MS2 bacteriophage coat protein.

In the case of any of the above described compositions or systems, the tracr sequence may be 30 or more nucleotides in length.

In the case of any of the above-described compositions or systems, the cell can be a eukaryotic cell or a prokaryotic cell; wherein the CRISPR complex is functional in the cell and, as a result, the enzyme in the CRISPR complex has a reduced ability to modify one or more off-target loci in the cell compared to an unmodified enzyme and/or, as a result, the enzyme in the CRISPR complex has an increased ability to modify one or more target loci compared to an unmodified enzyme.

The present invention also provides a CRISPR complex of any of the above-described compositions or from any of the above-described systems.

The present invention also provides an engineered CRISPR protein, complex, composition, system, vector, cell or cell line according to the present invention for use in therapy.

The present invention also provides a method of modifying a locus of interest in a cell, comprising contacting the cell with any of the above-described compositions or any of the above-described systems, or wherein the cell comprises any of the above-described CRISPR complexes present in the cell. In such methods, the cell may be a eukaryotic cell. In such methods, the organism may comprise the cell. In such methods, the organism may not be a human or other animal. The present invention also provides an engineered CRISPR protein, complex, composition, system, vector, cell, or cell line according to the present invention for use in modifying a locus of interest in a cell. Such modification preferably involves contacting the cell with any of the above-described compositions or any of the above-described systems. The present invention also provides the use of an engineered CRISPR protein, complex, composition, system, vector, cell, or cell line according to the present invention in the preparation of a medicament for modifying a locus of interest in a cell.

Any such method may be carried out ex vivo or in vitro.

Any such method of said modification may involve modulating gene expression. Said modulating gene expression may involve activating gene expression and/or repressing gene expression.

The present invention also provides an engineered CRISPR protein, complex, composition, system, vector, cell or cell line according to the present invention for use in modifying a locus of interest in a cell. The present invention also provides the use of an engineered CRISPR protein, complex, composition, system, vector, cell or cell line according to the present invention in the preparation of a medicament for modifying a locus of interest in a cell. Said modification preferably involves contacting the cell with any of the above-described compositions or systems. The present invention also provides a method of treating a disease, disorder or infection in an individual in need thereof, comprising administering an effective amount of any of the above-described compositions, systems or CRISPR complexes. The disease, disorder or infection may include a viral infection. The viral infection may be an HBV infection.

The present invention also provides an engineered CRISPR protein, complex, composition, system, vector, cell or cell line according to the present invention for use in the treatment of a disease, disorder or infection in an individual in need thereof. The disease, disorder or infection may include a viral infection. The viral infection may be an infection caused by HBV. The present invention also provides the use of an engineered CRISPR protein, complex, composition, system, vector, cell or cell line according to the present invention in the preparation 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.

The present invention also provides for the use of any of the CRISPR compositions, systems or complexes described above with respect to gene or genome editing.

The present invention also provides any of the CRISPR compositions, systems or complexes described above for use as a therapeutic agent. The therapeutic agent may be for gene or genome editing or gene therapy.

In one aspect of the present invention, there is provided a method for modifying an organism or non-human organism by manipulating a target sequence in a locus of interest in the genome of an HSC, for example, where the locus of interest in the genome is associated with a mutation associated with aberrant protein expression or with a disease state or disease course, comprising:

delivery to the HSC, such as by contacting the HSC with a particle comprising a non-naturally occurring or engineered composition comprising:

I. a polynucleotide sequence of a chimeric RNA (chiRNA) of the CRISPR-Cas system, containing:

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

(b) the paired tracr sequence and

(c) tracr sequence, and

II. a CRISPR enzyme, optionally comprising at least one or more nuclear localization sequences,

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

wherein the CRISPR complex comprises a CRISPR enzyme in a complex with (1) a guide sequence that is hybridized to a target sequence, and (2) a tracr mate sequence that is hybridized to the tracr sequence; and

the method may optionally also comprise delivering an HDR matrix, such as by said particle being contacted with an HSC that comprises an HDR matrix, or by contacting the HSC with another particle that comprises an HDR matrix, wherein the HDR matrix provides for expression of a normal or less aberrant form of the protein; wherein "normal" refers to the wild type and "aberrant" may refer to expression of the protein that results in a pathological condition or disease state; and

optionally, the method may include isolating or obtaining HSC from an organism or non-human organism, optionally expanding the population of HSC, contacting the particle(s) with the HSC to obtain a population of modified HSC, optionally expanding the population of modified HSC, and optionally introducing the modified HSC into the organism or non-human organism.

The present invention also provides an engineered CRISPR protein, complex, composition, system, vector, cell or cell line according to the present invention for use in modifying an organism or non-human organism by manipulating a target sequence in a locus of interest of the HSC genome. Said modification preferably comprises

delivery to the HSC, such as by contacting the HSC with a particle comprising a non-naturally occurring or engineered composition comprising:

I. a polynucleotide sequence of a chimeric RNA (chiRNA) of the CRISPR-Cas system, containing:

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

(b) the paired tracr sequence and

(c) tracr sequence, and

II. a CRISPR enzyme, optionally comprising at least one or more nuclear localization sequences,

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

wherein the CRISPR complex comprises a CRISPR enzyme in a complex with (1) a guide sequence that is hybridized to a target sequence, and (2) a tracr mate sequence that is hybridized to the tracr sequence. Said modification also optionally comprises delivering an HDR template, such as by said particle being contacted with an HSC that contains the HDR template, or by contacting the HSC with another particle that contains the HDR template, wherein the HDR template provides for expression of a normal or less aberrant form of the protein; wherein "normal" refers to the wild type, and "aberrant" may refer to protein expression that results in a pathological condition or disease state. Said modification also optionally includes isolating or obtaining HSC from an organism or non-human organism, optionally propagating a population of HSC, contacting the particle(s) with HSC to obtain a population of modified HSC, optionally propagating the population of modified HSC, and optionally introducing the modified HSC into an organism or non-human organism.

In one aspect of the present invention, there is provided a method of modifying an organism or non-human organism by manipulating a target sequence in a genomic locus of interest of an HSC, e.g., wherein the genomic locus of interest is associated with a mutation associated with aberrant protein expression or with a disease state or disease course, comprising: delivering to the HSC, e.g., by contacting the HSC with a particle comprising a non-naturally occurring or engineered composition comprising: I. (a) a guide sequence capable of hybridizing to a target sequence in the HSC, and (b) at least one or more tracr mate sequences, II. a CRISPR enzyme optionally with one or more NLS, and III. a polynucleotide sequence comprising a tracr sequence, wherein a tracr mate sequence hybridizes to a tracr sequence and a guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence, and wherein the CRISPR complex comprises a CRISPR enzyme in complex with (1) a guide sequence that is hybridized to a target sequence and (2) a tracr mate sequence that is hybridized to the tracr sequence; and

the method may optionally also comprise delivering an HDR matrix, such as by said particle being contacted with an HSC that comprises an HDR matrix, or by contacting the HSC with another particle that comprises an HDR matrix, wherein the HDR matrix provides for expression of a normal or less aberrant form of the protein; wherein "normal" refers to the wild type and "aberrant" may refer to expression of the protein that results in a pathological condition or disease state; and

optionally, the method may comprise isolating or obtaining HSCs from an organism or non-human organism, optionally expanding the population of HSCs, contacting the particle(s) with the HSCs to obtain a population of modified HSCs, optionally expanding the population of modified HSCs, and optionally introducing the modified HSCs into the organism or non-human organism. The present invention also provides an engineered CRISPR protein, complex, composition, system, vector, cell or cell line according to the present invention for use in such modification of an organism or non-human organism by manipulating a target sequence at a genomic locus of interest of an HSC, for example, where the genomic locus of interest is associated with a mutation associated with aberrant protein expression or with a disease state or disease course.

The delivery may be delivery of one or more polynucleotides encoding any one or more or all of the CRISPR complexes preferably linked to one or more regulatory elements for in vivo expression, such as via a particle(s) comprising a vector comprising a polynucleotide(s) operably linked to a regulatory element(s). Any or all of the polynucleotide sequence encoding the CRISPR enzyme, the guide sequence, the tracr mate sequence or the tracr sequence may be RNA. It should be appreciated that when a polynucleotide is referred to as RNA and is said to "comprise" an element such as a tracr mate sequence, the RNA sequence includes the element. When a polynucleotide is DNA and is said to contain an element such as a tracr mate sequence, the DNA sequence is or can be transcribed into RNA containing the element in question. If the element is a protein, such as the CRISPR enzyme, then the DNA or RNA sequence in question is or can be translated (and in the case of DNA, first transcribed).

In certain embodiments, the present invention provides a method of modifying an organism, e.g., a mammal, including a human, or a non-human mammal or organism, by manipulating a target sequence in a genomic locus of interest of an HSC, e.g., wherein the genomic locus of interest is associated with a mutation associated with aberrant protein expression or with a disease state or disease course, comprising delivering, e.g., by contacting a non-naturally occurring or engineered composition to the HSC, wherein the composition comprises one or more particles comprising viral, plasmid, or nucleic acid molecule (e.g., RNA) vector(s) operably encoding the composition for expression thereof, wherein the composition comprises: (A) I. a first regulatory element operably linked to a polynucleotide sequence of a chimeric RNA (chiRNA) of the CRISPR-Cas system, wherein the polynucleotide sequence comprises (a) a guide sequence capable of hybridizing to the target sequence in a eukaryotic cell, (b) the paired tracr sequence and (c) the tracr sequence, and II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences (or optionally at least one or more nuclear localization sequences since some embodiments may not provide an NLS), wherein (a), (b) and (c) are arranged in a 5' to 3' orientation, wherein components I and II are in the same or different vectors of a system wherein, when transcribed, the tracr mate sequence hybridizes to a tracr sequence and the guide sequence directs sequence-specific binding of the CRISPR complex to a target sequence, and wherein the CRISPR complex comprises a CRISPR enzyme in complex with (1) a guide sequence that is hybridized to a target sequence and (2) a tracr mate sequence that is hybridized to a tracr sequence, or (B) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising I. a first regulatory element operably linked to (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, (b) at least one or more tracr mate sequences, II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and III. a third regulatory element operably linked to a tracr sequence, wherein components I, II and III are on the same or different vectors of a system wherein, when transcribed, the tracr mate sequence hybridizes to a tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence, and wherein the CRISPR complex comprises a CRISPR enzyme in complex with (1) a guide sequence that is hybridized to a target sequence and (2) a tracr mate sequence that is hybridized to the tracr sequence; wherein the method may optionally further comprise delivering an HDR matrix, such as by said particle being contacted with an HSC that comprises an HDR matrix or by contacting the HSC with another particle that comprises an HDR matrix, wherein the HDR matrix provides for expression of a normal or less aberrant form of the protein; wherein "normal" refers to the wild type and "aberrant" may denote expression of the protein that results in a pathological condition or disease state; and optionally the method may comprise isolating or obtaining HSC from an organism or non-human organism, optionally expanding the population of HSC, contacting the particle(s) with the HSC to obtain a population of modified HSC, optionally expanding the population of modified HSC, and optionally introducing the modified HSC into the organism or non-human organism. In some embodiments, components I, II, and III are on the same vector. In other embodiments, components I and II are on the same vector while component III is on a different vector. In other embodiments, components I and III are in the same vector, while component II is in a different vector. In other embodiments, components II and III are in the same vector, while component I is in a different vector. In other embodiments, components I, II, and III are each in separate vectors. The present invention also provides a viral or plasmid vector system as described herein. The present invention also provides an engineered CRISPR protein, complex, composition, system, vector, cell, or cell line according to the present invention for use in such modification of an organism, e.g., a mammal, including a human or a non-human mammal or organism, by manipulating a target sequence in a genomic locus of interest of an HSC, e.g., wherein the genomic locus of interest is associated with a mutation associated with aberrant protein expression or with a disease state or disease course, comprising delivery, e.g., by contacting a non-naturally occurring or engineered composition with an HSC.

By manipulation of a target sequence, the applicants also mean epigenetic manipulation of a target sequence. This may be carried out on the chromatin state of the target sequence, such as by modifying the methylation state of the target sequence (i.e. adding or removing methylation or methylation patterns or CpG islands), modifying histones, increasing or decreasing the accessibility of the target sequence, or by activating folding into a 3D structure. It should be noted that where reference is made to a method for modifying an organism or mammal, including a human or a non-human mammal or organism, by manipulating a target sequence at a genomic locus of interest, this may be applied to the organism (or mammal) as a whole or to only one cell or population of cells from the organism (if the organism is multicellular). For example, in the case of a human, the applicants envisage, 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 biological fluid sample may be necessary. Stem cells are also particularly preferred in this regard. But, of course, in vivo embodiments are also envisaged. And the present invention is particularly advantageous with respect to HSC.

In some embodiments, the present invention encompasses a method of modifying an organism or non-human organism by manipulating first and second target sequences on opposite strands of a DNA duplex at a genomic locus of interest in an HSC, e.g., where the genomic locus of interest is associated with a mutation associated with aberrant protein expression or with a disease state or disease course, comprising delivering, e.g., by contacting the HSC with particle(s) comprising a non-naturally occurring or engineered composition comprising:

I. a first polynucleotide sequence of a chimeric RNA (chiRNA) of the CRISPR-Cas system, wherein the first polynucleotide sequence comprises:

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

(b) the first paired tracr sequence and

(c) the first tracr sequence,

II. a second polynucleotide sequence of the chiRNA of the CRISPR-Cas system, wherein the second polynucleotide sequence comprises:

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

(b) the second paired tracr sequence and

(c) the second tracr sequence, and

III. a polynucleotide sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences and comprising one or more mutations, wherein (a), (b) and (c) are arranged in a 5'-3' orientation; or

IV. the expression product(s) of one or more of I. - III., e.g., the first and second paired tracr sequences, the CRISPR enzyme;

wherein, when transcribed, the first and second tracr mate sequences hybridize to the first and second tracr sequences, respectively, and the first and second guide sequences direct sequence-specific binding of the first and second CRISPR complexes to the first and second target sequences, respectively, wherein the first CRISPR complex comprises a CRISPR enzyme in complex with (1) a first guide sequence that is hybridized to the first target sequence and (2) a first tracr mate sequence that is hybridized to the first tracr sequence, wherein the second CRISPR complex comprises a CRISPR enzyme in complex with (1) a second guide sequence that is hybridized to the second target sequence and (2) a second tracr mate sequence that is hybridized to the second tracr sequence, wherein the polynucleotide sequence encoding the CRISPR enzyme is DNA or RNA, and wherein the first guide sequence directs the cleavage of one strand of a DNA duplex near the first target sequences, and the second guide sequence directs cleavage of the other strand near the second target sequence, inducing a double-strand break, thereby modifying the organism or non-human organism; and wherein the method may optionally further comprise delivering an HDR matrix, such as by said particle being contacted with an HSC that contains the HDR matrix, or by contacting the HSC with another particle that contains the HDR matrix, wherein the HDR matrix provides expression of a normal or less aberrant form of the protein; wherein "normal" refers to the wild type, and "aberrant" may refer to expression of the protein that results in a pathological condition or a disease state; and optionally the method may comprise isolating or obtaining HSC from an organism or non-human organism, optionally expanding the population of HSC, contacting the particle(s) with the HSC to obtain a population of modified HSC, optionally expanding the population of modified HSC, and optionally introducing the modified HSC into the organism or non-human organism. In some methods of the present invention, any or all of the polynucleotide sequence encoding the CRISPR enzyme, the first and second guide sequences, the first and second tracr mate sequences, or the first and second tracr sequences is/are RNA. In further embodiments of the present invention, the polynucleotides encoding the sequence encoding the CRISPR enzyme, the first and second guide sequences, the first and second tracr mate sequences, or the first and second tracr sequences are/are RNA and are delivered by liposomes, nanoparticles, exosomes, microvesicles, or a gene gun; but preferably, the delivery is by means of a particle. In certain embodiments of the present invention, the first and second tracr paired sequences have 100% identity, and/or the first and second tracr sequences have 100% identity. In some embodiments, the polynucleotides may be contained in a vector system comprising one or more vectors. In preferred embodiments of the present invention, the CRISPR enzyme is a Cas9 enzyme, such as SpCas9. In one aspect of the present invention, the CRISPR enzyme comprises one or more mutations in the catalytic domain, wherein the one or more mutations compared to SpCas9 are selected from the group consisting of a D10A, E762A, H840A, N854A, N863A and D986A mutation, such as a D10A mutation. In preferred embodiments, the first CRISPR enzyme has one or more mutations such that the enzyme is an enzyme that introduces a single-strand break in the complementary strand, and the second CRISPR enzyme has one or more mutations such that the enzyme is an enzyme that introduces a single-strand break in the non-complementary strand. Alternatively, the first enzyme may be an enzyme that introduces a single-strand break in the non-complementary strand, and the second enzyme may be an enzyme that introduces a single-strand break in the complementary strand. In preferred methods of the present invention, a first guide sequence that directs cleavage of one strand of a DNA duplex near a first target sequence and a second guide sequence that directs cleavage of the other strand near a second target sequence result in the formation of a 5' overhang. In embodiments of the present invention, the 5' overhang comprises no more than 200 base pairs, preferably no more than 100 base pairs, or more preferably no more than 50 base pairs. In embodiments of the present invention, the 5' overhang comprises at least 26 base pairs, preferably at least 30 base pairs, or more preferably 34-50 base pairs. The present invention also provides an engineered CRISPR protein, complex, composition, system, vector, cell or cell line according to the present invention for use in such modification of an organism or an organism other than a human by manipulating first and second target sequences on opposite strands of a DNA duplex at a genomic locus of interest in HSCs, for example, where the genomic locus of interest is associated with a mutation associated with aberrant protein expression or with a disease state or disease course, comprising delivery, for example, by contacting the HSCs with a particle(s) containing a non-naturally occurring or engineered composition.

In some embodiments, the present invention encompasses a method of modifying an organism or non-human organism by manipulating first and second target sequences on opposite strands of a DNA duplex at a genomic locus of interest in an HSC, e.g., where the genomic locus of interest is associated with a mutation associated with aberrant protein expression or with a disease state or disease course, comprising delivering, e.g., by contacting the HSC with particle(s) comprising a non-naturally occurring or engineered composition comprising:

I. the first regulatory element functionally associated with

(a) a first guide sequence capable of hybridizing to a first target sequence and

(b) at least one or more paired tracr sequences,

II. the second regulatory element, functionally associated with

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

(b) at least one or more paired tracr sequences,

III. a third regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and

IV. the fourth regulatory element, functionally linked to the tracr sequence,

V. the expression product(s) of one or more of I. - IV., e.g., the first and second paired tracr sequences, the CRISPR enzyme;

wherein components I, II, III and IV are located in the same or different vectors of the system, while being transcribed, the mate tracr sequence hybridizes to the tracr sequence, and the first and second guide sequences direct sequence-specific binding of the first and second CRISPR complexes to the first and second target sequences, respectively, wherein the first CRISPR complex comprises a CRISPR enzyme in complex with (1) a first guide sequence that is hybridized to the first target sequence, and (2) a mate tracr sequence that is hybridized to the tracr sequence, wherein the second CRISPR complex comprises a CRISPR enzyme in complex with (1) a second guide sequence that is hybridized to the second target sequence, and (2) a mate tracr sequence that is hybridized to the tracr sequence, wherein the polynucleotide sequence encoding the CRISPR enzyme is DNA or RNA, and wherein the first guide sequence directs the cleavage of one strands of a DNA duplex near a first target sequence, and a second guide sequence directs cleavage of the other strand near the second target sequence, inducing a double-strand break, thereby modifying the organism or non-human organism; and wherein the method may optionally further comprise delivering an HDR template, such as by said particle being contacted with an HSC that contains the HDR template, or by contacting the HSC with another particle that contains the HDR template, wherein the HDR template provides for expression of a normal or less aberrant form of the protein; wherein "normal" refers to the wild type, and "aberrant" may refer to expression of the protein that results in a pathological condition or a disease state; and optionally the method may comprise isolating or obtaining HSCs from an organism or non-human organism, optionally expanding the population of HSCs, contacting the particle(s) with the HSCs to obtain a population of modified HSCs, optionally expanding the population of modified HSCs, and optionally introducing the modified HSCs into the organism or non-human organism. The present invention also provides an engineered CRISPR protein, complex, composition, system, vector, cell or cell line according to the present invention for use in such modification of an organism or non-human organism by manipulating first and second target sequences on opposite strands of a DNA duplex at a genomic locus of interest in HSCs, for example, wherein the genomic locus of interest is associated with a mutation associated with aberrant protein expression or with a disease state or disease course, comprising delivery, for example, by contacting the HSCs with particle(s) comprising a non-naturally occurring or engineered composition.

The present invention also provides a vector system as described herein. The system may comprise one, two, three or four different vectors. Components I, II, III and IV may thus be in one, two, three or four different vectors, and all combinations of possible locations of the components are contemplated herein, such as: Components I, II, III and IV may be in the same vector; Components I, II, III and IV may each be in separate vectors; Components I, II, III and IV may be in a total of two or three different vectors, and all combinations of locations are contemplated, and the like. In some methods of the present invention, any or all of the polynucleotide sequences encoding the CRISPR enzyme, the first and second guide sequences, the first and second tracr mate sequences or the first and second tracr sequences is/are RNA. In further embodiments of the present invention, the first and second tracr mate sequences have 100% identity and/or the first and second tracr sequences have 100% identity. In preferred embodiments of the present invention, the CRISPR enzyme is a Cas9 enzyme, such as SpCas9. In one aspect of the present invention, the CRISPR enzyme comprises one or more mutations in the catalytic domain, wherein the one or more mutations compared to SpCas9 are selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A; such as the D10A mutation. In preferred embodiments, the first CRISPR enzyme has one or more mutations such that the enzyme is a complementary strand break introducing enzyme, and the second CRISPR enzyme has one or more mutations such that the enzyme is a non-complementary strand break introducing enzyme. Alternatively, the first enzyme may be an enzyme that introduces a single-strand break in the non-complementary strand, and the second enzyme may be an enzyme that introduces a single-strand break in the complementary strand. In a further embodiment of the present invention, one or more viral vectors are delivered via liposomes, nanoparticles, exosomes, microvesicles or a gene gun; but delivery via particles is advantageous.

In preferred methods of the present invention, a first guide sequence directing cleavage of one strand of a DNA duplex near a first target sequence and a second guide sequence directing cleavage of the other strand near a second target sequence cause the formation of a 5' overhang. In embodiments of the present invention, the 5' overhang comprises no more than 200 base pairs, preferably no more than 100 base pairs, or more preferably no more than 50 base pairs. In embodiments of the present invention, the 5' overhang comprises at least 26 base pairs, preferably at least 30 base pairs, or more preferably 34-50 base pairs.

In some embodiments, the present invention encompasses a method of modifying a genomic locus of interest in an HSC, such as where the genomic locus of interest is associated with a mutation associated with aberrant protein expression or with a disease state or course of a disease, by introducing into the HSC, such as by contacting the HSC with a particle(s) comprising a Cas protein with one or more mutations and two guide RNAs that target a first strand and a second strand of a DNA molecule, respectively, in the HSC, whereby the guide RNAs target the DNA molecule and the Cas protein introduces a single-strand break into each of the first strand and the second strand of the DNA molecule, whereby the target in the HSC is altered; and, wherein the Cas protein and the two guide RNAs do not occur together in nature, and wherein the method may optionally further comprise delivering an HDR template, such as by said particle being contacted with an HSC that comprises an HDR template, or by contacting the HSC with another particle that comprises an HDR template, wherein the HDR template provides for expression of a normal or less aberrant form of the protein; wherein "normal" refers to the wild type and "aberrant" may denote expression of the protein that results in the occurrence of a pathological condition or a disease state; and wherein optionally the method may comprise isolating or obtaining HSC from an organism or non-human organism, optionally expanding the population of HSC, contacting the particle(s) with the HSC to obtain a population of modified HSC, optionally expanding the population of modified HSC, and optionally introducing the modified HSC into the organism or non-human organism. In preferred methods of the present invention, the Cas protein introduces a single-strand break into each of the first strand and the second strand of the DNA molecule, resulting in the formation of a 5' overhang. In embodiments of the present invention, the 5' overhang comprises no more than 200 base pairs, preferably no more than 100 base pairs, or more preferably no more than 50 base pairs. In embodiments of the present invention, the 5' overhang comprises at least 26 base pairs, preferably at least 30 base pairs, or more preferably 34-50 base pairs. Embodiments of the present invention also encompass guide RNAs comprising a guide sequence fused to a tracr mate sequence and a tracr sequence. In one aspect of the present invention, the Cas protein is codon optimized for expression in a eukaryotic cell, preferably a mammalian cell or a human cell. In further embodiments of the present invention, the Cas protein is a protein of the Type II CRISPR-Cas system, such as the Cas 9 protein. In a particularly preferred embodiment, the Cas protein is a Cas9 protein, such as SpCas9 or SaCas9. In aspects of the present invention, the Cas protein has one or more mutations compared to SpCas9 selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A; such as the D10A mutation. Aspects of the present invention relate to decreasing the expression of a gene product, or further introducing a polynucleotide template into a DNA molecule encoding a gene product, or precisely excising an insertion sequence by allowing re-hybridization and ligation of two 5' sticky ends, or altering the activity or function of a gene product, or increasing the expression of a gene product. In one embodiment, the gene product is a protein.

In some embodiments, the present invention encompasses a method of modifying a genomic locus of interest in an HSC, such as where the genomic locus of interest is associated with a mutation associated with aberrant protein expression or with a disease state or disease course, by introducing into the HSC, such as by contacting the HSC with a particle(s) comprising:

a) a first regulatory element operably linked to each of two guide RNAs of the CRISPR-Cas system that target the first strand and the second strand, respectively, of a double-stranded DNA molecule of the HSC, and

b) a second regulatory element functionally linked to the Cas protein, or

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

wherein components (a) and (b) are in the same or different vectors of the system, whereby the guide RNAs target the HSC DNA molecule and the Cas protein introduces a single-strand break into each of the first strand and the second strand of the HSC DNA molecule; and wherein the Cas protein and the two guide RNAs do not occur together in nature; and wherein the method may optionally also comprise delivering an HDR template, such as by said particle being contacted with the HSC that contains the HDR template or by contacting the HSC with another particle containing the HDR template, wherein the HDR template provides for expression of a normal or less aberrant form of the protein; wherein "normal" refers to the wild type and "aberrant" may mean expression of the protein that results in a pathological condition or a disease state; and optionally the method may comprise isolating or obtaining HSCs from an organism or non-human organism, optionally expanding the population of HSCs, contacting the particle(s) with the HSCs to obtain a population of modified HSCs, optionally expanding the population of modified HSCs, and optionally introducing the modified HSCs into the organism or non-human organism. In aspects of the present invention, the guide RNAs may comprise a guide sequence fused to a tracr mate sequence and a tracr sequence. In one embodiment of the present invention, the Cas protein is a type II CRISPR-Cas system protein. In one aspect of the present invention, the Cas protein is codon optimized for expression in a eukaryotic cell, preferably a mammalian cell or a human cell. In further embodiments of the present invention, the Cas protein is a type II CRISPR-Cas system protein, such as Cas 9 protein. In a particularly preferred embodiment, the Cas protein is a Cas9 protein, such as SpCas9 or SaCas9. In aspects of the present invention, the Cas protein has one or more mutations compared to SpCas9 selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A; for example, the D10A mutation. Aspects of the present invention relate to decreasing the expression of the gene product, or to further introducing a polynucleotide template into the DNA molecule encoding the gene product, or to precisely excising the insertion sequence by providing re-hybridization and ligation of two "sticky" 5'-ends, or to altering the activity or function of the gene product, or to increasing the expression of the gene product. In one embodiment of the present invention, the gene product is a protein. In preferred embodiments of the present invention, the vectors of the system are viral vectors. In a further embodiment, the vectors of the system are delivered via liposomes, nanoparticles, exosomes, microvesicles or a gene gun; particles being preferred. In one aspect of the present invention, there is provided a method for modifying a target polynucleotide in an HSC. In some embodiments, the method comprises causing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide, thereby effecting modification of the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence in said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence, which in turn hybridizes to the tracr sequence. In some embodiments, said cleavage comprises cleavage of one or two strands at a certain position of the target sequence by said CRISPR enzyme. In some embodiments, said cleavage results in reduced transcription of the target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous polynucleotide template, wherein said repair results in a mutation involving an insertion, deletion or substitution of one or more nucleotides in said target polynucleotide. In some embodiments, said mutation results in a change in one or more amino acids in a protein expressed from a gene comprising the target sequence. In some embodiments, the method further comprises delivering one or more vectors or expression product(s) thereof, such as via a particle(s), to said HSC, wherein the one or more vectors direct the expression of one or more of: a CRISPR enzyme, a guide sequence associated with a tracr mate sequence, and a tracr sequence. In some embodiments, said vectors are delivered to an HSC located in the subject's body. In some embodiments, said modification occurs in said HSC located in cell culture. In some embodiments, the method further comprises isolating said HSC from the subject prior to said modification. In some embodiments, the method further comprises returning said HSC and/or cells derived therefrom to said subject.

In one aspect of the present invention, there is provided a method for producing an HSC comprising a mutated gene responsible for developing a disease. In some embodiments, the gene responsible for developing a disease is any gene associated with an increased risk of having or developing a disease. In some embodiments, the method comprises (a) introducing one or more vectors or expression product(s) thereof, such as via a particle(s), into the HSC, wherein the one or more vectors direct the expression of one or more of: a CRISPR enzyme, a guide sequence associated with a tracr mate sequence, and a tracr sequence; and (b) causing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide in said disease-causing gene, wherein the CRISPR complex comprises a CRISPR enzyme in complex with (1) a guide sequence that is hybridized to a target sequence in the target polynucleotide and (2) a tracr mate sequence that is hybridized to the tracr sequence, thereby producing an HSC comprising a mutated disease-causing gene. In some embodiments, said cleavage comprises cleavage of one or two strands at a specific position of the target sequence by said CRISPR enzyme. In some embodiments, said cleavage results in reduced transcription of the target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous polynucleotide template, wherein said repair results in a mutation involving an insertion, deletion or substitution of one or more nucleotides in said target polynucleotide. In some embodiments, said mutation results in a change in one or more amino acids in the expression of a protein from a gene comprising the target sequence. In some embodiments, the modified HSC is administered to an animal, thereby producing an animal model.

In one aspect, the present invention provides methods for modifying a target polynucleotide in an HSC. Also provided is an engineered CRISPR protein, complex, composition, system, vector, cell, or cell line according to the present invention for use in modifying a target polynucleotide in an HSC. In some embodiments, the method comprises causing a CRISPR complex to bind to a target polynucleotide to effect cleavage of said target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence that is in turn hybridized to a tracr sequence. In other embodiments, the present invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell that is derived from an HSC. The method comprises increasing or decreasing expression of a target polynucleotide by using a CRISPR complex that binds to a polynucleotide in an HSC; The CRISPR complex is primarily delivered via particle(s).

In some methods, the target polynucleotide may be inactivated to effect modification of expression in the cell. For example, after the CRISPR complex binds to the target sequence in the cell, the target polynucleotide is inactivated, causing the sequence to not be transcribed, the encoded protein not being produced, or the sequence not functioning as a wild-type sequence.

In some embodiments, the RNA from the CRISPR-Cas system, such as a guide or sgRNA, can be modified; for example, to include an aptamer or a functional domain. An aptamer is a synthetic oligonucleotide that binds to a specific target molecule; for example, a nucleic acid molecule that has been engineered through repeated rounds of in vitro selection or SELEX (systematic evolution of ligands by exponential enrichment) to bind to a variety of molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms. Aptamers are useful in that they provide molecular recognition properties that make them competitors to antibodies. In addition to their ability to differentially recognize, aptamers provide advantages over antibodies, including that they cause little or no immunogenicity when used therapeutically. Accordingly, in practicing the present invention, either or both of the enzyme or the RNA may comprise a functional domain.

In some embodiments, the functional domain is a transcriptional activation domain, preferably VP64. In some embodiments, the functional domain is a transcriptional repression domain, preferably KRAB. In some embodiments, the transcriptional repression domain is SID or SID concatemers (e.g., SID4X). In some embodiments, the functional domain is an epigenetic modification domain, such that an epigenetic modification enzyme is provided. In some embodiments, the functional domain is an activation domain, which may be a P65 activation domain. In some embodiments, the functional domain provides nuclease activity. In one such embodiment, the functional domain provides Fok1.

The present invention also provides an in vitro or ex vivo cell comprising any of the modified CRISPR enzymes, compositions, systems or complexes described above or from any of the methods described above. The cell may be a eukaryotic cell or a prokaryotic cell. The present invention also provides a progeny of such cells. The present invention also provides a product of any such cell or any such progeny, wherein the product is a product of said one or more target loci modified with a modified CRISPR enzyme of a CRISPR complex. The product may be a peptide, a polypeptide or a protein. Some such products may be modified with a modified CRISPR enzyme of a CRISPR complex. In the case of some such modified products, the product of the target locus is physically different from the product of said target locus that has not been modified with said modified CRISPR enzyme.

The present invention also provides a polynucleotide molecule comprising a polynucleotide sequence encoding any of the non-naturally occurring CRISPR enzymes described above.

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

In the case of any such polynucleotide that comprises one or more regulatory elements, the one or more regulatory elements may be operably configured to express a non-naturally occurring CRISPR enzyme in a eukaryotic cell. The eukaryotic cell may be a human cell. The eukaryotic cell may be a rodent cell, optionally 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.

In the case of any such polynucleotide that comprises one or more regulatory elements, the one or more regulatory elements may be operably configured to express a non-naturally occurring CRISPR enzyme in a prokaryotic cell.

In the case of any such polynucleotide that comprises one or more regulatory elements, the one or more regulatory elements may be operably configured to express a non-naturally occurring CRISPR enzyme in an in vitro system.

The present invention also provides an expression vector comprising any of the above-described polynucleotide molecules. The present invention also provides such a polynucleotide molecule(s), for example, such polynucleotide molecules functionally configured to provide expression of a protein and/or component(s) based on a nucleic acid, as well as such a vector(s).

The present invention further provides a method for producing mutations in Cas9 or a mutated or modified Cas9 that is an orthologue of SaCas9 and/or SpCas9, comprising identifying an amino acid(s) that may be in close proximity to or may touch a nucleic acid molecule, such as DNA, RNA, sgRNA, etc., in such an orthologue, and/or an amino acid(s) similar to or corresponding to the amino acid(s) identified herein in SaCas9 and/or SpCas9, to effect the modification and/or mutation, and synthesizing or producing or expressing an orthologue comprising, consisting of, or consisting essentially of the modification(s) and/or mutation(s), or effecting the mutation, as discussed herein, such as by modifying, such as changing or mutating, a neutral amino acid to a charged, such as a positively charged amino acid, such as alanine, such as into lysine. The ortholog modified in this manner can be used in CRISPR-Cas systems; 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 system, as discussed in this document.

The present invention also provides an engineered CRISPR protein, complex, composition, system, vector, cell or cell line according to the present invention for use in making mutations in Cas9 or a mutated or modified Cas9 that is an ortholog of SaCas9 and/or SpCas9, comprising identifying amino acid(s) that may be in close proximity to or may touch a nucleic acid molecule, e.g., DNA, RNA, sgRNA, and/or amino acid(s) similar to or corresponding to the amino acid(s) identified herein in SaCas9 and/or SpCas9, to make the modification and/or mutation, and synthesizing or producing or expressing an ortholog comprising, consisting of, or consisting essentially of the modification(s) and/or mutation(s), or making the mutation, as discussed herein, e.g., by modifying, e.g., altering or mutating, a neutral amino acids into a charged, for example, positively charged amino acid, for example, from alanine, for example, into lysine.

In one aspect, the present invention provides efficient on-target activity and minimized off-target activity. In one aspect, the present invention provides efficient on-target cleavage by a CRISPR protein and minimized off-target cleavage by a CRISPR protein. In one aspect, the present invention provides target-specific binding of a CRISPR protein at a gene locus without cleaving DNA. In one aspect, the present invention provides efficient guide sequence-driven target binding of a CRISPR protein at a gene locus and minimized off-target binding of a CRISPR protein. Accordingly, in one aspect, the present invention provides target-specific gene regulation. In one aspect, the present invention provides target-specific binding of a CRISPR enzyme at a gene locus without cleaving DNA. Accordingly, one aspect of the present invention provides for cleavage at one gene locus and gene regulation at another gene locus using one CRISPR enzyme. One aspect of the present invention provides for orthogonal activation and/or inhibition and/or cleavage of multiple targets using one or more CRISPR proteins and/or enzymes.

In another aspect, the present invention provides a method for functionally screening genes in a genome in a cell pool ex vivo or in vivo, comprising administering or expressing a library comprising multiple CRISPR-Cas guide RNAs (sgRNAs), and wherein the screening further comprises using a CRISPR enzyme, wherein the CRISPR complex is modified to comprise a heterologous functional domain. In one aspect, the present invention provides a method for screening a genome, comprising administering a library to a host or expressing it in a host in vivo. In one aspect, the present invention provides a method as discussed herein, further comprising an activator administered to the host or expressed in the host. In one aspect, the present invention provides a method as discussed herein, wherein the activator is attached to a CRISPR protein. In one aspect, the present invention provides a method as discussed herein, wherein the activator is attached to the N-terminus or the C-terminus of the CRISPR protein. In one aspect of the present invention, there is provided a method as discussed herein, wherein the activator is attached to a loop of an sgRNA. In one aspect of the present invention, there is provided a method as discussed herein, further comprising a repressor introduced into the host or expressed in the host. In one aspect of the present invention, there is provided a method as discussed herein, wherein the screening comprises targeting and detecting gene activation, gene inhibition, or cleavage at a locus.

In one aspect, the present invention provides a method as discussed herein, wherein the host is a eukaryotic cell. In one aspect, the present invention provides a method or use as discussed herein, wherein the host is a mammalian cell. In one aspect, the present invention provides a method as discussed herein, wherein the host is a cell of a eukaryotic organism other than a human. In one aspect, the present invention provides a method as discussed herein, wherein the cell of a eukaryotic organism other than a human is a cell of a mammal other than a human. In one aspect, the present invention provides a method as discussed herein, wherein the cell of a mammal other than a human can be, including but not limited to, a cell of a primate, bovine, ovine, porcine, canine, rodent, Leporidae, such as a monkey, cow, sheep, pig, dog, rabbit, rat, or mouse. In one aspect, the present invention provides a method or use as discussed herein, wherein the cell may be a eukaryotic cell from an organism other than a mammal, such as a cell from a poultry (e.g., chicken), a vertebrate fish (e.g., salmon), or a shellfish (e.g., oyster, clam, lobster, shrimp). In one aspect, the present invention provides a method or use as discussed herein, wherein the cell from a eukaryotic organism other than a human is a plant cell. The plant cell may be derived from a monocot or dicot plant, or from an agricultural or cereal plant, such as cassava, corn, sorghum, soybean, wheat, oats, or rice. The plant cell may also be derived from an alga, a tree, or a productive plant, fruit, or vegetable (e.g., trees such as citrus trees, such as orange, grapefruit, or lemon trees; peach or nectarine trees; apple or pear trees; nut trees, such as almond or walnut or pistachio trees; nightshade plants; plants of the genus Brassica; plants of the genus Lactuca; plants of the genus Spinacia; plants of the genus Capsicum; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc.).

In one aspect, the present invention provides a method as discussed herein, comprising delivering CRISPR-Cas complexes, or component(s) thereof, or nucleic acid molecule(s) encoding them, wherein said nucleic acid molecule(s) are operably linked to regulatory sequence(s) and expressed in vivo. In one aspect, the present invention provides a method as discussed herein, wherein the in vivo expression is by a lentivirus, adenovirus, or AAV. In one aspect, the present invention provides a method as discussed herein, wherein the delivery is by a cell penetrating particle, nanoparticle, lipid, or peptide (CPP).

In one aspect of the present invention, there is provided a pair of CRISPR-Cas complexes, each comprising a guide RNA (sgRNA) comprising a guide sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell, wherein at least one loop of each sgRNA is modified by insertion of a different RNA sequence(s) that binds to one or more adaptor proteins, and wherein the adaptor protein is associated with one or more functional domains, wherein each sgRNA of each CRISPR-Cas comprises a functional domain characterized by DNA cleavage activity. In one aspect of the present invention, there are provided the paired CRISPR-Cas complexes discussed herein, wherein the DNA cleavage activity is mediated by the Fok1 nuclease.

In one aspect, the present invention provides a method of cleaving a target sequence at a genomic locus of interest, comprising delivering to a cell CRISPR-Cas complexes, or component(s) thereof, or nucleic acid molecule(s) encoding the same, wherein said nucleic acid molecule(s) are operably linked to a regulatory sequence(s) and expressed in vivo. In one aspect, the present invention provides a method as discussed herein, wherein delivery is via a lentivirus, an adenovirus, or an AAV. In one aspect, the present invention provides a method as discussed herein, or paired CRISPR-Cas complexes as discussed herein, wherein the target sequence for the first complex of the pair is on a first strand of double-stranded DNA, and the target sequence for the second complex of the pair is on a second strand of double-stranded DNA. In one aspect, the present invention provides a method as discussed herein, or paired CRISPR-Cas complexes as discussed herein, wherein the target sequences of the first and second complexes are located close to each other such that the DNA is cut in a manner that facilitates homologous repair. In one aspect, the method as set forth herein may further comprise introducing a DNA template into a cell. In one aspect, the method as set forth herein may involve paired CRISPR-Cas complexes as set forth herein, wherein each CRISPR-Cas complex has a CRISPR enzyme that is mutated such that it has no more than about 5% of the nuclease activity of a CRISPR enzyme that is not mutated.

In one aspect of the present invention, there is provided a library, method, or complex as discussed herein, wherein the sgRNA is modified such that it has at least one non-coding functional loop, such as wherein the at least one non-coding functional loop is repressor; such as wherein the at least one non-coding functional loop comprises Alu.

In one aspect, the present invention provides a method for altering or modifying the expression of a gene product. The method may comprise introducing into a cell comprising and expressing a DNA molecule encoding the gene product an engineered, non-naturally occurring CRISPR-Cas system comprising a Cas protein and a guide RNA that targets the DNA molecule, whereby the guide RNA targets the DNA molecule encoding the gene product, and the Cas protein cleaves the DNA molecule encoding the gene product, whereby expression of the gene product is altered; and wherein the Cas protein and the guide RNA do not occur together in nature. The present invention encompasses a guide RNA that includes a guide sequence fused to a tracr sequence. The present invention further encompasses 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 a more preferred embodiment, the mammalian cell is a human cell. In a further embodiment of the present invention, expression of the gene product is reduced.

The present invention also provides an engineered CRISPR protein, complex, composition, system, vector, cell or cell line as defined herein for use in altering the expression of a genomic locus of interest in a mammalian cell. The present invention also provides the use of an engineered CRISPR protein, complex, composition, system, vector, cell or cell line for the preparation of a medicament for altering the expression of a genomic locus of interest in a mammalian cell. Said alteration preferably involves contacting the cell with an engineered CRISPR protein, complex, composition, system, vector, cell or cell line of the present invention, thereby delivering the vector and allowing the formation of a CRISPR-Cas complex and its binding to a target. Said alteration also preferably involves determining whether the expression of the genomic locus has been altered.

In one aspect, the present invention provides altered cells and progeny of such cells, as well as products produced by the cells. The CRISPR-Cas9 proteins and systems of the present invention are used to produce cells comprising a modified target locus. In some embodiments, the method may include causing a nucleic acid targeting complex to bind to a target DNA or RNA to effect cleavage of said target DNA or RNA, thereby modifying the target DNA or RNA, wherein the nucleic acid targeting complex comprises a nucleic acid targeting effector protein in complex with a guide RNA hybridized to a target sequence within said target DNA or RNA. In one aspect, the present invention provides a method of repairing a gene locus in a cell. In another aspect, the present invention provides a method of modifying the expression of DNA or RNA in a eukaryotic cell. In some embodiments, the method comprises causing a nucleic acid targeting complex to bind to DNA or RNA, such that said binding results in increased or decreased expression of said DNA or RNA; wherein the nucleic acid targeting complex comprises a nucleic acid targeting effector protein in complex with a guide RNA. Similar considerations and conditions apply to the methods of modifying a target DNA or RNA set forth above. Indeed, these sampling, culturing and reintroduction options are encompassed by aspects of the present invention. In one aspect, the present invention provides methods of modifying a target DNA or RNA in a eukaryotic cell, which can be performed in vivo, ex vivo or in vitro. In some embodiments, the method comprises selecting a cell or a population of cells from a human or non-human animal and modifying the cell or cells. The culturing can be performed at any stage ex vivo. Such cells may be, but are not limited to, plant cells, animal cells, specific cell types of any organism, including stem cells, immune cells, T cells, B cells, dendritic cells, cardiovascular cells, epithelial cells, stem cells, and the like. The cells may be modified according to the present invention to produce gene products, for example, in controlled amounts, which may be increased or decreased, depending on the application, and/or mutated. In certain embodiments, the gene locus in the cell is repaired. The cell or cells may even be reintroduced into a non-human animal or into a plant. With respect to the cells reintroduced, it may be preferable that the cells are stem cells.

In one aspect, the present invention provides cells that transiently contain CRISPR systems or components thereof. For example, CRISPR proteins or enzymes, as well as nucleic acids, are transiently provided to a cell and a gene locus is altered, after which a decrease in the amount of one or more components of the CRISPR system occurs. Subsequently, cells, progeny of cells, and organisms that contain cells that have acquired a CRISPR-mediated genetic change contain reduced amounts of one or more components of the CRISPR system, or no longer contain one or more components of the CRISPR system. One non-limiting example is a self-inactivating CRISPR-Cas system, such as those further described herein. Thus, the present invention provides cells and organisms, and progeny of cells and organisms that contain one or more genetic loci altered by a CRISPR-Cas system, but substantially lack one or more components of the CRISPR system. In certain embodiments, components of the CRISPR system are substantially absent. Such cells, tissues and organisms preferably contain the desired or selected genetic alteration but have lost CRISPR-Cas components or remnants thereof that could potentially act non-specifically, leading to safety concerns or complicating regulatory approval. Among other things, the present invention provides products produced by cells, organisms and progeny of cells and organisms.

The present invention also provides a method for improving the specificity of a CRISPR system by providing an engineered CRISPR protein according to the present invention. Preferably, an engineered CRISPR protein is provided, wherein

the protein combines into a complex with a nucleic acid molecule containing RNA to form the CRISPR complex,

where, when in a CRISPR complex, a nucleic acid molecule is targeted to one or more target polynucleotide loci,

wherein the protein contains at least one modification compared to the unmodified protein,

wherein the CRISPR complex comprising the modified protein is characterized by altered activity compared to the complex comprising the unmodified protein. Said at least one modification is preferably in the RuvC and/or HNH domains as described herein, or in the binding groove between the HNH and RuvC domains. Preferred modifications are mutations described herein.

The present invention further provides the use of an engineered CRISPR protein according to the present invention to improve the specificity of the CRISPR system. Preferably, an engineered CRISPR protein is provided,

where the protein is complexed with a nucleic acid molecule containing RNA to form the CRISPR complex,

where, when in a CRISPR complex, a nucleic acid molecule is targeted to one or more target polynucleotide loci,

wherein the protein is modified to contain at least one modification compared to the unmodified protein,

wherein the CRISPR complex comprising the modified protein is characterized by altered activity compared to the complex comprising the unmodified protein. Said at least one modification is preferably in the RuvC and/or HNH domains as described herein, or in the binding groove between the HNH and RuvC domains. Preferred modifications are mutations described herein.

Brief description of graphic materials

The novel features of the present invention are set forth with characteristic features in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description, which sets forth illustrative embodiments in which the principles of the present invention are used, and to the accompanying drawings.

Figure 1A-1B provides a summary diagram, it being understood that the applicant(s)/inventor(s) do not necessarily commit to any particular theory set forth herein or in any particular figure, including Figure 1. The figure illustrates a mutation of positively charged residues that affects binding to the non-targetable strand of gDNA, resulting in improved specificity. The data in the table in the summary diagram are as follows, and relate to SpCas9 mutations:

In accordance with the SpCas9 numbering, Figure 1A illustrates alanine mutations that improve specificity located along the groove for the non-targetable strand, e.g., Arg780, Lys810, Lys855, Lys848, Lys1003, Arg1060, Arg976, His982. Without being bound by any particular theory, the proposed mechanism is that the nuclease activity is inactivated until the non-targetable strand of DNA triggers a steric change in the conformation of HNH; whereby binding of the non-targetable strand to the groove between HNH and RuvC depends on RNA:DNA pairing; whereby mutated residues for DNA binding in the groove require more energy for proper RNA:DNA pairing (Figure 1B). Using the information in this document, including the information in Figure 1, one skilled in the art can readily generate mutants of other Cas9s (e.g., other than SpCas9) that exhibit enhanced or reduced off-target effects. For example, the documents cited in this document provide information on numerous orthologs of SpCas9 and SaCas9, which are exemplified herein. Based on such information, including the sequence information of such other Cas9s, one skilled in the art, using the information in the present invention, can readily generate similar mutants with reduced off-target effects in Cas9 orthologs, in addition to SpCas9 and SaCas9, which are exemplified herein. In addition, the documents referenced in this document provide information on the crystal structure of Cas9s, such as SpCas9; and structural comparisons can be readily made between crystal structures, such as between the crystal structure of SpCas9 and the crystal structure of its ortholog, to also readily, without undue experimentation, obtain similar mutants characterized by reduced off-target effects in Cas9 orthologs in addition to SpCas9. Accordingly, the present invention is broadly applicable to modification(s) or mutation(s) aimed at reducing off-target effects in various Cas9 orthologs, including but not limited to SpCas9 and SaCas9. As further discussed herein, additional or further modification of the above-described Cas9 enzymes can readily be provided, resulting in an enzyme in a CRISPR complex characterized by an increased ability to modify one or more target loci compared to an unmodified enzyme.

Figure 2A shows the activity of modified SpCas9 enzymes measured by % insertion/deletion formation. 49 point mutants of SpCas9 are depicted. The target sequence EMX1.3 is the EMX1 gene sequence, and the activity is compared to the activity against a related non-target sequence (OT 46).

Figure 2B shows the activity of modified SpCas9 enzymes measured by % insertion/deletion formation. 49 point mutants of SpCas9 are depicted. The target sequence is the VEGFA gene sequence, and the activity is compared with the activity against two related non-target sequences (OT 1 and OT 2).

Figure 2C shows the activity of modified SpCas9 enzymes measured by % insertion/deletion formation. The target sequence is the VEGFA gene sequence, and the activity is compared to the activity against three related non-target sequences (OT 1, OT 4, and OT 18).

Figure 3 shows the activity of modified SpCas9 enzymes measured by % insertion/deletion formation. Point mutants showing specificity for non-target sequences are depicted. The target sequences are sequences from the EMX1 and VEGFA genes, and the activity is compared with the activity against nine related non-target sequences.

Figure 4A shows the activity of modified SpCas9 enzymes measured by % insertion/deletion formation. Double mutants of SpCas9 are shown. The target sequence is the EMX1 gene sequence, and the activity is compared to the activity toward two related non-target sequences.

Figure 4B shows the activity of modified SpCas9 enzymes measured by % insertion/deletion formation. Double mutants of SpCas9 are shown. The target sequence is the VEGFA gene sequence, and the activity is compared to the activity against three related non-target sequences.

Figure 5 shows the activity of modified SpCas9 enzymes measured by % insertion/deletion formation. Fourteen triple mutants of SpCas9 are shown. The target sequences are sequences from the EMX1 and VEGFA genes, and the activity is compared with the activity against four related non-target sequences (OT 46, OT 1, OT 4, and OT 18).

Figure 6 shows the activity of the modified SaCas9 enzymes measured by % insertion/deletion formation. The target sequence EMX101 is the EMX1 gene sequence, and the activity is compared with the activity against three related non-target sequences (OT1, OT2, and OT3).

Figure 7 shows the activity of the modified SaCas9 enzymes measured by % insertion/deletion formation. The target sequence EMX101 is the sequence from EMX1, and the activity is compared to the activity against a related non-target sequence (OT3).

Figure 8A-8D shows a phylogenetic tree of Cas genes; based on the information in this document and the knowledge of the state of the art, the mutation(s) or modification(s) of SpCas9 and SaCas9 given as an example can be applied to other Cas9.

Figure 9A-9F shows a phylogenetic analysis that identified five families of Cas9, including three groups of large Cas9 (~1400 amino acids) and two groups of small Cas9 (~1100 amino acids); based on the teachings herein and the prior art, the mutation(s) or modification(s) of SpCas9 and SaCas9 exemplified herein may be applied to other Cas9s (and thus the present invention encompasses the modification(s) or mutation(s) exemplified herein with respect to SpCas9 and SaCas9 with respect to all Cas9s and the families and groups of Cas9 depicted in Figure 9).

Figure 10A-10D show the activity of the modified SpCas9 enzymes as measured by % insertion/deletion formation. Figure 10A-C shows the activity against the target sequences EMX101, EMX1.1, EMX1.2, EMX1.3, EMX1.8, EMX1.10, DNMT1.1, DNMT1.2, DNMT1.4, DNMT1.7, VEGFA4, VEGFA5, and VEGFA3. Figure 10D shows the activity against VEGFA3 compared to the activity against the non-target sequence OT4.

Figure 11A-11D show the activity of the modified SpCas9 enzymes as measured by % insertion/deletion formation. Figure 11A-C shows the activity against the target sequences EMX101, EMX1.1, EMX1.2, EMX1.3, EMX1.8, EMX1.10, DNMT1.1, DNMT1.2, DNMT1.4, DNMT1.7, VEGFA4, VEGFA5, and VEGFA3. Figure 11D shows the activity against VEGFA3 compared to the activity against the non-target sequence OT4.

Figure 12 shows the activity of the modified SpCas9 enzymes, measured by % insertion/deletion formation. The target sequence is VEGFA3, and the activity is compared to the activity against four related non-target sequences (OT1, OT2, OT4, and OT18).

Figure 13 shows the activity of the modified SpCas9 enzymes, measured by % insertion/deletion formation. The target sequence is VEGFA3, and the activity is compared to the activity against four related non-target sequences (OT1, OT2, OT4, and OT18).

Figure 14 shows the activity of modified SpCas9 enzymes measured by % insertion/deletion formation. Fourteen point mutants of SpCas9 are depicted. The target sequence is EMX1.3 and the activity is compared to the activity against five related non-target sequences (OT14, OT23, OE35, OT46 and OT53).

Figure 15A–15F show aspects of the SpCas9 structure and improved specificity. Panel A is a model of target sequence unwinding. The nt-groove between the RuvC (teal) and HNH (magenta) domains stabilizes DNA unwinding through non-specific interactions with the non-complementary DNA strand. RNA:cDNA and Cas9:ncDNA interactions drive DNA unwinding (upper arrow) competing with cDNA:ncDNA rehybridization (lower arrow). Panel B: Structure of SpCas9 (PDB ID 4UN3) showing the nt-groove located between the HNH (magenta) and RuvC (teal) domains. The non-targetable DNA strand (red) was modeled within the nt-groove (inset) manually. Panel C: Screening of alanine point mutants for improved specificity. Panel D: Evaluation of the best point mutants for additional off-target loci. The top five mutants for specificity are highlighted in red. Panel E: Combination mutants have improved specificity compared to single point mutants. eSpCas9(1.0) and eSpCas9(1.1) are highlighted in red. Panel F: Screening of the best point mutants and combination mutants for 10 target loci for on-target cleavage efficiency. SpCas9(K855A), eSpCas9(1.0), and eSpCas9(1.1) are highlighted in red.

Figure 16A-16C show the retention of target efficiency in spCas9 mutants. Panel A shows the estimated target cleavage efficiency of SpCas9 mutants compared to SpCas9 for 24 sgRNAs targeting 9 genomic loci. Panel B is a Tukey plot of normalized target insertion/deletion rates for SpCas9(K855A), eSpCas9(1.0), and eSpCas9(1.1) mutants. Panel C is the results of Western blot analysis of SpCas9 and mutants using anti-SpCas9 antibody.

Figure 17A-17C show the sensitivity of spCas9 and the K855A, eSpCas9(1.0), and eSpCas9(1.1) mutants to single and double base mismatches between the guide RNA and the target DNA. Panel A depicts guide sequences with mismatches compared to the VEGFA target. Panel B shows heat maps for spCas9 and the three mutants showing the % insertions/deletions for guide sequences with a single base mismatch. Panel C shows the formation of insertions/deletions for guide sequences containing consecutive intersecting mismatches. Compared to the wild-type enzyme, eSpCas9(1.0) contains K810A, K1003A, R1060A; eSpCas9(1.1) contains K848A, K1003A, R1060A.

Figure 18A–18F show unbiased genome-wide off-target activity profiles of SpCas9(K855A) and eSpCas9(1.1) mutants. Panel A is a schematic overview of the BLESS (direct in situ breakpoint labeling, streptavidin enrichment, and next-generation sequencing) technology. Panel B shows a representative BLESS sequencing run of forward (red) and reverse (blue) reads mapped to the genome. Read mapping to Cas9 cut sites differs in shape from DSB hotspots. Panels C and D show Manhattan plots of genome-wide DSB clusters generated by each SpCas9 mutant using guides targeting EMX1(1) (panel C) and VEGFA(1) (panel D). Panels E and F show validation by targeted deep sequencing of off-target sites identified in BLESS. Off-target sites were categorized by DSB score (blue heatmap). Green heatmaps indicate sequence similarity between target and off-target sequences.

Figure 19 shows a schematic of sgRNA-directed DNA targeting and unwinding. Cas9 cleaves the target DNA through a series of coordinated steps. First, the PAM-interacting domain recognizes the NGG sequence at the 5′ end of the target DNA. Following PAM binding, the first 10-12 nucleotides of the target sequence (the seed sequence) are tested for sgRNA:DNA complementarity, a process mediated by separation of the DNA duplex. If the seed sequence nucleotides are complementary to the sgRNA, the remainder of the DNA is unwound and the entire length of the sgRNA hybridizes to the target DNA strand. In this model, the nt groove between the RuvC (teal) and HNH (magenta) domains stabilizes the non-targeted DNA strand and facilitates unwinding through non-specific interactions with the positive charges of the DNA phosphate backbone. RNA:cDNA and Cas9:ncDNA interactions drive DNA unwinding (upper arrow) in competition with cDNA:ncDNA rehybridization (lower arrow).

Figure 20 depicts the electrostatic properties of SpCas9 revealing the groove for the non-targetable strand. (A) Crystal structure (4UN3) of SpCas9 paired with sgRNA and target DNA, colored according to electrostatic potential to highlight positively charged regions. Scale bar represents -10 to 1 keV. (B) Structure identical to that in panel (A) except that the HNH domain has been removed to reveal the sgRNA:DNA heteroduplex. (C) Crystal structure (same orientation as (A)) colored according to domain: HNH (magenta), RuvC (turquoise), and PAM-interacting (PI) (beige).

Figures 21A-21D show off-target binding analysis of the resulting mutants. Twenty-nine SpCas9 point mutants were generated and tested for specificity for (A) the EMX1 target site and (B) two VEGFA target sites. Mutants combining the best residues that improve specificity were further tested for (C) EMX1 and (D) VEGFA.

Figure 22A-22C shows the annotated amino acid sequence of SpCas9. SpCas9 mutations that alter charges in the groove of the non-targetable strand were located predominantly in the RuvC and HNH domains (highlighted in yellow). The RuvC (blue), bridging helix (BH, green), REC (gray), HNH (magenta), and PI (beige) domains are annotated as in Nishmasu et al.

Figure 23 shows a comparison of the specificity of K855A, eSpCas9(1.0), and eSpCas9(1.1) using truncated sgRNAs and highlights that SpCas9(1.0) and eSpCas9(1.1) overcome the effect of truncated sgRNAs as a strategy to improve specificity. The insertion/deletion frequency at three loci (EMX1(1), VEGFA(1), and VEGFA(5)) was tested for the top annotated and predicted off-target sites. For both VEGFA target sites, the truncated sgRNAs increased the insertion/deletion frequency at some off-target sites and resulted in insertions/deletions at off-target sequences not observed with the wild-type sequences. The number of off-target sites detected by NGS for each SpCas9 mutant is shown below the heatmap.

Figure 24 shows that increasing the positive charge in the nt groove can lead to increased cleavage at off-target sites. Point mutants SpCas9(S845K) and SpCas9(L847R) exhibited lower specificity than wild-type SpCas9 at the EMX1(1) target site.

Figures 25A-25D depict the generation of eSaCas9 by nt-groove mutagenesis. A version of SaCas9 with improved specificity was generated similarly to eSpCas9. (A,B) Single and double amino acid residue mutants in the groove between the RuvC and HNH domains were screened for reduced off-target cleavage. (C) The mutants with improved specificity were combined to generate a SaCas9 variant that retained on-target cleavage of EMX site 7 and had significantly reduced off-target cleavage. (D) Crystal structure of SaCas9 showing the groove between the HNH and RuvC domains.

Figure 26 shows the determination of target efficiency characteristics for certain mutants with improved specificity. Three SpCas9 mutants had mutations in the phosphate acquisition loop (Lys1107, Glu1108, Ser1109) in the PI domain that conferred specificity to bases 1 and 2 of the sgRNA proximal to the PAM. These included a point mutant (K1107A) and two mutants in which the Lys-Glu-Ser sequence was replaced by the dipeptides Lys-Gly (KG) and Gly-Gly (GG), respectively. Our data indicate that these mutants can significantly reduce the efficiency of target cleavage.

Figure 27 shows that eSpCas9(1.1) is not cytotoxic to human cells. HEK293T cells were transfected with WT or eSpCas9(1.1) enzyme and incubated for 72 hours before cell viability was measured using the CellTiter-Glo assay, which detects fluorescence due to ATP production in living cells.

Figure 28 shows analysis of Nt groove mutants using truncated guide RNAs. Truncated guide RNAs (Tru) were combined with single amino acid mutants of SpCas9 and targeted (A) EMX1(1) or (B) VEGFA(1). While most mutants targeting EMX1 using an 18-nt guide retained targeting efficiency, mutants targeting VEGFA(1) using a 17-nt guide were significantly attenuated.

Figures 29A-29B show selected single and double amino acid residue mutants. As in the case of SpCas9, reducing the number of positive charges in the groove on the non-targetable strand enhances specificity. Reducing the number of positive charges can be achieved by replacing positively charged amino acids with neutral or negatively charged amino acids (A) or by shifting the position of the positively charged amino acid within the groove (B). The mutant of interest is K572.

Figure 30 shows the improved specificity of the selected mutants. CM2 exhibits a strong reduction in off-target activity while fully maintaining on-target activity. CM1: R499A; Q500K; K572A. CM2: R499A; Q500K; R654A; G655R. CM3: K572A; R654A; G655R.

Figure 31 shows activation of the HBG1 gamma globin locus by spCas9 complexes or spCas9 mutants with 15 bp, 17 bp, and 20 bp guides. Cas9(px165) represents unmutated spCas9. dCas9 denotes inactive spCas9. The single mutants ("SM") shown are R780A, K810A, and K848A. The double mutants ("SM") shown are R780A/K810A and R780A/K855A.

Figure 32 shows a comparison of different programmable nuclease platforms.

Figure 33 shows the types of therapeutic genome modifications. The specific type of genome editing therapy depends on the nature of the disease-causing mutation. a. In the case of a gene dysfunction, the pathogenic protein function is silenced by targeting the locus with NHEJ. Insertion/deletion formation in the gene of interest often results in frameshift mutations that result in premature stop codons and a nonfunctional protein product or nonsense-mediated transcript decay, resulting in suppression of gene function. b. HDR-mediated gene correction can be used to correct a deleterious mutation. DSB formation is targeted near the site of the mutation in the presence of an exogenously provided HDR correction template. HDR repair of the breakpoint with the exogenous template corrects the mutation, restoring gene function. c. An alternative to gene correction is gene addition. In this treatment, a therapeutic transgene is introduced into a safe locus in the genome. The DSB is targeted at the safe locus, and an HDR template with homology to the breakpoint, a promoter, and a transgene are introduced into the nucleus. HDR repair copies the promoter-transgene cassette into the safe locus, restoring gene function, albeit without true physiological control over gene expression.

Figure 34 shows ex vivo and in vivo editing therapies. In ex vivo editing therapies, cells are collected from a person, edited, and then replanted (upper panel). For this therapy to be successful, the target cells must be able to survive outside the body and be rehomed into target tissues after transplantation. In vivo therapies involve editing the genome of cells in situ (lower panels). In vivo systemic therapies, delivery vehicles that are relatively cell-species or state-neutral would be used to deliver editing to a wide range of tissue types. Although this type of editing therapy may be feasible in the future, there are currently no delivery systems that are efficient enough to make it practical. In vivo targeted therapies, in which patients are administered delivery vehicles that have tropism for specific organ systems, are practical using clinically relevant viral vectors.

Figures 35A-35B show a schematic representation of gene therapy using homologous recombination (HR) vectors using Cas9.

Figure 36 shows a diagram of the attachment of sugars for targeted delivery of a protein or guide, in particular using GalNac.

Figures 37A, 37B, and 37C together illustrate the sequence alignment of SaCas9 and SpCas9. The annotations of the RUVC and HNH domains of these two proteins are also shown in these three figures.

The figures shown in this document are for illustrative purposes only and are not necessarily drawn to scale.

Detailed description of the invention

Before describing the methods of the present invention, it should be noted that the present invention is not limited to the specific methods, components, products or combinations described, as such methods, components, products and combinations may, of course, vary. It should also be noted that the terminology used herein is not intended to be limiting, as the scope of the present invention will be limited only by the appended claims.

As used herein, the singular forms "a" and "an" include references to both singular and plural entities unless the context clearly requires otherwise.

As used herein, the terms "comprising," "comprises," and "contained" are synonymous with "including," "includes," or "comprising," "comprises," and are inclusive or open-ended and do not exclude additional, unreferenced parts, elements, or method steps. It should be understood that as used herein, the terms "comprising," "comprises," and "contained" include the terms "consisting of," "consists of," and "consists of," as well as the terms "consisting essentially of," "consists essentially of," and "consists essentially of." It should be noted that in the present invention, and in particular in the claims and/or paragraphs, terms such as "comprises," "comprised," "comprising," etc. may have the meaning ascribed to them in the U.S. patent law, for example, they may mean "includes," "included," "including," etc., and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in the U.S. patent law, for example, they allow for elements not expressly stated, but exclude elements that are found in the prior art or that affect the essential or novel characteristics of the present invention. In practicing the present invention, compliance with Article 53(c) EPC and Rule 28(b) as well as (c) EPC is preferred. Nothing contained herein is intended as an obligation.

Describing a range of numeric values using endpoints includes all numbers and fractions that fall within the ranges, as well as the endpoints specified.

As used herein, the term "about" or "approximately" when referring to a measurable quantity such as a parameter, quantity, time period, etc., is intended to encompass variations of +/- 20% or less, preferably +/- 10% or less, more preferably +/- 5% or less, and even more preferably +/- 1% or less, from the stated value, to the extent that such variations are suitable for implementation in the disclosed invention. It should be understood that the value to which the modifier "about" or "approximately" refers is also specifically disclosed, and preferably, on its own.

Although the terms "one or more" or "at least one", such as one or more or at least one element(s) of a group of elements, are self-explanatory in further examples, the term encompasses, inter alia, a reference to any one of the said elements, or to any two or more of the said elements, such as, for example, any ≥3, ≥4, ≥5, ≥6 or ≥7 etc. of the said elements, up to and including all of the said elements.

All references cited in this disclosure are hereby incorporated by reference in their entirety. In particular, it is expressly indicated that the teachings of all literature references in this document are incorporated by reference.

Unless otherwise defined, all terms used in the disclosure of the present invention, including technical and scientific terms, have the meaning commonly understood by one of ordinary skill in the art to which the present invention pertains. Definitions of terms are included for additional guidance to better understand the concept of the present invention.

In the following sections, various aspects of the present invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects, unless expressly stated otherwise. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Standard reference works covering the general principles of recombinant DNA technology include Molecular Cloning: A Laboratory Manual, 2nd ed., vol. 1–3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic revisions) ("Ausubel et al. 1992"); the Methods in Enzymology series (Academic Press, Inc.); Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press: San Diego, 1990; PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995); Harlow and Lane, eds. (1988) Antibodies, a Laboratory Manual; and Animal Cell Culture (R.I. Freshney, ed. (1987). General principles of microbiology are presented, for example, in Davis, B. D. et al., Microbiology, 3rd edition, Harper & Row, publishers, Philadelphia, Pa. (1980).

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, when the phrases "in one embodiment" or "in an embodiment" appear in various places throughout this specification, they are not necessarily all referring to the same embodiment, but may be. Moreover, particular features, structures, or characteristics may be combined in any suitable manner, as would be apparent to one skilled in the art based on the present invention, in one or more embodiments. Moreover, although some embodiments described herein include some, but not other, features included in other embodiments, it is intended that combinations of features from different embodiments are within the scope of the present invention and form different embodiments, as would be appreciated by those skilled in the art. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

In this description of the present invention, reference is made to the accompanying figures which form a part hereof and which show by way of illustration only specific embodiments by which the present invention may be carried out in practice. It is to be understood that other embodiments may be used and structural or logical changes may be made without departing from the scope of the present invention. The description is therefore not to be understood in a limiting sense and the scope of the present invention is defined by the appended claims.

It is not the intent of the present invention to cover within the scope of the present invention any previously known product, process for making a product, or method of using a product, so applicants reserve the right to and hereby disclaim any previously known product, process, or method. It should be further noted that the present invention is not intended to cover within the scope of the present invention any product, process for making a product, or method of using a product that does not conform to the written description and sufficient disclosure requirements of the USPTO (first paragraph of 35 USC § 112) or the EPO (EPC Article 83), so applicants reserve the right to and hereby disclaim any previously described product, process for making a product, or method of using a product.

Preferred statements (features) and embodiments of the present invention are set forth herein below. Each of the statements and embodiments of the present invention so defined may be combined with any other statement and/or embodiment unless explicitly stated otherwise. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features or statements indicated as being preferred or advantageous.

As used herein, the term "non-human organism" or "cell of a non-human organism" means an organism other than Homo sapiens or a cell not derived from such organism. As used herein, the term "non-human eukaryotic organism" or "cell of a non-human eukaryotic organism" means a eukaryotic organism other than Homo sapiens or a cell not derived from such organism. In preferred embodiments, such a eukaryotic organism (cell) is a non-human animal (cell), such as (a cell or population of cells) from a non-human mammal, a non-human primate, an ungulate, a rodent (preferably a mouse or rat), a rabbit, a canine, a dog, a cow, a bovine, a sheep, an ovine, a goat, a pig, a wild bird, a poultry, a chicken, a fish, an insect, or an arthropod, preferably a mammal such as a rodent, in particular a mouse. In some embodiments of the present invention, the organism or subject or cell may be (a cell or population of cells is derived from) an arthropod, such as an insect, or a nematode. In some methods of the present invention, the organism or subject or cell is a plant (cell). In some methods of the present invention, the organism or subject or cell is (is derived from) an alga, including a microscopic alga, or a fungus. One skilled in the art will appreciate that eukaryotic cells that can be transplanted or introduced into a non-human eukaryotic organism according to the methods mentioned herein are preferably obtained or derived from the same species as the eukaryotic organism into which they are transplanted. For example, a mouse cell is transplanted into a mouse in a certain embodiment according to the methods of the present invention described herein. In certain embodiments, the eukaryotic organism is an immunocompromised eukaryotic organism, i.e., a eukaryotic organism in which the immune system is partially or completely disabled. For example, the immunocompromised mice described herein can be used in the methods of the present invention. Examples of mice with a weakened immune system include, but are not limited to, nude mice, RAG -/- mice, SCID (severe combined immunodeficiency) mice, SCID-Beige mice, NOD (insulin-dependent diabetes)-SCID mice, NOG or NSG mice, etc.

It will be understood that the CRISPR-Cas system described herein does not naturally occur in the cell in question, i.e., is engineered or exogenous to the cell in question. The CRISPR-Cas system referred to herein has been introduced into the cell in question. Methods for introducing a CRISPR-Cas system into a cell are known in the art and are further described elsewhere in this document. A cell comprising a CRISPR-Cas system or having an introduced CRISPR-Cas system according to the present invention comprises or is capable of expressing individual components of the CRISPR-Cas system to create a functional CRISPR complex capable of modifying (such as cleaving) a target DNA sequence. Accordingly, as mentioned herein, a cell comprising a CRISPR-Cas system may be a cell comprising individual components of a CRISPR-Cas system for creating a functional CRISPR complex capable of modifying (such as cleaving) a target DNA sequence. Alternatively, as mentioned herein, and preferably, a cell comprising a CRISPR-Cas system may be a cell comprising one or more nucleic acid molecules encoding individual components of a CRISPR-Cas system that can be expressed in the cell for creating a functional CRISPR complex capable of modifying (such as cleaving) a target DNA sequence.

As used herein, the term "crRNA" or "guide RNA" or "single guide RNA" or "sgRNA" or "one or more nucleic acid components" of a Type V or Type VI CRISPR-Cas locus effector protein includes any polynucleotide sequence that has sufficient complementarity to a target nucleic acid sequence to hybridize to the target nucleic acid sequence and direct sequence-specific binding of a nucleic acid targeting complex to the target nucleic acid sequence. In some embodiments, when optimally aligned using a suitable alignment algorithm, the degree of complementarity is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. An optimal alignment can be determined using any suitable sequence alignment algorithm, including, but not limited to, the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transform (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). The ability of a guide sequence (within a nucleic acid targeting guide RNA) to direct sequence-specific binding of a nucleic acid targeting complex to a target nucleic acid sequence can be assessed using any suitable assay. For example, nucleic acid targeting components of a CRISPR system sufficient to form a nucleic acid targeting complex, including a guide sequence to be tested, can be provided to a host cell having a corresponding target nucleic acid sequence, such as by transfection with vectors encoding components of the nucleic acid targeting complex, followed by assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence, such as by the Surveyor nuclease assay described herein. Similarly, cleavage of a target nucleic acid sequence can be determined in vitro by providing a target nucleic acid sequence, components of a nucleic acid targeting complex, including a guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing the binding or extent of cleavage of the target sequence in reactions with the test and control guide sequences. Other assays are possible and can be performed by those skilled in the art. The guide sequence, and hence the guide RNA for targeting the nucleic acid, can be selected to target any target nucleic acid sequence. The target sequence can be DNA. The target sequence can be any RNA sequence. In some embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro RNA (miRNA), small interfering RNA (siRNA), 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 (scRNA). In some preferred embodiments, the target sequence may be a sequence within 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 within an RNA molecule selected from the group consisting of ncRNA and lncRNA. In some more preferred embodiments, the target sequence may be a sequence within an mRNA molecule or a pre-mRNA molecule.

In some embodiments, the nucleic acid targeting guide RNA is selected to reduce the proportion of secondary structure within the nucleic acid targeting guide RNA. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less of the nucleotides of the nucleic acid targeting guide RNA participate in self-complementary base pairing during optimal folding. Optimal folding can be determined using any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimum Gibbs free energy. An example of one such algorithm is mFold, which is described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example of a folding algorithm is the online web server RNAfold, developed at the Institute for Theoretical Chemistry at the University of Vienna, which uses a centroid-based structure prediction algorithm (see, e.g., A.R. Gruber et al., 2008, Cell 106(1): 23–24; and P.A. Carr and G.M. Church, 2009, Nature Biotechnology 27(12): 1151–62).

In certain embodiments, a guide RNA or crRNA may comprise, consist essentially of, or consist of a direct repeat (DR) sequence and a guide sequence or spacer sequence. In certain embodiments, a guide RNA or crRNA may comprise, consist essentially of, or consist of a direct repeat sequence fused to or linked to a guide sequence or spacer sequence. In certain embodiments, the direct repeat sequence may be located upstream (i.e., in the 5' direction) of the guide sequence or spacer sequence. In other embodiments, the direct repeat sequence may be located downstream (i.e., in the 3' direction) of the guide sequence or spacer sequence.

In certain embodiments, the crRNA comprises a stem-loop, preferably one stem-loop. In certain embodiments, the direct repeat sequence forms a stem-loop, preferably one stem-loop.

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

A "tracrRNA" sequence or similar terms includes any polynucleotide sequence that has sufficient complementarity with a crRNA sequence to allow hybridization. In some embodiments, the degree of complementarity between a tracrRNA sequence and a crRNA sequence over the entire length of the shorter of the two when optimally aligned is about or greater than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or more. In some embodiments, the tracr sequence is about or greater than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the crRNA sequence and the tracr mate sequence are contained in a single transcript such that hybridization between the two produces a transcript with a secondary structure such as a hairpin. In one embodiment of the invention, 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, the transcript has no more than five hairpins. In the hairpin structure, the portion of the sequence in the 5' direction relative to the terminal "N" and upstream of the loop corresponds to the tracr mate sequence, and the portion of the sequence in the 3' direction relative to the loop corresponds to the tracr sequence.

In general, a CRISPR-Cas, CRISPR-Cas9 or CRISPR system may be as used in the above-mentioned documents such as WO 2014/093622 (PCT/US2013/074667) and refer collectively to transcripts and other elements involved in the expression or control of the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, in particular the Cas9 gene in the case of CRISPR-Cas9, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or active partial tracrRNA), a tracr mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system) or “RNA” as that term is used herein (e.g., RNA for Cas9 directions, such as CRISPR RNA and trans-activating (tracr) RNA, or single guide RNA (sgRNA) (chimeric RNA), or other sequences and transcripts from the CRISPR locus. In general, a CRISPR system is characterized by elements that facilitate the formation of a CRISPR complex at the site of a target sequence (also called a protospacer in the context of the endogenous CRISPR system). In the context of CRISPR complex formation, a "target sequence" refers to a sequence to which a guide sequence is designed to be complementary, whereby hybridization between the target sequence and the guide sequence facilitates the formation of the CRISPR complex. The stretch of the guide sequence over which complementarity with the target sequence is important for cleavage activity is referred to herein as the seed sequence. The target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, the target sequence is in the nucleus or cytoplasm of the cell, and may include nucleic acids in or from mitochondria, organelles, vesicles, liposomes, or particles present within the cell. In some embodiments, particularly for non-nuclear applications, NLSs are not preferred. In some embodiments, direct repeats can be identified in silico by searching for repeat motifs that meet any or all of the following criteria: 1. are within a 2 kb window of genomic sequence flanking the Type II CRISPR locus; 2. span between 20 and 50 bp; and 3. are interspersed with 20-50 bp fragments. In some embodiments, 2 of these criteria may apply, such as 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all 3 criteria may apply.

In embodiments of the present invention, the terms guide sequence and guide RNA, i.e., RNA capable of directing Cas to a target locus in the genome, are used interchangeably, as in the above-mentioned cited documents such as WO 2014/093622 (PCT/US2013/074667). In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize to the target sequence and direct sequence-specific binding of a CRISPR 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 about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. An optimal alignment can be determined using any suitable sequence alignment algorithm, including, but not limited to, the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transform (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 length of the guide sequence is about or more 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 some embodiments, the length of the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides. Preferably, the length of the guide sequence is 10-30 nucleotides. The ability of the guide sequence to direct sequence-specific binding of the CRISPR complex to a target sequence can be assessed using any suitable assay. For example, components of a CRISPR system sufficient to form a CRISPR complex, including a guide sequence to be tested, can be provided to a host cell having an appropriate target sequence, such as by transfection with vectors encoding components of the CRISPR sequence, followed by assessment of preferential cleavage within the target sequence, such as by the Surveyor nuclease assay described herein. Similarly, cleavage of a target polynucleotide sequence can be determined in vitro by providing a target sequence, components of a CRISPR complex, including a guide sequence to be tested, and a control guide sequence different from the test guide sequence, and comparing binding or the extent of cleavage of the target sequence in reactions with the test and control guide sequences. Other assays are possible and can be performed by those skilled in the art.

In some embodiments of the CRISPR-Cas systems, the degree of complementarity between a guide and its corresponding target sequence can be about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%; wherein the length of the guide RNA or sgRNA can be 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; or the length of the guide RNA or sgRNA may be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides; and preferably the length of the tracrRNA is 30 or 50 nucleotides. However, an aspect of the present invention is directed to reducing off-target interactions, for example, reducing the interaction of the guide with a target sequence characterized by low complementarity. Indeed, the examples show that the present invention provides mutations that cause the CRISPR-Cas system to be able to recognize target and non-target sequences that are characterized by greater than 80% to about 95% complementarity, such as 83%-84%, or 88-89%, or 94-95% complementarity (e.g., distinguishing between an 18 nucleotide long target sequence and an 18 nucleotide long non-target sequence with 1, 2, or 3 mismatches). Accordingly, in the context of the present invention, the degree of complementarity between a guide sequence and its corresponding target sequence is more than 94.5%, or 95%, or 95.5%, or 96%, or 96.5%, or 97%, or 97.5%, or 98%, or 98.5%, or 99%, or 99.5%, or 99.9%, or 100%. The non-target locus has less than 100%, or 99.9%, or 99.5%, or 99%, or 99%, or 98.5%, or 98%, or 97.5%, or 97%, or 96.5%, or 96%, or 95.5%, or 95%, or 94.5%, or 94%, or 93%, or 92%, or 91%, or 90%, or 89%, or 88%, or 87%, or 86%, or 85%, or 84%, or 83%, or 82%, or 81%, or 80% complementarity between its sequence and the guide, wherein it is preferable that the non-target locus has 100%, or 99.9%, or 99.5%, or 99%, or 99%, or 98.5%, or 98%, or 97.5%, or 97%, or 96.5%, or 96%, or 95.5%, or 95%, or 94.5% complementarity between its sequence and the guide.

In particularly preferred embodiments according to the present invention, the guide RNA (capable of directing Cas to a target locus) may comprise (1) a guide sequence capable of hybridizing to a target locus of the genome in a eukaryotic cell; (2) a tracr sequence; and (3) a tracr mate sequence. All of (1)-(3) may be in a single RNA, i.e., an sgRNA (located in a 5'-3' orientation), or the tracrRNA may be an RNA different from the RNA containing the guide and tracr sequence. The tracr sequence hybridizes to the tracr mate sequence and directs the CRISPR/Cas complex to the target sequence.

The methods of the present invention described herein encompass inducing one or more mutations in a eukaryotic cell (in vitro, i.e., in an isolated eukaryotic cell) as discussed herein, comprising delivering to the cell a vector as discussed herein. The mutation(s) may comprise the introduction, deletion, or substitution of one or more nucleotides in each target sequence of the cell(s) using a guide(s) RNA or sgRNA. The mutations may comprise the introduction, deletion, or substitution of 1-75 nucleotides in each target sequence of said cell(s) using a guide(s) RNA or sgRNA. Mutations may include the introduction, deletion or substitution of 1, 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 or 75 nucleotides in each target sequence of the specified cell(s) using guide RNA or sgRNA. Mutations may include the introduction, deletion or substitution of 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 or 75 nucleotides in each target sequence of the specified cell(s) using guide RNA or sgRNA. Mutations include the introduction, deletion or substitution of 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 or 75 nucleotides in each target sequence of the specified cell(s) using guide(s) RNA or sgRNA. The mutations may include the introduction, deletion or substitution of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or 75 nucleotides in each target sequence of said cell(s) using guide(s) RNA or sgRNA. The mutations may include the introduction, deletion or substitution of 40, 45, 50, 75, 100, 200, 300, 400 or 500 nucleotides in each target sequence of said cell(s) using guide(s) RNA or sgRNA.

To minimize toxicity and off-target effects, it is recommended to control the concentration of the delivered Cas mRNA and guide RNA. Optimal concentrations of Cas mRNA and guide RNA can be determined by testing different concentrations in a cellular or non-human animal-eukaryotic model and using deep sequencing to analyze the degree of modification at potential off-target loci in the genome. Alternatively, to minimize the level of toxicity and off-target effects, Cas nickase mRNA (e.g., S. pyogenes Cas9 with the D10A mutation) can be delivered with a pair of guide RNAs targeting the site of interest. Guide sequences and strategies to minimize toxicity and off-target effects can be as in WO 2014/093622 (PCT/US2013/074667); or by mutation as described herein.

Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (containing a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands at or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs) the target sequence. Without wishing to be bound by theory, it is believed that a tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridizing at least a portion of the tracr sequence to all or a portion of a mate tracr sequence that is operably linked to a guide sequence.

The location of the RuvCI, RuvCII, RuvCIII and HNH domains is shown in Figure 22A-C. As used herein, the term "RuvCI domain" preferably refers to a domain comprising amino acids 1-60 of Streptococcus pyogenes Cas9 (SpCas9) or the corresponding region in another Cas9 ortholog or CRISPR nuclease other than Cas9. As used herein, the term "RuvCII domain" preferably refers to a domain comprising amino acids 718-775 of Streptococcus pyogenes Cas9 (SpCas9) or the corresponding region in another Cas9 ortholog or CRISPR nuclease other than Cas9. As used herein, the term "RuvCIII domain" preferably refers to a domain comprising amino acids 909-1099 of Streptococcus pyogenes Cas9 (SpCas9) or the corresponding region in another Cas9 ortholog or CRISPR nuclease other than Cas9. As used herein, the term "HNH domain" preferably refers to a domain comprising amino acids 776-908 of Streptococcus pyogenes Cas9 (SpCas9) or the corresponding region in another Cas9 ortholog or CRISPR nuclease other than Cas9. The groove between the RuvC and HNH domains refers to the groove between these domains in the three-dimensional structure of the non-naturally occurring CRISPR enzyme described herein. Figure 25D shows the crystal structure of SaCas9, which shows the groove between the HNH and RuvC domains in the three-dimensional structure of SaCas9.

Aptamers

One guide with the first aptamer/RNA-binding protein pair can be linked or fused to an activator, while the second guide with the second aptamer/RNA-binding protein pair can be linked or fused to a repressor. The guides are designed for different targets (loci), so this provides the ability to activate one gene and repress another gene. For example, this approach is shown in the following diagram:

guide 1-aptamer MS2-------RNA-binding protein MS2-------VP64 activator; and

guide 2 - aptamer PP7-------RNA-binding protein PP7-------repressor SID4x.

The present invention also relates to orthogonal gene targeting by PP7/MS2. In this example, an sgRNA targeting different loci is modified with distinct RNA loops to recruit MS2-VP64 or PP7-SID4X, which respectively activate and repress their target loci. PP7 is an RNA-binding protein of the Pseudomonas bacteriophage coat. Like MS2, it binds a specific sequence and secondary structure of RNA. The RNA recognition motif of PP7 differs from that of MS2. Therefore, PP7 and MS2 can be multiplexed to mediate distinct effects at different loci of the genome simultaneously. For example, an sgRNA targeting locus A can be modified by MS2 loops recruiting MS2-VP64 activators, while another sgRNA targeting locus B can be modified by PP7 loops recruiting PP7-SID4X repressor domains. Thus, dCas9 can mediate orthogonal locus-specific modifications in the same cell. This principle can be extended to include other orthogonal RNA-binding proteins such as Q-beta.

An alternative method of orthogonal repression involves the introduction of non-coding RNA loops with trans-acting repressor function into the guide (either at positions analogous to the MS2/PP7 loops integrated into the guide or at the 3' end of the guide sequence). For example, guides with non-coding (but known to be repressor) RNA loops have been designed (e.g., using the Alu repressor (in RNA), which disrupts RNA polymerase II function in mammalian cells). The Alu RNA sequence was placed: in place of the MS2 RNA sequences used here (e.g., in a tetraloop and/or stem-loop 2); and/or at the 3' end of the guide. This allows for possible combinations of MS2, PP7 or Alu at the tetraloop and/or stem-loop 2 positions, as well as the optional addition of Alu at the 3' end of the guide (with or without a linker).

The use of two different aptamers (different RNAs) allows the use of an activator-adaptor protein fusion and a repressor-adaptor protein fusion with different targeting elements to activate the expression of one gene while repressing the other. These, together with their different targeting elements, can be administered together or substantially together in a multiplexing approach. A plurality of such modified targeting elements can be used simultaneously, for example 10 or 20 or 30 etc., while only one (or at least a minimal amount) of Cas9 needs to be delivered, since a relatively small amount of Cas9 can be used with a large number of modified targeting elements. The adapter protein can be associated (preferably linked or fused) to one or more activators or one or more repressors. For example, the adapter protein can be associated with a first activator and a second activator. The first and second activators can be the same, but preferably they are different activators. For example, one might be VP64 while another might be p65, although these are just examples and other transcriptional activators are envisaged. Three or more, or even four or more activators (or repressors) may be used, but the size of the package may be a limitation so that the number does not exceed 5 different functional domains. Linkers are preferably used rather than direct fusion to the adaptor protein, with two or more functional domains associated with the adaptor protein. Suitable linkers may include a GlySer linker.

It is also envisaged that the enzyme-targeting element complex as a whole may be associated with two or more functional domains. For example, two or more functional domains may be associated with an enzyme, or two or more functional domains may be associated with a targeting element (via one or more adaptor proteins), or one or more functional domains may be associated with an enzyme and one or more functional domains may be associated with a targeting element (via one or more adaptor proteins).

The fusion between the adaptor protein and the activator or repressor may include a linker. For example, GlySer, GGGS linkers may be used. They may be used in repeats of 3 ((GGGGS) ₃ ) or 6, 9 or even 12 or more to provide the appropriate length, if necessary. Linkers may be used between the RNA-binding protein and the functional domain (activator or repressor), or between the CRISPR enzyme (Cas9) and the functional domain (activator or repressor). Linkers are used to construct a molecule with a sufficient degree of "mechanical flexibility".

Non-functional guides: The present invention may employ guide RNAs containing a non-functional guide sequence.

In one aspect of the present invention, there are provided guide sequences that are modified in a manner that allows formation of a CRISPR complex and successful binding to a target, while at the same time not providing successful nuclease activity (i.e., lacking nuclease activity/no insertion/deletion activity). For explanations, such modified guide sequences are referred to as "non-functional guides" or "non-functional guide sequences". These non-functional guides or non-functional guide sequences can be considered catalytically inactive or conformationally inactive in terms of nuclease activity. Nuclease activity can be measured using the Surveyor nuclease assay or deep sequencing commonly used in the art, preferably the Surveyor nuclease assay. Likewise, non-functional guide sequences may not sufficiently participate in productive base pairing in terms of the ability to facilitate catalytic activity or discriminate between target and non-target binding activity. Briefly, the Surveyor Nuclease assay involves purification and amplification of a CRISPR target site in a gene and the formation of heteroduplexes with primers that amplify the CRISPR target site. Following rehybridization, products were treated with SURVEYOR Nuclease and SURVEYOR S Enhancer (Transgenomics) according to the manufacturer's recommended protocols, analyzed on gels, and quantified based on the relative intensity of band staining.

Accordingly, in a related aspect of the present invention, there is provided a non-naturally occurring or engineered composition of a Cas9-based CRISPR-Cas system comprising a functional Cas9 as described herein and a guide RNA (gRNA), wherein the gRNA provides a non-functional guide sequence such that the gRNA is capable of hybridizing to a target sequence such that the Cas9-based CRISPR-Cas system is targeted to a genomic locus of interest in a cell, while lacking detectable insertion/deletion forming activity resulting from the nuclease activity of a wild-type Cas9 enzyme of the system, as determined by the SURVEYOR nuclease assay. Briefly, a gRNA comprising a non-functional guide sequence, whereby the gRNA is capable of hybridizing to a target sequence such that a Cas9-based CRISPR-Cas system is directed to a genomic locus of interest in a cell, while lacking detectable insertion/deletion forming activity resulting from the nuclease activity of a wild-type Cas9 enzyme of the system, as determined by the SURVEYOR nuclease assay, is referred to herein as a "non-functional gRNA". It should be understood that any of the gRNAs of the present invention described elsewhere herein can be used as non-functional gRNAs/gRNAs comprising a non-functional guide sequence described herein below. Any of the methods, products, compositions, and uses described elsewhere herein are equally applicable to non-functional gRNAs/gRNAs comprising a non-functional guide sequence, as further detailed below. For further guidance, the following specific aspects and implementation options are provided.

The ability of a non-functional guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence can be assessed using any suitable assay. For example, components of a CRISPR system sufficient to form a CRISPR complex, including a non-functional guide sequence to be tested, can be provided to a host cell having an appropriate target sequence, such as by transfection with vectors encoding components of the CRISPR sequence, followed by assessment of preferential cleavage within the target sequence, such as by the Surveyor nuclease assay described herein. Similarly, cleavage of a target polynucleotide sequence can be determined in vitro by providing a target sequence, components of a CRISPR complex, including a non-functional guide sequence to be tested, and a control guide sequence different from the non-functional guide sequence being tested, and comparing the binding or degree of cleavage of the target sequence in reactions with the test and control guide sequences. Other assays are possible and can be performed by those skilled in the art. A non-functional guide sequence can be selected to target any target sequence. In some embodiments, the target sequence is a sequence within the genome of the cell.

As further explained in this paper, several design parameters ensure that the correct scaffold is achieved in such non-functional guides. The non-functional guide sequences are shorter than the corresponding guide sequences that drive active specific insertion/deletion formation by Cas9. The non-functional guides are 5%, 10%, 20%, 30%, 40%, 50% shorter than the corresponding guides that drive the same Cas9, leading to active specific insertion/deletion formation by Cas9.

As explained below and known in the art, one aspect of the specificity of gRNA-Cas9 is determined by the direct repeat sequence, which must be suitably linked to such guides. In particular, this means that the direct repeat sequences are designed depending on the organism from which Cas9 is derived. Thus, the structural data available for the validated non-functional guide sequences can be used to design Cas9-specific equivalents. Structural similarities between, for example, the orthologous RuvC nuclease domains of two or more Cas9 effector proteins can be used to transfer the design to equivalent non-functional guides. Thus, the non-functional guide herein can be suitably modified in length and sequence to match such Cas9-specific equivalents, allowing for CRISPR complex formation and successful target binding, while not allowing for successful nuclease activity.

The use of non-functional guides in the context of this document, as well as the existing state of the art, provide a surprising and unforeseen platform for network biology and/or systems biology in in vitro, ex vivo and in vivo applications simultaneously, allowing multiplexed gene targeting and, in particular, bidirectional multiplexed gene targeting. Before the use of non-functional guides, targeting multiple targets, for example, to activate, repress and/or silence gene activity, was challenging and in some cases impossible. With the use of non-functional guides, it is possible to target multiple targets and thus multiple activities, for example, in the same cell, in the same animal, or in the same patient. Such multiplexing can occur at the same time or be distributed over a desired period of time.

For example, non-functional guides now provide for the first time the ability to use gRNA as a means for gene targeting without the consequences of nuclease activity, while still providing targeted means for activation or repression. A guide RNA providing a non-functional guide can be modified to further include elements that allow for the activation or repression of gene activity, in particular adaptor proteins (e.g., aptamers) described elsewhere in this document that allow for the functional placement of gene effectors (e.g., activators or repressors of gene activity). One example is the introduction of aptamers, as explained herein and in the prior art. By engineering a gRNA that includes a non-functional guide and incorporating protein-interacting aptamers (Konermann et al., "Genome-scale transcription activation by an engineered CRISPR-Cas9 complex," doi:10.1038/nature14136, incorporated herein by reference), a synthetic transcriptional activation complex can be assembled consisting of multiple distinct effector domains. These can be modeled after natural transcriptional activation processes. For example, an aptamer that selectively binds an effector (e.g., an activator or repressor; dimerized bacteriophage MS2 coat proteins as fusion proteins with an activator or repressor) or a protein that itself binds an effector (e.g., an activator or repressor) can be attached to the tetraloop and/or stem-loop 2 of a non-functional gRNA. In the case of MS2, the MS2-VP64 fusion protein binds to tetraloop and/or stem-loop 2 and in turn mediates transcriptional activation, for example for Neurog2. Other transcriptional activators include VP64, P65, HSF1, and MyoD1. As a pure example of this concept, replacing MS2 stem-loops with PP7-interacting stem-loops can be used to recruit repressor elements.

Thus, one aspect of the present invention is a gRNA that provides a non-functional guide, wherein the gRNA further comprises modifications that allow for gene activation or repression as described herein. Non-functional gRNAs may comprise one or more aptamers. The aptamers may be specific for gene effectors, gene activators, or gene repressors. Alternatively, the aptamers may be specific for a protein that in turn is specific for and recruits/binds gene effectors, gene activators, or gene repressors. If there are multiple sites for recruiting activators or repressors, it is preferred that the sites are specific for either activators or repressors. If there are multiple sites for binding activators or repressors, the sites may be specific for the same activators or the same repressors. The sites may also be specific for different activators or different repressors. Gene effectors, gene activators, and gene repressors may be present in the form of fusion proteins.

In one embodiment, the non-functional gRNA described herein or the Cas9-based CRISPR-Cas complex described herein comprises a non-naturally occurring or engineered composition comprising two or more adaptor proteins, wherein each protein is associated with one or more functional domains, and wherein the adaptor protein binds to a distinct RNA sequence(s) inserted into at least one loop of the non-functional gRNA.

Therefore, in one aspect, there is provided a non-naturally occurring or engineered composition comprising a guide RNA (gRNA) comprising a non-functional guide sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell, wherein the non-functional guide sequence is defined herein, a Cas9 comprising at least one or more nuclear localization sequences, wherein the Cas9 optionally comprises at least one mutation, wherein at least one loop of the non-functional gRNA is modified by insertion of a different RNA sequence(s) that binds to one or more adaptor proteins, and wherein the adaptor protein is associated with one or more functional domains; or wherein the non-functional gRNA is modified such that it has at least one non-coding functional loop, and wherein the composition comprises two or more adaptor proteins, wherein each protein is associated with one or more functional domains.

In certain embodiments, the adapter protein is a fusion protein comprising a functional domain, wherein the fusion protein optionally comprises a linker between the adapter protein and the functional domain, wherein the linker optionally comprises a GlySer linker.

In certain embodiments, at least one loop of the non-functional gRNA is not modified by insertion of a different RNA sequence(s) that binds to two or more adaptor proteins.

In certain embodiments, one or more functional domains associated with the adapter protein is a transcriptional activation domain.

In certain embodiments, one or more functional domains associated with the adapter protein are a transcriptional activation domain comprising VP64, p65, MyoD1, HSF1, RTA, or SET7/9.

In certain embodiments, one or more functional domains associated with the adaptor protein are a transcriptional repression domain.

In certain embodiments, the transcriptional repression domain is a KRAB domain.

In certain embodiments, the transcriptional repression domain is a NuE domain, a NcoR domain, a SID domain, or a SID4X domain.

In certain embodiments, at least one of the one or more functional domains associated with the adapter protein is characterized by one or more activities comprising methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcript release factor activity, histone modification activity, DNA integration activity, RNA cleavage activity, DNA cleavage activity, or nucleic acid binding activity.

In certain embodiments, the DNA cleavage activity is mediated by the Fok1 nuclease.

In certain embodiments, the non-functional gRNA is modified such that after the non-functional gRNA binds the adaptor protein and subsequently binds to Cas9 and the target, the functional domain is in a spatial orientation that allows the functional domain to perform its intended function.

In certain embodiments, at least one loop of the non-functional gRNA is a tetraloop and/or loop 2. In certain embodiments, the tetraloop and loop 2 of the non-functional gRNA are modified by insertion of different RNA sequence(s).

In certain embodiments, the insertion of a distinct RNA sequence(s) that binds to one or more adapter proteins is an aptamer sequence. In certain embodiments, the aptamer sequence is two or more aptamer sequences specific for the same adapter protein. In certain embodiments, the aptamer sequence is two or more aptamer sequences specific for different adapter proteins.

In certain embodiments, the adapter protein comprises MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, fCb5, fCb8r, fCb12r, fCb23r, 7s, PRR1.

In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell, optionally a mouse cell. In certain embodiments, the mammalian cell is a human cell.

In certain embodiments, the first adapter protein associates with the p65 domain and the second adapter protein associates with the HSF1 domain.

In certain embodiments, the composition comprises a Cas9-based CRISPR-Cas complex having at least three functional domains, at least one of which associates with Cas9 and at least two of which associate with a non-functional gRNA.

In certain embodiments, the composition further comprises a second gRNA, wherein the second gRNA is a functional gRNA capable of hybridizing to a second target sequence such that the second Cas9-based CRISPR-Cas system is directed to a second genomic locus of interest in a cell, wherein there is detectable insertion/deletion activity in the second genomic locus resulting from the nuclease activity of the Cas9 enzyme of the system.

In certain embodiments, the composition further comprises multiple non-functional gRNAs and/or multiple functional gRNAs.

One aspect of the present invention is to take advantage of the modularity and tunability of the gRNA scaffold to create a series of gRNA scaffolds with different binding sites (particularly aptamers) to recruit different types of effectors in an orthogonal manner. Again, by way of example and illustration of the broader concept, the replacement of the MS2 stem loops with PP7 interacting stem loops can be used to bind/recruit repressor elements to provide multiplex bidirectional transcriptional control. Thus, in general, a gRNA providing a non-functional guide can be used to provide multiplex transcriptional control and preferably bidirectional transcriptional control. Such transcriptional control is most advantageous for genes. For example, one or more gRNAs providing non-functional guide(s) can be used to target the activation of one or more target genes. At the same time, one or more gRNAs providing a non-functional guide(s) can be used to target the repression of one or more target genes. Such a sequence can be used in a variety of different combinations, for example, target genes are initially repressed and then after an appropriate period of time other targets are activated, or selected genes are repressed while selected genes are activated, followed by additional activation and/or repression. As a result, multiple components of one or more biological systems can be advantageously targeted together.

In one aspect, the present invention provides a nucleic acid molecule(s) encoding a non-functional gRNA, or a Cas9-based CRISPR-Cas complex, or a composition as described herein.

In one aspect of the present invention, there is provided a vector system comprising: a nucleic acid molecule encoding a non-functional guide RNA as defined herein. In certain embodiments, the vector system further comprises a nucleic acid molecule(s) encoding Cas9. In certain embodiments, the vector system further comprises a nucleic acid molecule(s) encoding a (functional) gRNA. In certain embodiments, the nucleic acid molecule or vector further comprises a regulatory element(s) functional in a eukaryotic cell operably linked to a nucleic acid molecule encoding a guide sequence (gRNA) and/or a nucleic acid molecule encoding Cas9 and/or optional nuclear localization sequence(s).

In another aspect, structural analysis can be used to study the interactions between the non-functional guide and the active Cas9 nuclease that mediate DNA binding but not DNA cleavage. This identifies amino acids that are important for the Cas9 nuclease activity. Modification of these amino acids provides improved Cas9 enzymes for gene editing.

A further aspect is the combination of the use of non-functional guides as described herein with other uses of CRISPR as described herein and as known in the art. For example, a gRNA providing non-functional guide(s) for targeted multiplex gene activation or repression or targeted multiplex bidirectional gene activation/repression can be combined with a gRNA providing guides that support nuclease activity as described herein. Such gRNAs providing guides that support nuclease activity may further comprise modifications that provide for repression of gene activity (e.g., aptamers), or may not comprise such modifications. Such gRNAs providing guides that support nuclease activity may further comprise modifications that provide for activation of gene activity (e.g., aptamers), or may not comprise such modifications. Thus, additional means for multiplex gene control are introduced (e.g. multiplex targeted activation of a gene without nuclease activity/without insertion/deletion activity can be provided simultaneously or in combination with targeted repression of a gene with nuclease activity).

For example, 1) the use of one or more gRNAs (e.g. 1-50, 1-40, 1-30, 1-20, preferably 1-10, more preferably 1-5) providing non-functional guide(s) targeting one or more genes and further modified with appropriate aptamers to recruit gene activators; 2) can be combined with one or more gRNAs (e.g. 1-50, 1-40, 1-30, 1-20, preferably 1-10, more preferably 1-5) providing non-functional guide(s) targeting one or more genes and further modified with appropriate aptamers to recruit gene repressors. 1) and/or 2) can then be combined with 3) one or more gRNAs (e.g. 1-50, 1-40, 1-30, 1-20, preferably 1-10, more preferably 1-5) targeting one or more genes. This combination can then be performed in order as 1) + 2) + 3) with 4) one or more gRNAs (e.g. 1-50, 1-40, 1-30, 1-20, preferably 1-10, more preferably 1-5) targeting one or more genes and further modified with appropriate aptamers to recruit gene activators. This combination can then be carried out in order as 1) + 2) + 3) + 4) with 5) one or more gRNAs (e.g. 1-50, 1-40, 1-30, 1-20, preferably 1-10, more preferably 1-5) targeting one or more genes and further modified with appropriate aptamers to recruit gene repressors. As a result, various uses and combinations are included in the present invention. For example, a combination of 1) + 2); combination 1) + 3); combination 2) + 3); combination 1) + 2) + 3); combination 1) + 2) + 3) +4); combination 2) + 3) +4); combination 1) + 2) + 4); combination 1) + 2) + 3) +4) + 5); combination 1) + 3) + 4) +5); combination 2) + 3) +4) +5); combination 1) + 2) + 4) +5); combination 1) + 2) +3) + 5); combination 1) + 3) +5); combination 2) + 3) +5); combination 1) + 2) +5).

In one aspect of the present invention, there is provided an algorithm for designing, evaluating or selecting a targeting sequence based on a non-functional guide RNA (non-functional guide sequence) for directing a Cas9-based CRISPR-Cas system to a target gene locus. In particular, it was determined that the specificity of the non-functional guide RNA depends on i) the GC content and ii) the length of the targeting sequence and can be optimized by varying these parameters. In one aspect of the present invention, there is provided an algorithm for designing or evaluating a targeting sequence based on a non-functional guide RNA that minimizes off-target binding or interaction of the non-functional guide RNA. In one embodiment of the present invention, an algorithm for selecting a targeting sequence based on a non-functional guide RNA for targeting a CRISPR system to a gene locus in an organism comprises a) determining the location of one or more CRISPR motifs in a gene locus, analyzing a 20-nucleotide sequence downstream of each CRISPR motif by i) determining the GC content of the sequence; and ii) determining whether there are off-target matches in the 15 nucleotides downstream closest to the CRISPR motif in the genome of the organism, and c) selecting the 15-nucleotide sequence for use in a non-functional guide RNA if the GC content of the sequence is 70% or less and no off-target matches are identified. In one embodiment, the sequence is selected as a targeting sequence if the GC content is 60% or less. In certain embodiments, a sequence is selected as a targeting sequence if the GC content is 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, or 30% or less. In one embodiment, two or more sequences of a gene locus are analyzed and the sequence with the lowest GC content, or the sequence with the second lowest GC content, or the sequence with the third lowest GC content, is selected. In one embodiment, a sequence is selected as a targeting sequence if no off-target matches are identified in the genome of the organism. In one embodiment, a targeting sequence is selected if no off-target matches are identified in the genome of the organism.

In one aspect, the present invention provides a method for selecting a targeting sequence based on a non-functional guide RNA for directing a functionalized CRISPR system to a gene locus in an organism, the method comprising: a) determining the location of one or more CRISPR motifs in the gene locus; b) analyzing a 20 nucleotide sequence downstream of each CRISPR motif by: i) determining the GC content of the sequence; and ii) determining whether there are off-target matches to the first 15 nucleotides of the sequence in the genome of the organism; c) selecting the sequence for use in the guide RNA if the GC content of the sequence is 70% or less and no off-target matches are identified. In one embodiment, the sequence is selected if the GC content is 50% or less. In one embodiment, the sequence is selected if the GC content is 40% or less. In one embodiment, the sequence is selected if the GC content is 30% or less. In one embodiment, two or more sequences are analyzed and the sequence with the lowest GC content is selected. In one embodiment, off-target matches are determined in regulatory sequences of the organism. In one embodiment, the gene locus is a regulatory region. In one aspect, a non-functional guide RNA is provided that provides a targeting sequence selected according to the above methods.

In one aspect of the present invention, a non-functional guide RNA is provided for targeting a functionalized CRISPR system to a gene locus in an organism. In one embodiment of the present invention, the non-functional guide RNA provides a targeting sequence, wherein the CG content of the target sequence is 70% or less, and the first 15 nucleotides of the targeting sequence do not match a non-target sequence located downstream of a CRISPR motif in a regulatory sequence from another gene locus in the organism. In certain embodiments, the GC content of the targeting sequence is 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, or 30% or less. In certain embodiments, the GC content of the targeting sequence is from 70% to 60%, or from 60% to 50%, or from 50% to 40%, or from 40% to 30%. In one embodiment, the targeting sequence has the lowest CG content among potential targeting sequences for a locus.

In one embodiment of the present invention, the first 15 nucleotides of the non-functional guide match the target sequence. In another embodiment, the first 14 nucleotides of the non-functional guide match the target sequence. In another embodiment, the first 13 nucleotides of the non-functional guide match the target sequence. In another embodiment, the first 12 nucleotides of the non-functional guide match the target sequence. In another embodiment, the first 11 nucleotides of the non-functional guide match the target sequence. In another embodiment, the first 10 nucleotides of the non-functional guide match the target sequence. In one embodiment, the first 15 nucleotides of the non-functional guide do not match a non-target sequence located downstream of the CRISPR motif in the regulatory region of another gene locus. In other embodiments, the first 14 nucleotides, or the first 13 nucleotides of the non-functional guide, or the first 12 nucleotides of the guide, or the first 11 nucleotides of the non-functional guide, or the first 10 nucleotides of the non-functional guide do not match a non-target sequence located downstream of the CRISPR motif in a regulatory region of another gene locus. In other embodiments, the first 15 nucleotides, or 14 nucleotides, or 13 nucleotides, or 12 nucleotides, or 11 nucleotides of the non-functional guide do not match a non-target sequence located downstream of the CRISPR motif in the genome.

In certain embodiments, the non-functional guide RNA includes additional nucleotides at the 3' end that do not match the target sequence. Thus, a non-functional guide RNA that includes the first 15 nucleotides, or 14 nucleotides, or 13 nucleotides, or 12 nucleotides, or 11 nucleotides located downstream of the CRISPR motif can be extended in length at the 3' end by 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, or more.

The present invention provides a method for directing a Cas9-based CRISPR-Cas system, including but not limited to a non-functional Cas9-based system (dCas9) or a functionalized Cas9-based system (which may include a functionalized Cas9 or a functionalized guide), to a gene locus. In one aspect, the present invention provides a method for selecting a targeting sequence based on a non-functional guide RNA and directing the functionalized CRISPR system to a gene locus in an organism. In one aspect, the present invention provides a method for selecting a targeting sequence based on a non-functional guide RNA and performing gene regulation of a target gene locus using a functionalized Cas9-based CRISPR-Cas system. In certain embodiments, the method is used to perform regulation of a target gene while minimizing off-target effects. In one aspect of the present invention, there is provided a method for selecting two or more targeting sequences based on non-functional guide RNAs and gene regulation of two or more target gene loci using a functionalized Cas9-based CRISPR-Cas system. In certain embodiments, the method is used to regulate two or more target gene loci while minimizing off-target effects.

In one aspect of the present invention, there is provided a method for selecting a targeting sequence based on a non-functional guide RNA for directing a functionalized Cas9 to a gene locus in an organism, which comprises: a) determining the location of one or more CRISPR motifs in the gene locus; b) analyzing a sequence located downstream of each CRISPR motif by: i) selecting 10-15 nucleotides adjacent to the CRISPR motif, ii) determining the GC content of the sequence; and c) selecting a sequence of 10-15 nucleotides in length as a targeting sequence for use in the guide RNA if the GC content of the sequence is 40% or greater. In one embodiment, the sequence is selected if the GC content is 50% or greater. In one embodiment, the sequence is selected if the GC content is 60% or greater. In one embodiment, the sequence is selected if the GC content is 70% or greater. In one embodiment, two or more sequences are analyzed and the sequence with the highest GC content is selected. In one embodiment, the method further comprises adding to the 3' end of the selected sequence of nucleotides that do not match the sequence located downstream of the CRISPR motif. In one aspect, a non-functional guide RNA is provided that provides a targeting sequence selected in accordance with the above-mentioned methods.

In one aspect of the present invention, a non-functional guide RNA is provided for directing a functionalized CRISPR system to a gene locus in an organism, wherein the targeting sequence based on the non-functional guide RNA consists of 10-15 nucleotides adjacent to a CRISPR motif in the gene locus, wherein the CG content of the target sequence is 50% or more. In certain embodiments, the non-functional guide RNA further comprises nucleotides added to the 3' end of the targeting sequence that do not match a sequence located downstream of the CRISPR motif in the gene locus.

In one aspect of the present invention, a single effector is provided that is targeted to one or more, or two or more gene loci. In certain embodiments, the effector is associated with Cas9, and one or more, or two or more selected non-functional guide RNAs are used to target the Cas9-associated effector to one or more, or two or more selected target gene loci. In certain embodiments, the effector is associated with one or more, or two or more selected non-functional guide RNAs, wherein each selected non-functional guide RNA, when complexed with a Cas9 enzyme, causes its associated effector to be located on the target of the non-functional guide RNA. One non-limiting example of such CRISPR systems modulates the activity of one or more, or two or more gene loci that are subject to regulation by the same transcription factor.

In one aspect, the present invention provides two or more effectors to be directed to one or more gene loci. In certain embodiments, two or more non-functional guide RNAs are used, wherein each of the two or more effectors is associated with a selected non-functional guide RNA, wherein each of the two or more effectors is located at a selected target for its non-functional guide RNA. One non-limiting example of such CRISPR systems modulates the activity of one or more, or two or more gene loci to be regulated by different transcription factors. Thus, in one non-limiting embodiment, the two or more transcription factors are located in different regulatory sequences of a single gene. In another non-limiting embodiment, the two or more transcription factors are located in different regulatory sequences of different genes. In certain embodiments, one transcription factor is an activator. In certain embodiments, one transcription factor is an inhibitor. In certain embodiments, one transcription factor is an activator and the other transcription factor is an inhibitor. In certain embodiments, gene loci expressing different components of the same regulatory pathway are regulated. In certain embodiments, gene loci expressing components of different regulatory pathways are regulated.

In one aspect of the present invention, there is also provided a method and algorithm for designing and selecting non-functional guide RNAs that are specific for target DNA cleavage or target binding and gene regulation mediated by an active Cas9-based CRISPR-Cas system. In certain embodiments, a Cas9-based CRISPR-Cas system provides orthogonal gene control using an active Cas9 that cleaves target DNA at one gene locus while simultaneously binding to and regulating another gene locus.

In one aspect of the present invention, there is provided a method for selecting a targeting sequence based on a non-functional guide RNA for directing a functionalized Cas9 to a gene locus in an organism without cleavage thereof, which comprises: a) determining the location of one or more CRISPR motifs in the gene locus; b) analyzing the sequence located downstream of each CRISPR motif by: i) selecting 10-15 nucleotides adjacent to the CRISPR motif, ii) determining the GC content of the sequence; and c) selecting a sequence of 10-15 nucleotides in length as a targeting sequence for use in a non-functional guide RNA if the GC content of the sequence is 30% or more, 40% or more. In certain embodiments, the GC content of the targeting sequence is 35% or greater, 40% or greater, 45% or greater, 50% or greater, 55% or greater, 60% or greater, 65% or greater, or 70% or greater. In certain embodiments, the GC content of the targeting sequence is from 30% to 40%, or from 40% to 50%, or from 50% to 60%, or from 60% to 70%. In one embodiment of the present invention, two or more sequences at a gene locus are analyzed and the sequence with the highest GC content is selected.

In one embodiment of the present invention, the portion of the targeting sequence in which the GC content is assessed is 10-15 contiguous nucleotides of the 15 target nucleotides closest to the PAM. In one embodiment of the present invention, the portion of the guide in which the GC content is taken into account is 10-11 nucleotides, or 11-12 nucleotides, or 12-13 nucleotides, or 13, or 14, or 15 contiguous nucleotides of the 15 nucleotides closest to the PAM.

In one aspect of the present invention, an algorithm is further provided for identifying non-functional guide RNAs that promote cleavage of a gene locus of a CRISPR system without allowing functional activation or inhibition. It is observed that increased GC content in non-functional guide RNAs of 16-20 nucleotides in length coincides with increased DNA cleavage and decreased functional activation.

It is also demonstrated herein that the efficiency of a functionalized Cas9 can be improved by adding nucleotides to the 3' end of the guide RNA that do not match the target sequence downstream of the CRISPR motif. For example, for non-functional guide RNAs that are 11-15 nucleotides long, shorter guides may be less likely to promote target cleavage, but they are also less effective in promoting CRISPR system binding and functional control. In certain embodiments, adding nucleotides that do not match the target sequence to the 3' end of a non-functional guide RNA improves activation efficiency while increasing unwanted target cleavage. In one aspect, the present invention also provides a method and algorithm for identifying improved non-functional guide RNAs that effectively promote the function of the CRISPR system in DNA binding and gene regulation while not promoting DNA cleavage. Thus, in certain embodiments of the present invention, a non-functional guide RNA is provided that includes the first 15 nucleotides, or 14 nucleotides, or 13 nucleotides, or 12 nucleotides, or 11 nucleotides located downstream of a CRISPR motif, and is extended in length at the 3' end by nucleotides that do not match the target by 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides or more.

In one aspect, the present invention provides a method for performing selective orthogonal gene control. As will be appreciated from the disclosure herein, the selection of a non-functional guide according to the present invention, taking into account the guide length and GC content, provides for efficient and selective transcriptional control by a functional Cas9-based CRISPR-Cas system, such as regulation of transcription of a gene locus by activation or inhibition and minimization of off-target effects. Accordingly, by providing efficient regulation of individual target loci, the present invention also provides for efficient orthogonal regulation of two or more target loci.

In certain embodiments, orthogonal gene control is performed by activating or inhibiting two or more target loci. In certain embodiments, orthogonal gene control is performed by activating or inhibiting one or more target loci and cleaving one or more target loci.

In one aspect, the present invention provides a cell comprising a non-naturally occurring Cas9-based CRISPR-Cas system comprising one or more non-functional guide RNAs disclosed or produced according to the method or algorithm described herein, wherein the expression of one or more gene products has been altered. In one embodiment, the present invention alters the expression of two or more gene products in the cell. The present invention also provides a cell line derived from such a cell.

In one aspect of the present invention, there is provided a multicellular organism comprising one or more cells comprising a non-naturally occurring Cas9-based CRISPR-Cas system comprising one or more non-functional guide RNAs disclosed or produced according to the method or algorithm described herein. In one aspect of the present invention, there is provided a product obtained from a cell, cell line, or multicellular organism comprising a non-naturally occurring Cas9-based CRISPR-Cas system comprising one or more non-functional guide RNAs disclosed or produced according to the method or algorithm described herein.

A further aspect of the present invention is the use of a gRNA comprising the non-functional guide(s) described herein, optionally in combination with a gRNA comprising the guide(s) described herein or in the prior art, in combination with systems (e.g., cells, transgenic animals, transgenic mice, inducible transgenic animals, inducible transgenic mice) that are engineered to either overexpress Cas9 or, preferably, a Cas9 knockin. As a result, a single system (e.g., transgenic animal, cell) can serve as a basis for multiplexed gene modifications in systems/network biology. With the non-functional guides, this can now be done in vitro, ex vivo and in vivo.

For example, after providing Cas9, one or more non-functional gRNAs can be provided to direct multiplex gene regulation, and preferably multiplex bidirectional gene regulation. The one or more non-functional gRNAs can be provided in a spatially and temporally appropriate manner (e.g., by tissue-specific induction of Cas9 expression), if necessary or desired. Because the transgenic/inducible Cas9 is provided (e.g., expressed) in a cell, tissue, animal of interest, both gRNAs providing non-functional guides and gRNAs providing guides are equally effective. Similarly, a further aspect of the present invention is the use of a gRNA comprising the non-functional guide(s) described herein, optionally in combination with a gRNA comprising the guide(s) described herein or in the art, in combination with systems (e.g., cells, transgenic animals, transgenic mice, inducible transgenic animals, inducible transgenic mice) that are engineered to knock out Cas9-based CRISPR-Cas.

As a result, the combination of the non-functional guides described herein with the CRISPR applications described herein and CRISPR applications known in the art provides a highly efficient and accurate means for multiplexed screening of systems (e.g., network biology). Such screening, for example, enables the identification of specific combinations of gene activities to identify genes responsible for diseases (e.g., "on/off" combinations), in particular genetic diseases. A preferred application of such screening is cancer. Likewise, screening for the treatment of such diseases is included in the present invention. Cells or animals can be exposed to abnormal conditions that lead to a disease or disease-like effects. Candidate compositions can be provided and screened for effect in a desired multiplexed environment. For example, a patient's cancer cells can be screened for which gene combinations will cause their death, and this information can then be used to develop appropriate therapies.

In one aspect of the present invention, a kit is provided comprising one or more components described herein. The kit may include non-functional guides described herein, with or without guides described herein.

The structural information provided herein enables detailed study of the interaction of the non-functional gRNA with target DNA and Cas9, providing the ability to engineer or modify the structure of the non-functional gRNA to optimize the functionality of the overall Cas9-based CRISPR-Cas system. For example, loops in the non-functional gRNA can be increased without overlapping with the Cas9 protein by inserting adaptor proteins that can bind to the RNA. Such adaptor proteins can further recruit effector proteins or fusion products that contain one or more functional domains.

In some preferred embodiments, the functional domain is a transcriptional activation domain, preferably VP64. In some embodiments, the functional domain is a transcriptional repression domain, preferably KRAB. In some embodiments, the transcriptional repression domain is SID or SID concatemers (e.g., SID4X). In some embodiments, the functional domain is an epigenetic modification domain, such that an epigenetic modification enzyme is provided. In some embodiments, the functional domain is an activation domain, which may be a P65 activation domain.

One aspect of the present invention is that the above-mentioned elements are contained in a single composition or are contained in separate compositions. These compositions can advantageously be applied to a host to induce a functional effect at the genome level.

In general, non-functional gRNAs are modified in a manner that provides specific binding sites (e.g., aptamers) for adaptor proteins containing one or more functional domains (e.g., through formation of a fusion protein) to bind. The modified non-functional gRNAs are modified such that once the non-functional gRNA forms a CRISPR complex (i.e., Cas9 binds to the non-functional gRNA and the target), binding of the adaptor proteins occurs and the functional domain on the adaptor protein is positioned in a spatial orientation that is advantageous for the efficiency of its inherent function. For example, if the functional domain is a transcriptional activator (e.g., VP64 or p65), the transcriptional activator is positioned in a spatial orientation that allows it to influence the transcription of the target. Similarly, a transcriptional repressor will be positioned preferentially to affect transcription of the target, and a nuclease (e.g., Fok1) will be positioned preferentially to cleave or partially cleave the target.

It will be appreciated by those skilled in the art that modifications in a non-functional gRNA that allow binding of the adapter + functional domain but do not allow proper placement of the adapter + functional domain (e.g., due to steric hindrance in the three-dimensional structure of the CRISPR complex) are modifications that are not intended. The one or more modified non-functional gRNAs may be modified at a tetraloop, stem-loop 1, stem-loop 2, or stem-loop 3 described herein, preferably at either the tetraloop or stem-loop 2, and most preferably at both the tetraloop and stem-loop 2.

As explained herein, the functional domains may be, for example, one or more domains from the group consisting of domains with methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcript release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switches (e.g., light-inducible). In some cases, it is preferable that at least one NLS is additionally provided. In some cases, the position of the NLS at the N-terminus is preferable. When more than one functional domain is included, the functional domains may be the same or different.

The non-functional gRNA can be designed to include multiple binding recognition sites (e.g., aptamers) specific for the same or different adaptor proteins. The non-functional gRNA can be designed to bind to a promoter region located -1000 to +1 nucleotides upstream of the transcription start site (i.e., TSS), preferably -200 nucleotides. This placement improves functional domains that affect gene activation (e.g., transcriptional activators) or gene inhibition (e.g., transcriptional repressors). The modified non-functional gRNA can be one or more modified non-functional gRNAs (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) targeting one or more target loci contained in the composition.

The adaptor protein may be any of a number of proteins that bind to an aptamer or recognition site introduced into the modified non-functional gRNA and that allows one or more functional domains to be correctly positioned after the non-functional gRNA has been incorporated into the CRISPR complex to act on the target within its intended function. As explained in detail in this application, they may be coat proteins, preferably bacteriophage coat proteins. Functional domains associated with such adapter proteins (e.g. in the form of a fusion protein) may comprise, for example, one or more domains from the group consisting of domains with methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcript release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity and molecular switches (e.g. inducible by light). Preferred domains are Fok1, VP64, P65, HSF1, MyoD1. In case the functional domain is a transcriptional activator or a transcriptional repressor, it is advantageous to additionally provide at least an NLS and preferably at the N-terminus. When including more than one functional domain, the functional domains may be the same or different. Known linkers can be used in the adapter protein to attach such functional domains.

Thus, the modified non-functional gRNA, the inactivated Cas9 (with or without functional domains) and the binding protein with one or more functional domains may each be separately contained in a composition and may be administered to the host separately or together. Alternatively, these components may be provided in a single composition for administration to the host. Administration to the host may be accomplished using viral vectors known to the skilled artisan or described herein for delivery to the host (e.g., a lentiviral vector, an adenoviral vector, an AAV-based vector). As explained herein, the use of different selection markers (e.g., for lentiviral gRNA selection) and different gRNA concentrations (e.g., depending on whether multiple gRNAs are used) may be advantageous to induce an improved effect.

Based on this concept, several options are suitable for inducing a transformation at a genomic locus, including DNA cleavage, gene activation, or gene inactivation. Using the provided compositions, one skilled in the art will be able to efficiently and specifically target one or multiple loci using the same or different functional domains to induce one or more transformations at a genomic locus. The compositions can be used in a variety of methods for screening libraries in cells and functional modeling in vivo (e.g., lincRNA gene activation and function identification; gain-of-function mutation modeling; loss-of-function mutation modeling; use of the compositions of the present invention to generate cell lines and transgenic animals for optimization and screening purposes).

The present invention encompasses the use of the compositions of the present invention for the creation and use of transgenic cells/animals with conditionally dependent or inducible CRISPR that could not have been expected prior to the practice of the present invention or application. For example, the target cell comprises a conditionally dependent or inducible Cas9 (e.g., in the form of Cre-dependent constructs) and/or a conditionally dependent or inducible adapter protein, and when the vector introduced into the target cell is expressed, the vector expresses that which induces or provides a condition for the expression of the Cas9 enzyme and/or the expression of the adapter in the target cell. By using the teachings and compositions of the present invention with a known method for creating a CRISPR complex, inducible genome transformations that are affected by the functional domains are also an aspect of the present invention. One example of this is the generation of a CRISPR knockin/conditionally expressing transgenic animal (e.g., a mouse containing, e.g., a Lox-Stop-polyA-Lox (LSL) cassette) and subsequent delivery of one or more compositions providing one or more modified non-functional gRNAs (e.g., -200 nucleotides to the TSS in a target gene of interest for gene activation purposes) described herein (e.g., modified non-functional gRNAs with one or more aptamers recognized by coat proteins, e.g., MS2), one or more adaptor proteins described herein (MS2 binding protein linked to one or more VP64), and means for inducing the animal to have conditionally expressing (e.g., Cre recombinase to provide inducible expression of Cas9). Alternatively, the adaptor protein can be provided as a condition-dependent or inducible element with condition-dependent or inducible Cas9 to provide an efficient model for screening purposes that advantageously requires only minimal engineering and introduction of specific non-functional gRNAs for a variety of applications.

In another aspect, the non-functional guides are further modified to improve specificity. Protected non-functional guides can be synthesized, wherein a secondary structure is introduced into the 3' end of the non-functional guide to improve its specificity. A protected guide RNA (pgRNA) comprises a guide sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell, and a protection strand, wherein the protection strand is optionally complementary to the guide sequence, and wherein the guide sequence can hybridize to some extent to the protection strand. The pgRNA optionally includes an extension sequence. The thermodynamics of pgRNA-target DNA hybridization is determined by the number of bases complementary between the guide RNA and the target DNA. Using "thermodynamic protection", the specificity of a non-functional gRNA can be improved by adding a protection sequence. For example, in one method, a complementary protection strand of variable length is added to the 3' end of a guide sequence within a non-functional gRNA. As a result, the protection strand binds to at least a portion of the non-functional gRNA and provides a protected gRNA (pgRNA). In turn, the non-functional gRNAs mentioned herein can be readily protected using the described embodiments to produce pgRNA. The protection strand can be either a separate RNA transcript or strand, or a chimeric version, attached to the 3' end of a guide sequence based on a non-functional gRNA.

Tandem guides and application options in the multiplex (tandem) targeting approach

The inventors have shown that the CRISPR enzymes defined herein can use more than one guide RNA without loss of activity. This enables the use of the CRISPR enzymes, systems, or complexes defined herein to target multiple DNA targets, genes, or gene loci with a single enzyme, system, or complex defined herein. The guide RNAs can be arranged in tandem, optionally separated by a nucleotide sequence, such as a direct repeat defined herein. The position of the different guide RNAs in tandem does not affect activity. It should be noted that the terms "CRISPR-Cas system," "CRISP-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, CRISP-Cas enzyme or Cas enzyme is Cas9 or any of its modified or mutated variants described elsewhere herein.

In one aspect, the present invention provides 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 Cas9 as described elsewhere herein, used for tandem or multiplex targeting. It will be appreciated that any of the CRISPR (or CRISPR-Cas or Cas) enzymes, complexes or systems of the present invention described elsewhere herein may be used in such an approach. Any of the methods, products, compositions and uses described elsewhere herein are equally applicable to the multiplex or tandem targeting approach described in further detail below. For purposes of additional guidance, the following specific aspects and embodiments are provided.

In one aspect of the present invention, there is provided the use of the Cas9 enzyme, complex or system as defined herein for targeting multiple gene loci. In one embodiment, this can be accomplished by using multiple (tandem or multiplex) guide RNA (gRNA) sequences.

In one aspect of the present invention, methods are provided for using one or more elements of a Cas9 enzyme, complex or system as defined herein for tandem or multiplex targeting, wherein said CRISP system comprises multiple guide RNA sequences. Preferably, said gRNA sequences are separated by a nucleotide sequence, such as a direct repeat, as defined elsewhere herein.

The Cas9 enzyme, system or complex defined herein provides effective means for modifying multiple target polynucleotides. The Cas9 enzyme, system or complex defined herein is characterized by a wide variety of applications, including modification (e.g., formation of deletions, insertions, translocations, inactivation, activation) of one or more target polynucleotides in a variety of cell types. In this regard, the Cas9 enzyme, system or complex defined herein of the present invention has a wide range of applications, such as in gene therapy, drug screening, diagnosis and determination of disease prognosis, including targeting multiple gene loci within a single CRISPR system.

In one aspect, the present invention provides a Cas9 enzyme, system or complex as defined herein, i.e., a Cas9-based CRISPR-Cas complex comprising a Cas9 protein having at least one destabilization domain associated therewith and multiple guide RNAs that target multiple nucleic acid molecules, such as DNA molecules, whereby each of said multiple guide RNAs specifically targets its respective nucleic acid molecule, such as a DNA molecule. Each target nucleic acid molecule, such as a DNA molecule, can encode a gene product or span a gene locus. Therefore, the use of multiple guide RNAs allows targeting multiple gene loci or multiple genes. In some embodiments, the Cas9 enzyme can cleave a DNA molecule encoding a gene product. In some embodiments, expression of the gene product is altered. The Cas9 protein and the guide RNAs do not occur together in nature. The present invention encompasses guide RNAs that provide tandemly arranged guide sequences. The present invention further encompasses coding sequences for a Cas9 protein that is 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 Cas9 enzyme may form part of a CRISPR system or complex that further comprises tandemly arranged 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 capable of specifically hybridizing to a target sequence at a genomic locus of interest in a cell. In some embodiments, a functional Cas9-based CRISPR system or complex binds to multiple target sequences. In some embodiments, a functional CRISPR system or complex can edit multiple target sequences, for example, the target sequences can provide a genomic locus, and in some embodiments, a change in gene expression can be provided. In some embodiments, a functional CRISPR system or complex can contain additional functional domains. In some embodiments, the present invention provides a method for changing or modifying the expression of multiple gene products. The method can include introducing them into a cell containing said target nucleic acids, such as DNA molecules, or containing and expressing a target nucleic acid, such as DNA molecules; for example, the target nucleic acids can encode gene products or provide for the expression of gene products (e.g., regulatory sequences).

In preferred embodiments, the CRISPR enzyme used for multiplex targeting is Cas9, or the CRISPR system or complex comprises Cas9. In some embodiments, the CRISPR enzyme used for multiplex targeting is AsCas9, or the CRISPR system or complex used for multiplex targeting comprises AsCas9. In some embodiments, the CRISPR enzyme is LbCas9, or the CRISPR system or complex comprises LbCas9. In some embodiments, the Cas9 enzyme used for multiplex targeting cleaves both strands of DNA to form a double-strand break (DSB). In some embodiments, the CRISPR enzyme used for multiplex targeting is a nickase. In some embodiments, the Cas9 enzyme used for multiplex targeting is a dual nickase. In some embodiments, the Cas9 enzyme used for multiplex targeting is a Cas9 enzyme, such as a DD Cas9 enzyme, as defined elsewhere herein.

In embodiments, Cas9 can be paired, such as as a pair of nickases, such as SaCas9 nickase (eSaCas9 nickase). Additionally, Cas9 can be packaged with one or two or more guides in an AAV-based vector. This can be accomplished as described in Friedland AE et al, Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications, Genome Biol. 2015 Nov 24;16:257. doi: 10.1186/s13059-015-0817-8., the disclosure of which is hereby incorporated by reference.

In some general embodiments, the Cas9 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 a non-functional Cas9, as defined elsewhere herein.

In one aspect of the present invention, there are provided means for delivering a Cas9 enzyme, system or complex for use in multiple targeting as defined herein, or polynucleotides as defined herein. Non-limiting examples of such delivery means are, for example, a particle(s) delivering a component(s) of the complex, a vector(s) comprising the polynucleotide(s) discussed herein (e.g., encoding a CRISPR enzyme, providing nucleotides encoding a CRISPR complex). In some embodiments, the vector may be a plasmid or a viral vector, such as AAV or lentivirus. Transient transfection with plasmids, such as HEK cells, may be advantageous, especially given the size limitations of AAV and that, although Cas9 fits into AAV, an upper limit may be reached in the case of additional guide RNAs.

Also provided is a model in which the Cas9 enzyme, complex or system used herein is constitutively expressed for use in multiplex targeting. The organism may be transgenic and may be transfected with the vectors of the present invention or may be a progeny of an organism so transfected. In a further aspect, the present invention provides compositions comprising the CRISPR enzyme, system and complex defined herein or the polynucleotides or vectors described herein. Also provided are Cas9-based CRISPR systems or complexes comprising multiple guide RNAs, preferably in a tandem arrangement format. Said different guide RNAs may be separated by nucleotide sequences such as direct repeats.

Also provided is a method of treating a subject, such as a subject in need thereof, comprising inducing gene editing by transforming the subject with a polynucleotide encoding a Cas9-based CRISPR system or complex, or any of the polynucleotides or vectors described herein, and administering them to the subject. A suitable repair template may also be provided, such as delivered by a vector containing said repair template. Also provided is a method of treating a subject, such as a subject in need thereof, comprising inducing transcriptional activation or repression of multiple target gene loci by transforming the subject with the polynucleotides or vectors described herein, wherein said polynucleotide or vector encodes or comprises a Cas9 enzyme, complex or system comprising multiple guide RNAs, preferably arranged in tandem. In case any treatment is carried out ex vivo, such as in cell culture, it should be understood that the term "subject" may be replaced by the phrase "cell or cell culture".

Also provided are compositions comprising a Cas9 enzyme, complex or system comprising multiple guide RNAs, preferably arranged in tandem, or a polynucleotide or vector encoding or comprising said Cas9 enzyme, complex or system comprising multiple guide RNAs, preferably arranged in tandem, for use in the treatment methods defined elsewhere herein. A kit of parts comprising such compositions may be provided. Also provided is the use of said composition in the manufacture of a medicament for such treatment methods. The present invention also provides the use of a Cas9-based CRISPR system in screening, such as gain-of-function mutation screens. Cells that are artificially overexpressing a gene may reduce gene expression over time (re-equilibration), such as by negative feedback. By the time screening begins, the expression level of the unregulated gene may be reduced again. The use of an inducible Cas9 activator allows transcription to be induced immediately prior to screening, thereby minimizing the likelihood of false negative hits. Accordingly, by using the present invention in screening, such as screening for a gain-of-function mutation, the likelihood of false negative results can be minimized.

In one aspect of the present invention, there is provided an engineered, non-naturally occurring CRISPR system comprising a Cas9 protein and multiple guide RNAs, each of which specifically targets a DNA molecule encoding a gene product in a cell, whereby each of the multiple guide RNAs targets its specific DNA molecule encoding a gene product, and the Cas9 protein cleaves the target DNA molecule encoding the gene product, whereby the expression of the gene product is altered; and wherein the CRISPR protein and the guide RNAs do not occur together in nature. The present invention encompasses multiple guide RNAs comprising multiple guide sequences, preferably separated by a nucleotide sequence such as a direct repeat, and optionally fused to a tracr sequence. In one embodiment of the present invention, the CRISPR protein is a CRISPR-Cas protein of type V or VI, and in a more preferred embodiment, the CRISPR protein is a Cas9 protein. The present invention further encompasses a Cas9 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 present invention, expression of the gene product is reduced.

In another aspect of the present invention, there is provided an engineered, non-naturally occurring vector system comprising one or more vectors comprising a first regulatory element operably linked to multiple guide RNAs of a Cas9-based CRISPR system, each of which specifically targets a DNA molecule encoding a gene product, and a second regulatory element operably linked to a sequence encoding a CRISPR protein. Both regulatory elements can be in the same vector or in 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 can cleave one or both strands or exhibit virtually no nuclease activity), resulting in altered expression of the multiple gene products; and wherein the CRISPR protein and multiple guide RNAs do not occur together in nature. In a preferred embodiment, the CRISPR protein is a Cas9 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 present invention, the expression of each of the multiple gene products is altered, preferably reduced.

In one aspect of the present invention, a vector system is provided, comprising one or more vectors. In some embodiments, the system comprises: (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 upstream or downstream (as appropriate) of the direct repeat sequence, wherein upon expression, the one or more guide sequences direct sequence-specific binding of a CRISPR complex to one or more target sequences in a eukaryotic cell, wherein the CRISPR complex comprises a Cas9 enzyme in complex with one or more guide sequences that hybridize to the one or more target sequences; and (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said Cas9 enzyme, preferably comprising at least one nuclear localization sequence and/or at least one NES; wherein components (a) and (b) are in the same or different vectors of the system. If applicable, a tracr sequence may also be provided. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences directs sequence-specific binding of a Cas9-based CRISPR complex to its target sequence in a eukaryotic cell. In some embodiments, the CRISPR complex comprises one or more nuclear localization sequences and/or one or more NESs that are sufficiently efficient to direct accumulation of said Cas9-based CRISPR complex in a detectable amount in or outside the nucleus of a eukaryotic cell. 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 length of each of the guide sequences is at least 16, 17, 18, 19, 20, 25 nucleotides, or 16-30, or 16-25, or 16-20 nucleotides.

Recombinant expression vectors may comprise polynucleotides encoding a Cas9 enzyme, system or complex for use in the multiple targeting defined herein in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors comprise one or more regulatory elements, which may be selected based on the host cells intended to be used for expression, which are operably linked to the nucleic acid sequence intended to be expressed. In the context of a recombinant expression vector, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) such that expression of the nucleotide sequence is allowed (e.g., in an in vitro transcription/translation system or in a host cell upon introduction of the vector into the host cell).

In some embodiments, the host cell is transiently or non-transiently transfected with one or more vectors comprising polynucleotides encoding a Cas9 enzyme, system, or complex for use in multiplex targeting as defined herein. In some embodiments, the cell is transfected while it is in vivo in a subject. In some embodiments, the cell that is transfected is derived from the subject. In some embodiments, the cell is derived from cells derived from the subject, such as cell lines. A variety of cell lines useful for tissue culture are known in the art, and examples are provided elsewhere herein. Cell lines are available from a variety of sources known to those skilled in the art (see, for example, the American Type Culture Collection (ATCC) (Manassas, VA)). In some embodiments, a cell transfected with one or more vectors comprising polynucleotides encoding a Cas9 enzyme, system, or complex for use in multiple targeting as defined herein is used to generate a new cell line comprising one or more vector-derived sequences. In some embodiments, a cell transiently transfected with components of a Cas9-based CRISPR system or complex for use in multiple targeting as described herein (such as by transient transfection with one or more vectors, or transfection with RNA) and modified by the activity of the Cas9-based CRISPR system or complex is used to generate a new cell line comprising cells comprising the modification but lacking any other exogenous sequence. In some embodiments, cells transiently or non-transiently transfected with one or more vectors containing polynucleotides encoding a Cas9 enzyme, system, or complex for use in multiplex targeting as defined herein, or cell lines derived from such cells, are used in the evaluation of one or more test compounds.

The term "regulatory elements" is defined in other sections of this document.

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

In one aspect of the present invention there is provided a eukaryotic host cell comprising (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for insertion of one or more guide RNA sequences upstream or downstream (as appropriate) of the direct repeat sequence, wherein upon expression the guide sequence(s) direct(s) sequence-specific binding of a Cas9-based CRISPR complex to a corresponding target sequence(s) in a eukaryotic cell, wherein the Cas9-based CRISPR complex comprises a Cas9 enzyme in complex with one or more guide sequences that hybridize to the corresponding target sequence(s); and/or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said Cas9 enzyme, preferably comprising at least one nuclear localization sequence and/or NES. In some embodiments, the host cell comprises components (a) and (b). If applicable, a tracr sequence may also be provided. In some embodiments, component (a), component (b), or components (a) and (b) are stably integrated into the genome of a eukaryotic host cell. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element and optionally separated by a direct repeat, wherein when expressed, each of the two or more guide sequences directs sequence-specific binding of the Cas9-based CRISPR complex to its target sequence in a eukaryotic cell. In some embodiments, the Cas9 enzyme comprises one or more nuclear localization sequences and/or nuclear export sequences or NESs characterized by sufficient efficiency to direct the accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell and/or outside thereof.

In some embodiments, the Cas9 enzyme is a type V or type VI CRISPR system enzyme. In some embodiments, the Cas9 enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is derived from the Cas9 of Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, or Porphyromonas macacae, and may include additional Cas9 alterations or mutations defined elsewhere herein, and may be a chimeric Cas9. In some embodiments, the Cas9 enzyme is codon optimized for expression in a eukaryotic cell. In some embodiments, the CRISPR enzyme directs single or double strand cleavage at a specific position in a 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, the length of one or more guide sequences is (or the length of each is) at least 16, 17, 18, 19, 20, 25 nucleotides, or 16-30, or 16-25, or 16-20 nucleotides. When multiple guide RNAs are used, they are preferably separated by a direct repeat sequence. In one aspect of the present invention, a eukaryotic organism other than a human is provided; preferably a multicellular eukaryotic organism comprising a eukaryotic host cell according to any of the described embodiments. In other aspects of the present invention, a eukaryotic organism is provided, preferably a multicellular eukaryotic organism comprising a eukaryotic host cell according to any of the described embodiments. In some embodiments of these aspects, the organism may be an animal; for example, a mammal. The organism may also be an arthropod, such as an insect. The organism may also be a plant. In addition, the organism may be a fungus.

In one aspect of the present invention, a kit is provided comprising one or more components described herein. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the vector system comprises (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 upstream or downstream (as appropriate) of the direct repeat sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a Cas9-based CRISPR complex to a target sequence in a eukaryotic cell, wherein the Cas9-based CRISPR complex comprises a Cas9 enzyme in complex with a guide sequence that hybridizes to the target sequence; and/or (b) a second regulatory element operably linked by an enzyme-coding sequence encoding said Cas9 enzyme comprising a nuclear localization sequence. If applicable, a tracr sequence may also be provided. In some embodiments, the kit comprises components (a) and (b) located in the same or different vectors of the system. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences directs sequence-specific binding of a CRISPR complex to its target sequence in a eukaryotic cell. In some embodiments, the Cas9 enzyme comprises one or more nuclear localization sequences that are sufficiently efficient to direct 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 type VI CRISPR system enzyme. In some embodiments, the CRISPR enzyme is a Cas9 enzyme. In some embodiments, the Cas9 enzyme is derived from the Cas9 of Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, or Porphyromonas macacae (e.g., modified to have or have the ability to associate with at least one DD), and may include an additional alteration or mutation of Cas9 and may be a chimeric Cas9. In some embodiments, the DD-CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the DD-CRISPR enzyme directs cleavage of one or both strands at a specific position in a target sequence. In some embodiments, the DD-CRISPR enzyme does not have or has substantially no DNA strand cleavage activity (e.g., has no more than 5% of the nuclease activity of the wild-type enzyme or the enzyme without a mutation or alteration that reduces nuclease 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 length of the guide sequence is at least 16, 17, 18, 19, 20, 25 nucleotides, or 16-30, or 16-25, or 16-20 nucleotides.

In one aspect, the present invention provides a method for modifying multiple target polynucleotides in a host cell, such as a eukaryotic cell. In some embodiments, the method comprises causing a Cas9-based CRISPR complex to bind to multiple target polynucleotides, such as to cleave said multiple target polynucleotides, thereby causing the multiple target polynucleotides to be modified, wherein the Cas9-based CRISPR complex comprises a Cas9 enzyme in complex with multiple guide sequences, each of which hybridizes to a specific target sequence within said target polynucleotide, wherein said multiple guide sequences are linked to a direct repeat sequence. If applicable, a tracr sequence may also be provided (e.g., to provide a single guide RNA, sgRNA). In some embodiments, said cleavage comprises cleaving one or both strands at a specific position of each target sequence using said Cas9 enzyme. In some embodiments, said cleavage results in reduced transcription of multiple target genes. In some embodiments, the method further comprises repairing one or more of said cleaved target polynucleotides by homologous recombination with an exogenous polynucleotide template, 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 mutation results in a change in one or more amino acids in 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 direct expression of one or more of: a Cas9 enzyme and multiple guide RNA sequences linked to a direct repeat sequence. If applicable, a tracr sequence may also be provided. In some embodiments, said vectors are delivered to a eukaryotic cell in a subject. 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 the subject's body prior to performing said modification. In some embodiments, the method further comprises returning said eukaryotic cell and/or cells derived therefrom to said subject.

In one aspect of the present invention, there is provided a method for modifying the expression of multiple polynucleotides in a eukaryotic cell. In some embodiments, the method comprises causing a Cas9-based CRISPR complex to bind to multiple polynucleotides, such that said binding results in increased or decreased expression of said polynucleotides; wherein the Cas9-based CRISPR complex comprises a Cas9 enzyme in a complex with multiple guide sequences, each of which specifically hybridizes to its own target sequence within said polynucleotide, wherein said guide sequences are linked to a direct repeat sequence. If applicable, a tracr sequence may also be provided. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cells, wherein the one or more vectors direct the expression of one or more of: a Cas9 enzyme and multiple guide sequences linked to direct repeat sequences. If applicable, a tracr sequence may also be provided.

In one aspect of the present invention, there is provided a recombinant polynucleotide comprising multiple guide RNA sequences upstream or downstream (as appropriate) of a direct repeat sequence, wherein each of the guide sequences, when expressed, directs sequence-specific binding of a Cas9-based CRISPR complex to its corresponding target sequence present in a eukaryotic cell. In some embodiments, the target sequence is a viral sequence present in a eukaryotic cell. If applicable, a tracr sequence may also be provided. In some embodiments, the target sequence is a proto-oncogene or an oncogene.

Aspects of the present invention include a non-naturally occurring or engineered composition that may comprise 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 Cas9 enzyme as defined herein, which may comprise at least one or more nuclear localization sequences.

One aspect of the present invention encompasses methods of modifying a genomic locus of interest to alter gene expression in a cell by introducing into the cell any of the compositions described herein.

One aspect of the present invention is that the above-mentioned elements are contained in a single composition or are contained in separate compositions. These compositions can advantageously be applied to a host to induce a functional effect at the genome level.

As used herein, the term "guide RNA" or "gRNA" has the meaning used elsewhere herein and includes any polynucleotide sequence that has sufficient complementarity to a target nucleic acid sequence to hybridize to the target nucleic acid sequence and direct sequence-specific binding of a nucleic acid targeting complex to the target nucleic acid sequence. Each gRNA may be designed to include multiple binding recognition sites (e.g., aptamers) specific for the same or different adaptor proteins. Each gRNA may be designed to bind to a promoter region located -1000 to +1 nucleotides upstream of a transcription start site (i.e., TSS), preferably -200 nucleotides. This placement enhances functional domains that affect gene activation (e.g., transcriptional activators) or gene inhibition (e.g., transcriptional repressors). The modified gRNA may be one or more modified gRNAs (e.g., at least 1 gRNA, at least 2 gRNAs, at least 5 gRNAs, at least 10 gRNAs, at least 20 gRNAs, at least 30 gRNAs, at least 50 gRNAs) targeting one or more target loci contained in the composition. Said sequences of multiple gRNAs may be arranged in tandem and are preferably separated by a direct repeat.

Thus, each of the gRNA, the CRISPR enzyme defined herein may be individually contained in a composition and may be administered to the host individually or together. Alternatively, these components may be provided in a single composition for administration to the host. Administration to the host may be accomplished via viral vectors known to the skilled artisan or described herein for delivery to the host (e.g., a lentiviral vector, an adenoviral vector, an AAV-based vector). As explained herein, the use of different selection markers (e.g., for lentiviral sgRNA selection) and different gRNA concentrations (e.g., depending on whether multiple gRNAs are used) may be advantageous to induce an improved effect. Based on this concept, several options are suitable for inducing transformation at a genomic locus, including DNA cleavage, gene activation, or gene inactivation. Using the provided compositions, a person skilled in the art will be able to efficiently and specifically target one or multiple loci using the same or different functional domains to induce one or more transformations at a genomic locus. The compositions can be used in a variety of methods for screening libraries in cells and functional modeling in vivo (e.g., lincRNA gene activation and function identification; gain-of-function modeling; loss-of-function modeling; using the compositions of the present invention to create cell lines and transgenic animals for optimization and screening purposes).

The present invention encompasses the use of the compositions of the present invention to generate and use transgenic cells/animals with conditionally or inducibly expressed CRISPR; see, e.g., Platt et al., Cell (2014), 159(2):440-455, or the PCT patent publications cited therein, such as WO 2014/093622 (PCT/US2013/074667). For example, cells or animals, such as non-human animals, e.g., vertebrates or mammals, such as rodents, e.g., mice, rats, or other laboratory or non-laboratory animals, e.g., cats, dogs, sheep, etc., can be characterized by a "knock-in" state, whereby the animal conditionally or inducibly expresses Cas9, as described in Platt et al. Thus, the target cell or animal contains a conditionally dependent or inducible (e.g., in the form of Cre-dependent constructs) CRISPR enzyme (e.g., Cas9) (e.g., in the form of Cre-dependent constructs), when expressing the vector introduced into the target cell, the vector expresses that which induces or provides a condition for the expression of the CRISPR enzyme (e.g., Cas9) in the target cell. Using the ideas and compositions defined in this document with a known method for creating a CRISPR complex, inducible genome transformations are also an aspect of the present invention. Examples of such inducible transformations have been described in other sections of this document.

In some embodiments, the phenotypic change is preferably the result of modification of the genome in targeting a genetic disease, particularly in therapeutic methods, and preferably if a repair template is provided to correct or alter the phenotype.

In some embodiments, diseases that can be targeted include diseases that are caused by pathogenic splicing defects.

In some embodiments, cellular targets include hematopoietic stem/progenitor cells (CD34+); human T cells, and ocular cells (retinal cells), such as photoreceptor progenitor cells.

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

In some embodiments, the target diseases also include cancer; sickle cell anemia (caused by a point mutation); HBV, HIV; beta-thalassemia; and an ophthalmologic or eye disease, such as Leber congenital amaurosis (LCA), caused by a splicing defect.

In some embodiments, delivery methods include cationic lipid-mediated "direct" delivery of the enzyme-targeting (ribonucleoprotein) complex and electroporation of plasmid DNA.

The methods, products, and uses described herein may be used for purposes other than therapy. In addition, any of the methods described herein may be used in vitro and ex vivo.

In one aspect, there is provided a non-naturally occurring or engineered composition comprising:

I. two or more polynucleotide sequences of the CRISPR-Cas system, providing

(a) a first guide sequence capable of hybridizing to a first target sequence at a polynucleotide locus,

(b) a second guide sequence capable of hybridizing to a second target sequence at the polynucleotide locus,

(c) direct repeat sequence,

And

II. the Cas9 enzyme or a second polynucleotide sequence encoding it,

wherein, when transcribed, the first and second guide sequences direct sequence-specific binding of the first and second Cas9-based CRISPR complexes to the first and second target sequences, respectively,

wherein the first CRISPR complex comprises a Cas9 enzyme in complex with a first guide sequence that can hybridize to a first target sequence,

wherein the second CRISPR complex comprises a Cas9 enzyme in complex with a second guide sequence that can hybridize to a second target sequence, and

wherein the first guide sequence directs cleavage of one strand of a DNA duplex near a first target sequence, and the second guide sequence directs cleavage of the other strand near a second target sequence, thereby inducing a double-strand break, thereby providing modification of an organism, either non-human or non-animal. Similarly, compositions may be provided that contain more than two guide RNAs, for example, each specific for one target, and they are arranged in tandem in the CRISPR composition or system or complex described herein.

In another embodiment, Cas9 is delivered to the cell as a protein. In another and particularly preferred embodiment, Cas9 is delivered to the cell as a protein or as a nucleotide sequence encoding it. Delivery to the cell as a protein may include delivery of a ribonucleoprotein (RNP) complex in which the protein is in a complex with multiple guides.

In one aspect, host cells and cell lines modified with or containing the compositions, systems or modified enzymes of the present invention are provided, including stem cells and their progeny.

In one aspect, methods of cell therapy are provided in which, for example, a single cell or population of cells is isolated or cultured, wherein such cell or cells are or have been modified ex vivo as described herein, and then returned (selected cells) or introduced (cultured cells) into the body. Stem cells, whether embryonic, induced pluripotent or totipotent stem cells, are also particularly preferred in this regard. But, of course, in vivo embodiments are also contemplated.

The methods according to the present invention may further comprise delivery of templates, such as repair templates, which may be dsODN or ssODN, see below. Delivery of templates may be carried out simultaneously or separately from delivery of any or all of the CRISPR enzyme or guide RNAs and by the same or different delivery mechanisms. In some embodiments, it is preferred that the template is delivered together with guide RNAs and, preferably, also with the CRISPR enzyme. An example is an AAV-based vector, wherein the CRISPR enzyme is AsCas9 or LbCas9.

The methods of the present invention may further comprise: (a) delivering to a cell a double-stranded oligodeoxynucleotide (dsODN) comprising sticky ends complementary to the sticky ends created by said double-strand break, wherein said dsODN integrates into a locus of interest; or (b) delivering to a cell a single-stranded oligodeoxynucleotide (ssODN), wherein said ssODN acts as a template for homologous repair of said double-strand break. The methods of the present invention may be used to prevent or treat a disease in an individual, optionally wherein said disease is caused by a defect in said locus of interest. The methods of the present invention may be performed in vivo in an individual or ex vivo on a cell removed from an individual, optionally wherein said cell is returned to the individual.

The present invention also encompasses products obtained by using a CRISPR enzyme, or a Cas enzyme, or a Cas9 enzyme, or a CRISPR-CRISPR enzyme, or a CRISPR-Cas system, or a CRISPR-Cas9 system, for use in tandem or in multiple targeting, as defined herein.

Guided RNAs for a Cas9-based CRISPR-Cas system according to the present invention

In one aspect, the present invention provides chaperoned Cas9-based CRISPR-Cas systems or complexes, in particular such a system comprising a chaperoned guide of a Cas9-based CRISPR-Cas system. By "chaperoned" is meant that the Cas9-based CRISPR-Cas system or complex or guide is delivered at a specific time or location in the cell such that the activity of the Cas9-based CRISPR-Cas system or complex or guide is spatially or temporally controlled. For example, the activity and location of the Cas9-based CRISPR-Cas system or complex or guide can be controlled by an aptamer sequence of the chaperone RNA that has a binding affinity for an aptamer ligand, such as a cell surface protein or other localized cellular component. Alternatively, the chaperone aptamer, for example, may respond to an effector of the aptamer on or in the cell, such as a transient effector such as an external energy source that is applied to the cell at a specific time.

Cas9-based CRISPR-Cas escort systems or complexes have a gRNA with a functional structure designed to improve the gRNA structure, architecture, stability, genetic expression, or any combination thereof. Such a structure may include an aptamer.

Aptamers are biomolecules that can be designed or selected to bind tightly to other ligands, for example using a technique called systematic evolution of ligands by exponential enrichment (SELEX; Tuerk C, Gold L: "Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase." Science 1990, 249:505-510). Nucleic acid-based aptamers, for example, can be selected from pools of randomly sequenced oligonucleotides that exhibit high binding affinity and specificity for a variety of biomedically relevant targets, suggesting a range of therapeutic uses for aptamers (Keefe, Anthony D., Supriya Pai, and Andrew Ellington. "Aptamers as therapeutics." Nature Reviews Drug Discovery 9.7 (2010): 537-550). These characteristics also suggest a variety of applications for aptamers as drug delivery vehicles (Levy-Nissenbaum, Etgar, et al. "Nanotechnology and aptamers: applications in drug delivery." Trends in biotechnology 26.8 (2008): 442-449; and Hicke BJ, Stephens AW. "Escort aptamers: a delivery service for diagnosis and therapy." J Clin Invest 2000, 106:923-928.). It is also possible to design aptamers that function as molecular switches that respond to signal quenching by changing properties, such as RNA aptamers that link fluorophores to mimic the activity of green fluorescent protein (Paige, Jeremy S., Karen Y. Wu, and Samie R. Jaffrey. "RNA mimics of green fluorescent protein." Science 333.6042 (2011): 642–646). It has also been suggested that aptamers could be used as components of targeted siRNA therapeutic delivery systems, for example to target cell surface proteins (Zhou, Jiehua, and John J. Rossi. "Aptamer-targeted cell-specific RNA interference." Silence 1.1 (2010): 4).

Accordingly, provided herein is a gRNA modified, for example, with one or more aptamers designed to improve delivery of the gRNA, including delivery across the cell membrane, into intracellular compartments or into the nucleus. Such a structure may include, either in addition to one or more aptamers or without such one or more aptamers, a moiety(s) that (impart) impart to the guide the ability to deliver, induce or respond to a selected effector. Accordingly, the present invention encompasses a gRNA that responds to normal or pathological physiological conditions, including but not limited to pH, hypoxia, _O2 concentration, temperature, protein concentration, enzyme concentration, lipid structure, exposure to light, mechanical disruption (e.g., ultrasound waves), magnetic fields, electric fields or electromagnetic radiation.

In one aspect of the present invention, there is provided a non-naturally occurring or engineered composition comprising an escorted guide RNA (egRNA) comprising:

a guide RNA sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell; and

a chaperone aptamer RNA sequence, wherein the chaperone aptamer has a binding affinity for an aptamer ligand on or in a cell, or the chaperone aptamer is responsive to a localized effector of the aptamer on or in a cell, wherein the presence of the aptamer ligand or effector on or in a cell is limited in time or space.

A chaperone aptamer, for example, can change conformation in response to interaction with a ligand or an aptamer effector in the cell.

The chaperone aptamer may be characterized by a specific binding affinity for the aptamer ligand.

The aptamer ligand may be localized to a specific location or compartment of the cell, such as on or in the cell membrane. Accordingly, binding of the chaperone aptamer to the aptamer ligand may direct the egRNA to a location of interest in the cell, such as the interior of the cell, via binding to the aptamer ligand, which is a cell surface ligand. In this way, targeting of various spatially restricted locations within the cell, such as the cell nucleus or mitochondria, may be achieved.

Once the intended changes have been introduced, such as by editing the intended copies of a gene in the cell's genome, continued CRISPR/Cas9 expression in that cell is no longer necessary. Indeed, long-term expression would be undesirable in the event of off-target effects at sites in the genome not intended for editing, etc. Thus, time-limited expression would be desirable. Inducible expression is one approach, but the applicants have also constructed a self-inactivating Cas9-based CRISPR-Cas system that relies on the use of a non-coding targeting guide sequence within the CRISPR vector itself. Thus, once expression has begun, the CRISPR system will cause its own destruction, but before the destruction is complete, it will have sufficient time to edit the genomic copies of the target gene (which, in terms of a normal point mutation in a diploid cell, would require no more than two edits). In simple terms, the self-inactivating CRISPR-Cas system using Cas9 comprises an additional RNA (i.e., a guide RNA) that targets the coding sequence of the CRISPR enzyme itself or that targets one or more non-coding guide target sequences that are complementary to unique sequences present in one or more of the following: (a) within a promoter driving expression of non-coding RNA elements, (b) within a promoter driving expression of the Cas9 gene, (c) within 100 bp of the ATG translation start codon in the Cas9 coding sequence, (d) within an inverted terminal repeat (iTR) of a viral delivery vector, such as in the AAV genome.

An egRNA may include an aptamer RNA binding sequence that operably links the chaperone RNA sequence to the guide RNA sequence.

In embodiments, the egRNA may include one or more photolabile linkages or non-naturally occurring residues.

In one aspect, the chaperone aptamer RNA sequence may be complementary to a target miRNA, which may or may not be present in the cell, such that only when the target miRNA is present is binding of the chaperone aptamer RNA sequence to the target miRNA observed, resulting in cleavage of the egRNA by the RNA-induced silencing complex (RISC) in the cell.

In embodiments, the length of the chaperone RNA aptamer sequence may be, for example, from 10 to 200 nucleotides, and the egRNA may include more than one chaperone RNA aptamer sequence.

It should be understood that any of the guide RNA sequences described elsewhere herein can be used in the egRNA described herein. In certain embodiments of the present invention, a guide RNA or mature crRNA comprises, consists essentially of, or consists of a direct repeat sequence and a guide sequence or spacer sequence. In certain embodiments, a guide RNA or mature crRNA comprises, consists essentially of, or consists of a direct repeat sequence associated with a guide sequence or spacer sequence. In certain embodiments, a guide RNA or mature crRNA comprises 19 nucleotides of a partial direct repeat followed by 23-25 nucleotides of a guide sequence or spacer sequence. In certain embodiments, the effector protein is an FnCas9 effector protein, and a guide sequence of at least 16 nucleotides in length is required to achieve detectable DNA cleavage, and a guide sequence of at least 17 nucleotides in length is required to achieve efficient DNA cleavage in vitro. In certain embodiments, the direct repeat sequence is located upstream (i.e., in the 5' direction) of the guide sequence or spacer sequence. In a preferred embodiment, the seed sequence (i.e., a sequence critical for recognition and/or hybridization to a sequence at a target locus) of the FnCas9 guide RNA is within approximately the first 5 nucleotides at the 5' end of the guide sequence or spacer sequence.

The egRNA may be included in a non-naturally occurring or engineered composition with a Cas9-based CRISPR-Cas complex together with a Cas9 that may include at least one mutation, such as a mutation that results in the Cas9 having no more than 5% of the nuclease activity of a Cas9 lacking the at least one mutation, such as having a nuclease activity that is reduced by at least 97% or 100% compared to a Cas9 lacking the at least one mutation. The Cas9 may also include one or more nuclear localization sequences. Mutated Cas9 enzymes with modulated activity, such as reduced nuclease activity, are described elsewhere herein.

The engineered Cas9-based CRISPR-Cas composition can be provided in a cell such as a eukaryotic cell, a mammalian cell, or a human cell.

In embodiments, the compositions described herein comprise a Cas9-based CRISPR-Cas complex with at least three functional domains, at least one of which associates with Cas9 and at least two of which associate with egRNA.

The compositions described herein can be used to introduce a genomic locus transformation into a host cell, such as a eukaryotic cell, in particular a mammalian cell or a non-human eukaryotic organism, in particular a non-human mammal such as a mouse, in vivo. Transformation of a genomic locus can involve affecting gene activation, gene inhibition, or cleavage at the locus. The compositions described herein can also be used to modify a genomic locus of interest to alter gene expression in the cell. Methods for introducing a genomic locus transformation into a host cell using the Cas9 enzyme provided herein are described in detail elsewhere in this document. Delivery of the composition can be accomplished, for example, by delivering a nucleic acid molecule(s) encoding the composition, wherein the nucleic acid molecule(s) is(are) operably linked to a regulatory sequence(s), and expressing the nucleic acid molecule(s) in vivo, for example, using a lentivirus, adenovirus, or AAV.

The present invention provides compositions and methods by which gRNA-mediated gene editing activity can be tailored. The present invention provides gRNA secondary structures that improve cleavage efficiency by increasing gRNA and/or increasing the amount of RNA delivered to a cell. gRNA may include light-labile or inducible nucleotides.

To improve the efficiency of gRNA, such as gRNA delivered via viral or non-viral technology, applicants have added secondary structures to gRNA that enhance its stability and improve gene editing. Additionally, to overcome the lack of efficient delivery, applicants have modified gRNA with cell-penetrating RNA aptamers; wherein the aptamers bind to cell surface receptors and facilitate the entry of gRNA into cells. Notably, cell-penetrating aptamers can be engineered to target specific cellular receptors to mediate cell-specific delivery. Applicants have also created guides that are inducible.

Light sensitivity of the inducible system can be achieved by activation and binding of cryptochrome-2 and CIB1. Blue light stimulation induces an activating conformational change in cryptochrome-2, which leads to recruitment of its binding partner CIB1. This binding is rapid and reversible, reaching saturation <15 sec after a stimulation pulse and returning to baseline <15 min after the end of stimulation. These rapid binding kinetics result in the system being time-limited only by the rate of transcription/translation and transcript/protein decay, rather than by the uptake and clearance of inducing agents. Cryptochrome-2 activation is also highly sensitive, allowing the use of low-intensity light stimulation and reducing the risks of phototoxicity. Moreover, in a context such as the intact mammalian brain, varying light intensities can be used to control the size of the stimulated region, allowing for greater precision than vector delivery alone can provide.

The present invention provides energy sources such as electromagnetic radiation, sound energy or thermal energy for inducing the guide. Advantageously, the electromagnetic radiation is a component of visible light. In a preferred embodiment, the light is blue light with a wavelength of about 450 to about 495 nm. In a particularly preferred embodiment, the wavelength is about 488 nm. In another preferred embodiment, the stimulation with light is carried out by pulsed irradiation. The light power can vary in the range of about 0-9 mW/cm ² . In a preferred embodiment, a stimulation pattern consisting of no more than 0.25 seconds of stimulation every 15 seconds should lead to maximum activation.

The cells envisaged in the practice of the present invention may be a prokaryotic cell or a eukaryotic cell, preferably an animal cell, a plant cell or a yeast cell, more preferably a mammalian cell.

A chemically sensitive or energy-sensitive guide may undergo a conformational change upon induction by binding of a chemical source or by exposure to energy, which allows it to act as a guide and function in a Cas9-based CRISPR-Cas system or complex. The present invention may provide for the use of a chemical source or an energy source to ensure the function of the guide and the Cas9-based CRISPR-Cas system or complex; and optionally further determining that the expression of the genomic locus is altered.

There are several different designs of this chemically inducible system: 1. the ABI-PYL-based system, inducible by abscisic acid (ABA) (see, e.g., http://stke.sciencemag.org/cgi/content/abstract/sigtrans;4/164/rs2), 2. the FKBP-FRB-based system, inducible by rapamycin (or related rapamycin-based chemicals) (see, e.g., http://www.nature.com/nmeth/journal/v2/n6/full/nmeth763.html), 3. the GID1-GAI-based system, inducible by gibberellin (GA) (see, e.g., http://www.nature.com/nchembio/journal/v8/n5/full/nchembio.922.html).

Another system envisaged by the present invention is a chemically inducible system based on a change in subcellular localization. Applicants have also developed a system in which the polypeptide comprises a DNA-binding domain comprising at least five or more transcription activator-like effector (TALE) monomers and at least one or more semi-monomers specifically positioned to target a genomic locus of interest associated with at least one or more effector domains, further linked to a chemically or energy-sensitive protein. This protein will result in a change in subcellular localization of the entire polypeptide (i.e., transport of the entire polypeptide from the cytoplasm to the nucleus of cells) upon binding of a chemical or upon energy transfer with the chemically or energy-sensitive protein. Such transport of the entire polypeptide from one subcellular compartment or organelle in which its activity is blocked due to the absence of a substrate for the effector domain to another in which the substrate is present will allow the entire polypeptide to come into contact with its required substrate (i.e., genomic DNA in the mammalian nucleus), and this leads to activation or repression of target gene expression.

A system of this type could also be used to induce cleavage of a genomic locus of interest in a cell where the effector domain is a nuclease.

The chemically inducible system may be an estrogen receptor (ER)-based system induced by 4-hydroxytamoxifen (4OHT) (see, e.g., http://www.pnas.org/content/104/3/1027.abstract). The mutated ligand binding domain of the estrogen receptor, called ERT2, translocates to the nucleus of cells upon binding of 4-hydroxytamoxifen. In further embodiments of the present invention, any naturally occurring or engineered derivative of any nuclear receptor, thyroid hormone receptor, retinoic acid receptor, estrogen receptor, estrogen-related compound receptor, glucocorticoid receptor, progesterone receptor, androgen receptor may be used in inducible systems similar to the ER-based inducible system.

Another inducible system is based on a design using a transient receptor potential (TRP) ion channel system induced by energy, heat, or radio waves (see, for example, http://www.sciencemag.org/content/336/6081/604). These TRP proteins respond to a variety of stimuli, including light and heat. When this protein is activated by light or heat, the ion channel will open and allow ions, such as calcium, to enter the plasma membrane. This influx of ions will bind to intracellular ion-interacting partners associated with the polypeptide, including the guide and other components of the Cas9-based CRISPR-Cas complex or system, and the binding will induce a change in the subcellular localization of the polypeptide, causing the entire polypeptide to enter the cell nucleus. Once inside the nucleus, the targeting protein and other components of the Cas9-based CRISPR-Cas complex will be active and will modulate the expression of the target gene in cells.

A system of this type could also be used to induce cleavage of a genomic locus of interest in a cell; and in this regard, it should be noted that the Cas9 enzyme is a nuclease. Light can be generated by laser or other forms of energy sources. Heat can be generated by increasing the temperature caused by the energy source or by nanoparticles that release heat after absorbing energy from the energy source, delivered in the form of radio waves.

Although activation by light may be an advantageous embodiment, it may sometimes be disadvantageous, particularly for in vivo applications where light cannot penetrate the skin or other organs. In this case, methods are envisaged for using other types of activation energy, particularly electric field energy and/or ultrasound, which will have a similar effect.

The electric field energy is preferably applied, essentially as described in the prior art, by applying one or more electric pulses of about 1 Volt/cm to about 10 kVolt/cm under in vivo conditions. Instead of or in addition to pulses, the electric field can be delivered in a continuous manner. The electric pulse can be applied for 1 μs to 500 milliseconds, preferably 1 μs to 100 milliseconds. The electric field can be applied continuously or in a pulsed manner for about 5 minutes.

As used herein, "electric field energy" is electrical energy that is applied to a cell. Preferably, the electric field has a strength of about 1 volt/cm to about 10 kvolt/cm or more under in vivo conditions (see WO97/49450).

As used herein, the term "electric field" includes one or more pulses of varying capacitance and voltage, and includes exponential and/or square waveforms, and/or modulated waveforms and/or modulated square waveforms. References to electric fields and electricity should be taken to include reference to the presence of an electrical potential difference in the environment of the cell. Such an environment may be created by static electricity, alternating current (AC), direct current (DC), etc., as is known in the art. The electric field may be uniform, non-uniform, or otherwise, and may vary in strength and/or direction in a time-dependent manner.

Single or multiple applications of the electric field, as well as single or multiple applications of ultrasound in any order and in any combination, are also possible. Ultrasound and/or the electric field can be delivered as single or multiple continuous applications or as pulses (pulsed delivery).

Electroporation has been used in both in vitro and in vivo procedures to introduce foreign material into living cells. In in vitro applications, a living cell sample is first mixed with the agent of interest and placed between electrodes, such as parallel plates. The electrodes then apply an electric field to the cell/injection mixture. Examples of systems that perform in vitro electroporation include the Electro Cell Manipulator ECM600 and the Electro Square Porator T820, both manufactured by the BTX Division of Genetronics, Inc. (see U.S. Patent No. 5,869,326).

Known electroporation techniques (both in vitro and in vivo) operate by applying a short high voltage pulse to electrodes positioned around the treatment site. The electric field generated between the electrodes temporarily creates pores in the cell membranes, causing molecules of the agent of interest to penetrate the cells. In known electroporation applications, this electric field provides a single square wave pulse of about 1000 V/cm, lasting approximately 100 μs. Such a pulse can be generated, for example, in known applications of the Electro Square Porator T820.

Preferably, the electric field has a strength of from about 1 V/cm to about 10 kV/cm under in vitro conditions. Thus, the electric field can have a strength of 1 V/cm, 2 V/cm, 3 V/cm, 4 V/cm, 5 V/cm, 6 V/cm, 7 V/cm, 8 V/cm, 9 V/cm, 10 V/cm, 20 V/cm, 50 V/cm, 100 V/cm, 200 V/cm, 300 V/cm, 400 V/cm, 500 V/cm, 600 V/cm, 700 V/cm, 800 V/cm, 900 V/cm, 1 kV/cm, 2 kV/cm, 5 kV/cm, 10 kV/cm, 20 kV/cm, 50 kV/cm or more. More preferably, from about 0.5 kV/cm to about 4.0 kV/cm in vitro. Preferably, the electric field has a strength of from about 1 V/cm to about 10 kV/cm in vivo. However, the electric field strength can be reduced if the number of pulses delivered to the target site is increased. Thus, pulsed delivery of electric fields at lower field strengths is envisaged.

Preferably, the electric field is applied in the form of multiple pulses, such as double pulses of the same strength and capacitance or successive pulses of different strength and/or capacitance. The term "pulse" as used herein includes one or more electric pulses at varying capacitance and voltage and includes exponential and/or square waveforms and/or modulated waveforms/square waveforms.

Preferably, the electrical pulse is delivered in the form of a waveform selected from an exponential waveform, a square waveform, a modulated waveform, and a modulated square waveform.

In a preferred embodiment, direct current at low voltage is used. Thus, the applicants disclose the use of an electric field applied to a cell, tissue or tissue mass at a field strength of 1 V/cm to 20 V/cm for a period of 100 milliseconds or more, preferably 15 minutes or more.

Ultrasound is preferably used at a power level of approximately 0.05 W/ ^cm2 to approximately 100 W/ ^cm2 . Diagnostic or therapeutic ultrasound or a combination of both may be used.

As used in this document, the term "ultrasound" refers to a form of energy that consists of mechanical vibrations whose frequency is so high that they are beyond the range of human hearing. The lower frequency limit of the ultrasound spectrum as a whole may be taken as approximately 20 kHz. Most diagnostic applications of ultrasound use frequencies in the range of 1 to 15 MHz (from Ultrasonics in Clinical Diagnosis, P. N. T. Wells, ed., 2nd. Edition, Publ. Churchill Livingstone [Edinburgh, London & NY, 1977]).

Ultrasound has been used in both diagnostic and therapeutic applications. When used as a diagnostic tool ("diagnostic ultrasound"), ultrasound is typically used at energy densities in the range of up to approximately 100 mW/ ^cm2 (FDA recommendation), although energy densities up to 750 mW/ ^cm2 have been used. In physical therapy, ultrasound is typically used as an energy source in the range of up to approximately 3-4 W/ ^cm2 (WHO recommendation). Other therapeutic applications may use higher ultrasound intensities, such as HIFU with parameters of 100 W/cm2 to 1 kW/ ^cm2 (or even higher) for short periods of time. As used herein, the term "ultrasound" is intended to encompass diagnostic, therapeutic, and focused ultrasound.

Focused ultrasound (FUS) provides delivery of thermal energy without an invasive probe (see Morocz et al 1998 Journal of Magnetic Resonance Imaging Vol.8, No. 1, pp.136-142. Another form of focused ultrasound is high-intensity focused ultrasound (HIFU), reviewed by Moussatov et al in Ultrasonics (1998) Vol.36, No.8, pp.893-900 and TranHuuHue et al in Acustica (1997) Vol.83, No.6, pp.1103-1106.

Preferably, a combination of diagnostic ultrasound and therapeutic ultrasound is used. However, this combination is not intended to be limiting, and the skilled reader will recognize that any of a variety of ultrasound combinations may be used. In addition, the energy density, the frequency of ultrasound oscillation, and the period of exposure may be varied.

Preferably, the ultrasonic energy source is applied at a power flux density of from about 0.05 to about 100 W cm ^-2 . Even more preferably, the ultrasonic energy source is applied at a power flux density of from about 1 to about 15 W cm ^-2 .

Preferably, the ultrasonic energy source is applied at an oscillation frequency of about 0.015 to about 10.0 MHz. More preferably, the ultrasonic energy source is applied at an oscillation frequency of about 0.02 to about 5.0 MHz or about 6.0 MHz. Most preferably, the ultrasound is applied at an oscillation frequency of 3 MHz.

Preferably, the exposure occurs for a period of time of about 10 milliseconds to about 60 minutes. Preferably, the exposure occurs for a period of time of about 1 second to about 5 minutes. More preferably, the ultrasound is applied for about 2 minutes. However, depending on the specific target cell to be destroyed, the exposure may have a longer duration, such as 15 minutes.

Preferably, the target tissue is exposed to an ultrasonic energy source at an acoustic flux density of from about 0.05 W cm ^-2 to about 10 W cm ^-2 , with an oscillation frequency in the range of from about 0.015 to about 10 MHz (see WO 98/52609). However, alternatives are also possible, such as exposure to an ultrasonic energy source at an acoustic flux density of more than 100 W cm ^-2 , but for shorter periods of time, such as 1000 W cm ^-2 for periods in the range of milliseconds or less.

Preferably, the ultrasound is applied in the form of multiple pulses; thus, both continuous waves and pulsed waves (pulsed ultrasound delivery) can be used in any combination. For example, continuous wave ultrasound can be applied followed by pulsed wave ultrasound, or vice versa. This can be repeated any number of times, in any order and combination. Pulsed wave ultrasound can be applied on top of continuous wave ultrasound, and any number of pulses can be applied in any number of groups.

Preferably, the ultrasound may comprise pulsed wave ultrasound. In a very preferred embodiment, the ultrasound is applied at a power flux density of 0.7 W cm ^-2 or 1.25 W cm ^-2 in the form of a continuous wave. Higher power flux densities may be used when using pulsed wave ultrasound.

The use of ultrasound is advantageous because, like light, it can be focused precisely on a target. Moreover, ultrasound is advantageous because it can be focused deeper into tissue than light. It is therefore better suited for therapy that penetrates an entire tissue (such as, for example, an unlimited lobe of the liver) or an entire organ (such as, for example, an unlimited whole liver or an entire muscle, such as the heart). Another important advantage is that ultrasound is a non-invasive treatment that is used in a wide variety of diagnostic and therapeutic applications. For example, ultrasound is well known in medical imaging techniques and, in addition, in orthopedic therapy. In addition, devices suitable for applying ultrasound to a subject-vertebral body to be treated are widely available and their use is well known in the art.

The rapid transcriptional response and endogenous targeting of the present invention make it an ideal system for studying transcriptional dynamics. For example, the present invention can be used to study the dynamics of variant production following induced expression of a target gene. At the other end of the transcription cycle, mRNA decay studies are often conducted in response to a strong extracellular stimulus that causes changes in the expression levels of multiple genes. The present invention can be used to reversibly induce transcription of an endogenous target, after which the punctate stimulation can be stopped and the kinetics of degradation of a single target can be monitored.

The temporal precision of the present invention may enable time-dependent genetic regulation that is consistent with experimental interventions. For example, targets putatively affecting long-term potentiation (LTP) may be modulated in organ-like or dissociated neuronal cultures, but only during the stimulus to induce LTP, so as not to interfere with normal cell development. Similarly, in cellular models that exhibit disease phenotypes, targets putatively affecting the efficacy of a particular therapy may be modulated only during treatment. Conversely, genetic targets may be modulated only during a pathological stimulus. Any number of experiments in which the timing of the triggering of genetic changes in response to external experimental stimuli is important may potentially benefit from the present invention.

The in vivo context provides similarly broad possibilities for controlling gene expression within the scope of the present invention. Photoinducibility provides the potential for spatial precision. Taking advantage of developments in optrode technology, a stimulating fiber optic electrode can be placed in a specific region of the brain. The size of the stimulation region can then be adjusted using light intensity. This can be done in combination with delivery of the Cas9-based CRISPR-Cas system or complex of the present invention, or, in the case of Cas9 transgenic animals, the guide RNA of the present invention can be delivered and optrode technology can provide the ability to modulate gene expression in specific regions of the brain. A light-transmitting organism expressing Cas9 can have the guide RNA of the present invention introduced into it and very precise localized laser-induced changes in gene expression can then be induced.

The culture medium for culturing the host cells includes a medium commonly used for tissue culture, such as M199-Earle's base, Eagle's MEM (E-MEM), Dulbecco's MEM (DMEM), SC-UCM102, UP-SFM (GIBCO BRL), EX-CELL302 (Nichirei), EX-CELL293-S (Nichirei), TFBM-01 (Nichirei), ASF104, among others. Suitable culture media for specific cell types can be found in the American Type Culture Collection (ATCC) or the European Collection of Cell Cultures (ECACC). The culture media can be supplemented with amino acids such as L-glutamine, salts, antifungal or antibacterial agents such as Fungizone®, penicillin-streptomycin, animal serum, etc. The cell culture medium may optionally be serum-free.

The present invention can also provide important temporal precision in vivo. The present invention can be used to alter gene expression during a specific stage of development. The present invention can be used to select the time point for initiating genetic changes at a specific time during an experiment. For example, genes involved in a conditioned response can be overexpressed or repressed only during the delivery of a conditioned stimulus to a specific region of the intact brain of a rodent or primate. In addition, the present invention can be used to induce changes in gene expression only during specific stages of disease development. For example, an oncogene can be overexpressed only after a tumor has reached a specific size or metastatic stage. Conversely, proteins suspected of influencing the development of Alzheimer's disease can be knocked down only at specific times in an animal's life and within a specific region of the brain. Although these examples are not an exhaustive list of potential applications of the present invention, they indicate some areas in which the present invention may represent a promising technology.

Protected guides: The enzymes of the present invention can be used in combination with protected guide RNAs.

In one aspect, the present invention aims to further enhance the specificity of individual guide RNAs with respect to Cas9 by thermodynamically controlling the binding specificity of the guide RNA to the target DNA. This is a general approach to introducing mismatches, extensions or truncations into the guide sequence to increase/decrease the number of complementary bases relative to the mismatched bases shared by the genomic target and its potential non-target loci, in order to provide a thermodynamic advantage to the target loci of the genome over the non-target loci of the genome.

In one aspect of the present invention, a guide sequence is provided that is modified with a secondary structure to enhance the specificity of the Cas9-based CRISPR-Cas system, and as a result, the secondary structure can protect against exonuclease activity and allow for additions at the 3' end of the guide sequence.

In one aspect of the present invention, a "guard RNA" is hybridized to a guide sequence, wherein the "guard RNA" is a strand of RNA complementary to the 5' end of a guide RNA (gRNA), thereby forming a partially double-stranded gRNA. In one embodiment of the present invention, protection of mismatched bases by a perfectly complementary guard sequence reduces the likelihood of binding of target DNA to mismatched base pairs at the 3' end. In embodiments of the present invention, additional sequences may also be present to provide increased length.

Guide RNA (gRNA) extensions that match a genomic target provide protection and enhance gRNA specificity. Extension of gRNA with a matching sequence distal to the end of a spacer seed sequence for individual genomic targets is predicted to provide enhanced specificity. Matched gRNA extensions that enhance specificity were observed in cells without truncation. Prediction of the gRNA structure accompanying these stable extensions revealed that stable forms arise from protected states where the extension forms a closed loop with the gRNA seed sequence due to complementary sequences in the spacer extension and the spacer seed sequence. These results demonstrate that the protected guide concept also includes sequences matching the target genomic sequence distal to the 20-mer spacer binding site. Thermodynamic prediction can be used to predict fully matched or partially matched guide extensions that result in protected states of gRNA. This extends the concept of protected gRNAs to the interaction between X and Z, where X will typically be 17-20 nucleotides long and Z will be 1-30 nucleotides long. Thermodynamic prediction can be used to determine the optimal extension state for Z, potentially introducing small amounts of mismatches in Z to promote the formation of protected conformations between X and Z. Throughout this application, the terms "X" and "seed" sequence length (SL) are used interchangeably with the term "open length" (EpL), which refers to the number of nucleotides available for binding to target DNA; the terms "Y" and "protection length" (PL) are used interchangeably to refer to the length of the protection sequence; and the terms "Z", "E", "E'" and "EL" are used interchangeably and correspond to the term "extension" (ExL), which denotes the number of nucleotides by which the target sequence is extended.

The extension sequence, which corresponds to the extension (ExL), may optionally be attached directly to the guide sequence at the 3' end of the protected guide sequence. The length of the extension sequence may be 2-12 nucleotides. Preferably, ExL may be 0, 2, 4, 6, 8, 10 or 12 nucleotides in length. In a preferred embodiment, ExL is 0 or 4 nucleotides in length. In a more preferred embodiment, ExL is 4 nucleotides in length. The extension sequence may or may not be complementary to the target sequence.

The extension sequence may also optionally be attached directly to the guide sequence at the 5' end of the protected guide sequence, as well as at the 3' end of the protector sequence. As a result, the extension sequence serves as a linking sequence between the protected sequence and the protector sequence. Without being bound by theory, it is believed that such a linking may position the protector sequence near the protector sequence to improve the binding of the protector sequence to the protector sequence.

Addition of gRNA mismatches at the distal end of the gRNA can demonstrate enhanced specificity. Introduction of unprotected distal mismatches into Y or extension of the gRNA with distal mismatches (Z) can demonstrate enhanced specificity. This concept is mentioned to be related to the X, Y, and Z components used in protected gRNAs. The unprotected mismatch concept can be further generalized to the X, Y, and Z concepts described for protected guide RNAs.

Cas9Cas9 In one aspect of the present invention, enhanced specificity of Cas9Cas9 is provided, wherein the double-stranded 3' end of the protected guide RNA (pgRNA) allows for two possible outcomes: (1) the guide RNA-protective RNA will be exchanged for the guide RNA-target DNA strand and the guide will fully bind to the target, or (2) the guide RNA will fail to fully bind to the target, and since Cas9-mediated target cleavage is a multi-step kinetic reaction that requires guide RNA:target DNA binding to activate Cas9-catalyzed DSB formation, Cas9-mediated cleavage does not occur if the guide RNA is not bound correctly. According to certain embodiments, the protected guide RNA improves target binding specificity compared to a naturally occurring CRISPR-Cas system. According to particular embodiments, the protected modified guide RNA improves stability compared to naturally occurring CRISPR-Cas. According to particular embodiments, the length of the protection sequence is from 3 to 120 nucleotides and comprises 3 or more contiguous nucleotides complementary to another sequence of the guide or protection sequence. According to particular embodiments, the protection sequence forms a hairpin. According to particular embodiments, the guide RNA further comprises a protected sequence and an opening sequence. According to particular embodiments, the opening sequence has 1-19 nucleotides. More particularly, the opening sequence is at least 75%, at least 90%, or about 100% complementary to the target sequence. According to particular embodiments, the guide sequence is at least 90% or about 100% complementary to the protection strand. According to particular embodiments, the guide sequence is at least 75%, at least 90%, or about 100% complementary to the target sequence. According to particular embodiments, the guide RNA further comprises an extension sequence. More particularly, the extension sequence is operably linked to the 3' end of the protected guide sequence, and optionally directly linked to the 3' end of the protected guide sequence. According to particular embodiments, the extension sequence has 1-12 nucleotides. According to particular embodiments, the extension sequence is operably linked to the guide sequence at the 3' end of the protected guide sequence and the 5' end of the guard strand, and optionally directly linked to the 3' end of the protected guide sequence and the 3' end of the guard strand, wherein the extension sequence is a linking sequence between the protected sequence and the guard strand. According to particular embodiments, the extension sequence is not 100% complementary to the guard strand, optionally is not at least 95% complementary to the guard strand, at least 90% complementary to the guard strand, at least 80% complementary to the guard strand, at least 70% complementary to the guard strand, at least 60% complementary to the guard strand, or at least 50% complementary to the guard strand. According to particular embodiments, the guide sequence further comprises mismatched bases attached to the end of the guide sequence, wherein the mismatched bases thermodynamically optimize specificity.

In one aspect, the present invention provides an engineered, non-naturally occurring CRISPR-Cas system comprising a Cas9 protein and a protected guide RNA that targets a DNA molecule encoding a gene product in a cell, whereby the protected guide RNA targets the DNA molecule encoding the gene product, and the Cas9 protein cleaves the DNA molecule encoding the gene product, whereby expression of the gene product is altered; and wherein the Cas9 protein and the protected guide RNA do not occur together in nature. The present invention encompasses a protected guide RNA that includes a guide sequence fused at the 3' end to a direct repeat sequence. The present invention further encompasses a Cas9 protein that is 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 present invention, expression of the gene product is reduced. In some embodiments, the Cas9 enzyme is Cas9 of Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium or Francisella Novicida and may include a mutated Cas9 derived from these organisms. The enzyme may be an additional homolog or ortholog of Cas9. In some embodiments, the nucleotide sequence encoding the Cfp1 enzyme is codon optimized for expression in a eukaryotic cell. In some embodiments, the Cas9 enzyme directs single or double strand cleavage 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. Generally and throughout this specification, 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, do not contain free ends (e.g., circular); 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 circular loop of double-stranded DNA into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication-deficient retroviruses, adenoviruses, replication-deficient adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) integrate into the host cell genome upon introduction into the host cell and thus replicate along with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are functionally linked. Such vectors are referred to herein as "expression vectors." Common expression vectors suitable for recombinant DNA techniques are often in the form of plasmids.

Recombinant expression vectors may comprise a nucleic acid of the present invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors comprise one or more regulatory elements, which may be selected taking into account the host cells that are intended to be used for expression, which are operably linked to the nucleic acid sequence that is intended to be expressed. In the context of a recombinant expression vector, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) such that expression of the nucleotide sequence is allowed (e.g. in an in vitro transcription/translation system or in a host cell upon introduction of the vector into the host cell).

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

In one aspect of the present invention, there is provided 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 sequences downstream of the direct repeat sequence, wherein, when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in the eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme in complex with a guide RNA providing a guide sequence that hybridizes to the target sequence, and/or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said Cas9 enzyme comprising a nuclear localization sequence. In some embodiments, the host cell comprises 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 guide sequences operably linked to the first regulatory element, wherein, when expressed, each of the two or more guide sequences directs sequence-specific binding of the CRISPR complex to its target sequence in a eukaryotic cell. In some embodiments, the Cas9 enzyme directs cleavage of one or two strands at a specific position of the target sequence. In some embodiments, the Cas9 enzyme does not have DNA strand cleavage 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 one aspect of the present invention, a eukaryotic organism other than a human is provided; preferably a multicellular eukaryotic organism comprising a eukaryotic host cell according to any of the described embodiments. In other aspects of the present invention, a eukaryotic organism is provided, preferably a multicellular eukaryotic organism comprising a eukaryotic host cell according to any of the described embodiments. In some embodiments of these aspects, the organism may be an animal; for example, a mammal. The organism may also be an arthropod, such as an insect. The organism may also be a plant or yeast. In addition, the organism may be a fungus.

In one aspect of the present invention, a kit is provided comprising one or more components as described herein above. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the vector system comprises (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 downstream of the direct repeat sequence, wherein, when expressed, the guide sequence directs sequence-specific binding of a Cas9-based CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a Cas9 enzyme in complex with a protected guide RNA comprising a guide sequence that hybridizes to the target sequence, and/or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said Cas9 enzyme comprising a nuclear localization sequence. In some embodiments, the kit comprises components (a) and (b) located in the same or different vectors of the system. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences directs sequence-specific binding of the CRISPR complex to its target sequence in a eukaryotic cell. In some embodiments, the Cas9 enzyme comprises one or more nuclear localization sequences characterized by sufficient efficiency to direct accumulation of said Cas9 enzyme in a detectable amount in the nucleus of a eukaryotic cell. In some embodiments, the Cas9 enzyme is Cas9 of Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, or Francisella tularensis 1 Novicida, and may include a mutated Cas9 derived from these organisms. The enzyme may be a homolog or ortholog of Cas9. In some embodiments, the CRISPR 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 CRISPR enzyme does not have DNA strand cleavage 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 one aspect of the present invention, there is provided a method for modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method comprises causing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide, thereby effecting modification of the target polynucleotide, wherein the CRISPR complex comprises a Cas9 enzyme in a complex with a protected guide RNA that provides a guide sequence hybridizable to a target sequence within said target polynucleotide. In some embodiments, said cleavage comprises cleavage of one or two strands at a specific position of the target sequence using said Cas9 enzyme. In some embodiments, said cleavage results in reduced transcription of the target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide using non-homologous end joining (NHEJ) gene insertion mechanisms, more particularly using an exogenous polynucleotide template, wherein said repair results in a mutation involving an insertion, deletion or substitution of one or more nucleotides in said target polynucleotide. In some embodiments, said mutation results in a change in one or more amino acids in a protein expressed from a gene comprising 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 direct the expression of one or more of: a Cas9 enzyme, a protected guide RNA comprising a guide sequence linked to a direct repeat sequence. In some embodiments, said vectors are delivered to a eukaryotic cell in a subject. 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 the subject's body prior to performing said modification. In some embodiments, the method further comprises returning said eukaryotic cell and/or cells derived therefrom to said subject.

In one aspect of the present invention, there is provided a method for modifying the expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises causing a Cas9-based CRISPR complex to bind to the polynucleotide, such that said binding results in increased or decreased expression of said polynucleotide; wherein the CRISPR complex comprises a Cas9 enzyme in a complex with a protected guide RNA that provides a guide sequence hybridizable to a target sequence within said polynucleotide. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cells, wherein the one or more vectors direct the expression of one or more of: the Cas9 enzyme and the protected guide RNA.

In one aspect of the present invention, there is provided a method for producing a model eukaryotic cell comprising a mutated gene responsible for developing a disease. In some embodiments, the gene responsible for developing a disease is any gene associated with an increased risk of having or developing a disease. In some embodiments, the method comprises (a) introducing one or more vectors into a eukaryotic cell, wherein the one or more vectors direct the expression of one or more of: a Cas9 enzyme and a protected guide RNA providing a guide sequence linked to a direct repeat sequence; and (b) causing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide within said gene responsible for developing a disease, wherein the CRISPR complex comprises a Cas9 enzyme in a complex with a guide RNA providing a sequence that hybridizes to a target sequence within the target polynucleotide, thereby producing a model eukaryotic cell comprising a mutated gene responsible for developing a disease. In some embodiments, said cleavage comprises cleaving one or two strands at a specific position of a target sequence using said Cas9 enzyme. In some embodiments, said cleavage results in reduced transcription of a target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide using non-homologous end joining (NHEJ) gene insertion mechanisms using an exogenous polynucleotide template, 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 a change in one or more amino acids in the expression of a protein from a gene comprising the target sequence.

In one aspect of the present invention, there is provided a method for developing a biologically active agent that modulates a cellular signaling event associated with a disease-promoting gene. In some embodiments, the disease-promoting gene is any gene associated with an increased risk of having or developing a disease. In some embodiments, the method comprises (a) contacting a test compound with a model cell according to any of the described embodiments; and (b) detecting a change in a readout that indicates a decrease or increase in a cellular signaling event associated with said mutation in said disease-promoting gene, thereby obtaining said biologically active agent that modulates said cellular signaling event associated with said disease-promoting gene.

In one aspect of the present invention, there is provided a recombinant polynucleotide comprising a protected guide sequence downstream of a direct repeat sequence, wherein, when expressed, the protected guide sequence directs sequence-specific binding of a CRISPR complex to a 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.

In one aspect of the present invention, there is provided a method of selecting one or more cells by introducing one or more mutations into a gene in one or more cells, the method comprising: introducing one or more vectors into the cell(s), wherein the one or more vectors direct the expression of one or more of: a Cas9 enzyme, a protected guide RNA providing a guide sequence, and an editing template; wherein the editing template comprises one or more mutations that abolish cleavage by the Cas9 enzyme; providing non-homologous end joining (NHEJ) gene insertion mechanisms of the editing template to a target polynucleotide in the cell(s) to be selected; allowing the CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide within said gene, wherein the CRISPR complex comprises a Cas9 enzyme in complex with a protected guide RNA providing a guide sequence that hybridizes to a target sequence within the target polynucleotide, wherein the binding of the CRISPR complex to the target polynucleotide induces cell death, thereby allowing selection of one or more cells into which one or more mutations have been introduced. In a preferred embodiment of the present invention, the cell to be selected may be a eukaryotic cell. Aspects of the present invention provide for the selection of specific cells without the need for a selection marker or a two-step method that may include a negative selection system.

With respect to mutations in the Cas9 enzyme, if the enzyme is not FnCas9, the mutations may be as described elsewhere herein; a conservative substitution for any of the amino acids that are substituted is also provided. In one aspect, the present invention provides for any or each or all embodiments discussed herein that the CRISPR enzyme comprises at least one or more, or at least two or more mutations, wherein the at least one or more mutations or the at least two or more mutations are selected from the mutations described elsewhere herein.

In a further aspect, the present invention relates to a computer-assisted method for identifying or designing potential compounds for insertion or binding to a CRISPR-Cas9 system or a functional part thereof, or vice versa (a computer-assisted method for identifying or designing potential CRISPR-Cas9 systems or a functional part thereof for binding to desired compounds) or to a computer-assisted method for identifying or designing potential CRISPR-Cas9 systems (e.g., in terms of predicting regions of a CRISPR-Cas9 system that can be manipulated, e.g., based on crystal structure data or based on Cas9 ortholog data, or taking into account where a functional group such as an activator or a repressor can be attached to a CRISPR-Cas9 system, or in relation to Cas9 truncations or in relation to the design of nickases), said method comprising:

the use of a computer system, such as a stored program computer, comprising a processor, a data storage system, an input device, and an output device, the stages of:

(a) inputting into a computer with a stored program, via said input device, data comprising three-dimensional coordinates of a subset of atoms from or associated with a crystal structure of CRISPR-Cas9, for example in a binding domain of the CRISPR-Cas9 system or alternatively or additionally in domains that vary based on differences among Cas9 orthologs or concerning Cas9 or concerning niccases or concerning functional groups, optionally with information on the structure of the CRISPR-Cas9 system complex(es), thereby obtaining a data set;

(b) comparing, using said processor, said data set with a computer database of structures stored in said computer data storage system, such as structures of compounds that bind, or are predicted to bind, or are claimed to bind, to the CRISPR-Cas9 system, or to Cas9 orthologues (e.g., Cas9s or to domains or regions that vary among Cas9 orthologues), or to a crystal structure of CRISPR-Cas9, or to nickases or to functional groups;

(c) selecting from said database, using computational methods, structure(s), such as CRISPR-Cas9 structures, that can bind to desired structures, desired structures that can bind to specific CRISPR-Cas9 structures, parts of the CRISPR-Cas9 system that can be manipulated, such as based on data on other parts of the CRISPR-Cas9 crystal structure and/or on Cas9 orthologs, truncated Cas9, novel nicks or specific functional groups, or positions for attachment of functional groups or functional group-CRISPR-Cas9 systems;

(d) designing, using computer methods, a model of the selected structure(s) and

(e) outputting data on the selected structure(s) to the specified output device;

and not necessarily the synthesis of one or more selected structures;

and additionally optional testing of the said synthesized selected structure(s) as a CRISPR-Cas9 system or such(s) in it;

or the method comprises: providing coordinates of at least two atoms of a CRISPR-Cas9 crystal structure, such as at least two atoms from a table of crystal structures for the CRISPR-Cas9 crystal structure set forth herein, or coordinates of at least a subdomain of a CRISPR-Cas9 crystal structure ("selected coordinates") that provide the structure of a candidate molecule comprising a binding molecule or parts of the CRISPR-Cas9 system that can be manipulated, such as from data on other parts of the CRISPR-Cas9 crystal structure and/or from Cas9 orthologs or the structure of functional groups, and fitting the structure of the candidate molecule to the selected coordinates, thereby obtaining product data comprising CRISPR-Cas9 structures that can bind to desired structures, desired structures that can bind to certain CRISPR-Cas9 structures, parts of the CRISPR-Cas9 system that can be manipulated, truncated Cas9, novel nickases or specific functional groups, or positions for attachment of functional groups or functional group-CRISPR-Cas9 systems, with output data about them; and optionally synthesizing the compound(s) based on said product data and further optionally including testing said synthesized compound(s) as a CRISPR-Cas9 system or the same therein.

Testing may include analysis of the CRISPR-Cas9 system obtained from the said synthesized selected structure(s), for example, with respect to binding or performance of the required function.

The output information in the above-mentioned methods may include data transmission, such as transmission of information via telecommunication, telephone, video conference, mass media, such as a presentation such as a computer presentation (e.g. POWERPOINT), the Internet, e-mail, a message using documents such as a computer program document (e.g. WORD), etc. Accordingly, the present invention also encompasses computer-readable media containing data on atomic coordinates in accordance with the crystal structure provided herein, wherein said data define a three-dimensional structure of CRISPR-Cas9 or at least one subdomain thereof, or data on a structure factor for CRISPR-Cas9, wherein said data on a structure factor can be obtained based on the data on atomic coordinates from the crystal structure provided herein. Computer-readable media may also contain any data from the above-mentioned methods. The present invention further encompasses methods using a computer system for obtaining or performing rational design as in the above-mentioned methods, providing either data on atomic coordinates in accordance with the crystal structure provided herein, wherein said data defines a three-dimensional structure of CRISPR-Cas9 or at least one subdomain thereof, or data on a structure factor for CRISPR-Cas9, wherein said data on a structure factor is obtainable based on the data on atomic coordinates from the crystal structure provided herein. The present invention further encompasses a method of conducting business, including providing a user with a computer system, or a storage medium, or data on a three-dimensional structure of CRISPR-Cas9, or at least one subdomain thereof, or a structure factor for CRISPR-Cas9, wherein said structure is set forth in the form of data on atomic coordinates from the crystal structure provided herein, or said structure factor is obtainable based on such data, or an electronic storage medium provided herein, or a data transmission provided herein.

A "binding site" or "active site" refers to, or consists essentially of, or consists of, a site (such as an atom, a functional group of an amino acid residue, or a plurality of such atoms and/or groups) in a binding cavity or region that can bind to a compound, such as a nucleic acid molecule, that is involved in the binding.

By "fitting" is meant determining, by automated or semi-automated means, the interactions between one or more atoms of a candidate molecule and at least one atom of a structure of the present invention and calculating the degree of stability of such interactions. The interactions include attractions and repulsions due to charge, steric aspects, etc. Various computer-aided fitting methods are described below.

By "root mean square (or rms) deviation" is meant the square root of the arithmetic mean of the squares of the deviations from the mean.

By "computer system" is meant hardware, software, and data storage used to analyze the data by atomic coordinates. The minimum hardware of the computer systems of the present invention typically comprises a central processing unit (CPU), input devices, output devices, and data storage. For visualization of the data by structure, it is desirable to provide a display or monitor. The data storage means may be RAM or means for accessing the machine-readable media of the present invention. Examples of such systems are a computer and tablet devices running Unix, Windows, or Apple operating systems.

By "machine-readable media" is meant any medium or media that can be read and accessed directly or indirectly by a computer, such as such that the media are suitable for use in the aforementioned computer system. Such media include, but are not limited to, magnetic storage devices such as floppy disks, hard disk storage devices, and magnetic tape; optical storage devices such as optical disks or CD-ROMs; electrical storage devices such as RAM and ROM; flash drives; cloud storage devices, and hybrids of these categories such as magnetic/optical storage devices.

The present invention encompasses the use of the protected guides described herein above in the optimized functional CRISPR-Cas enzyme systems described herein.

RISC formation through guide design

In some embodiments, the guide may be a protected guide (e.g., pgRNA) or a chaperoned guide (e.g., esgRNA), as described herein. In some embodiments, both are used in RISC. RISC is a key component of RNAi. RISC (RNA-induced silencing complex) is a multiprotein, more specifically a ribonucleoprotein, complex that includes one strand of a double-stranded RNA (dsRNA) fragment, such as a small interfering RNA (siRNA) or a microRNA (miRNA), which acts as a template for RISC to recognize a complementary messenger RNA (mRNA) transcript. The mRNA is then cleaved by one of the RISC components.

In this regard, the formation of RISC is advantageous in some embodiments. Guide RNAs according to various aspects of the present invention, including but not limited to protected and/or escorted guide RNAs, can be adapted to include RNA nucleotides that promote the formation of RISC, for example in combination with siRNA or miRNA, which can be provided or can, for example, already be expressed in the cell. This can be useful, for example, as a self-inactivating system for removing or destroying the guide.

In this case, the guide RNA may comprise a sequence complementary to a target miRNA or siRNA, which may or may not be present in the cell. Thus, only when the miRNA or siRNA is present, for example, as a result of expression (in the cell or through human intervention), is binding of the RNA sequence to the miRNA or siRNA observed, which then leads to cleavage of the guide RNA by the RNA-induced silencing complex (RISC) in the cell. Therefore, in some embodiments, the guide RNA comprises an RNA sequence complementary to the target miRNA or siRNA, and binding of the guide RNA sequence to the target miRNA or siRNA leads to cleavage of the guide RNA by the RNA-induced silencing complex (RISC) in the cell.

This is further explained below with specific reference to both protected and accompanied guides.

RISC formation using protected guides

For example, a protected guide may be described in the following aspect: an engineered, non-naturally occurring composition comprising a clustered regularly interspaced short palindromic repeats (Cas)-associated CRISPR system (CRISPR-Cas) with a polynucleotide sequence protected by a guide RNA (pgRNA) comprising (a) a guard sequence, (b) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, (c) a tracr mate sequence, and (d) a tracr sequence, wherein (a), (b), (c), and (d) are arranged in a 5' - 3' orientation, wherein the guard sequence comprises two or more nucleotides that are non-complementary to the target sequence, wherein, when transcribed, the tracr mate sequence hybridizes to the tracr sequence, and the guide sequence directs sequence-specific binding of the CRISPR complex to the target a sequence wherein the CRISPR complex comprises a type II Cas9 protein in a complex with (1) a guide sequence that hybridizes to a target sequence and (2) a tracr mate sequence that hybridizes to the tracr sequence, and wherein the polynucleotide sequence of one or more of the guide, tracr, and tracr mate sequence is modified.

In one aspect, the present protected guide system is used to provide secondary structure protection of 5' extensions of sgRNA. For example, applicants extend an sgRNA, thereby introducing a miRNA binding site, to make the sgRNA active only after the miRNA binding site is processed and cut by the RISC machinery. This would not be possible without secondary structure protection, since exonuclease processing would start at the 5' end and cut in the direction of the sgRNA. By adding a small loop with secondary structure in the 5' direction of the added miRNA site, the miRNA can be protected from cutting by exonucleases.

RISC formation using guided traces

In another example, a chaperoned guide can be described. In particular, esgRNAs inducible by miRNA are provided. In this case, the aptamer sequence of the chaperoned RNA is complementary to the target miRNA, so that if the target miRNA included in the RNA-induced silencing complex (RISC) is present in the cell, the aptamer sequence of the chaperoned RNA binds to the target miRNA, which leads to cleavage of the esgRNA by the RNA-induced silencing complex (RISC) in the cell.

In alternative embodiments, a number of primary and secondary structures may be provided at the 5' end of the esgRNA designed such that the RISC complex can gain access to the miRNA binding site. The esgRNA may have a first and second linker sequence located 5' of the guard sequence. In alternative embodiments, the length of each of linkers 1 and 2, for example, may independently be 0, 1, 2, 3, or 4 nucleotides, wherein the length of the guard sequence is 0, 1, or 2 nucleotides.

In an exemplary embodiment, the induction of esgRNA targeting can be demonstrated using miR-122 in a HEK.293 cell system in which miR-122 is not natively expressed. In the absence of exogenous miR-122, protected esgRNAs do not mediate targeted nuclease activity toward EMX1.3. Upon addition of exogenous miR-122 (100 ng/well), targeted cleavage of EMX1.3 was observed (as distinct cleavage products visible as electrophoretic variants on gels). This demonstrates that highly expressed endogenous miRNAs can be used in systems that provide genetically inducible sgRNAs. Any miRNA can be used in place of miRNA122, and the appropriate sequence is easily determined.

For example, an sgRNA can bind to an aptamer sequence of a "chaperone" RNA that is complementary to an endogenous target miRNA. The target miRNA can form a RNA-induced silencing complex (RISC) in the cell. If the target miRNA is present in the cell, the aptamer sequence of the chaperone RNA binds to the target miRNA, causing the cellular RISC to cleave the esgRNA. Cleavage of the chaperone sequence releases the active sgRNA.

For example, a protected guide may be described in the following aspect: a non-naturally occurring or engineered composition comprising an escorted single guide RNA (esgRNA) of CRISPR-Cas9 comprising:

a guide RNA sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell; and

aptamer sequence of the chaperoned RNA,

wherein the aptamer sequence of the chaperoned RNA provides binding affinity for an aptamer ligand on or in a cell, or the aptamer sequence of the chaperoned RNA responds to a localized effector of the aptamer on or in a cell,

wherein the presence of the ligand or effector of the aptamer on or in the cell is limited in time or space.

The chaperone aptamer RNA sequence may be complementary to a target miRNA, which may or may not be present in the cell, such that only when the target miRNA is present is binding of the chaperone aptamer RNA sequence to the target miRNA observed, resulting in cleavage of the esgRNA by the RNA-induced silencing complex (RISC) in the cell. Therefore, in some embodiments, the aptamer sequence of the chaperone RNA is complementary to the target miRNA, and binding of the aptamer sequence of the chaperone RNA to the target miRNA results in cleavage of the esgRNA by the RNA-induced silencing complex (RISC) in the cell.

According to the present invention, a nucleotide sequence encoding at least one of said guide RNA or Cas protein is operably linked in a cell to a regulatory element comprising a promoter of a gene of interest, whereby expression of at least one component of the CRISPR-Cas system is driven by the promoter of the gene of interest. "Operably linked" is intended to mean that the nucleotide sequence encoding the guide RNA and/or Cas is linked to the regulatory element(s) in a manner that allows expression of the nucleotide sequence, as also described elsewhere herein. The term "regulatory elements" is also described elsewhere herein. According to the present invention, the regulatory element comprises a promoter of a gene of interest, such as, preferably, a promoter of an endogenous gene of interest. In certain embodiments, the promoter is in its endogenous position in the genome. In such embodiments, the nucleic acid encoding the CRISPR and/or Cas is under the transcriptional control of the promoter of the gene of interest at its native location in the genome. In certain other embodiments, the promoter is provided on a (separate) nucleic acid molecule, such as a vector or plasmid, or other extrachromosomal nucleic acid, i.e., the promoter is not provided at its native location in the genome. In certain embodiments, the promoter is integrated into the genome at a non-native location in the genome.

In certain embodiments, a nucleic acid encoding a guide RNA is operably linked in a cell to a regulatory element providing a promoter of a gene of interest. In certain embodiments, a nucleic acid encoding a Cas is operably linked in a cell to a regulatory element providing a promoter of a gene of interest. In certain embodiments, a nucleic acid encoding a guide RNA is operably linked in a cell to a regulatory element providing a promoter of a gene of interest, and a nucleic acid encoding a Cas is operably linked in a cell to a regulatory element providing a promoter of a gene of interest. In the latter case, the promoter directing the expression of the guide RNA and Cas may be the same or may be different. In certain embodiments, a nucleic acid encoding a guide RNA and/or Cas is integrated into the genome. In certain embodiments, a nucleic acid encoding a guide RNA and/or Cas is extrachromosomal or episomal. The nucleic acid encoding the guide RNA and the nucleic acid encoding Cas may be located on the same or different nucleic acid molecules.

The selected DNA sequences targeted by the guide RNA(s) of the present invention may be endogenous DNA sequences or exogenous DNA sequences. The selected DNA sequences, such as exogenous DNA sequences targeted by the guide RNA(s) of the present invention, may be integrated into the genome or may be extrachromosomal (e.g., provided in a plasmid or vector). In certain embodiments, the methods described herein comprise introducing a vector or plasmid into a cell by means known in the art, as described elsewhere herein, wherein said vector or plasmid comprises said selected DNA sequence, and said method comprises detecting a modification of said selected DNA sequence in said vector. It will be understood that said vector or plasmid or at least a selected DNA sequence contained therein may integrate into the genome, such as by random integration or by homologous recombination. If the selected target DNA sequence is an endogenous sequence, it is preferred that the sequence is selected such that the modification does not affect the (normal) functioning of the cell or has a minimal effect. Such sequences are readily identified by a person skilled in the art by conventional analysis or experimentation. In any case, it is preferred that such a selected endogenous target DNA sequence is not located in the coding sequence or ORF of a gene and/or is not located in regulatory sequences of a gene (such as promoters, enhancers, silencers, etc.).

As described elsewhere in this document, the selected target DNA sequence is modified by a functional CRISPR complex (i.e., the guide RNA is in a complex with a Cas protein, wherein the guide RNA comprises a guide sequence, a tracr mate sequence, and a tracr sequence in a 5' to 3' orientation, wherein the tracr sequence may or may not be located on the same nucleic acid molecule as the guide sequence and the tracr mate sequence). As used herein, the term "modified" is generally understood to mean "mutated," i.e., the nucleic acid sequence of the target DNA sequence is altered as described elsewhere in this document, such as by point mutations, deletions, substitutions, or insertions of one or more nucleotides.

However, as described elsewhere herein, it will also be appreciated that in certain embodiments, the term "modified" corresponds to alterations of target loci, such as activation or repression of gene transcription, methylation or demethylation of CpG sites, and the like, which may not require point mutations, deletions, substitutions, or insertions of one or more nucleotides. Moreover, as described elsewhere herein, it will also be appreciated that reference to a CRISPR-Cas enzyme as "altering" or "modifying" one or more target polynucleotide loci encompasses a direct alteration or modification, such as by the catalytic activity of the enzyme itself, but also an indirect alteration or modification, such as by a catalytic activity associated with the CRISPR-Cas enzyme, such as that of a heterologous functional domain, such as a transcriptional activation domain. In addition, as will be appreciated, it is contemplated that one or more target polynucleotide loci that are "altered" or "modified" by the action of a CRISPR-Cas enzyme may be contained within or near a polynucleotide sequence complementary to a portion of a guide RNA that is a guide sequence, such as in embodiments where the alteration or modification is accomplished by the catalytic activity of the CRISPR-Cas enzyme itself, such as cleavage of DNA by the nuclease activity of the CRISPR-Cas enzyme. However, embodiments are also encompassed where one or more target loci to be "altered" or "modified" are located at a specific position other than a sequence complementary to a portion of a guide RNA that is a guide sequence, such as in embodiments where the alteration or modification is accomplished by a heterologous functional domain associated with the CRISPR-Cas enzyme, such as activation or repression of gene transcription. In this regard, "altering" or "modifying" (or similar terms) a target locus means that this is achieved through the direct or indirect action of the CRISPR-Cas enzyme, and furthermore, the "target locus" to be altered or modified and the "target sequence" that is complementary to the portion of the guide RNA that is the guide sequence may or may not be the same.

In certain embodiments, in the methods of the invention described herein, the CRISPR-Cas system is multiplexed, i.e., multiple different guide RNAs can be provided. Each guide RNA can target (i.e., hybridize to) a different selected target DNA. The expression of the different guide RNAs can be driven by promoters of different genes of interest. Accordingly, in certain embodiments, the methods of the invention described herein are methods of detecting the expression of more than one, such as at least two, genes of interest in a cell, comprising providing a cell comprising a CRISPR-Cas system, wherein the CRISPR-Cas system comprises more than one, such as at least two, guide RNAs that target a different selected DNA sequence, and the Cas protein is capable of modifying the selected DNA sequence; wherein each guide RNA is operably linked in the cell to a regulatory element that provides a promoter of a different gene of interest; and determining the expression of said genes of interest based on the detection of a modification of said respective selected DNA sequences. In certain embodiments, more than one different guide RNA may be operably linked in a cell to a regulatory element providing a promoter of the same gene of interest. The different guide RNAs may be provided on different nucleic acid molecules or on the same nucleic acid molecule. The respective guide RNAs may be designed such that only a modification of a first selected target DNA creates or destroys a second selected target DNA.

In certain embodiments, one or more components of a CRISPR-Cas system may be expressed in a cell in a conditionally dependent manner (e.g., tissue-specific or cell type-specific expression) and/or in an inducible manner (e.g., chemically inducible expression). Conditionally inducible expression systems are described elsewhere herein. In particular embodiments, one or more guide RNAs may be expressed in a cell in a conditionally dependent manner and/or in an inducible manner. In particularly preferred embodiments, Cas may be expressed in a cell in a conditionally dependent manner and/or in an inducible manner.

As used herein, the term "targeting" a selected DNA sequence means that the guide RNA is capable of hybridizing to the selected DNA sequence. As used herein, "hybridization" or "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. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogsteen bonding, or any other sequence-specific method. The complex may comprise two strands forming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combination thereof. The hybridization reaction may be a step in a larger process, such as the initial step of PGR or the cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing to a given sequence is referred to as a "complementary sequence" for the sequence.

As used herein, the term "expression" or "causing expression" means the process by which a polynucleotide is transcribed from a DNA template (such as to form mRNA or other RNA transcripts) and/or the process by which the transcribed mRNA is further translated to form peptides, polypeptides, or proteins. The transcripts and encoded polypeptides may be referred to collectively as the "gene product." If the polynucleotide is derived from genomic DNA, expression may involve splicing of mRNA in a eukaryotic cell. As used herein, "expression" of a gene or nucleic acid encompasses not only the expression of genes in cells, but also the transcription and translation of nucleic acid(s) in cloning systems and in any other context.

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, it may contain modified amino acids and its structure may be interrupted by components other than amino acids. The terms also encompass a polymer of amino acids that has been modified; for example, by disulfide bonding, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation such as coupling with a labeling component. As used herein, the term "amino acid" includes natural and/or non-natural or synthetic amino acids, including glycine and both the D- and I-optical isomers, and amino acid analogs and peptidomimetics.

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 included are tissues, cells, and progeny thereof, of a biological organism, whether obtained in vivo or cultured in vitro.

In certain embodiments, the methods and cells of the present invention described herein can be used in methods for screening therapeutic agents and/or diagnostic methods. Candidate therapeutic agents can have different effects on temporal expression profiles that can be recorded according to the methods described herein.

The terms "therapeutic agent", "agent producing a therapeutic effect", or "agent for treating" are used interchangeably and mean a molecule or compound that produces some beneficial effect when administered to a subject. Beneficial effects include the ability to make diagnostic determinations; alleviate a disease, symptom, disorder, or condition; reduce or prevent the onset of a disease, symptom, disorder, or condition; and generally counteract a disease, symptom, disorder, or condition.

As used herein, "treating" or "treating" or "alleviating" or "alleviating" are used interchangeably. These terms refer to an approach to obtaining beneficial or desired results, including but not limited to a therapeutic effect and/or a prophylactic effect. A therapeutic effect is understood to mean any therapeutically significant improvement or effect with respect to one or more diseases, conditions or symptoms being treated. For a prophylactic effect, the compositions may be administered to a subject at risk of developing a particular disease, condition or symptom or to a subject who reports one or more physiological symptoms of the disease, even though the disease, condition or symptom may not have manifested yet.

As used herein, the terms "chimeric RNA", "chimeric guide RNA", "guide RNA", "single guide RNA" and "synthetic guide RNA" refer to a polynucleotide sequence comprising a guide sequence, a tracr sequence and a tracr mate sequence. The term "guide sequence" refers to a sequence of approximately 20 bp within the guide RNA that defines a target site and can be used interchangeably with the terms "guide" or "spacer". The term "tracr mate sequence" can also be used interchangeably with the term "direct repeat(s)". The guide sequence, tracr and tracr mate sequence can be provided on a single nucleic acid molecule. Alternatively, the guide and tracr mate sequences may be provided on a single nucleic acid molecule, while the tracr sequence is provided on a separate nucleic acid molecule.

In general, a CRISPR-Cas or CRISPR system is as used in the above-mentioned documents such as WO 2014/093622 (PCT/US2013/074667), and refers collectively to transcripts and other elements involved in the expression or control of the activity of CRISPR-associated ("Cas") genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or active partial tracrRNA), a tracr mate sequence (encompassing a "direct repeat" and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a "spacer" in the context of an endogenous CRISPR system), or "RNA" as that term is used herein (e.g., RNA for targeting Cas such as Cas9, e.g., CRISPR RNA and trans-activating (tracr) RNA or single guide RNA (sgRNA) (chimeric RNA)), or other sequences and transcripts from a CRISPR locus. In general, a CRISPR system is characterized by elements that facilitate the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of the endogenous CRISPR system). In the context of CRISPR complex formation, a "target sequence" refers to a sequence to which a guide sequence is designed to be complementary, wherein hybridization between the target sequence and the guide sequence facilitates the formation of a CRISPR complex. The target sequence may comprise any polynucleotide, such as DNA or RNA 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 searching for repeat motifs that meet any or all of the following criteria: 1. are within a 2 kb window of genomic sequence flanking a Type II CRISPR locus; 2. span from 20 to 50 bp; and 3. alternate with fragments of 20-50 bp. In some embodiments, 2 of these criteria may apply, such as 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all 3 criteria may apply.

In embodiments of the present invention, the terms guide sequence and guide RNA, i.e., RNA capable of directing Cas to a target locus in the genome, are used interchangeably, as in the above-mentioned cited documents such as WO 2014/093622 (PCT/US2013/074667). In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize to the target sequence and direct sequence-specific binding of a CRISPR 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 about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. An optimal alignment can be determined using any suitable sequence alignment algorithm, including, but not limited to, the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transform (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 length of the guide sequence is about or more 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 some embodiments, the length of the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides. Preferably, the length of the guide sequence is 10-30 nucleotides. The ability of the guide sequence to direct sequence-specific binding of the CRISPR complex to a target sequence can be assessed using any suitable assay. For example, components of a CRISPR system sufficient to form a CRISPR complex, including a guide sequence to be tested, can be provided to a host cell having an appropriate target sequence, such as by transfection with vectors encoding components of the CRISPR sequence, followed by assessment of preferential cleavage within the target sequence, such as by the Surveyor nuclease assay described herein. Similarly, cleavage of a target polynucleotide sequence can be determined in vitro by providing a target sequence, components of a CRISPR complex, including a guide sequence to be tested, and a control guide sequence different from the test guide sequence, and comparing binding or the extent of cleavage of the target sequence in reactions with the test and control guide sequences. Other assays are possible and can be performed by those skilled in the art.

A guide sequence, i.e., an RNA capable of directing Cas to a target locus in the genome, can be selected to target any target sequence. In some embodiments, the target sequence is a sequence within the genome of a cell. Exemplary target sequences include sequences that are unique in the target genome. For example, for S. pyogenes Cas9, a unique target sequence in the genome can include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGG, where NNNNNNNNNNNNXGG (N is A, G, T, or C; and X can be any) is characterized by a single occurrence in the genome. A unique target sequence in the genome may include a target site for S. pyogenes Cas9 of the form MMMMMMMMMNNNNNNNNNNNXGG, where NNNNNNNNNNNXGG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. For S. thermophilus CRISPR1 Cas9, a unique target sequence in the genome may include a target site for Cas9 of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW, where NNNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T) has a single occurrence in the genome. A unique target sequence in the genome may include a target site for S. thermophilus CRISPR1 Cas9 of the form MMMMMMMMMNNNNNNNNNNNXXAGAAW, where NNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T) has a single occurrence in the genome. For S. pyogenes Cas9, a unique target sequence in the genome may include a target site for Cas9 of the form MMMMMMMMNNNNNNNNNNNNXGGXG, where NNNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. A unique target sequence in a genome may include a target site for S. pyogenes Cas9 in the form MMMMMMMMMNNNNNNNNNNNXGGXG, where NNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be any) has a single occurrence in the genome. In each of these sequences, "M" may be A, G, T, or C and should not be considered in identifying the sequence as unique. In some embodiments, the guide sequence is selected to reduce the proportion of secondary structure within the guide sequence. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less of the nucleotides of the guide sequence participate in self-complementary base pairing upon optimal folding. Optimal folding can be determined using any suitable polynucleotide folding algorithm. Some programs are based on the calculation of the minimum Gibbs free energy. An example of one such algorithm is mFold, which is described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133–148). Another example of a folding algorithm is the online web server RNAfold, developed at the Institute for Theoretical Chemistry at the University of Vienna, which uses a centroid-based structure prediction algorithm (see, e.g., A. R. Gruber et al., 2008, Cell 106(1): 23–24; and P. A. Carr and G. M. Church, 2009, Nature Biotechnology 27(12): 1151–62).

In general, a tracr mate sequence includes any sequence that has sufficient complementarity with a tracr sequence to facilitate one or more of the following: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at a target sequence, wherein the CRISPR complex contains a tracr mate sequence that is hybridized to the tracr sequence. In general, the degree of complementarity is specified with respect to an optimal alignment of the tracr mate sequence and the tracr sequence over the entire length of the shorter of the two sequences. The optimal alignment may be determined using any suitable alignment algorithm and may additionally take into account secondary structures, such as a region of self-complementarity within either the tracr sequence or the tracr mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and the tracr mate sequence over the entire length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more. In some embodiments, the length of the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 or more nucleotides. In some embodiments, the tracr sequence and the tracr mate sequence are contained in a single transcript such that hybridization between the two produces a transcript with a secondary structure such as a hairpin. In one embodiment of the invention, 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, the transcript has no more than five hairpins. In the hairpin structure, the portion of the sequence in the 5' direction relative to the terminal "N" and upstream of the loop corresponds to the tracr mate sequence, and the portion of the sequence in the 3' direction relative to the loop corresponds to the tracr sequence. Additional non-limiting examples of individual polynucleotides comprising a guide sequence, a tracr mate sequence, and a tracr sequence are the following (listed from 5' to 3'), wherein "N" is the base of the guide sequence, the first block of lowercase letters is the tracr mate sequence, the second block of lowercase letters is the tracr sequence, and the final poly-T sequence is a transcription terminator: (1) NNNNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: X); (2) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT; (SEQ ID NO: X) (3) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccgaaatcaacaccctgtcattttatggcagggtgtTTTTTT; (SEQ ID NO: X) (4) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTT; (SEQ ID NO: X) (5) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaacttgaaaaagtgTTTTTTT; and (SEQ ID NO: X) (6) NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTTTTT (SEQ ID NO: X). In some embodiments, sequences (1) - (3) are used in combination with Cas9 from S. thermophilus CRISPR1. In some embodiments, sequences (4) - (6) are used in combination with Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a transcript separate from the transcript comprising the tracr mate sequence.

In some embodiments, a candidate tracrRNA can subsequently be predicted using sequences that meet any or all of the following criteria: 1. sequence homology to direct repeats (motif search using Geneious software with mismatches of up to 18 bp); 2. the presence of a predicted Rho-independent transcription terminator in the direction of transcription; and 3. a stable hairpin secondary structure between the tracrRNA and the direct repeat. In some embodiments, 2 of these criteria may apply, such as 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all 3 criteria may apply.

In some embodiments, chimeric synthetic guide RNA (sgRNA) constructs may include at least a 12 bp duplex structure between the direct repeat and the tracrRNA.

RNAs for targeting Cas, such as Cas9, may comprise a CRISPR RNA and a transactivating (tracr) RNA. The tracr and tracr mate sequences may be joined to form a transactivating (tracr) sequence. The tracr and tracr mate sequences may optionally be designed to form a single guide RNA (sgRNA). Indeed, it is advantageous that the RNAs for targeting Cas may comprise a chimeric single guide RNA (sgRNA). The tracr sequence and the tracr mate sequence over the entire length of the shorter of the two, when optimally aligned, may be complementary by about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more. The length of the tracr sequence may be about or greater than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50 or more nucleotides.

In classical CRISPR-Cas systems, the degree of complementarity between a guide and its corresponding target sequence may be about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%; wherein the length of the guide RNA or sgRNA may be 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; or the length of the guide RNA or sgRNA may be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides; and preferably the length of the tracrRNA is 30 or 50 nucleotides. However, an aspect of the present invention is directed to reducing off-target interactions, for example, reducing the interaction of the guide with a target sequence characterized by low complementarity. Indeed, the examples show that the present invention provides mutations that cause the CRISPR-Cas system to be able to recognize target and non-target sequences that are characterized by greater than 80% to about 95% complementarity, such as 83%-84%, or 88-89%, or 94-95% complementarity (e.g., distinguishing between an 18 nucleotide long target sequence and an 18 nucleotide long non-target sequence with 1, 2, or 3 mismatches). Accordingly, in the context of the present invention, the degree of complementarity between a guide sequence and its corresponding target sequence is more than 94.5%, or 95%, or 95.5%, or 96%, or 96.5%, or 97%, or 97.5%, or 98%, or 98.5%, or 99%, or 99.5%, or 99.9%, or 100%. The non-target locus has less than 100%, or 99.9%, or 99.5%, or 99%, or 99%, or 98.5%, or 98%, or 97.5%, or 97%, or 96.5%, or 96%, or 95.5%, or 95%, or 94.5%, or 94%, or 93%, or 92%, or 91%, or 90%, or 89%, or 88%, or 87%, or 86%, or 85%, or 84%, or 83%, or 82%, or 81%, or 80% complementarity between its sequence and the guide, wherein it is preferable that the non-target locus has 100%, or 99.9%, or 99.5%, or 99%, or 99%, or 98.5%, or 98%, or 97.5%, or 97%, or 96.5%, or 96%, or 95.5%, or 95%, or 94.5% complementarity between its sequence and the guide.

In particularly preferred embodiments according to the present invention, the guide RNA (capable of directing Cas to a target locus) may comprise (1) a guide sequence capable of hybridizing to a target locus of the genome in a eukaryotic cell; (2) a tracr sequence; and (3) a tracr mate sequence. All of (1)-(3) may be in a single RNA, i.e., an sgRNA (located in a 5'-3' orientation), or the tracrRNA may be an RNA different from the RNA containing the guide and tracr sequence. The tracr sequence hybridizes to the tracr mate sequence and directs the CRISPR/Cas complex to the target sequence.

The methods of the present invention described herein encompass inducing one or more mutations in a eukaryotic cell (in vitro, i.e., in an isolated eukaryotic cell) as discussed herein, comprising delivering to the cell a vector as discussed herein. The mutation(s) may comprise the introduction, deletion, or substitution of one or more nucleotides in each target sequence of the cell(s) using a guide(s) RNA or sgRNA. The mutations may comprise the introduction, deletion, or substitution of 1-75 nucleotides in each target sequence of said cell(s) using a guide(s) RNA or sgRNA. Mutations may include the introduction, deletion or substitution of 1, 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 or 75 nucleotides in each target sequence of the specified cell(s) using guide RNA or sgRNA. Mutations may include the introduction, deletion or substitution of 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 or 75 nucleotides in each target sequence of the specified cell(s) using guide RNA or sgRNA. Mutations include the introduction, deletion or substitution of 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 or 75 nucleotides in each target sequence of the specified cell(s) using guide(s) RNA or sgRNA. The mutations may include the introduction, deletion or substitution of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or 75 nucleotides in each target sequence of said cell(s) using guide(s) RNA or sgRNA. The mutations may include the introduction, deletion or substitution of 40, 45, 50, 75, 100, 200, 300, 400 or 500 nucleotides in each target sequence of said cell(s) using guide(s) RNA or sgRNA.

To minimize toxicity and off-target effects, it will be important to control the concentration of Cas mRNA and guide RNA delivered. Optimal concentrations of Cas mRNA and guide RNA can be determined by testing different concentrations in a cellular or non-human animal-eukaryotic model and using deep sequencing to analyze the degree of modification at potential off-target loci in the genome. Alternatively, to minimize the level of toxicity and off-target effects, Cas nickase mRNA (e.g., S. pyogenes Cas9 with the D10A mutation) can be delivered with a pair of guide RNAs targeting the site of interest. Guide sequences and strategies to minimize toxicity and off-target effects can be as in WO 2014/093622 (PCT/US2013/074667); or by mutation as described herein.

In some embodiments, the CRISPR system is predominantly derived from a type II CRISPR system. In some embodiments, one or more elements of the CRISPR system are derived from a particular organism that contains an endogenous CRISPR system, such as Streptococcus pyogenes. In preferred embodiments of the present invention, the CRISPR system is a type II CRISPR system and the Cas enzyme is Cas9, which catalyzes DNA cleavage. Non-limiting examples of Cas proteins include 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, homologs thereof, or modified versions thereof. A preferred Cas enzyme may be identified as Cas9, as it may refer to a general class of enzymes that are homologous to the largest multiple nuclease domain nuclease of the Type II CRISPR system. Most preferably, the Cas9 enzyme is derived from or is derived from SpCas9 or SaCas9. It should be understood that SpCas9 or SaCas9 is derived from or is derived from S. pyogenes or S. aureus Cas9. By derived, applicants mean that the derived enzyme is essentially based on the wild-type enzyme in the sense that it has a high degree of sequence homology to that enzyme, but has been mutated (modified) in some way, as described herein. It should be understood that the terms Cas enzyme and CRISPR are generally used interchangeably herein, unless it is obvious otherwise. The Cas enzyme may be, for example, any naturally occurring bacterial Cas9, as well as any chimeric forms, mutant forms, homologues or orthologues. Most of the residue numbering variations used herein refer to the Cas9 enzyme from the Streptococcus pyogenes type II CRISPR locus (alternatively annotated as SpCas9 or spCas9). However, it should be understood that the present invention includes many other Cas9s from other microbial species, such as orthologs of SpCas9, or Cas9s derived from microorganisms other than S. pyogenes, such as SaCas9 from S. aureus, St1Cas9 from S. thermophilus, etc. One skilled in the art will be able to determine suitable corresponding residues in Cas9 enzymes other than SpCas9 by comparing the relevant amino acid sequences. Thus, where a particular amino acid substitution is indicated using SpCas9 numbering, unless the context clearly indicates that it is not intended to apply to other Cas9 enzymes, the present invention is intended to cover corresponding modifications in other Cas9 enzymes.

In some embodiments, the unmodified Cas has DNA cleavage activity similar to Cas9. In some embodiments, the Cas directs cleavage of one or both strands at a specific position of a target sequence, such as within the target sequence and/or within a sequence complementary to the target sequence. In some embodiments, the Cas directs cleavage of one or both strands within about 1, 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 vector encodes a Cas that is mutated compared to the corresponding wild-type enzyme such that the mutated Cas lacks the ability to cleave one or both strands of a target polynucleotide containing the target sequence. For example, replacing aspartate with alanine (D10A) in the catalytic domain of RuvC I Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves one strand). Other examples of mutations that make Cas9 a nickase include, but are not limited to, H840A, N854A, and N863A. As a further example, two or more of the catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III, or the HNH domain) can be mutated to produce a mutated Cas9 that substantially completely lacks DNA cleavage activity. In some embodiments, the D10A mutation is combined with one or more of the H840A, N854A, or N863A mutations to produce a Cas9 enzyme that substantially completely lacks DNA cleavage activity. In some embodiments, a Cas enzyme is considered to have substantially no DNA cleavage activity if the DNA cleavage activity of the mutated enzyme is no more than about 25%, 10%, 5%, 1%, 0.1%, 0.01% or less relative to the DNA cleavage activity of the unmutated form of the enzyme; an example would be where the DNA cleavage activity of the mutated form is absent or insignificant compared to the unmutated form. Thus, Cas may contain one or more mutations and may be used as a universal DNA binding protein, fused or not fused to a functional domain. The mutations may be artificially introduced mutations or gain-function or loss-of-function mutations. The mutations may include, but are not limited to, mutations in one of the catalytic domains (e.g., D10 and H840) in the RuvC and HNH catalytic domains, respectively; or the CRISPR enzyme may comprise one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A, or D986A, and/or one or more mutations in the RuvC1 or HNH domain of Cas, or have another mutation discussed herein. In one aspect of the present invention, the Cas enzyme may be fused to a protein, such as a TAG, and/or an inducible/controllable domain, such as a chemically inducible/controllable domain. In the present invention, the Cas may be chimeric Cas proteins, such as a Cas that has enhanced function due to being a chimera. The chimeric Cas proteins may be novel Cas that contain fragments of more than one naturally occurring Cas. They may comprise fusion products of the N-terminal fragment(s) of one Cas9 homologue with the C-terminal fragment(s) of another Cas homologue. Cas may be delivered to the cell in the form of mRNA. Expression of Cas may be under the control of an inducible promoter. If the enzyme is not SpCas9, mutations may be made at any or all of the residues corresponding to positions 10, 762, 840, 854, 863, and/or 986 of SpCas9 (which may be determined, for example, using standard sequence comparison tools). In particular, any or all of the following mutations are preferred for SpCas9: D10A, E762A, H840A, N854A, N863A, and/or D986A; and conservative substitution of any of the substitutable amino acids is also contemplated. The same (or conservative substitutions for these mutations) are also preferred in the corresponding positions of other Cas9. Particularly preferred are D10 and H840 in SpCas9. However, in other Cas9, residues corresponding to D10 and H840 of SpCas9 are also preferred. It is obvious that the purpose of the present invention is not to cover known mutations. That is, mutations that are known in the art to cause Cas9 to become a nickase or Cas9 to become "non-functional", such as having little, such as 5%, or less than 5%, such as less than 4%, 3%, 2% or 1%, nuclease activity or no nuclease activity compared to unmutated Cas9, are intended to be excluded from the scope of mutations in Cas9 that reduce or eliminate the interaction between the target and non-target nucleic acid molecules, and applicant reserves the right to use a disclaimer to exclude such a known mutation that causes a "nickase" or "non-functional" Cas9. Indeed, the phrase "whereby an enzyme in a CRISPR complex has a reduced ability to modify one or more off-target loci compared to an unmodified enzyme, and/or whereby an enzyme in a CRISPR complex has an increased ability to modify one or more target loci compared to an unmodified enzyme" (or similar expressions) is not intended to encompass mutations that result only in nickase or non-functional Cas9, or known Cas9 mutations. HOWEVER, this does not mean that the modification(s) or mutation(s) of the present invention "whereby the enzyme in the CRISPR complex has a reduced ability to modify one or more off-target loci compared to an unmodified enzyme and/or whereby the enzyme in the CRISPR complex has an increased ability to modify one or more target loci compared to an unmodified enzyme" (or similar expressions) cannot be combined with mutations that cause the enzyme to be a nickase or to be non-functional. Such a non-functional enzyme may be an improved nucleic acid binding agent. And such a nickase may be an improved nickase. For example, changing a neutral amino acid(s) in and/or near a groove and/or other charged residues at other specific positions in Cas9 that are in close proximity to a nucleic acid (e.g., DNA, cDNA, RNA, sgRNA) to a positively charged amino acid(s) may result in a situation "whereby an enzyme in a CRISPR complex has a reduced ability to modify one or more off-target loci compared to an unmodified enzyme and/or whereby an enzyme in a CRISPR complex has an increased ability to modify one or more target loci compared to an unmodified enzyme," such as a higher number of cuts. Since these can be either enhanced on-target or off-target cuts (over-cutting Cas9), the use of such an enzyme with what is known in the art as truncated guide or truncated sgRNAs (see, e.g., Fu et al., "Improving CRISPR-Cas nuclease specificity using truncated guide RNAs," Nature Biotechnology 32, 279–284 (2014) doi:10.1038/nbt.2808, received 17 November 2013, accepted 06 January 2014, published online 26 January 2014, corrected online 29 January 2014) to provide enhanced on-target activity without increasing the number of off-target cuts, or to produce over-cutting nickases, or to combine with a mutation that renders Cas9 non-functional for the superbinder.

In practicing the present invention, orthologs of SpCas9 can be used. The Cas enzyme can be identified as Cas9 because it can denote a general class of enzymes that have homology to the largest multiple nuclease domain nuclease of the type II CRISPR system. Most preferably, the Cas9 enzyme is derived from or is derived from SpCas9 (Cas9 of S. pyogenes) or SaCas9 (Cas9 of S. aureus). "StCas9" refers to the wild-type Cas9 from S. thermophilus, and its protein sequence is provided in the SwissProt database under the accession number G3ECR1. Similarly, Cas9 of S. pyogenes or SpCas9 is included in SwissProt under the accession number Q99ZW2. By "derived" we mean that the derived enzyme is essentially based on the wild-type enzyme in the sense that it has a high degree of sequence homology to that enzyme, but has been mutated (modified) in some way as described herein. It should be noted that the terms "Cas" and "CRISPR enzyme" are generally used interchangeably herein unless otherwise obvious. As mentioned above, most of the residue numbering variations used herein refer to the Cas9 enzyme from the CRISPR II locus of Streptococcus pyogenes. However, it should be noted that the present invention includes many other Cas9s from other microbial species, such as SpCas9, SaCa9, St1Cas9, etc. The enzymatic action of Streptococcus pyogenes-derived Cas9 or any closely related Cas9 results in the formation of double-strand breaks in target site sequences that hybridize to 20 nucleotides of a guide sequence and that contain a protospacer adjacent motif (PAM) sequence (examples include NGG/NRG or a PAM that can be defined as described herein) following 20 nucleotides of the target sequence. The CRISPR activity of Cas9, which consists of site-specific recognition and cleavage of DNA, is determined by a guide sequence, a tracr sequence, which partially hybridizes to the guide sequence and the PAM sequence. Without being bound by theory, it is believed that the target sequence must be associated with a PAM (protospacer adjacent motif); that is, a short sequence recognized by the CRISPR complex. The requirements for the accuracy and length of the PAM sequence vary depending on the Cas enzyme used, but PAMs are typically 2-5 base pair long sequences adjacent to the protospacer (i.e., the target sequence). In some embodiments, the method includes causing a CRISPR complex to bind to a target polynucleotide to effect cleavage of said target polynucleotide, thereby causing the target polynucleotide to be modified, wherein the CRISPR complex comprises a Cas enzyme complexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence that is in turn hybridized to the tracr sequence. More aspects of the CRISPR system are described in Karginov and Hannon, The CRISPR system: small RNA-guided defense in bacteria and archaea, Mole Cell 2010, January 15; 37(1): 7. The type II CRISPR locus is derived from Streptococcus pyogenes SF370, which contains a cluster of four genes, Cas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements, tracrRNA, and a characteristic array of repetitive sequences (direct repeats) interspersed with short fragments of non-repetitive sequences (spacers, approximately 30 bp each). In this system, a targeted double-strand break (DSB) in DNA is formed in four sequential steps. First, two non-coding RNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the direct repeats in the pre-crRNA, which is then processed into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas to a target DNA consisting of a protospacer and an appropriate PAM by forming a heteroduplex between the spacer region of the crRNA and the protospacer DNA. Finally, Cas mediates cleavage of the target DNA upstream of the PAM to form a DSB within the protospacer. A pre-crRNA array consisting of a single spacer flanked by two direct repeats (DRs) is also encompassed by the term "paired tracr sequences." In certain embodiments, the presence of Cas may be constant, or its presence may be induced, or its presence may be conditionally dependent, or it may be introduced or delivered. Optimization of Cas may be used to enhance function or to develop new functions, and chimeric Cas proteins may be produced. Cas may also be used as a universal DNA binding protein.

Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (containing a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands at or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs) the target sequence. Without wishing to be bound by theory, it is believed that a tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridizing at least a portion of the tracr sequence to all or a portion of a mate tracr sequence that is operably linked to a guide sequence.

In the present invention, the term "Cas" may mean "Cas9" or a CRISPR enzyme. In the context of the present invention, Cas9 or Cas or a CRISPR enzyme is mutated or modified "whereby the enzyme in the CRISPR complex has a reduced ability to modify one or more off-target loci compared to an unmodified enzyme" (or similar expressions); and when reading the present description, the terms "Cas9" or "Cas" or "CRISPR enzyme, etc." are intended to include mutated or modified Cas9 or Cas or a CRISPR enzyme according to the present invention, i.e. "whereby the enzyme in the CRISPR complex has a reduced ability to modify one or more off-target loci compared to an unmodified enzyme" (or similar expressions).

Codon optimization and codon usage for Cas protein expression

A nucleic acid molecule encoding Cas is preferably codon-optimized for Cas. In this case, an example of a codon-optimized sequence is a sequence optimized for expression in a eukaryote, such as humans (i.e., is optimized for expression in humans), or in another eukaryote, animal, or mammal discussed herein; see, for example, the codon-optimized human SaCas9 sequence in WO 2014/093622 (PCT/US2013/074667). Although this is preferred, it should be understood that other examples are possible and that codon optimization for a host species other than humans or codon optimization for specific organs is known. In some embodiments, the Cas encoding enzyme sequence is codon-optimized for expression in certain cells, such as eukaryotic cells. Eukaryotic cells may be cells of or derived from a particular organism, such as cells of a mammal, including but not limited to a human or non-human eukaryote, or an animal or mammal discussed herein, such as a mouse, rat, rabbit, dog, cattle, or non-human mammal or primate. In some embodiments, methods of modifying the genetic identity of the germline of a human and/or methods of modifying the genetic identity of animals that are likely to cause them suffering without any significant medical benefit to the human or animal, as well as animals resulting from such methods, may be excluded. In general, codon optimization refers to a method of modifying a nucleic acid sequence to enhance expression levels in host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of such host cell, while maintaining the native amino acid sequence. Different species exhibit a certain "preference" for particular codons for a particular amino acid. Codon "preference" (differences in codon usage between organisms) is often correlated with the efficiency of translation of messenger RNA (mRNA), which in turn is believed to depend on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The prevalence of selected tRNAs in a cell generally indicates the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism by codon optimization. Codon usage tables are publicly available, such as in the Codon Usage Database, available on the Internet at www.kazusa.orjp/codon/, and these tables can be adapted in a variety 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 the codons of a given sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA). 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 the Cas encoding sequence correspond to the most frequently used codon for a particular amino acid.

In certain embodiments, the methods described herein may comprise providing a Cas transgenic cell, wherein one or more nucleic acids encoding one or more guide RNAs are provided or introduced operably linked into the cell to a regulatory element comprising a promoter of one or more genes of interest. As used herein, the term "Cas transgenic cell" refers to a cell, such as a eukaryotic cell, into whose genome a Cas gene has been integrated. The nature, type, or origin of the cell is not particularly limited according to the present invention. Also, the method by which the Cas transgene is introduced into the cell may vary and may be any method known in the art. In certain embodiments, the Cas transgenic cell is produced by introducing the Cas transgene into an isolated cell. In certain other embodiments, the Cas transgenic cell is produced by isolating cells from a Cas transgenic organism. By way of example and without limitation, a Cas transgenic cell referred to herein can be derived from a Cas transgenic eukaryote, such as a Cas knockin eukaryote. Reference is made to WO 2014/093622 (PCT/US13/74667), which is incorporated herein by reference. The methods of U.S. Patent Application Publication Nos. 20120017290 and 20110265198 assigned to Sangamo BioSciences, Inc. relating to targeting the Rosa locus can be modified to use the CRISPR-Cas system of the present invention. The methods of U.S. Patent Application Publication No. 20130236946 assigned to Cellectis relating to targeting the Rosa locus can also be modified to use the CRISPR-Cas system of the present invention. As a further example, reference is made to Platt et. al. (Cell; 159(2):440-455 (2014)), describing a Cas9 knockin mouse, which is incorporated herein by reference. The Cas transgene may further comprise a Lox-Stop-polyA-Lox (LSL) cassette, thereby allowing for induction of Cas expression by Cre recombinase. Alternatively, a Cas transgenic cell can be produced by introducing a Cas transgene into an isolated cell. Delivery systems for transgenes are well known in the art. As an example, the Cas transgene can be delivered, for example, to a eukaryotic cell via vector (e.g., AAV, adenovirus, lentivirus) and/or particle and/or nanoparticle delivery, as also described elsewhere herein.

It will be appreciated by those skilled in the art that a cell, such as a Cas transgenic cell as referred to herein, may contain additional alterations in the genome in addition to the presence of an integrated Cas gene or mutations arising from the sequence-specific action of Cas when complexed with RNA capable of directing Cas to a target locus, such as, for example, one or more oncogenic mutations, as described by way of example and without limitation in Platt et al. (2014), Chen et al. (2014), or Kumar et al. (2009).

Cas protein with one or more NLS

In some embodiments, the Cas sequence is fused to one or more nuclear localization sequences (NLS), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs. In some embodiments, the Cas comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the amino terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more NLSs at or near the carboxy terminus, or a combination thereof (e.g., none or at least one or more NLSs at the amino terminus and none or one or more NLSs at the carboxy terminus). In cases where more than one NLS is present, each may be selected independently of the others, such that one NLS may be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies. In a preferred embodiment of the present invention, the Cas comprises no more than 6 NLSs. In some embodiments, an NLS is considered to be near the N- or C-terminus when the closest amino acid to the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more amino acids along the polypeptide chain from the N- or C-terminus. Non-limiting examples of NLSs include an NLS sequence derived from an NLS from the large T antigen of the SV40 virus with the amino acid sequence PKKKRKV (SEQ ID NO: X); NLS from nucleoplasmin (e.g., the two-part NLS from nucleoplasmin with the sequence KRPAATKKAGQAKKKK) (SEQ ID NO: X); NLS from c-myc with the amino acid sequence PAAKRVKLD (SEQ ID NO: X) or RQRRNELKRSP (SEQ ID NO: X); NLS from hRNPA1 M9 with the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: X); the sequences RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: X) of the IBB domain of importin alpha; the sequences VSRKRPRP (SEQ ID NO: X) and PPKKARED (SEQ ID NO: X) of the myoma T protein; the sequences POPKKKPL (SEQ ID NO: X) of human p53; the SALIKKKKKMAP (SEQ ID NO: X) sequences of mouse c-abl IV; the DRLRR (SEQ ID NO: X) and PKQKKRK (SEQ ID NO: X) sequences from influenza virus NS1; the RKLKKKIKKL (SEQ ID NO: X) sequences from the hepatitis virus delta antigen; the REKKKFLKRR (SEQ ID NO: X) sequences from the mouse Mx1 protein; the KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: X) sequence from human poly(ADP-ribose) polymerase; and the RKCLQAGMNLEARKTKK (SEQ ID NO: X) sequences from the steroid hormone receptors (human) glucocorticoids. In general, one or more NLSs are sufficiently efficient to direct the accumulation of Cas in detectable amounts in the nucleus of a eukaryotic cell. In general, the degree of nuclear localization activity may be a result of the following: the number of NLSs in the Cas, the specific NLS(s) used, or a combination of these factors. Detection of nuclear accumulation may be accomplished by any suitable technique. For example, a detectable marker may be fused to the Cas so that cellular localization may be visualized, such as in combination with a means for detecting nuclear localization (e.g., a nuclear-specific stain such as DAPI). Cell nuclei may also be isolated from the cells, and their contents may then be analyzed by any suitable method for protein detection, such as immunohistochemistry, Western blotting, or enzyme activity assay. Nuclear accumulation can also be determined indirectly, such as by analyzing the effect of CRISPR complex formation (e.g., analyzing DNA cleavage or mutation in a target sequence, or analyzing gene expression activity altered by CRISPR complex formation and/or Cas activity) compared to a control that is not exposed to Cas or the complex or exposed to Cas that lacks one or more NLSs. In some embodiments, no NLSs are added or fused to the Cas protein.

Delivery of the CRISPR system

By utilizing this disclosure and the prior art, a CRISPR-Cas system, particularly the novel CRISPR systems described herein, or components thereof, or nucleic acid molecules thereof (including, for example, an HDR template), or nucleic acid molecules encoding or comprising components thereof, can be delivered using a delivery system described herein, both generally and in detail.

General information about vectors

In general and throughout this specification, 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, no free ends (e.g., circular); nucleic acid molecules that contain DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular loop of double-stranded DNA into which additional segments of DNA can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally derived DNA or RNA sequences are present in the vector for packaging into the virus (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by the virus for transfection into a host cell. Certain vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., mammalian vectors other than episomal vectors) integrate into the host cell genome upon introduction into the host cell and are thereby replicated along with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are functionally linked. Such vectors are referred to herein as "expression vectors." Vectors for and providing expression in a eukaryotic cell may be referred to herein as "eukaryotic expression vectors". Commonly used expression vectors suitable for recombinant DNA techniques are often in the form of plasmids.

Recombinant expression vectors may comprise a nucleic acid of the present invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors comprise one or more regulatory elements, which may be selected taking into account the host cells that are intended to be used for expression, which are operably linked to the nucleic acid sequence that is intended to be expressed. In the context of a recombinant expression vector, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) such that expression of the nucleotide sequence is allowed (e.g. in an in vitro transcription/translation system or in a host cell upon introduction of the vector into the host cell).

The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., 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 direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct expression preferentially in a target tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a time-dependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, which may or may not also be tissue-specific or cell type-specific. In some embodiments, the vector comprises one or more pol III promoters (e.g., 1, 2, 3, 4, 5 or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5 or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5 or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, the U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase gene promoter, the β-actin gene promoter, the glycerophosphate kinase (PGK) gene promoter, and the EF1α promoter. Also encompassed by the term "regulatory element" are enhancer elements such as the WPRE; CMV; the R-U5' segment of the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); the SV40 enhancer; and the intronic sequence between exons 2 and 3 of the rabbit β-globin gene (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981). Those skilled in the art will appreciate that the configuration of the expression vector may depend on factors such as the choice of host cell to be transformed, the desired expression level, etc. The vector can be introduced into host cells to thereby produce transcripts, proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids described herein (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).

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

In some embodiments, one or more vectors that direct the expression of one or more elements of a nucleic acid targeting system are introduced into a host cell such that expression of the elements of the nucleic acid targeting system directs the formation of a nucleic acid targeting complex at one or more target sites. For example, each of the nucleic acid targeting effector enzyme and the nucleic acid targeting guide RNA can be operably linked to separate regulatory elements in separate vectors. The nucleic acid targeting RNAs can be delivered to an animal or mammal transgenic for a nucleic acid targeting effector protein, such as an animal or mammal that continuously, or induced, or under certain conditions expresses the nucleic acid targeting effector protein; or an animal or mammal that otherwise expresses a nucleic acid targeting effector protein or has cells containing a nucleic acid targeting effector protein, such as by previously introducing therein a vector or vectors encoding and expressing in vivo a nucleic acid targeting effector protein. Alternatively, two or more elements expressed from the same or different regulatory elements may be combined in 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 elements of the nucleic acid targeting system that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5'-end ("upstream") of the second element or 3'-end ("downstream") of the second element. The coding sequence of one element may be located on the same or opposite strand relative to the coding sequence of the second element and oriented in the same or opposite direction. In some embodiments, one promoter drives the expression of a transcript 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 a single intron). In some embodiments, the nucleic acid targeting effector protein and the nucleic acid targeting guide RNA may be operably linked to and 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 as used in the foregoing documents, such as WO 2014/093622 (PCT/US2013/074667). In some embodiments, the vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a "cloning site"). In some embodiments, the one or more insertion sites (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more insertion sites) are upstream and/or downstream of one or more sequence elements of one or more vectors. In some embodiments, the vector comprises an insertion site upstream of the tracr mate sequence and optionally downstream of a regulatory element operably linked to the tracr mate sequence, such that upon insertion of the guide sequence into the insertion site and upon expression, the guide sequence directs sequence-specific binding of the nucleic acid targeting complex to a target sequence in a eukaryotic cell. In some embodiments, the vector comprises two or more insertion sites to allow insertion of a guide sequence into each site. In such an arrangement, the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or combinations thereof. In cases where multiple distinct guide sequences are used, a single expression construct may be used to direct nucleic acid targeting activities to multiple distinct corresponding target sequences in a cell. For example, one vector can contain about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more targeting sequences. In some embodiments, vectors containing about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more such targeting sequences can be produced and optionally delivered to a cell. In some embodiments, the vector comprises a regulatory element operably linked to an enzyme-coding sequence that encodes an effector protein for targeting a nucleic acid. The effector protein for targeting a nucleic acid or the guide RNA or guide RNAs for targeting a nucleic acid can be delivered separately; and advantageously, at least one of them is delivered via a particle-based or nanoparticle-based complex. The nucleic acid targeting effector protein mRNA may be delivered prior to the nucleic acid targeting guide RNA to allow time for the nucleic acid targeting effector protein to be expressed. The nucleic acid targeting effector protein mRNA may be administered 1-12 hours (preferably about 2-6 hours) prior to administration of the nucleic acid targeting guide RNA. Alternatively, the nucleic acid targeting effector protein mRNA and the nucleic acid targeting guide RNA may be administered together. Advantageously, the second booster dose of guide RNA may be administered 1-12 hours (preferably about 2-6 hours) after the first administration of nucleic acid targeting effector protein mRNA + guide RNA. Administration of additional doses of effector protein mRNA for targeting nucleic acid and/or guide RNA may be useful to achieve the most efficient levels of genome modification.

General information regarding vector delivery

Certain aspects of the present invention relate to vectors, for example, for the delivery or introduction into a cell of Cas and/or RNA capable of directing Cas to a target locus (i.e., guide RNA), as well as for increasing the amount of these components (e.g., in prokaryotic cells). As used herein, a "vector" is a vehicle that enables or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another segment of DNA can be inserted to thereby effect replication of the inserted segment. Typically, a vector is capable of replication if associated with appropriate control elements. In general, the term "vector" refers to a nucleic acid molecule capable of transferring 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, do not contain free ends (e.g., circular); nucleic acid molecules that contain DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular loop of double-stranded DNA into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, where virally derived DNA or RNA sequences are present in the vector for packaging into the virus (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses (AAV)). Viral vectors also include polynucleotides carried by the virus for transfection into a host cell. Certain vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal vectors for mammals). Other vectors (e.g., non-episomal mammalian vectors) integrate into the host cell genome upon introduction into the host cell and are thus replicated along with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are functionally linked. Such vectors are referred to herein as "expression vectors." Common expression vectors suitable for recombinant DNA techniques are often in the form of plasmids.

Recombinant expression vectors may comprise a nucleic acid of the present invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors comprise one or more regulatory elements, which may be selected taking into account the host cells that are intended to be used for expression, which are operably linked to the nucleic acid sequence that is intended to be expressed. In the context of a recombinant expression vector, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) such that expression of the nucleotide sequence is allowed (e.g. in an in vitro transcription/translation system or in a host cell upon introduction of the vector into the host cell). With respect to recombination and cloning methods, reference should be made to U.S. Patent Application No. 10/815,730, published September 2, 2004 as US 2004-0171156 A1, the contents of which are incorporated herein by reference in their entirety.

The vector(s) may comprise a regulatory element(s), such as a promoter(s). The vector(s) may comprise Cas coding sequences and/or one, but may also comprise at least 3, or 8, or 16, or 32, or 48, or 50 guide RNA (e.g. sgRNA) coding sequences, such as 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 RNAs (e.g. sgRNA). There may be a promoter for each RNA (e.g. sgRNA) in one vector, advantageously if up to about 16 RNAs (e.g. sgRNA) are present; and if there are more than 16 RNAs (e.g., sgRNAs) in a single vector, then one or more promoters can drive the expression of more than one RNA (e.g., sgRNA), for example, if there are 32 RNAs (e.g., sgRNAs), then each promoter can drive the expression of two RNAs (e.g., sgRNAs), and if there are 48 RNAs (e.g., sgRNAs), then each promoter can drive the expression of three RNAs (e.g., sgRNAs). By simple arithmetic and conventional cloning protocols, as well as the teachings of the present disclosure, one skilled in the art will be able to readily practice the present invention with respect to RNAs (e.g., sgRNAs) for a suitable exemplary vector, such as AAV, and a suitable promoter, such as the U6 promoter, e.g., U6-sgRNA. For example, the packaging limit of AAV is ~4.7 kb. The length of one U6-sgRNA (plus restriction sites for cloning) is 361 bp. Therefore, one skilled in the art can easily accommodate about 12-16, such as 13, U6-sgRNA cassettes in a single vector. It can be assembled using any suitable method, such as the Golden Gate strategy used for TALE assembly (http://www.genome-engineering.org/taleffectors/). One skilled in the art can also use a tandem guide strategy to increase the number of U6-sgRNA by about 1.5-fold, such as to increase the number of U6-sgRNA from 12 to 16, such as from 13 to about 18-24, such as about 19. Therefore, one skilled in the art can easily obtain about 18-24, such as about 19, promoter RNAs, such as U6-sgRNA, in a single vector, such as an AAV-based vector. An additional way to increase the number of promoters and RNAs, e.g., sgRNAs, in a vector is to use a single promoter (e.g., U6) to express an array of RNAs, e.g., sgRNAs, separated by cleavable sequences. Yet another additional way to increase the number of promoters-RNAs, e.g., sgRNAs, in a vector is to express an array of promoters-RNAs, e.g., sgRNAs, separated by cleavable sequences within an intron of a coding sequence or gene; in this case, it is advantageous to use a polymerase II promoter, which may have increased expression and the ability to transcribe long RNA in a tissue-specific manner. (See, e.g., http://nar.oxfordjournals.org/content/34/7/e53.short, http://www.nature.com/mt/journal/v16/n9/abs/mt2008144a.html). In an advantageous embodiment, the AAV can package U6 in tandem with an sgRNA targeting up to and including approximately 50 genes. Accordingly, based on the knowledge of the prior art and the teachings of the present disclosure, one skilled in the art can readily prepare and use vector(s), such as a single vector, expressing multiple RNAs or guides or sgRNAs under the control of one or more promoters, or functionally effective or operably linked to them, particularly with respect to the number of RNAs or guides or sgRNAs discussed herein, without undue experimentation.

The coding guide RNAs, e.g. sgRNAs, sequences and/or Cas coding sequences may be functionally or functionally effectively linked to a regulatory element(s), and hence the regulatory element(s) controls expression. The promoter(s) may be a constitutive promoter(s) and/or a conditionally activated 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, the retroviral LTR promoter of Rous sarcoma virus (RSV), the cytomegalovirus (CMV) promoter, the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the glycerophosphate kinase (PGK) promoter, and the EF1α promoter. A preferred promoter is the U6 promoter.

Vector delivery

Vector delivery, e.g., plasmid, viral delivery. The CRISPR enzyme, e.g., Cas9, and/or any of the RNAs of the present invention, e.g., guide RNA, can be delivered using any suitable vector, e.g., plasmid or viral vectors such as adeno-associated virus (AAV), lentivirus, adenovirus or other types of viral vectors, or combinations thereof. Cas9 and one or more guide RNAs can be packaged in one or more vectors, e.g., plasmid or viral vectors. In some embodiments, the vector, e.g., plasmid or viral vector, is delivered to the tissue of interest by, e.g., intramuscular injection, while in other cases, delivery is via intravenous, transdermal, intranasal, oral, transmucosal or other delivery methods. Such delivery can be in the form of a single dose or multiple doses. One skilled in the art will appreciate that the actual dose to be delivered herein may vary significantly depending on a number of factors such as the choice of vector, the target cell, organism or tissue, the general condition of the subject being treated, the degree of transformation/modification required, the route of administration, the route of administration, the type of transformation/modification required, etc.

Such a dose may further comprise, for example, a carrier (water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, a pharmaceutically acceptable carrier (e.g., phosphate buffered saline), a pharmaceutically acceptable excipient, and/or other compounds known in the art. The dose may further comprise one or more pharmaceutically acceptable salts, such as, for example, a salt of an inorganic acid, such as hydrochloride, hydrobromide, phosphate, sulfate, etc.; and salts of organic acids, such as acetates, propionates, malonates, benzoates, etc. Additionally, it may also contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gels or gelling materials, flavorings, coloring agents, microspheres, polymers, suspending agents, etc. In addition, one or more other conventional pharmaceutical ingredients such as preservatives, humectants, suspending agents, surfactants, antioxidants, anti-caking agents, fillers, chelating agents, coating agents, chemical stabilizers, etc. may also be present, especially if the dosage form is a form to be reconstituted. Suitable illustrative ingredients include microcrystalline cellulose, sodium carboxymethylcellulose, 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 detailed discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991), incorporated herein by reference.

In one embodiment according to this document, delivery is carried out via adenovirus, which can be in a single booster dose containing at least 1 x 10 ⁵ particles (also referred to as particle units, pu) of adenoviral vector. In one embodiment according to this document, the dose is preferably at least about 1 x 10 ⁶ particles (e.g., about 1 x 10 ⁶ - 1 x 10 ¹² particles), more preferably at least about 1 x 10 ⁷ particles, more preferably at least about 1 x 10 ⁸ particles (e.g., about 1 x 10 ⁸ - 1 x 10 ¹¹ particles or about 1 x 10 ⁸ - 1 x 10 ¹² particles) and most preferably at least about 1 x 10 ⁰ particles (e.g., about 1 x 10 ⁹ - 1 x 10 ¹⁰ particles or about 1 x 10 ⁹ - 1 x 10 ¹² particles) or even at least about 1 x 10 ¹⁰ particles (e.g., about 1 x 10 ¹⁰ - 1 x 10 ¹² particles) of adenoviral vector. Alternatively, the dose contains no more than about 1 x 10 ¹⁴ particles, preferably no more than about 1 x 10 ¹³ particles, even more preferably no more than about 1 x 10 ¹² particles, even more preferably no more than about 1 x 10 ¹¹ particles, and most preferably no more than about 1 x 10 ¹⁰ particles (e.g., no more than about 1 x 10 ⁹ particles). Thus, the dose may comprise a single dose of adenoviral vector, for example, with about 1 x 10 ⁶ particle units (pu), about 2 x 10 ⁶ pu, about 4 x 10 ⁶ pu, about 1 x 10 ⁷ pu, about 2 x 10 ⁷ pu, about 4 x 10 ⁷ pu, about 1 x 10 ⁸ pu, about 2 x 10 ⁸ pu, about 4 x 10 ⁸ pu, about 1 x 10 ⁹ pu, about 2 x 10 ⁹ pu, about 4 x 10 ⁹ pu, about 1 x 10 ¹⁰ pu, about 2 x 10 ¹⁰ pu, about 4 x 10 ¹⁰ pu, about 1 x 10 ¹¹ pu, about 2 x 10 ¹¹ pu, about 4 x 10 ¹¹ pu, about 1 x 10 ¹² pu, about 2 x 10 ¹² pu, or about 4 x 10 ¹² pu of an adenovirus vector. See, for example, the adenovirus vectors in U.S. Patent No. 8,454,972 B2 to Nabel, et al., issued June 4, 2013; incorporated herein by reference, and the doses at column 29, lines 36-58 of that patent. In one embodiment according to this document, the adenovirus is delivered via multiple doses.

In one embodiment according to the present document, delivery is carried out via AAV. It is believed that a therapeutically effective dose for in vivo delivery of AAV to a human is in the range of about 20 to about 50 ml of saline solution containing about 1 x 10 ¹⁰ to about 1 x 10 ¹⁰ functional AAV particles/ml of solution. The dose can be adjusted to balance the therapeutic benefit and any side effects. In one embodiment according to the present document, the dose of AAV is typically in the concentration range of about 1 x 10 ⁵ to 1 x 10 ⁵⁰ AAV genomes, about 1 x 10 ⁸ to 1 x 10 ²⁰ AAV genomes, about 1 x 10 ¹⁰ to about 1 x 10 ¹⁶ genomes, or about 1 x 10 ¹¹ to about 1 x 10 ¹⁶ AAV genomes. A human dose may be about 1 x 10 ¹³ AAV genomes. Such concentrations may be delivered in a carrier solution of about 0.001 ml to about 100 ml, about 0.05 ml to about 50 ml, or about 10 ml to about 25 ml. Other effective doses may be readily determined by one of ordinary skill in the art through routine dose-response testing. See, for example, U.S. Patent No. 8,404,658 B2 to Hajjar, et al., issued March 26, 2013, at column 27, lines 45-60.

General principles of packaging and use of promoters

The following methods exist for packaging Cas9-encoding nucleic acid molecules, such as DNA, into vectors, such as viral vectors, to mediate genome modification in vivo.

To achieve NHEJ-mediated gene knockout, the following is required.

One viral vector

A vector containing two or more expression cassettes:

promoter - a nucleic acid molecule encoding the Cas9 terminator;

promoter-gRNA1-terminator;

promoter-gRNA2-terminator;

promoter-gRNA(N)-terminator (up to the maximum vector size).

Two viral vectors

Vector 1 containing one expression cassette to drive Cas9 expression:

promoter - a nucleic acid molecule encoding the Cas9 terminator;

vector 2 containing one or more expression cassettes for directing the expression of one or more guide RNAs:

promoter-gRNA1-terminator;

promoter-gRNA(N)-terminator (up to the maximum vector size).

To mediate repair involving homologous recombination.

In addition to the single and dual viral vector approaches described above, an additional vector can be used to deliver a template for repair involving homologous recombination.

The promoter used to drive expression of the nucleic acid molecule encoding Cas9 may include the following.

The AAV ITR can serve as a promoter: this is advantageous in that it eliminates the need for an additional promoter element (which may take up space in the vector). The freed up extra space can be used to drive the expression of additional elements (gRNA, etc.). Also, the ITR activity is relatively weaker, so it can be used to reduce the potential toxicity caused by Cas9 overexpression.

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

For expression in the brain or other part of the CNS, the following promoters can be used: the synapsin I gene for all neurons, the CaMKII-alpha gene for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc.

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

SP-B can be used for expression in the lungs.

For endothelial cells, ICAM can be used.

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

For osteoblasts, OG-2 can be used.

The promoter used to drive the guide RNA may include the following:

promoters for Pol III, such as U6 or H1.

Use of a Pol II promoter and intronic cassettes for gRNA expression.

Crystallization and structure of CRISPR-Cas9

Crystallization of CRISPR-Cas9 and characterization of the crystal structure. Crystals can be prepared using techniques used in protein crystallography, including batch processing, liquid bridge, dialysis, vapor diffusion, and hanging drop methods. Typically, crystals are grown by dissolving nearly pure CRISPR-Cas9 and the nucleic acid molecule to which it binds in an aqueous buffer containing a precipitant at a concentration just below that required for precipitation. Water is removed by controlled evaporation to provide precipitation conditions, which are maintained until crystal growth ceases. See, Nishimasu et al.

Applications of Crystals, Crystal Structure Coordinates, and Atomic Structure. Crystals, and in particular the atomic structure coordinates derived therefrom, have a wide range of applications. Crystals and structure coordinates are particularly useful for identifying compounds (nucleic acid molecules) that bind to CRISPR-Cas9 and CRISPR-Cas9 that can bind to specific compounds (nucleic acid molecules). Thus, the structure coordinates described herein can be used as phasing models in determining crystal structures of additional synthetic or mutated CRISPR-Cas9, Cas9, nickase, binding domains. Providing a crystal structure of CRISPR-Cas9 complexed with a nucleic acid molecule can provide one skilled in the art with insight into CRISPR-Cas9. Such insight enables the development of modified CRISPR-Cas9, such as by attaching a functional group such as a repressor or activator. Although a functional group such as a repressor or activator can be attached to the N- or C-terminus of CRISPR-Cas9, the crystal structure shows that the N-terminus appears to be obstructed or buried, while the C-terminus is more accessible to a functional group such as a repressor or activator. Furthermore, the crystal structure shows that there is a flexible loop between residues corresponding to approximately 534-676 of CRISPR-Cas9 (S. pyogenes), which is suitable for the attachment of a functional group such as an activator or repressor. The attachment can be accomplished through a linker such as a flexible glycine-serine (GlyGlyGlySer) or (GGGS)3 or a rigid alpha-helical linker such as (Ala(GluAlaAlaAlaLys)Ala). In addition to the flexible loop, there is also a nuclease or H3 region, an H2 region, and a helical region. By "helix" or "helical" is meant a helix as known in the art, including but not limited to an alpha helix. In addition, the term "helix" or "helical" may also be used to refer to a C-terminal helical element with an N-terminal turn.

Providing a crystal structure of CRISPR-Cas9 complexed with a nucleic acid molecule provides a new approach for finding new drugs or compounds, identifying and developing compounds that can bind to CRISPR-Cas9, and thus the present invention provides tools useful for diagnosing, treating or preventing conditions or diseases in multicellular organisms, such as algae, plants, vertebrates, fish, amphibians, reptiles, birds, mammals; such as domestic plants, animals (e.g., food animals such as pig, ruminant, chicken; companion animal such as feline, canine, rodent (rabbit, gerbil, hamster); laboratory animals such as mouse, rat) and humans. Accordingly, the present invention provides a method for rationally designing CRISPR-Cas9 complexes based on the use of a computer. Such rational design may include providing a structure of a CRISPR-Cas9 complex defined by some or all (e.g., at least 2 or more, such as at least 5, preferably at least 10, more preferably at least 50, and even more preferably at least 100 atoms of the structure) of the coordinates from a table of crystal structures and/or shown in a figure(s) relating to the crystal structure; see Nishimasu et al.; providing a structure of a desired nucleic acid molecule for which a CRISPR-Cas9 complex is desired; and adjusting the structure of the CRISPR-Cas9 complex defined by some or all of the coordinates to a desired nucleic acid molecule included in said adjustment to obtain a putative modification(s) of the CRISPR-Cas9 complex defined by some or all of the coordinates so that said desired nucleic acid molecule binds to the CRISPR-Cas9 complex(es) comprising the desired nucleic acid molecule. The method or a fit according to the method may use coordinates of atoms of interest of a CRISPR-Cas9 complex defined by some or all of the coordinates that correspond to a region proximate to the active site or binding site (e.g., at least 2 or more, such as at least 5, preferably at least 10, more preferably at least 50, and even more preferably at least 100 atoms of the structure) to model the region proximate to the active site or binding site. These coordinates may be used to define a space that is then screened in silico for a desired or candidate nucleic acid molecule. Thus, the present invention provides a computer-based method for rationally designing CRISPR-Cas9 complexes. The method may comprise providing coordinates of at least two atoms from a table of crystal structures ("selected coordinates"); see Nishimasu et al.; providing a structure of a candidate or desired nucleic acid molecule and fitting the structure of the candidate to the selected coordinates. In this way, the skilled person can also fit a functional group and a candidate or desired nucleic acid molecule. For example, providing a structure of a CRISPR-Cas9 complex defined by some or all (e.g. at least 2 or more, such as at least 5, preferably at least 10, more preferably at least 50 and even more preferably at least 100 atoms of the structure) coordinates; providing a structure of a desired nucleic acid molecule for which a CRISPR-Cas9 complex is required; fitting the structure of the CRISPR-Cas9 complex defined by some or all coordinates from a table of crystal structures and/or shown in figures relating to the crystal structure; see Nishimasu et al.; to a desired nucleic acid molecule included in said match, to obtain putative modification(s) of a detectable CRISPR-Cas9 complex so that said desired nucleic acid molecule binds to a CRISPR-Cas9 complex(es) comprising the desired nucleic acid molecule; selecting a putative match of the CRISPR-Cas9 complex(es)-desired nucleic acid molecule, matching such putative match of the CRISPR-Cas9 complex(es)-desired nucleic acid molecule to a functional group (e.g., an activator, a repressor), such as with respect to locations for accommodating the functional group (e.g., positions within a flexible loop) and/or putative modifications of the putative match of the CRISPR-Cas9 complex(es)-desired nucleic acid molecule to obtain locations for accommodating the functional group.

However, the crystal structure information of SpCas9 (see Nishimasu et al.) does not predict the reduction in off-target effects achieved by the mutations of the present invention; or that particular mutations may result in the reduction in off-target effects disclosed herein. However, by providing information on mutations that provide or by which the reduction in off-target effects is achieved, one skilled in the art will be able to readily apply the teachings of this document in combination with the crystal structure information of SpCas9 and the sequence information of Cas9 to perform sequence and structure analyses of Cas9 to determine similar amino acids that can be mutated or modified in a similar manner to produce additional mutated or modified Cas9s in which the mutation or modification results in the reduction in off-target effects.

Thus, the present invention together with the SpCas9 crystal information can be practiced using coordinates corresponding to a region adjacent to the mutation(s) or modification(s) disclosed herein, or an active site or binding site located in close proximity to such mutation(s) or modification(s); and therefore, methods for determining additional mutations or modifications in SpCas9 or similar mutations or modifications in Cas9 orthologs can use an aspect, such as a comparative aspect, of the subdomain(s) of interest of the CRISPR-Cas9 complex. Methods for determining additional mutations or modifications in SpCas9 or similar mutations or modifications in Cas9 orthologs can be practiced using the domain or subdomain coordinates. The methods may optionally comprise synthesizing a candidate or desired nucleic acid molecule and/or CRISPR-Cas9 systems according to the "in silico" results and testing "practical" or actual mutations or modifications for binding and/or activity and/or reduction of off-target effects. CRISPR-Cas9 systems comprising mutations or modifications may optionally comprise a functional group. Such methods may comprise examining a cell or an organism comprising a cell for a desired response, such as reducing the manifestation of symptoms or a condition or disease, preferably comprising reducing off-target effects. Obtaining the structure of a candidate nucleic acid molecule may comprise selecting a compound by computer screening a database containing data on nucleic acid molecules, such as data that are relevant to conditions or diseases. The 3-D identifier with respect to binding of the candidate nucleic acid molecule may be derived from geometric and functional constraints derived from the structure and chemical nature of the CRISPR-Cas9 complex or its domains or regions in the crystal structure, taking into account the mutations or modifications disclosed herein. In fact, the identifier may be the type of actual modification(s) of the crystal structure of the CRISPR-Cas9 complex according to this document to ensure binding of CRISPR-Cas9 to the candidate or desired nucleic acid molecule. Herein, the "practical" steps may then be performed using the identifier and nucleic acid molecules that are suspected of having good binding.

"Fitting" may mean determining interactions between at least one atom of the candidate and at least one atom of the CRISPR-Cas9 complex by automated or semi-automated means and calculating the stability of such interactions. The interactions may include attraction, repulsion, charge-based, steric aspects, etc. "Subdomain" may mean at least one, such as one, two, three, or four, complete secondary structure element(s). Particular regions or domains of CRISPR-Cas9 include those identified in the table of crystal structures and the figures corresponding thereto; see Nishimasu et al.

In any case, the three-dimensional structure of the CRISPR-Cas9 complex (e.g. S. pyogenes Cas9; see Nishimasu et al.) provides in the context of the present invention an additional tool for identifying additional mutations in Cas9 orthologs in the form of positions for the mutations/modifications identified herein that can be applied to Cas9 orthologs based on sequence and structural position comparison using the crystal structure of the CRISPR-SpCas9 complex. The crystal structure can also be the basis for the design of new and specific Cas9, such as those having the mutation(s) or modification(s) according to this document, and comprising, or having as a fusion partner, or having associated therewith, any one or more of a variety of functional groups, such as a transcriptional repressor, a transcriptional activator, a nuclease domain, a DNA methyltransferase, a protein acetyltransferase, a protein deacetylase, a protein methyltransferase, a protein deaminase, a protein kinase, and a protein phosphatase; and in some aspects, the functional domain is an epigenetic regulator; see, for example, Zhang et al., U.S. Patent No. 8,507,272, and again this and all documents cited therein and all documents cited in the present application are hereby incorporated by reference, by modifying Cas9 with the novel nickases. Based on the present disclosure and knowing the three-dimensional crystal structure of CRISPR-Cas9 (S. pyogenes Cas9), computer modeling programs can be used to design or identify various molecules that are expected to interact with candidate or confirmed sites, such as binding sites or other structural or functional features of the CRISPR-Cas9 system (e.g., S. pyogenes Cas9). A compound that potentially binds (a "binder") can be examined by applying computer modeling using a docking program. Docking programs are known; for example, GRAM, DOCK, or AUTODOCK (see Walters et al. Drug Discovery Today, vol. 3, no. 4 (1998), 160-178, and Dunbrack et al. Folding and Design 2 (1997), 27-42). This procedure may involve computer fitting of potential binders to determine how well the shape and chemical structure of the potential binder will facilitate binding to the CRISPR-Cas9 system (e.g., S. pyogenes Cas9). The active site or binding site of the CRISPR-Cas9 system (e.g., S. pyogenes Cas9) can be manually inspected by computer. Programs such as GRID (P. Goodford, J. Med. Chem, 1985, 28, 849-57), which is a program that identifies likely interaction sites between molecules with different functional groups, can also be used to analyze the active site or binding site to predict partial structures of binders. Computer programs can be used to evaluate the attraction, repulsion, or steric hindrance of two binding partners, such as a CRISPR-Cas9 system (e.g., S. pyogenes Cas9) and a candidate nucleic acid molecule, or a nucleic acid molecule and a candidate CRISPR-Cas9 system (e.g., S. pyogenes Cas9); and the crystal structure of CRISPR-Cas9 (S. pyogenes Cas9) facilitates such methods. In general, the tighter the fit, the less steric hindrance, and the greater the attractive forces, the stronger the binding potential, since these properties are consistent with a sharper binding constant. Moreover, the more specificity in the design of a candidate CRISPR-Cas9 system (e.g., S. pyogenes Cas9), the more likely it will not interact with additionally non-target molecules. The present invention also allows "practical" methods. For example, in one aspect of the present invention, there is provided a method for determining the structure of a binder (e.g., a target nucleic acid molecule) of a candidate CRISPR-Cas9 system (e.g., S. pyogenes Cas9) associated with a candidate CRISPR-Cas9 system (e.g., S. pyogenes Cas9), wherein said method comprises (a) providing a first crystal of a candidate CRISPR-Cas9 system (e.g., S. pyogenes Cas9) according to the present invention or a second crystal of a candidate CRISPR-Cas9 system (e.g., S. pyogenes Cas9), (b) contacting the first crystal or the second crystal with said binder under conditions resulting in the formation of a complex; and (c) determining the structure of said candidate (e.g., a CRISPR-Cas9 system (e.g., S. pyogenes Cas9) or a complex of the CRISPR-Cas9 system (e.g., S. pyogenes Cas9)). The second crystal may have essentially the same coordinates discussed in this paper, but due to minor changes in the CRISPR-Cas9 system (e.g., the Cas9 in such a system is, e.g., S. pyogenes Cas9, as opposed to S. pyogenes Cas9), where "e.g., S. pyogenes Cas9" indicates that the Cas9 is Cas9 and may be from or derived from S. pyogenes or an ortholog thereof), the crystal may form in a different symmetry space group. The present invention further provides, instead of or in addition to the "in silico" methods, other "practical" methods comprising high-throughput screening of a binder (e.g., a target nucleic acid molecule) and a candidate CRISPR-Cas9 system (e.g., S. pyogenes Cas9), or a candidate binder (e.g., a target nucleic acid molecule) and a CRISPR-Cas9 system (e.g., S. pyogenes Cas9), or a candidate binder (e.g., a target nucleic acid molecule) and a candidate CRISPR-Cas9 system (e.g., S. pyogenes Cas9) (the aforementioned CRISPR-Cas9 system(s) with or without one or more functional groups), to select compounds with binding activity. Such pairs of the binder and the CRISPR-Cas9 system that exhibit binding activity can be selected and further crystallized with a CRISPR-Cas9 crystal having the structure according to the present document, for example, by co-crystallization or wetting, for X-ray analysis. After the design, identification or selection of candidate pairs of the binder and the CRISPR-Cas9 system by determining those that have favorable suitable properties, for example, a predicted strong attraction based on the crystal structure data of the binder and CRISPR-Cas9 pairs according to the present document, these candidate pairs can then be screened for activity using "practical" methods. Accordingly, in one aspect, the present invention may comprise preparing or synthesizing candidate pairs and contacting a binder (e.g., a target nucleic acid molecule) and a candidate CRISPR-Cas9 system (e.g., S. pyogenes Cas9), or a candidate binder (e.g., a target nucleic acid molecule) and a CRISPR-Cas9 system (e.g., S. pyogenes Cas9), or a candidate binder (e.g., a target nucleic acid molecule) and a candidate CRISPR-Cas9 system (e.g., S. pyogenes Cas9) (the aforementioned(s) CRISPR-Cas9 system(s) with or without one or more functional groups) to determine the binding ability. In the latter step, the contacting is preferably performed under conditions that allow the function to be determined. Instead of or in addition to performing such an analysis, the present invention may comprise preparing or synthesizing a complex(es) as a result of said contacting and analyzing the complex(es), for example, by X-ray diffraction, or NMR, or other methods to determine binding or interaction properties. Detailed structural information regarding binding can then be obtained, and the structure or function of the candidate CRISPR-Cas9 system or its components can be adjusted based on this information. These steps can be repeated and re-performed as necessary. Alternatively or in addition, candidate CRISPR-Cas9 systems from or used in the above methods can be exposed to nucleic acid molecules in vivo, including without limitation by administration to an organism (including a non-human animal and a human) to determine or confirm function, including whether they result in a desired outcome (e.g., symptom amelioration, treatment).

The present invention further relates to a method for determining three-dimensional structures of CRISPR-Cas systems or complex(es) of unknown structure using the structure coordinates from the documents discussed herein, especially when corrected for the modification(s) or mutation(s) discussed herein. For example, if X-ray crystallographic or NMR spectroscopy data are obtained for a CRISPR-Cas system or complex of unknown crystal structure, the structure of the CRISPR-Cas9 complex can be used to interpret these data to obtain a probable structure for the unknown system or complex using techniques such as phase modeling in the case of X-ray crystallographic analysis. Thus, the method may include aligning a graphical representation of a CRISPR-Cas system or complex with an unknown crystal structure with a similar graphical representation of a CRISPR-Cas9 system or complex with a crystal structure according to the documents cited herein (preferably adjusted for modification(s) or mutation(s) according to this document), ensuring that homologous or similar regions (e.g., homologous or similar sequences) match; modeling the structure of the matched homologous or similar regions (e.g., sequences) of the CRISPR-Cas system or complex with an unknown crystal structure; and determining a conformation (e.g., taking into account that favorable interactions should form such that a low-energy conformation is formed) of the unknown crystal structure that substantially preserves the structure of the matched homologous regions. "Homologous regions" describe, for example, with respect to amino acids, amino acid residues in two sequences that are identical or have similar, e.g., aliphatic, aromatic, polar, negatively charged or positively charged chemical side chain groups. Homologous regions, with respect to nucleic acid molecules, may be characterized by at least 85%, or 86%, or 87%, or 88%, or 89%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% homology or identity. Identical and similar regions are sometimes described by those skilled in the art as "invariant" and "conserved," respectively. Advantageously, the first and third steps are performed using computer modeling. Homology modeling is a technique that is well known to those skilled in the art (see, for example, Greer, Science vol. 228 (1985) 1055, and Blundell et al. Eur J Biochem vol 172 (1988), 513). A computer representation of the conserved regions of the CRISPR-Cas9 crystal structure according to this document and those of a CRISPR-Cas system with an unknown crystal structure helps to predict and determine the crystal structure of a CRISPR-Cas system with an unknown crystal structure. Furthermore, aspects of the present invention that utilize the in silico crystal structure of CRISPR-Cas9 can be equally applied to the new mutation(s) or modification(s) according to this document and the CRISPR-Cas crystal structure. In this manner, a library of CRISPR-Cas crystal structures can be obtained. Thus, the present invention provides for rational design of a CRISPR-Cas system. For example, once a conformation or crystal structure of a CRISPR-Cas system or complex has been determined using the methods described herein (including taking into account prior art information based on documents cited herein), such conformation can be used in the computer-based methods of this document to determine the conformation or crystal structure of other CRISPR-Cas systems or complexes whose crystal structure is not yet known. All of these crystal structure data may be in a database, and the methods of this document may be more reliable if the comparisons of this document are made between a crystal structure or portions thereof from this document and one or more crystal structures from a library. The present invention further provides systems, such as computer systems, for obtaining structures and/or performing rational design of a CRISPR-Cas system or complex. The system may comprise or be obtained from atomic coordinate data, such as by modeling, wherein said data define a three-dimensional structure of the CRISPR-Cas system or complex or at least one domain or subdomain thereof, or their structure factor data, wherein said structure factor data is obtained from the atomic coordinate data. The present invention also relates to machine-readable media with atomic coordinate data, wherein said data define a three-dimensional structure of the CRISPR-Cas system or complex or at least one domain or subdomain thereof, or their structure factor data. "Machine-readable media" refers to any media that can be read and accessed directly by a computer, and includes, but is not limited to, magnetic storage; optical storage; electrical storage; cloud storage, and hybrids of these categories. By providing such machine-readable media, the atomic coordinate data can be accessed in a conventional manner for modeling or other "in silico" methods. The present invention further encompasses methods of conducting business by providing access to such computer-readable media, for example, on a subscription basis, via the Internet or a global communications computer network; or the computer system may be available to a user on a subscription basis. "Computer system" refers to hardware, software, and data storage used to analyze the atomic coordinate data of the present invention. Minimum hardware of the computer systems of the present invention may include a central processing unit (CPU), input devices, output devices, and data storage means. A display or monitor may be provided for visualizing the structure data. The present invention further encompasses methods of communicating the information presented herein or obtained by any method or step thereof described herein, for example, via telecommunications, telephone, mass communications, media, presentations, the Internet, e-mail, etc. The crystal structures of the present invention can be analyzed to obtain a Fourier electron density distribution map(s) of the CRISPR-Cas systems or complexes. Fourier electron density maps can be calculated from X-ray diffraction patterns. These maps can then be used to determine aspects of binding or other interactions. Electron density maps can be calculated using well-known programs such as those in the CCP4 computer package (Collaborative Computing Project, No. 4. The CCP4 Suite: Programs for Protein Crystallography, Acta Crystallographica, D50, 1994, 760-763). Programs such as "QUANTA" (1994, San Diego, Calif.: Molecular Simulations, Jones et al., Acta Crystallography A47 (1991), 110-119) can be used to visualize the maps and build models.

The crystal structure provided herein provides atomic coordinate data for CRISPR-Cas9 (S. pyogenes) and lists each atom with a unique number; the chemical element and its position for each amino acid residue (determined from electron density maps and antibody sequence comparisons), the amino acid residue at which the element is located, a chain identifier, a residue number, coordinates (e.g., X, Y, Z) that define the atomic position relative to the crystallographic axes (in angstroms) for the corresponding atom, the atomic occupancy of the corresponding position, "B", an isotopic substitution parameter (in angstroms ² ) that reflects the motion of the atom about its atomic center, and an atomic number.

In particular embodiments of the present invention, conformational changes in the crystal structures of the CRISPR-Cas9 system or CRISPR-Cas9 components provide important and relevant information about the flexibility or mobility of protein structural regions relative to nucleotide (RNA or DNA) structural regions that may be important for the function of the CRISPR-Cas system. The structural information provided for Cas9 (e.g., Cas9 S. pyogenes: Crystal structure 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, O. Cell Feb 27. (2014). 156(5):935-49; or Cas9 Sa: Crystal Structure of Staphylococcus aureus Cas9, Nishimasu et al., Cell 162, 1113-1126 (Aug. 27, 2015)) as a CRISPR enzyme in the present application can be used for further design and optimization of the CRISPR-Cas system, and can also be extrapolated to detailed structure-function studies in systems with another CRISPR enzyme. One aspect of the present invention relates to a crystal structure of S. pyogenes Cas9 in complex with sgRNA and its target DNA at a resolution of 2.4 Å. The structure reveals a bilobed arrangement consisting of a target recognition lobe and a nuclease lobe that ensure the placement of the sgRNA:DNA duplex in the positively charged groove at their interface. The recognition lobe is essential for the binding of sgRNA and DNA, and the nuclease lobe contains the HNH and RuvC nuclease domains properly positioned to cleave the complementary and non-complementary strands of the target DNA, respectively. This high-resolution structure and the functional analyses presented herein elucidate the molecular mechanism of RNA-guided DNA targeting by Cas9 and provide comprehensive information for the design of optimized CRISPR-Cas systems and their components.

In certain embodiments of the present invention, the crystal structure provides a necessary step towards understanding the molecular mechanism of RNA-guided DNA targeting by Cas9. The structural and functional analyses herein provide a useful basis for the rational design of Cas9-based genome modulation technologies and can provide guidance on Cas9-mediated recognition of PAM sequences on target DNA or mismatch tolerance between an sgRNA:DNA duplex. Aspects of the present invention also relate to truncation mutants, for example, a truncation mutant of S. pyogenes Cas9 can facilitate packaging of Cas9 into limited-size viral vectors for in vivo and therapeutic applications. Similarly, engineering of a PAM interacting (PI) domain can provide programming of PAM specificity, improve target site recognition accuracy, and increase the versatility of the Cas9 genome engineering platform. Furthermore, engineering of the PAM interacting (PI) domain may enable programming of PAM specificity, improve target site recognition accuracy, and increase the versatility of the Cas genome engineering platform, such as Cas9. Cas proteins, such as Cas9 proteins, can be engineered to alter their PAM specificity, such as described by Kleinstiver BP et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015 Jul 23;523(7561):481-5. doi: 10.1038/nature14592.

The present invention encompasses optimized functional CRISPR-Cas enzyme systems. In particular, the CRISPR enzyme comprises one or more mutations that convert it into a DNA-binding protein to which functional domains exhibiting a function of interest can be recruited or added or inserted or attached. In certain embodiments, the CRISPR enzyme comprises one or more mutations that include, but are not limited to, D10A, E762A, H840A, N854A, N863A, or D986A (based on the amino acid position numbering of S. pyogenes Cas9), and/or the one or more mutations are in the RuvC1 or HNH domain of the CRISPR enzyme or are another mutation discussed herein. In some embodiments, the CRISPR enzyme has one or more mutations in the catalytic domain, wherein upon transcription, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence, and wherein the enzyme further comprises a functional domain.

The structural information presented in this paper allows detailed study of the interaction of sgRNA (or chimeric RNA) with target DNA and CRISPR enzyme (e.g., Cas9), providing the ability to design or alter the structure of sgRNA to optimize the functionality of the overall CRISPR-Cas system. For example, loops in sgRNA can be enlarged without overlapping with the Cas9 protein by inserting different RNA loop(s) or different sequence(s) that can recruit adaptor proteins that can bind to the different RNA loop(s) or different sequence(s).

Functional variants of enzymes according to the present invention

In embodiments, the Cas9 protein referred to herein also encompasses a functional variant. A "functional variant" of a protein, as used herein, means a variant of such a protein that retains, at least in part, the activity of the protein. Functional variants may include mutants (which may be insertion, deletion, or substitution mutants), including polymorphs, and the like. Functional variants also include fusion products of such a protein with another, typically unrelated, nucleic acid, protein, polypeptide, or peptide. Functional variants may be naturally occurring or may be produced by man. Advantageous embodiments may involve an engineered or non-naturally occurring type II RNA-targeting effector protein, such as Cas9 or an ortholog or homolog thereof.

General information regarding protein mutation according to the present invention

The present invention encompasses a CRISPR-Cas complex comprising a CRISPR enzyme and a guide RNA (sgRNA), wherein the CRISPR enzyme comprises at least one mutation such that the CRISPR enzyme has no more than 5% of the nuclease activity of a CRISPR enzyme lacking the at least one mutation and optionally at least one or more nuclear localization sequences; the guide RNA (sgRNA) comprises a guide sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell; and wherein the CRISPR enzyme is associated with two or more functional domains; or at least one loop of the sgRNA is modified by insertion of different RNA sequence(s) that binds to one or more adaptor proteins, and wherein the adaptor protein is associated with two or more functional domains; or the CRISPR enzyme is associated with one or more functional domains, and at least one loop of the sgRNA is modified by insertion of a different RNA sequence(s) that binds to one or more adaptor proteins, and wherein the adaptor protein is associated with one or more functional domains.

Functional domains and adaptor proteins, aptamers

Adaptor proteins may include, but are not limited to, orthogonal RNA binding protein/aptamer combinations that are found in a variety of bacteriophage coat proteins. Such coat proteins include, but are not limited to, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, and PRR1. Such adaptor proteins or orthogonal RNA binding proteins may further recruit effector proteins or fusion products that contain one or more functional domains. In some embodiments, the functional domain may be selected from the group consisting of a transposase domain, an integrase domain, a recombinase domain, a resolvase domain, an invertase domain, a protease domain, a DNA methyltransferase domain, a DNA hydroxylmethylase domain, a DNA demethylase domain, a deaminase, a histone acetylase domain, a histone deacetylase domain, a nuclease domain, a repressor domain, an activator domain, nuclear localization signal domains, a transcription regulatory protein (or transcription complex recruitment) domain, a cellular uptake activity associated domain, a nucleic acid binding domain, an antibody presentation domain, a histone modifying enzyme, a histone modifying enzyme recruiter; inhibitor of histone modifying enzymes, histone methyltransferase, histone demethylase, histone kinase, histone phosphatase, histone ribosylase, histone deribosylase, histone ubiquitinase, histone deubiquitinase, histone biotinase and histone tail protease.

In some preferred embodiments, the functional domain is a transcriptional activation domain, preferably VP64. In some embodiments, the functional domain is a transcriptional repression domain, preferably KRAB. In some embodiments, the transcriptional repression domain is SID or concatemers of SID (e.g., SID4X). In some embodiments, the functional domain is an epigenetic modification domain, such that an epigenetic modification enzyme is provided. In some embodiments, the functional domain is an activation domain, which can be a P65 activation domain. In some embodiments, the functional domain is a deaminase, such as a cytidine deaminase. The cytidine deaminase can be directed to a target nucleic acid where it directs the conversion of cytidine to uridine, resulting in C to T (G to A in the complementary strand) substitutions. In such an embodiment, the nucleotide substitutions can be made without cleavage of the DNA.

In one aspect, a Surveyor nuclease assay is used to identify insertion/deletion/nuclease activity. In general, the Surveyor nuclease assay comprises extracting genomic DNA, PCR amplifying a region of the genome flanking a CRISPR target site, purifying the products, re-hybridizing to ensure formation of heteroduplexes. After re-hybridization, the products are treated with SURVEYOR nuclease and SURVEYOR S enhancer (Transgenomics) according to the manufacturer's recommended protocol. The assay can be performed using polyacrylamide gels according to known methods. Quantitative analysis can be based on relative band intensity values.

***Inducible enzyme and split enzyme ("Split-Cas9")

In one aspect of the present invention, there is provided a non-naturally occurring or engineered CRISPR-Cas system based on inducible Cas9, comprising:

the first Cas9-based fusion construct to which the first half of the inducible dimer is attached, and

a second Cas9-based fusion construct to which the second half of the inducible dimer is attached,

wherein the first Cas9-based fusion construct is operably linked to one or more nuclear localization signals,

wherein the second Cas9-based fusion construct is operably linked to one or more nuclear export signals,

where contact with an energy source, which is an inductor, ensures that the first and second halves of the induced dimer are brought together,

where bringing the first and second halves of the inducible dimer together allows the first and second Cas9-based fusion constructs to form a functional Cas9-based CRISPR-Cas system,

wherein the Cas9-based CRISPR-Cas system comprises 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

where a functional Cas9-based CRISPR-Cas system binds to a target sequence and optionally edits a genomic locus to alter gene expression.

In one aspect of the present invention, in the inducible Cas9-based CRISPR-Cas system, the inducible dimer is, or comprises, or consists essentially of, or consists of, an inducible heterodimer. In one aspect, in the inducible Cas9-based CRISPR-Cas system, the first half, or first portion, or first fragment of the inducible heterodimer is, or comprises, or consists of, or consists essentially of FKBP, optionally FKBP12. In one aspect of the present invention, in the inducible Cas9-based CRISPR-Cas system, the second half, or second portion, or second fragment of the inducible heterodimer is, or comprises, or consists of, or consists essentially of FRB. In one aspect of the present invention, in an inducible Cas9-based CRISPR-Cas system, the arrangement in the first Cas9-based fusion construct is, or comprises, or consists of, or consists essentially of, the N'-terminal portion of Cas9-FRB-NES. In one aspect of the present invention, in an inducible Cas9-based CRISPR-Cas system, the arrangement in the first Cas9-based fusion construct is, or comprises, or consists of, or consists essentially of, NES-N'-terminal portion of Cas9-FRB-NES. In one aspect of the present invention, in an inducible Cas9-based CRISPR-Cas system, the arrangement in the second Cas9-based fusion construct is, or comprises, or consists essentially of, or consists of, the C'-terminal portion of Cas9-FKBP-NLS. In one aspect, the present invention provides an inducible Cas9-based CRISPR-Cas system, wherein the arrangement in the second Cas9-based fusion construct is, or comprises, or consists of, or consists essentially of NLS-C'-terminal portion of Cas9-FKBP-NLS. In one aspect, in the inducible Cas9-based CRISPR-Cas system, a linker may be present that separates the Cas9 portion from the half or portion or fragment of the inducible dimer. In one aspect, in the inducible Cas9-based CRISPR-Cas system, the energy source that is an inducer is, or comprises, or consists essentially of, or consists of rapamycin. In one aspect, in the inducible Cas9-based CRISPR-Cas system, the inducible dimer is an inducible homodimer. In one aspect, in an inducible Cas9-based CRISPR-Cas system, the Cas9 is FnCas9. In one aspect, in an inducible Cas9-based CRISPR-Cas system, one or more functional domains are associated with one or both portions of the Cas9, for example, the functional domains optionally include a transcriptional activator, a transcriptional element, or a nuclease, such as a Fok1 nuclease. In one aspect of an inducible Cas9-based CRISPR-Cas system, a functional Cas9-based CRISPR-Cas system binds to a target sequence, and wherein the enzyme is a non-functional Cas9, which optionally has a nuclease activity that is reduced by at least 97% or 100% (or has no more than 3% and substantially 0% nuclease activity) compared to a Cas9 that does not have at least one mutation. The present invention further encompasses a polynucleotide encoding the inducible Cas9-based CRISPR-Cas system discussed herein, and it is provided in one aspect of the present invention.

In one aspect of the present invention, there is provided a vector for delivering a first Cas9-based fusion construct to which a first half or portion or fragment of an inducible dimer is attached and which is operably linked to one or more nuclear localization signals, as discussed herein. In one aspect of the present invention, there is provided a vector for delivering a second Cas9-based fusion construct to which a second half or portion or fragment of an inducible dimer is attached and which is operably linked to one or more nuclear export signals.

In one aspect of the present invention, there is provided a vector for delivering both a first Cas9-based fusion construct to which a first half, or portion, or fragment of an inducible dimer is attached and which is operably linked to one or more nuclear localization signals discussed herein; and a second Cas9-based fusion construct to which a second half, or portion, or fragment of an inducible dimer is attached and which is operably linked to one or more nuclear export signals discussed herein.

In one aspect, the vector may be a single plasmid or expression cassette.

In one aspect, the present invention provides a eukaryotic host cell or cell line transformed with any of the vectors discussed herein or expressing the inducible Cas9-based CRISPR-Cas system discussed herein.

In one aspect, the present invention provides a transgenic organism transformed with any of the vectors discussed herein or expressing the inducible Cas9-based CRISPR-Cas system discussed herein or a progeny thereof. In one aspect, the present invention provides a model organism that constitutively expresses the inducible Cas9-based CRISPR-Cas system discussed herein.

In one aspect of the present invention, there is provided a non-naturally occurring or engineered CRISPR-Cas system based on inducible Cas9, comprising:

the first Cas9-based fusion construct to which the first half of the inducible heterodimer is attached, and

a second Cas9-based fusion construct to which the second half of the inducible heterodimer is attached,

wherein the first Cas9-based fusion construct is operably linked to one or more nuclear localization signals,

where the second Cas9-based fusion construct is functionally linked to a nuclear export signal,

where contact with an energy source, which is an inductor, ensures that the first and second halves of the induced heterodimer are brought together,

where bringing the first and second halves of the inducible heterodimer together allows the first and second Cas9-based fusion constructs to form a functional Cas9-based CRISPR-Cas system,

wherein the Cas9-based CRISPR-Cas system comprises 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

where a functional Cas9-based CRISPR-Cas system edits a genomic locus to alter gene expression.

In one aspect, the present invention provides a method of treating a subject in need thereof, comprising inducing gene editing by transforming the subject with a polynucleotide as discussed herein or any of the vectors as discussed herein, and administering to the subject an energy source that is an inducer. The present invention encompasses ways of using such a polynucleotide or vector in the manufacture of a medicament, e.g., such a medicament, for treating a subject or for such a method of treating a subject. The present invention encompasses a polynucleotide as discussed herein or any of the vectors as discussed herein for use in a method of treating a subject in need thereof, comprising inducing gene editing, wherein the method further comprises administering to the subject an energy source that is an inducer. In one aspect, the method also provides a repair template, e.g., delivered by a vector containing said repair template.

The present invention also provides a method of treating a subject in need thereof, comprising inducing transcriptional activation or repression by transforming the subject with a polynucleotide discussed herein or any of the vectors discussed herein, wherein said polynucleotide or vector encodes or comprises a catalytically inactive Cas9 and one or more associated functional domains discussed herein; wherein the method further comprises administering to the subject an energy source that is an inducer. The present invention also provides a polynucleotide discussed herein or any of the vectors discussed herein for use in a method of treating a subject in need thereof, comprising inducing transcriptional activation or repression, wherein the method further comprises administering to the subject an energy source that is an inducer.

Accordingly, the present invention encompasses, inter alia, homodimers as well as heterodimers, non-functional Cas9 or Cas9 characterized by a substantial absence of nuclease activity, for example due to mutation, systems or complexes in which one or more NLS and/or one or more NES are present; functional domain(s) associated with split-Cas9; methods, including methods of treatment, and routes of use.

It should be understood that when Cas9, Cas9 protein or Cas9 enzyme are referred to herein, split-Cas9 according to the present invention is meant. In one aspect, the present invention provides a method of altering or modifying the expression of a gene product. The method may comprise introducing into a cell containing and expressing a DNA molecule encoding a gene product, an engineered, non-naturally occurring Cas9-based CRISPR-Cas system comprising a Cas9 protein and a guide RNA that targets the DNA molecule, whereby the guide RNA targets the DNA molecule encoding the gene product, and the Cas9 protein cleaves the DNA molecule encoding the gene product, whereby the expression of the gene product is altered; and wherein the Cas9 protein and the guide RNA do not occur together in nature. The present invention encompasses a guide RNA that provides a guide sequence associated with a direct repeat (DR) sequence. The present invention further encompasses a Cas9 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 present invention, expression of the gene product is reduced.

In one aspect of the present invention, there is provided an engineered, non-naturally occurring Cas9-based CRISPR-Cas system comprising a Cas9 protein and a guide RNA that targets a DNA molecule encoding a gene product in a cell, whereby the guide RNA targets the DNA molecule encoding the gene product, and the Cas9 protein cleaves the DNA molecule encoding the gene product, whereby expression of the gene product is altered; and wherein the Cas9 protein and the guide RNA do not occur together in nature; wherein there is provided a split-Cas9 according to the present invention. The present invention encompasses a guide RNA that comprises a guide sequence associated with a DR sequence. The present invention further encompasses a Cas9 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 present invention, expression of the gene product is reduced.

In another aspect, the present invention provides an engineered, non-naturally occurring vector system comprising one or more vectors comprising a first regulatory element operably linked to a guide RNA of a Cas9-based CRISPR-Cas system that targets a DNA molecule encoding a gene product, and a second regulatory element operably linked to a Cas9 protein; wherein split-Cas9 according to the present invention is provided. Components (a) and (b) can be located in the same or different vectors of the system. The guide RNA targets a DNA molecule encoding a gene product in a cell, and the Cas9 protein cleaves the DNA molecule encoding the gene product, resulting in altered expression of the gene product; and wherein the Cas9 protein and the guide RNA do not occur together in nature. The present invention encompasses a guide RNA that provides a guide sequence associated with a DR sequence. The present invention further encompasses a Cas9 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 present invention, expression of the gene product is reduced.

In one aspect of the present invention, a vector system is provided, comprising one or more vectors. In some embodiments, the system comprises: (a) a first regulatory element operably linked to a DR sequence and one or more insertion sites for inserting one or more guide sequences downstream of the DR sequence, wherein, when expressed, the guide sequence directs sequence-specific binding of a Cas9-based CRISPR-Cas complex to a target sequence in a eukaryotic cell, wherein the Cas9-based CRISPR-Cas complex comprises Cas9 in complex with (1) a guide sequence that hybridizes to the target sequence and (2) a DR sequence; and (b) a second regulatory element operably linked to an enzyme-coding sequence that encodes said Cas9 enzyme comprising a nuclear localization sequence; wherein components (a) and (b) are in the same or different vectors of the system; wherein split-Cas9 according to the present invention is provided. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein, when expressed, each of the two or more guide sequences directs sequence-specific binding of a Cas9-based CRISPR-Cas complex to its target sequence in a eukaryotic cell.

In some embodiments, the Cas9-based CRISPR-Cas complex comprises one or more nuclear localization sequences that are sufficiently efficient to direct the accumulation of said Cas9-based CRISPR-Cas complex in a detectable amount in the nucleus of a eukaryotic cell. Without being bound by theory, it is believed that the nuclear localization sequence is not necessary for the activity of the Cas9-based CRISPR-Cas complex in eukaryotes, but the inclusion of such sequences enhances the activity of the system, especially with respect to targeting nucleic acid molecules in the nucleus.

In some embodiments, the Cas9 enzyme is a Cas9 of a bacterial species selected from the group consisting of Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, and Porphyromonas macacae, and may include a mutated Cas9 derived from these organisms. The enzyme may be a homolog or ortholog of Cas9. In some embodiments, Cas9 is codon optimized for expression in a eukaryotic cell. In some embodiments, Cas9 directs a single or double strand cleavage at a specific position in a target sequence. In a preferred embodiment, the strand break is a staggered break that forms a 5' sticky end. 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 minimum length of the direct repeat is 16 nucleotides and comprises one stem loop. In further embodiments, the length of the direct repeat is greater than 16 nucleotides, preferably greater than 17 nucleotides, and comprises more than one stem loop or optimized secondary structures.

In one aspect of the present invention, there is provided a eukaryotic host cell comprising (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for insertion of one or more guide sequences downstream of a DR sequence, wherein, when expressed, the guide sequence directs sequence-specific binding of a Cas9-based CRISPR-Cas complex to a target sequence in a eukaryotic cell, wherein the Cas9-based CRISPR-Cas complex comprises Cas9 in complex with (1) a guide sequence that hybridizes to the target sequence and (2) a DR sequence; and/or (b) a second regulatory element operably linked to an enzyme-coding sequence that encodes said Cas9 enzyme comprising a nuclear localization sequence. In some embodiments, the host cell comprises components (a) and (b); wherein a split-Cas9 according to the present invention is provided. In some embodiments, component (a), component (b), or components (a) and (b) are stably integrated into the genome of a eukaryotic host cell. In some embodiments, component (a) further comprises two or more guide sequences operably linked to a first regulatory element, wherein when expressed, each of the two or more guide sequences directs sequence-specific binding of a Cas9-based CRISPR-Cas complex to its target sequence in a eukaryotic cell. In some embodiments, Cas9 is codon-optimized for expression in a eukaryotic cell. In some embodiments, Cas9 directs a single or double strand cleavage at a specific position of a target sequence. In a preferred embodiment, the strand break is a staggered break that forms a 5' sticky end. In some embodiments, Cas9 lacks DNA strand cleavage activity. In some embodiments, the first regulatory element is a polymerase III promoter. In some embodiments, the direct repeat has a minimum length of 16 nucleotides and comprises one stem-loop. In further embodiments, the direct repeat has a length of more than 16 nucleotides, preferably more than 17 nucleotides, and comprises more than one stem-loop or optimized secondary structures. In one aspect, the present invention provides a eukaryotic organism other than a human; preferably a multicellular eukaryotic organism comprising a eukaryotic host cell according to any of the described embodiments. In other aspects, the present invention provides a eukaryotic organism, preferably a multicellular eukaryotic organism comprising a eukaryotic host cell according to any of the described embodiments. In some embodiments of these aspects, the organism may be an animal; for example, a mammal. The organism may also be an arthropod, such as an insect. The organism may also be a plant. In addition, the organism may be a fungus.

In one aspect of the present invention, a kit is provided comprising one or more components described herein. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the vector system comprises (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 downstream of a DR sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a Cas9-based CRISPR-Cas complex to a target sequence in a eukaryotic cell, wherein the Cas9-based CRISPR-Cas complex comprises Cas9 in complex with (1) a guide sequence that hybridizes to the target sequence and (2) a DR sequence; and/or (b) a second regulatory element operably linked to an enzyme-coding sequence that encodes said Cas9 enzyme comprising a nuclear localization sequence, and advantageously it provides for split-Cas9 according to the present invention. In some embodiments, the kit comprises components (a) and (b) located in the same or different vectors of the system. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences directs sequence-specific binding of the Cas9-based CRISPR-Cas complex to its target sequence in a eukaryotic cell. In some embodiments, the Cas9 comprises one or more nuclear localization sequences that are sufficiently efficient to direct the accumulation of said Cas9 in a detectable amount in the nucleus of a eukaryotic cell. In some embodiments, the Cas9 enzyme is a Cas9 of a bacterial species selected from the group consisting of Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, and Porphyromonas macacae, and may include mutated Cas9 from these organisms. The enzyme may be a homolog or ortholog of Cas9. In some embodiments, Cas9 is codon optimized for expression in a eukaryotic cell. In some embodiments, Cas9 directs the cleavage of one or two strands at a specific position in a target sequence. In a preferred embodiment, the strand break is a staggered break that forms a 5' sticky end. In some embodiments, the CRISPR enzyme does not have DNA strand cleavage activity. In some embodiments, the direct repeat has a minimum length of 16 nucleotides and comprises one stem loop. In further embodiments, the direct repeat is greater than 16 nucleotides, preferably greater than 17 nucleotides, and comprises more than one stem loop or optimized secondary structures.

In one aspect of the present invention, there is provided a method for modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing a Cas9-based CRISPR-Cas complex to bind to a target polynucleotide to effect cleavage of said target polynucleotide, thereby effecting modification of the target polynucleotide, wherein the Cas9-based CRISPR-Cas complex comprises Cas9 in a complex with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a direct repeat sequence. In some embodiments, said cleavage comprises cleaving one or two strands at a certain position of the target sequence using said Cas9; wherein a split-Cas9 according to the present invention is provided. In some embodiments, said cleavage results in reduced transcription of the target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous polynucleotide template, wherein said repair results in a mutation involving an insertion, deletion or substitution of one or more nucleotides in said target polynucleotide. In some embodiments, said mutation results in a change in one or more amino acids in a protein expressed from a gene comprising 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 drive expression of one or more of Cas9 and a guide sequence associated with a DR sequence. In some embodiments, said vectors are delivered to a eukaryotic cell in a subject. 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 the subject prior to said modification. In some embodiments, the method further comprises returning said eukaryotic cell and/or cells derived therefrom to said subject.

In one aspect of the present invention, there is provided a method of modifying the expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises causing a Cas9-based CRISPR-Cas complex to bind to a polynucleotide, such that said binding results in increased or decreased expression of said polynucleotide; wherein the Cas9-based CRISPR-Cas complex comprises Cas9 in a complex with a guide sequence that hybridizes to a target sequence within said polynucleotide, wherein said guide sequence is linked to a direct repeat sequence; wherein a split-Cas9 according to the present invention is provided. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cells, wherein the one or more vectors direct the expression of one or more of Cas9 and a guide sequence linked to a DR sequence.

In one aspect, the present invention provides a method for producing a model eukaryotic cell comprising a mutated disease-causative gene. In some embodiments, the disease-causative gene is any gene associated with an increased risk of having or developing a disease. In some embodiments, the method comprises (a) introducing one or more vectors into a eukaryotic cell, wherein the one or more vectors direct expression of one or more of Cas9 and a guide sequence associated with a direct repeat sequence; and (b) allowing a Cas9-based CRISPR-Cas complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide within said disease-causative gene, wherein the Cas9-based CRISPR-Cas complex comprises Cas9 in complex with (1) a guide sequence that hybridizes to a target sequence within the target polynucleotide, and (2) a DR sequence, thereby producing a model eukaryotic cell comprising a mutated disease-causative gene; wherein a split-Cas9 according to the present invention is provided. In some embodiments, said cleavage comprises cleaving one or two strands at a certain position of the target sequence using said Cas9. In a preferred embodiment, the strand break is a staggered break forming a 5' sticky end. In some embodiments, said cleavage results in reduced transcription of the target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous polynucleotide template, wherein said repair results in a mutation involving an insertion, deletion or substitution of one or more nucleotides in said target polynucleotide. In some embodiments, said mutation results in a change in one or more amino acids in the expression of a protein from a gene comprising the target sequence.

In one aspect of the present invention, there is provided a method for developing a biologically active agent that modulates a cellular signaling event associated with a disease-promoting gene. In some embodiments, the disease-promoting gene is any gene associated with an increased risk of having or developing a disease. In some embodiments, the method comprises (a) contacting a test compound with a model cell according to any of the described embodiments; and (b) detecting a change in a readout that indicates a decrease or increase in a cellular signaling event associated with said mutation in said disease-promoting gene, thereby obtaining said biologically active agent that modulates said cellular signaling event associated with said disease-promoting gene.

In one aspect of the present invention, there is provided a recombinant polynucleotide comprising a guide sequence downstream of a direct repeat sequence, wherein the guide sequence, when expressed, directs sequence-specific binding of a Cas9-based CRISPR-Cas complex to a 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.

In one aspect of the present invention, there is provided a method of selecting a cell or more cells by introducing one or more mutations into a gene in one or more cells, the method comprising introducing one or more vectors into the cell(s), wherein the one or more vectors direct the expression of one or more of Cas9, a guide sequence associated with a direct repeat sequence, and an editing template; wherein the editing template comprises one or more mutations that provide for the termination of cleavage by Cas9; providing for homologous recombination of the editing template with a target polynucleotide in the cell(s) to be selected; allowing a Cas9-based CRISPR-Cas complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide within said gene, wherein the Cas9-based CRISPR-Cas complex comprises Cas9 in a complex with (1) a guide sequence that hybridizes to a target sequence within the target polynucleotide, and (2) a direct repeat sequence, wherein the binding of the Cas9-based CRISPR-Cas complex to the target polynucleotide induces cell death, thereby allowing selection of one or more cells into which one or more mutations have been introduced; wherein split-Cas9 according to the present invention is provided. In another preferred embodiment of the present invention, the cell to be selected may be a eukaryotic cell. Aspects of the present invention provide for the selection of specific cells without the need for a selection marker or a two-step method that may include a negative selection system.

Throughout this document, the phrase "wherein split-Cas9 according to the present invention is provided" or similar expression appears; and these indicate that the Cas9 in the embodiments provided herein may be the split-Cas9 discussed herein.

In one aspect, the present invention relates to a non-naturally occurring or engineered inducible Cas9-based CRISPR-Cas system comprising a first Cas9-based fusion construct to which a first half of an inducible heterodimer is attached and a second Cas9-based fusion construct to which a second half of the inducible heterodimer is attached, wherein the first Cas9-based fusion construct is operably linked to one or more nuclear localization signals, wherein the second Cas9-based fusion construct is operably linked to a nuclear export signal, wherein contact with an energy source that is an inducer causes the first and second halves of the inducible heterodimer to be brought together, wherein bringing the first and second halves of the inducible heterodimer together allows the first and second Cas9-based fusion constructs to form a functional Cas9-based CRISPR-Cas system, wherein the Cas9-based CRISPR-Cas system comprises 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 wherein the functional Cas9-based CRISPR-Cas system edits the genomic locus to alter gene expression. In one embodiment of the present invention, the first half of the inducible heterodimer is FKBP12, and the second half of the inducible heterodimer is FRB. In another embodiment of the present invention, the energy source that is an inducer is rapamycin.

An inducing energy source may be considered simply an inducer or a dimerizing agent. The term "inducing energy source" is used throughout this document for consistency. An inducing energy source (or inducer) functions with the reduction of Cas9. In some embodiments, an inducing energy source causes the two halves of Cas9 to be brought together by the action of the two halves of an inducible dimer. The two halves of the inducible dimer are therefore brought together in the presence of an inducing energy source. Without an inducing energy source, the two halves of the dimer will not form a dimer (dimerize).

Thus, the two halves of the inducible dimer interact with the energy source, which is the inducer, to dimerize into a dimer. In turn, this ensures the restoration of Cas9 by bringing the first and second parts of Cas9 together.

Each of the CRISPR enzyme fusion constructs contains one split-Cas9 portion. They are fused, preferably via a linker, such as the GlySer linker described herein, to one of the two halves of the dimer. The two halves of the dimer may be essentially two identical monomers that together form a homodimer, or they may be different monomers that together form a heterodimer. Thus, the two monomers can be considered one half of a complete dimer.

Cas9 is composite in the sense that the two parts of the Cas9 enzyme essentially comprise a functional Cas9. Such a Cas9 may function as a genome-editing enzyme (when complexed with target DNA and a guide) such as a nickase or nuclease (cleaving both strands of DNA), or it may be a non-functional Cas9, which is essentially a DNA-binding protein with little or no catalytic activity, typically due to mutation(s) in its catalytic domains.

The two parts of split-Cas9 can be thought of as the N'-terminal part and the C'-terminal part of split-Cas9. Fusion typically occurs at the point of splitting of Cas9. In other words, the C'-terminus of the N'-terminal part of split-Cas9 is fused to one half of the dimer, while the N'-terminus of the C'-terminal part is fused to the other half of the dimer.

Cas9 is not splittable in the sense that the gap is re-created. The split point is typically designed in silico and cloned into the construct. Together, the two parts of split-Cas9, the N'-terminal and C'-terminal parts, form a complete Cas9, containing 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, and most preferably at least 99% or more of the wild-type amino acids (or nucleotides encoding them). Some trimming may be possible, and mutants are envisaged. Non-functional domains can be completely deleted. What is important is that the two parts can be brought together and that the desired Cas9 function is restored or reestablished.

A dimer can be a homodimer or a heterodimer.

One or more, preferably two, NLSs can be used in operative linkage to the first Cas9-based construct. One or more, preferably two, NESs can be used in operative linkage to the first Cas9-based construct. The NLSs and/or NESs preferably flank the split-Cas9-dimer (i.e., half-dimer) fusion, i.e., one NLS can be located at the N'-terminus of the first Cas9-based construct, and one NLS can be at the C'-terminus of the first Cas9-based construct. Similarly, one NES can be located at the N'-terminus of the second Cas9-based construct, and one NES can be at the C'-terminus of the second Cas9-based construct. When referring to the N'- or C'-termini, it is understood that they correspond to the 5'- and 3'-termini in the corresponding nucleotide sequence.

A preferred arrangement is that the first Cas9-based construct is 5'-NLS-(N'-terminal portion of Cas9)-linker-(first half of the dimer)-NLS-3'. A preferred arrangement is that the second Cas9-based construct is 5'-NES-(second half of the dimer)-linker-(C'-terminal portion of Cas9)-NES-3'. A suitable promoter is preferably located upstream of each of these constructs. The two constructs can be delivered separately or together.

In some embodiments, one or all of the NESs for functionally linking to the second Cas9-based construct may be replaced with an NLS. However, this may not generally be preferred, and in other embodiments, the localization signal for functionally linking to the second Cas9 construct is one or more NESs.

It should also be understood that the NES may be operably linked to the N'-terminal fragment of split-Cas9, and that the NLS may be operably linked to the C'-terminal fragment of split-Cas9. However, an arrangement in which the NLS is operably linked to the N'-terminal fragment of split-Cas9 and the NES is operably linked to the C'-terminal fragment of split-Cas9 may be preferred.

The NES functions to localize the second Cas9-based fusion construct outside the nucleus, at least until an energy source that is an inducer is provided (e.g., at least until an energy source for the inducer to perform its function is provided). The presence of the inducer stimulates dimerization of the two Cas9-based fusion products in the cytoplasm and makes it thermodynamically favorable for the dimerized first and second Cas9-based fusion products to be translocated into the nucleus. Without being limited by theory, applicants believe that the NES ensures the sequestration of the second Cas9-based fusion product in the cytoplasm (i.e., outside the nucleus). The NLS in the first Cas9-based fusion product ensures its localization to the nucleus. In both cases, applicants use the NES or the NLS to shift the equilibrium (the nuclear transport equilibrium) in the desired direction. Dimerization typically occurs outside the nucleus (a very small fraction may occur in the nucleus), and the NLSs in the dimerized complex shift the nuclear transport equilibrium toward nuclear localization such that dimerized and hence reconstituted Cas9 enters the nucleus.

In fact, the applicants are able to restore the function of split-Cas9. As a proof of concept, transient transfection was used and dimerization occurred in the background in the presence of an energy source, which is an inducer. No activity was observed with individual Cas9 fragments. Stable expression was then achieved by lentiviral delivery to demonstrate this, and it was shown that the split-Cas9 approach could be feasible.

This split-Cas9 approach according to the present invention is advantageous because it allows for the induction of Cas9 activity, thus providing temporal control. Moreover, different localization sequences (i.e. NES and NLS as preferred) can be used to reduce the activity of the self-assembled complexes in the environment. Tissue-specific promoters, for example one for each of the first and second Cas9-based fusion constructs, can also be used for tissue-specific targeting, thus providing spatial control. Two different tissue-specific promoters can be used to provide a higher degree of control if necessary. The same approach can be used with stage-specific promoters, or there can be a mixture of stage-specific promoters and tissue-specific promoters, wherein one of the first and second Cas9-based fusion constructs is under the control of (i.e., is operably linked to or contains) a tissue-specific promoter, while the other of the first and second Cas9-based fusion constructs is under the control of (i.e., is operably linked to or contains) a stage-specific promoter.

The inducible Cas9-based CRISPR-Cas system comprises one or more nuclear localization sequences (NLS) described herein, for example, operably linked to a first Cas9-based fusion construct. These nuclear localization sequences are ideally characterized by sufficient efficiency to direct the accumulation of said first Cas9-based fusion construct in a detectable amount in the nucleus of a eukaryotic cell. Without being bound by theory, it is believed that the nuclear localization sequence is not necessary for the activity of the Cas9-based CRISPR-Cas complex in eukaryotes, but the inclusion of such sequences enhances the activity of the system, especially with respect to targeting nucleic acid molecules in the nucleus, and facilitates the function of the two-part system of the present invention.

Similarly, the second Cas9-based fusion construct is operably linked to a nuclear export sequence (NES). In fact, it can be linked to one or more nuclear export sequences. In other words, the number of export sequences used with the second Cas9-based fusion construct is preferably 1, or 2, or 3. Typically, 2 are preferred, but 1 is sufficient and is therefore preferred in some embodiments. Suitable examples of NLSs and NESs are known in the art. For example, a preferred nuclear export signal (NES) is human protein tyrosine kinase 2. Preferred signals will be species-specific.

If the FRB and FKBP system is used, the FKBP is preferably flanked by nuclear localization sequences (NLS). If the FRB and FKBP system is used, the preferred arrangement is N'-terminal Cas9-FRB-NES: C'-terminal Cas9-FKBP-NLS. Thus, the first Cas9 fusion will contain the C'-terminal portion of Cas9, and the second Cas9 fusion will contain the N'-terminal portion of Cas9.

Another advantageous aspect of the present invention is that it can be rapidly activated, i.e. has a rapid response. Without being limited by theory, it is believed that Cas9 activity can be induced by dimerization of existing (already present) fusion constructs (by bringing into contact with an energy source, which is an inducer) faster than by expression (especially translation) of new fusion constructs. Thus, the first and second Cas9-based fusion constructs can be expressed in the target cell in advance, i.e. before Cas9 activity is required. Cas9 activity can then be temporarily controlled and then quickly established by adding an energy source, which is an inducer, which ideally acts faster (by dimerizing the heterodimer and thereby providing Cas9 activity) than by expression (including inducing transcription) of Cas9 delivered, for example, by a vector.

The terms "Cas9" or "Cas9 enzyme" and "CRISPR enzyme" are used interchangeably in this document unless otherwise apparent.

Applicants have demonstrated that Cas9 can be separated into two components that, when brought back together, reconstitute a functional nuclease. Using rapamycin-sensitive dimerization domains, Applicants have generated chemically inducible Cas9 to transiently control Cas9-mediated genome editing and modulate transcription. In other words, Applicants have demonstrated that Cas9 can be chemically inducible when separated into two fragments and that rapamycin-sensitive dimerization domains can be used to controllably reassemble Cas9. Applicants have shown that reassembled Cas9 can be used to mediate genome editing (via nuclease/nickase activity) and to modulate transcription (as a DNA-binding domain, the so-called "non-functional Cas9").

Thus, the use of rapamycin-sensitive dimerization domains is preferred. Reassembly of Cas9 is preferred. Reassembly can be determined by reconstitution of binding activity. If Cas9 is a nickase or induces a double-strand break, then a suitable percentage comparison with wild type is performed as described herein.

The rapamycin treatment may be for 12 days. The dose may be 200 nM. This time and/or molar dosage is an example of an appropriate dose for human embryonic kidney 293FT (HEK293FT) cell lines and may also be used for other cell lines. This schedule may be extrapolated for in vivo therapeutic use, such as mg/kg. However, it is also envisaged here that the standard dosage is also used to administer rapamycin to a subject. By "standard dosage" is meant the dosage for rapamycin's usual therapeutic use or primary indication (i.e., the dose used when rapamycin is administered to prevent organ rejection).

It should be noted that the preferred order of the Cas9-FRB/FKBP moieties is separate and they are inactive until rapamycin-induced dimerization of FRB and FKBP results in reassembly of a functional full-length Cas9 nuclease. Thus, it is preferred that the first Cas9-based fusion construct to which the first half of the inducible heterodimer is attached is delivered separately and/or localized separately from the second Cas9-based fusion construct to which the first half of the inducible heterodimer is attached.

To ensure the isolation of the Cas9(N)-FRB fragment in the cytoplasm, where there is a lower probability of dimerization with the nuclear-localized Cas9(C)-FKBP fragment, it is preferable to use one nuclear export sequence (NES) from human protein tyrosine kinase 2 (Cas9(N)-FRB-NES) in Cas9(N)-FRB. In the presence of rapamycin, Cas9(N)-FRB-NES dimerizes with Cas9(C)-FKBP-2xNLS to reconstitute the complete Cas9 protein, which shifts the nuclear transport equilibrium toward nuclear import and enables DNA targeting.

High Cas9 dosage can increase the frequency of insertion-deletion formation in off-target (OT) sequences, which are characterized by multiple mismatched bases to the guide strand. Such sequences are particularly susceptible if the mismatched bases are non-sequential and/or are outside the seed region of the guide. Accordingly, transient control of Cas9 activity can be used to reduce dosage in long-term expression experiments, and hence result in reduced formation of off-target insertions/deletions compared to constitutively active Cas9.

Viral delivery is preferred. In particular, a lentivirus or AAV-based delivery vector is envisaged. Applicants have obtained a lentivirus-based split-Cas9 construct similar to the lentiCRISPR plasmid. The components must be small enough to meet the AAV size limit of ~4.7 kb.

Applicants have demonstrated that stable low copy number expression of split-Cas9 can be used to induce large insertions/deletions at a target locus without significant mutation at off-target sites. Applicants have cloned Cas9 fragments (2 parts based on split 5 described herein).

It is also possible to use non-functional Cas9 containing the VP64 transactivation domain, for example, added to Cas9(C)-FKBP-2xNLS (inactive Cas9(C)-FKBP-2xNLS-VP64). These fragments reconstitute a catalytically inactive Cas9-VP64 fusion product (non-functional Cas9-VP64). Transcriptional activation is induced by VP64 in the presence of rapamycin, inducing dimerization of the Cas9(C)-FKBP fusion product and the Cas9(N)-FRB fusion product. In other words, applicants tested the inducibility of split non-functional Cas9-VP64 and showed that transcriptional activation is induced by non-functional split-Cas9-VP64 in the presence of rapamycin. Thus, the inducible Cas9 of the present invention may be associated with one or more functional domains, such as a transcriptional activator or repressor or a nuclease (such as Fok1). The functional domain may be linked or fused to one part of split-Cas9.

A preferred arrangement is that the first Cas9-based construct is arranged as follows: 5'-first localization signal-(N'-terminal portion of Cas9)-linker-(first half of the dimer)-first localization signal-3', and the second Cas9-based construct is arranged as follows: 5'-second localization signal-(second half of the dimer)-linker-(C'-terminal portion of Cas9)-second localization signal-functional domain-3'. In this case, the functional domain is placed at the 3'-end of the second Cas9-based construct. Alternatively, the functional domain can be placed at the 5'-end of the first Cas9-based construct. One or more functional domains can be used at the 3'-end, or the 5'-end, or at both ends. A suitable promoter is preferably located upstream of each of these constructs. The two constructs can be delivered separately or together. Localization signals can be NLS or NES, provided that they are not mixed in each design.

In one aspect of the present invention, a CRISPR-Cas system based on inducible Cas9 is provided, wherein the Cas9 has a nuclease activity reduced by at least 97% or 100% compared to a Cas9 enzyme lacking at least one mutation.

Accordingly, it is also preferred that the Cas9 be a non-functional Cas9. Ideally, the partitioning should always be such that the catalytic domain(s) are not affected. The meaning of a non-functional Cas9 is that DNA binding occurs, but no cleavage or nicase activity occurs.

In one aspect, the present invention provides an inducible Cas9-based CRISPR-Cas system discussed herein, wherein one or more functional domains are associated with Cas9. Such a functional domain may be associated (i.e., linked or fused) with one or both parts of a split-Cas9. It may be associated with each of the two parts of a split-Cas9. Thus, they may typically be present as part of a first and/or second Cas9-based fusion construct, as fusion products within that construct. The functional domains are typically fused via a linker, such as the GlySer linker discussed herein. The one or more functional domains may be an activation domain or a transcriptional repression domain. Although they may be different domains, it is preferred that all functional domains are either activators or repressors, and that a mixture of the two is not used.

The transcriptional activation domain may include VP64, p65, MyoD1, HSF1, RTA, or SET7/9.

In one aspect of the present invention, there is provided a CRISPR-Cas system based on inducible Cas9 as discussed herein, wherein one or more functional domains associated with Cas9 are a transcriptional repression domain.

In one aspect of the present invention, there is provided a CRISPR-Cas system based on inducible Cas9 as discussed herein, wherein the transcriptional repression domain is a KRAB domain.

In one aspect of the present invention, there is provided a CRISPR-Cas system based on inducible Cas9 as discussed herein, wherein the transcriptional repression domain is a NuE domain, an NcoR domain, a SID domain, or a SID4X domain.

In one aspect of the present invention, there is provided an inducible Cas9-based CRISPR-Cas system as discussed herein, wherein one or more functional domains associated with the adaptor protein are characterized by one or more activities comprising methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcript release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, DNA integration activity, or nucleic acid binding activity.

Histone modifying domains are also preferred in some embodiments. Exemplary histone modifying domains are discussed below. Transposase domains, HR (homologous recombination) machinery domains, recombinase domains, and/or integrase domains are also preferred as functional domains of the present invention. In some embodiments, HR machinery domains, integrase domains, recombinase domains, and/or transposase domains have DNA integration activity.

In one aspect of the present invention, there is provided a CRISPR-Cas system based on the inducible Cas9 discussed herein, wherein the DNA cleavage activity is provided by a nuclease.

In one aspect of the present invention, there is provided a Cas9-based CRISPR-Cas system as discussed herein, wherein the nuclease comprises a Fok1 nuclease.

The use of such functional domains, which are preferred for the split-Cas9-based system of the present invention, is also discussed in detail in Konermann et al. (“Genome-scale transcriptional activation with an engineered CRISPR-Cas9 complex,” published in Nature on December 11, 2014).

The system according to the present invention can be used with any guide.

In certain embodiments, modified guides may be used. Particularly preferred are guides according to the teachings of the above-mentioned article by Konermann, published in Nature on December 11, 2014. These guides are modified by adding protein-binding moieties of RNA (such as aptamers). Such moiety(s) may replace a portion of the guide. The corresponding domains of the RNA binding protein may be used for subsequent recognition of the RNA and recruitment of functional domains, such as those described herein, to the guide. They are especially intended for use with non-functional Cas9, which results in activation or repression of transcription or cleavage of DNA by nucleases such as Fok1. The use of such guides in combination with non-functional Cas9 is effective, and is particularly effective if Cas9 itself is also associated with its own functional domain, as discussed herein. If non-functional Cas9 (with or without its own associated functional domain) is induced with reconstitution according to the present invention, i.e. is a split-Cas9, then the tool is particularly suitable.

A guide RNA (gRNA) also preferred for use in accordance with the present invention may comprise a guide sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell, wherein the gRNA is modified by inserting a different RNA sequence(s) that binds to one or more adaptor proteins, and wherein the adaptor protein is associated with one or more functional domains. Cas9 may comprise at least one mutation such that the Cas9 enzyme has no more than 5% of the nuclease activity of a Cas9 enzyme lacking the at least one mutation; and/or at least one or more nuclear localization sequences. Also provided is a non-naturally occurring or engineered composition comprising one or more guide RNAs (gRNA) comprising a guide sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell, a Cas9 enzyme comprising at least one or more nuclear localization sequences, wherein the Cas9 enzyme comprises at least one mutation such that the Cas9 enzyme has no more than 5% of the nuclease activity of a Cas9 enzyme lacking the at least one mutation, wherein the at least one gRNA is modified by insertion of a different RNA sequence(s) that binds to one or more adaptor proteins, and wherein the adaptor protein is associated with one or more functional domains.

The gRNA is preferably modified by inserting a different RNA sequence(s) that binds to one or more adaptor proteins. The insertion of a different RNA sequence(s) that binds to one or more adaptor proteins is preferably an aptamer sequence or two or more aptamer sequences specific for the same adaptor protein or different adaptor proteins. The adapter protein preferably comprises MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, fCb5, fCb8r, fCb12r, fCb23r, 7s, PRR1. Cell lines stably expressing, inter alia, non-functional split-Cas9 may be suitable.

Applicants have demonstrated that Cas9 can be split into two distinct fragments that, when brought back together by chemical induction, reconstitute a functional full-length Cas9 nuclease. The split-Cas9 structure will be suitable for a number of applications. For example, split-Cas9 may enable genetic strategies to restrict Cas9 activity to cell populations at an interface by placing each fragment under the control of different tissue-specific promoters. In addition, different chemically inducible dimerization domains, such as APA and gibberellin, may also be used.

The energy source, which is an inductor, is preferably chemical induction.

The split position or location is the point at which the first portion of the Cas9 enzyme is separated from the second portion. In some embodiments, the first portion will comprise or encode from 1 to X amino acids, while the second portion will comprise or encode from X+1 amino acids to the end. In this example, the numbering is continuous, but this may not always be necessary since amino acids (or nucleotides encoding them) can be trimmed from the end of either of the split ends, as long as sufficient DNA binding activity and, if necessary, nickase or DNA cleavage activity are retained, such as at least 40%, 50%, 60%, 70%, 80%, 90% or 95% of the activity of wild-type Cas9.

The illustrative numbering provided herein may be relative to a wild-type protein, preferably wild-type FnCas9. However, it is envisaged that mutants of wild-type Cas9, such as the FnCas9 protein, may be used. The numbering may also not exactly match the numbering of FnCas9, since, for example, some N'- or C'-terminal truncations or deletions may be used, but this can be resolved using standard sequence alignment tools. Orthologs are also preferred as a sequence alignment tool.

Thus, the splitting position can be selected by a person of ordinary skill in the art, for example, based on crystal structure data and/or computer-aided structure prediction results.

For example, computational analysis of the primary structure of Cas9 nucleases revealed three distinct regions (Fig. 1). The first is the C-terminal RuvC-like domain, which is the only functional domain characterized. The second is the N-terminal alpha-helical region, and the third is a region of mixed alpha and beta structure located between the RuvC-like domain and the alpha-helical region. Several short stretches of unstructured regions are predicted in the primary structure of Cas9. Unstructured regions that are solvent accessible and not conserved among different Cas9 orthologs may represent preferred sides for separation (Fig. 2 and Fig. 3).

The table below shows non-restrictive potential cleavage sites in As and LbCas9. A cleavage site within such a region may be suitable.

Separation area AsCas9 LbCas9 1 575-588 566-571 2 631-645 754-757 3 653-664 - 4 818-844 -

For Fn, As and Lb Cas9 mutants, it should be obvious that the appropriate position for the potential splitting site is, for example, based on sequence alignment. For enzymes other than Fn, As and Lb, the crystal structure of the ortholog can be used if there is a relatively high degree of homology between the ortholog and the putative Cas9, or computational prediction can be used.

Ideally, the split position should be located within a region or loop. Preferably, the split position is where interruption of the amino acid sequence does not result in partial or complete disruption of a structural element (e.g., alpha helices or beta sheets). Unstructured regions (regions that are not found in the crystal structure because these regions are not sufficiently structured to "freeze" in the crystal) are often preferred options. Applicants may, for example, perform splits in unstructured regions that are accessible on the surface of Cas9.

Applicants may follow the following procedure, which is provided as a preferred example and as a guide. Since the unstructured regions are not found in the crystal structure, applicants consider the surrounding amino acid sequence in the crystal with the primary amino acid sequence of Cas9. Each unstructured region may consist of, for example, approximately 3-10 amino acids that are not found in the crystal. Applicants therefore perform a split between these amino acids. To include a larger number of potential split ends, applicants include splits located in loops outside of Cas9 using the same criteria as with the unstructured regions.

In some embodiments, the splitting position is in an external loop of Cas9. In other preferred embodiments, the splitting position is in an unstructured region of Cas9. The unstructured region is typically a very flexible external loop whose structure is difficult to determine from an X-ray crystal pattern.

Once the split position has been identified, suitable designs can be developed.

Typically, the NES is located at the N'-terminus of the first part of the amino acid to be separated (or at the 5'-end of the nucleotide encoding it). In this case, the NLS is located at the C'-terminus of the second part of the amino acid to be separated (or at the 3'-end of the nucleotide encoding it). Thus, the first Cas9-based fusion construct can be operably linked to one or more nuclear export signals, and the second Cas9-based fusion construct can be operably linked to a nuclear localization signal.

Of course, the reverse order of arrangement can be envisaged, in which the NLS is located at the N'-terminus of the first part of the amino acid to be separated (or at the 5'-end of the nucleotide encoding it). In such a case, the NES is located at the C'-terminus of the second part of the amino acid to be separated (or at the 3'-end of the nucleotide encoding it). Thus, the first Cas9-based fusion construct can be operably linked to one or more nuclear localization signals, and the second Cas9-based fusion construct can be operably linked to a nuclear export signal.

Splits that ensure that the two parts (or the side of the split) are approximately the same length may be useful for packaging purposes. For example, it is thought to be easier to maintain stoichiometry between the two parts when the transcripts are approximately the same size.

In some examples, the N- and C-terminal portions of a codon-optimized human Cas9, such as FnCas9, are fused to the dimerization domains of FRB and FKBP, respectively. This arrangement may be preferred. They may be redirected (i.e., N'-terminus to FKBP and C'-terminus to FRB).

Linkers such as (GGGGS) ₃ are preferably used herein to separate the Cas9 fragment from the dimerization domain. (GGGGS) ₃ is preferred because it is a relatively long linker (15 amino acids). Glycine residues are the most flexible, and serine residues increase the likelihood that the linker will be on the outside of the protein. (GGGGS) ₆ , (GGGGS) ₉ or (GGGGS) ₁₂ can preferably be used as alternatives. Other preferred alternatives are (GGGGS) ₁ , (GGGGS) ₂ , (GGGGS) ₄ , (GGGGS) ₅ , (GGGGS) ₇ , (GGGGS) ₈ , (GGGGS) ₁₀ or (GGGGS) ₁₁ .

For example, (GGGGS) ₃ can be inserted between the N'-terminal fragment of Cas9 and FRB. For example, (GGGGS) ₃ can be inserted between FKB and the C'-terminal fragment of Cas9.

Alternative linkers are available, but highly flexible linkers are thought to be better at maximizing the ability to join the two parts of Cas9 and thus restore Cas9 activity. One alternative is that the NLS of nucleoplasmin can be used as a linker.

A linker can also be used between Cas9 and any functional domain. Again, in this case, the (GGGGS) ₃ linker (or its 6, 9 or 12 repeat variants) can be used, or the nucleoplasmin NLS can be used as a linker between Cas9 and the functional domain.

Alternatives to the FRB/FKBP system are envisaged. For example, the ABA and gibberellin system.

Accordingly, preferred examples of the FKBP family 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 coumermycin; GAI, which dimerizes with GID1 in the presence of gibberellin; or Snap-tag, which dimerizes with HaloTag in the presence of HaXS.

Alternatives within the FKBP family itself are also preferred. For example, an FKBP that homodimerizes (i.e. one FKBP dimerizes with another FKBP) in the presence of FK1012. Thus, a CRISPR-Cas system based on non-naturally occurring or engineered inducible Cas9 is also envisaged, comprising:

the first Cas9-based fusion construct to which the first half of the inducible homodimer is attached, and

a second Cas9-based fusion construct to which the second half of the inducible homodimer is attached,

wherein the first Cas9-based fusion construct is operably linked to one or more nuclear localization signals,

wherein the second Cas9-based fusion construct is operably linked to (optionally one or more) nuclear export signal(s),

where contact with an energy source, which is an inductor, brings the first and second halves of the induced homodimer together,

where bringing the first and second halves of the inducible homodimer together allows the first and second Cas9-based fusion constructs to form a functional Cas9-based CRISPR-Cas system,

wherein the Cas9-based CRISPR-Cas system comprises 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

where a functional Cas9-based CRISPR-Cas system binds to a target sequence and optionally edits a genomic locus to alter gene expression.

In one embodiment, the homodimer is preferably FKBP and the energy source that is an inducer is preferably FK1012. In another embodiment, the homodimer is preferably GryB and the energy source that is an inducer is preferably coumermycin. In another embodiment, the homodimer is preferably ABA and the energy source that is an inducer is preferably gibberellin.

In other embodiments, the dimer is a heterodimer. Preferred examples of heterodimers 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 in the presence of coumermycin; GAI, which dimerizes with GID1 in the presence of gibberellin; or Snap-tag, which dimerizes with HaloTag in the presence of HaXS.

The applicants used FKBP/FRB because it is well characterized and both domains are small enough (<100 amino acids) to facilitate packaging. Furthermore, rapamycin has been used for a long time and its side effects are well known. Large dimerization domains (>300 aa) should also work, but longer linkers may be required to ensure Cas9 reconstitution.

Paulmurugan and Gambhir (Cancer Res, August 15, 2005 65; 7413) discuss the basics of the FRB/FKBP/rapamycin system. Another useful reference is the paper by Crabtree et al. (Chemistry & Biology 13, 99-107, Jan 2006).

In this example, a single vector, an expression cassette (plasmid), is constructed. The gRNA is under the control of the U6 promoter. Two different split-Cas9 constructs are used. The split-Cas9 construct is based on a first NLS-flanked Cas9 fusion with FKBP fused to the C'-terminal portion of split-Cas9 via a GlySer linker; and a second NES-flanked Cas9 fusion with FRB fused to the N'-terminal portion of split-Cas9 via a GlySer linker. P2A is used to separate the first and second Cas9 fusions, allowing for transcriptional separation. Split-Cas9 exhibits insertion/deletion formation similar to wild-type in the presence of rapamycin, but significantly lower insertion/deletion formation than wild-type in the absence of rapamycin.

Accordingly, one vector is provided. The vector contains:

the first Cas9-based fusion construct to which the first half of the inducible dimer is attached, and

a second Cas9-based fusion construct to which the second half of the inducible dimer is attached,

wherein the first Cas9-based fusion construct is operably linked to one or more nuclear localization signals,

wherein the second Cas9-based fusion construct is operably linked to one or more nuclear export signals,

where bringing into contact with an energy source, which is an inductor, ensures that the first and second halves of the induced heterodimer are brought together,

where bringing the first and second halves of the inducible heterodimer together allows the first and second Cas9-based fusion constructs to form a functional Cas9-based CRISPR-Cas system,

wherein the Cas9-based CRISPR-Cas system comprises 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

wherein a functional Cas9-based CRISPR-Cas system binds to a target sequence and optionally edits a genomic locus to alter gene expression. These elements are preferably present in a single construct, such as an expression cassette.

The first Cas9-based fusion construct is preferably flanked by at least one nuclear localization signal at each end. The second Cas9-based fusion construct is preferably flanked by at least one nuclear export signal at each end.

A method for treating a subject in need thereof is also provided, comprising inducing gene editing by transforming the subject using a polynucleotide encoding the system or any of the vectors according to the present invention and administering to the subject an energy source which is an inducer. A suitable repair matrix may also be provided, for example delivered by a vector containing said repair matrix.

Also provided is a method for treating a subject in need thereof, comprising inducing transcriptional activation or repression by transforming the subject with a polynucleotide encoding the system according to the present invention or any of the vectors according to the present invention, wherein said polynucleotide or vector encodes or contains catalytically inactive Cas9 and one or more functional domains associated therewith; wherein the method further comprises administering to the subject an energy source that is an inducer.

Compositions comprising the system according to the present invention for use in said method of treatment are also provided. Use of the system according to the present invention in the manufacture of a medicinal product for such methods of treatment is also provided.

Examples of conditions that can be treated with the system of the present invention are described herein or in documents cited herein.

A single vector can contain a transcript splitter such as P2A. P2A splits the transcript in two, separating the first and second Cas9-based fusion constructs. The splitting occurs due to "ribosome skipping." Essentially, the ribosome skips an amino acid during translation, which breaks the protein chain and results in two separate polypeptides/proteins. A single vector is also suitable for applications where low background activity is unimportant, but high inducible activity is desired.

One example is the generation of clonal embryonic stem cell lines. A common procedure is transient transfection with plasmids encoding wt Cas9 or Cas9 nickase. These plasmids produce Cas9 molecules that remain active for several days and have a higher probability of off-target activity. Using a single expression vector for split-Cas9 allows for the "high" Cas9 activity to be confined to a shorter period of time (e.g., a single dose of an inducer such as rapamycin). Without continuous (daily) treatments with an inducer (e.g., rapamycin), the activity of individual split-Cas9 expression vectors is low and provides a reduced probability of unwanted off-target effects.

A peak of induced Cas9 activity is useful in some embodiments and can be most easily induced using a single delivery vector, but is also possible with a dual vector system (each vector delivering one half of the split-Cas9). The peak can represent high activity and last for a short period of time, typically the duration of the inducer.

Accordingly, a method for producing clonal lines of embryonic stem cells is provided, comprising transfecting one or more embryonic stem cells with a polynucleotide encoding the system according to the present invention or one of the vectors according to the present invention for expressing split-Cas9 according to the present invention and introducing into one or more stem cells an energy source, which is an inducer, according to the present invention or bringing them into contact with it to induce Cas9 repair. A repair matrix may be provided.

As with all methods described in this document, it should be understood that suitable gRNAs or guides will be required.

When functional domains and the like are "associated" with one or another part of an enzyme, they are typically fusion products. The term "associated with" is used herein to refer to one molecule "binding" to another, such as between parts of Cas9 and a functional domain. In the case of such protein-protein interactions, this association can be thought of in terms of recognition, such as an antibody recognizing an epitope. Alternatively, one protein can be associated with another protein by a fusion of the two, such as one subunit being fused to another subunit. Fusion typically occurs by the addition of one amino acid sequence to another, such as by splicing the nucleotide sequences that encode each protein or subunit. Alternatively, it can be thought of as essentially a linkage of two molecules, or a direct linkage, such as a fusion protein. In any case, the fusion protein may include a linker between the two subunits of interest (i.e., between the enzyme and the functional domain, or between the adaptor protein and the functional domain). Thus, in some embodiments, the Cas9 portion is associated with the functional domain by binding thereto. In other embodiments, the Cas9 is associated with the functional domain by the two being fused together, optionally through an intervening linker. Examples of linkers include the GlySer linkers discussed herein.

Other examples of inducers include light and hormones. For light, inducible dimers can be heterodimers and include a first light-inducible half-dimer and a second (and complementary) light-inducible half-dimer. A preferred example of a first and second light-inducible half-dimer is the CIB1 and CRY2 system. The CIB1 domain is the heterodimeric binding partner of light-sensitive cryptochrome 2 (CRY2).

In another example, the blue-light-sensitive Magnet dimerization system (pMag and nMag) can be fused to the two parts of the split-Cas9 protein. In response to light stimulation, pMag and nMag dimerize and Cas9 reassembles. For example, such a system is described together with Cas9 by Nihongaki et al. (Nat. Biotechnol. 33, 755–790, 2015).

The present invention contemplates that the energy source, which is an inducer, can be heat, ultrasound, electromagnetic energy or a chemical substance. In a preferred embodiment of the present invention, the energy source, which is an inducer, can be an antibiotic, a small molecule, a hormone, a hormone derivative, a steroid or a steroid derivative. In a more preferred embodiment, the energy source, which is an inducer, can be abscisic acid (ABA), doxycycline (DOX), cumate, rapamycin, 4-hydroxytamoxifen (4OHT), estrogen or ecdysone. The present invention contemplates that at least one "switch" can be selected from the group consisting of antibiotic-based inducible systems, electromagnetic energy-based inducible systems, small molecule-based inducible systems, nuclear receptor-based inducible systems and hormone-based inducible systems. In a more preferred embodiment, the at least one switch may be selected from the group consisting of tetracycline (Tet)/DOX inducible systems, light inducible systems, ABA inducible systems, cumate repressor/operator based systems, 4OHT/estrogen inducible systems, ecdysone based inducible systems, and FKBP12/FRAP (FKBP12-rapamycin complex) inducible systems. Such inducers are also discussed herein and in PCT/US2013/051418, incorporated herein by reference.

In general, any application that may involve Cas9, whether wt, nickase or non-functional Cas9 (with or without associated functional domains), can be implemented using the split-Cas9 approach of the present invention. The inducible nature of Cas9 activity remains an advantage.

As a further example, split-Cas9 fusion products with fluorescent proteins such as GFP can be produced. This will allow for visualization of genomic loci (see "Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System" Chen B. et al. Cell 2013), but in an inducible manner. Thus, in some embodiments, one or more portions of Cas9 can be associated with (and in particular fused to) a fluorescent protein, such as GFP.

Additional experiments examined whether there is a difference in off-target cleavage between wild-type (wt) and split-Cas9 at similar levels of off-target cleavage. To do this, we transiently transfected wt and split-Cas9 Cas9 plasmids and harvested at different time points. We determined off-target activation after identifying a subset of samples in which on-target cleavage was +/- 5%. We generated cell lines stably expressing wt or split-Cas9 Cas9 without guides (using lentivirus). Following antibiotic selection, guides were delivered using a separate lentivirus and harvested at different time points to measure on-target/off-target cleavage.

Applicants introduced 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)Cas9-NES fragment to facilitate faster degradation and hence reduce the stability of the non-functional split-Cas9-VP64 complex.

Such destabilizing sequences, described elsewhere in this document (including PEST), may be preferred for use with split-Cas9 systems.

Cell lines stably expressing non-functional split-Cas9-VP64 and MS2-p65-HSF1 + guide were generated. Screening for PLX resistance may demonstrate that irreversible, time-regulated transcriptional activation may be useful in drug screening. This approach may be advantageous if non-functional split-Cas9-VP64 is irreversible.

In one aspect of the present invention, there is provided a non-naturally occurring or engineered Cas9-based CRISPR-Cas system, which can comprise at least one "switch", wherein the activity of said Cas9-based CRISPR-Cas system is controlled with respect to the "switch" by contacting with at least one energy source that is an inducer. In one embodiment of the present invention, the control with respect to the at least one "switch" or the activity of said Cas9-based CRISPR-Cas system can be activated, enhanced, terminated or suppressed. Contacting with at least one energy source that is an inducer can result in a first effect and a second effect. The first effect may be one or more of nuclear import, nuclear export, recruitment of a secondary component (such as an effector molecule), a conformational change (of a protein, DNA, or RNA), cleavage, release of a cargo molecule (such as a protected molecule or cofactor), association, or dissociation. The second effect may be one or more of activation, enhancement, termination, or suppression of control over at least one "switch" or activity of said Cas9-based CRISPR-Cas system. In one embodiment, the first effect and the second effect may occur as a cascade.

In another aspect of the present invention, the Cas9-based CRISPR-Cas system may further comprise at least one or more of a nuclear localization signal (NLS), a nuclear export signal (NES), a functional domain, a flexible linker, a mutation, a deletion, an alteration, or a truncation. One or more of the NLS, NES, or functional domain may be conditionally activated or inactivated. In another embodiment, the mutation may be one or more of a mutation in a homologous region of a transcription factor, a mutation in a DNA-binding domain (such as mutated basic residues in a basic helix-loop-helix structure), a mutation in an endogenous NLS, or a mutation in an endogenous NES. The present invention contemplates that the energy source that is an inducer may be heat, ultrasound, electromagnetic energy, or a chemical substance. In a preferred embodiment of the present invention, the energy source that is an inducer may be an antibiotic, a small molecule, a hormone, a hormone derivative, a steroid, or a steroid derivative. In a more preferred embodiment, the energy source that is an inducer may be abscisic acid (ABA), doxycycline (DOX), cumate, rapamycin, 4-hydroxytamoxifen (4OHT), estrogen, or ecdysone. The present invention contemplates that at least one "switch" may be selected from the group consisting of antibiotic-based inducible systems, electromagnetic energy-based inducible systems, small molecule-based inducible 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 (Tet)/DOX inducible systems, light inducible systems, ABA inducible systems, cumate repressor/operator based systems, 4OHT/estrogen inducible systems, ecdysone based inducible systems, and FKBP12/FRAP (FKBP12-rapamycin complex) inducible systems.

The aspects of control detailed in this application relate to at least one or more "switches". The term "switch" as used herein refers to a system or set of components that act in concert to effect 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 comprise essential components in the form of gene regulatory proteins and specific DNA sequences that these proteins recognize. In one aspect, "switches" refer to inducible and repressible systems used in gene regulation. In general, an inducible system may be inactive until a specific molecule (called an inducer) is present that causes the gene to be expressed. The molecule is said to "induce expression". The manner in which this is accomplished depends on the control mechanisms as well as differences in cell type. A repressible system is active until a specific molecule (called a corepressor) is present that suppresses gene expression. The molecule is said to "repress expression." The manner in which this is accomplished depends on the control mechanisms as well as differences in cell type. The term "inducible" as used herein may encompass all aspects of a "switch" regardless of the molecular mechanism involved. Accordingly, a "switch" as contemplated in the present invention may include, but is not limited to, antibiotic-based inducible systems, electromagnetic energy-based inducible systems, small molecule-based inducible systems, nuclear receptor-based inducible systems, and hormone-based inducible systems. In preferred embodiments, the "switch" may be a tetracycline (Tet)/DOX-inducible system, light-inducible systems, abscisic acid-inducible system, cumate repressor/operator-based system, 4OHT/estrogen-inducible system, ecdysone-based inducible systems, and FKBP12/FRAP (FKBP12-rapamycin complex)-inducible system.

The Cas9-based CRISPR-Cas system of the present invention can be designed to modulate or alter the expression of individual endogenous genes in a spatially and temporally precise manner. The Cas9-based CRISPR-Cas system can be designed to bind to a promoter sequence of a gene of interest to alter gene expression. Cas9 can be split in two, with one half fused to one half of a cryptochrome heterodimer (cryptochrome-2 or CIB1), while the remaining cryptochrome is fused to the other half of Cas9. In some aspects, a transcriptional effector domain can also be included in the Cas9-based CRISPR-Cas system. Effector domains can be either activators, such as VP16, VP64, or p65, or repressors, such as KRAB, EnR, or SID. In the unstimulated state, one half of the Cas9-cryptochrome-2 protein localizes to the promoter region of the gene of interest but does not bind to the CIB1 effector protein. Upon stimulation with blue light, cryptochrome-2 is activated, undergoes a conformational change, and exposes its binding domain. CIB1, in turn, binds to cryptochrome-2, resulting in the localization of the other half of Cas9 to the promoter region of the gene of interest and initiation of genome editing that 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 entireties.

Activator and repressor domains that can further modulate function can be selected based on species, potency, mechanism, duration, size, or any number of other parameters. Preferred effector domains include, but are not limited to, a transposase domain, an integrase domain, a recombinase domain, a resolvase domain, an invertase domain, a protease domain, a DNA methyltransferase domain, a DNA demethylase domain, a histone acetylase domain, a histone deacetylase domain, a nuclease domain, a repressor domain, an activator domain, nuclear localization signal domains, a transcriptional protein recruitment domain, a cellular uptake activity associated domain, a nucleic acid binding domain, or an antibody presentation domain.

There are several different ways to generate inducible systems: 1. the ABI-PYL-based system inducible by abscisic acid (ABA) (see, for example, stke.sciencemag.org/cgi/content/abstract/sigtrans;4/164/rs2), 2. the FKBP-FRB-based system inducible by rapamycin (or rapamycin-related chemicals) (see, for example, nature.com/nmeth/journal/v2/n6/full/nmeth763.html), 3. the GID1-GAI-based system inducible by gibberellin (GA) (see, for example, nature.com/nchembio/journal/v8/n5/full/nchembio.922.html).

Another system contemplated by the present invention is a chemically inducible system based on a change in subcellular localization. Applicants also provide an inducible Cas9-based CRISPR-Cas system designed to target a genomic locus of interest, wherein the Cas9 enzyme is divided into two fusion constructs that are further linked to different parts of a chemically or energy-sensitive protein. Such a chemically or energy-sensitive protein will lead to a change in the subcellular localization of one of the halves of the Cas9 enzyme (i.e., the transport of one of the halves of the Cas9 enzyme from the cytoplasm to the nucleus of cells) upon binding a chemical or upon transfer of energy to the chemically or energy-sensitive protein. Such transport of the fusion constructs from one subcellular compartment or organelle in which its activity is limited due to the absence of a substrate for the reconstituted Cas9-based CRISPR-Cas system to another in which the substrate is present will allow the components to combine and restore functional activity, and then come into contact with their required substrate (i.e., genomic DNA in the mammalian cell nucleus) and result in the activation or repression of target gene expression.

Other inducible systems have been proposed, such as unlimited regulation by heavy metals [Mayo KE et al., Cell 1982, 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 1981, 78:2038-2042; Klock G et al., Nature (London) 1987, 329:734-736, and Lee F et al., Nature (London) 1981, 294:228-232], and heat shock [Nouer L: Heat Shock Response. Boca Raton, FL: CRC; 1991], and other reagents have been developed [Mullick A, Massie B: Transcription, translation and the control of gene expression. In Encyclopedia of Cell Technology, eds: Speir RE. Wiley; 2000:1140-1164, and Fussenegger M, Biotechnol Prog 2001, 17:1-51]. However, limitations exist with such mammalian inducible promoters, such as "leakage" in the "off" state and pleiotropic effects of inducers (heat shock, heavy metals, glucocorticoids, etc.). The use of insect hormones (ecdysone) has been proposed in the hope of reducing the antagonism of cellular processes in mammalian cells [No D et al., Proc Natl Acad Sci USA 1996, 93:3346-3351]. Another excellent system uses rapamycin as an inducer [Rivera VM et al., Nat Med 1996, 2:1028-1032], but rapamycin's role as an immunosuppressant was a major limitation of its use in vivo and it was therefore necessary to find a biologically inert compound [Saez E et al., Proc Natl Acad Sci USA 2000, 97:14512-14517] to control gene expression.

See also the section below on induced systems.

Destabilized enzyme: enzymes according to the present invention having or associated with destabilization domains

In one aspect, the present invention provides 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 preferably but not limited to Cas9 as described elsewhere herein, associated with at least one destabilization domain (DD); and, for brevity, such a non-naturally occurring or engineered CRISPR enzyme associated with at least one destabilization domain (DD) is referred to herein as a "DD-CRISPR enzyme". It is understood that any of the CRISPR enzymes of the present invention as described elsewhere herein may be used as having or associated with the destabilizing domains described herein below. Any of the methods, products, compositions, and uses described elsewhere herein are equally applicable to CRISPR enzymes associated with destabilizing domains, as described in detail below.

For further guidance, the following specific aspects and implementation options are provided.

Because the aspects and embodiments described in this section involve DD-CRISPR, DD-Cas, DD-Cas9Cas9, DD-CRISPR-Cas enzymes, or DD-CRISPR-Cas9 systems or complexes, the terms "CRISPR," "Cas," "Cas9, "CRISPR system," "CRISPR complex," "CRISPR-Cas," "CRISPR-Cas9," or the like without the "DD" prefix may be construed as having the DD prefix, particularly where the context permits, so that the present invention encompasses embodiments with DDs. Accordingly, in one aspect, the present invention provides methods of using one or more elements of a CRISPR system (which may be considered a DD-CRISPR system and/or a CRISPR system). The CRISPR complex of the present invention provides an effective means for modifying a target polynucleotide. The CRISPR complex of the present invention has broad utility, including modifying (e.g., causing a deletion, insertion, translocation, inactivation, activation) of a target polynucleotide in a variety of cell types. The CRISPR complex of the present invention as such has a wide range of applications, such as in gene therapy, drug screening, disease diagnosis and prognosis.

In one aspect of the present invention, there is provided an engineered, non-naturally occurring DD-CRISPR-Cas system comprising a DD-CRISPR enzyme, such as a DD-CRISPR enzyme, wherein the CRISPR enzyme is a Cas protein (herein referred to as a "DD-Cas protein", i.e., the "DD" before a term such as a "DD-CRISPR-Cas9 complex" means a CRISPR-Cas9 complex comprising a Cas9 protein having at least one destabilization domain associated therewith), preferably a DD-Cas protein, such as a Cas9 protein, associated with at least one destabilization domain (herein referred to as a "DD-Cas9 protein"), and a guide RNA that targets a nucleic acid molecule, such as a DNA molecule, whereby the guide RNA targets a nucleic acid molecule, such as a DNA molecule. A nucleic acid molecule, such as a DNA molecule, may encode a gene product. In some embodiments, a DD-Cas protein may cleave a DNA molecule encoding a gene product. In some embodiments, expression of the gene product is altered. The Cas protein and the guide RNA do not occur together in nature. The present invention encompasses a guide RNA that provides a guide sequence, optionally, if applicable, fused to a tracr sequence. The present invention further encompasses encoding 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 a more preferred embodiment, the mammalian cell is a human cell. Expression of the gene product may be reduced. The CRISPR enzyme may form part of a CRISPR-Cas system that further comprises a guide RNA (gRNA) that comprises a guide sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell. In some embodiments, a functional CRISPR-Cas system binds to a target sequence. In some embodiments, a functional CRISPR-Cas system may edit a target sequence, for example, the target sequence may comprise a genomic locus, and in some embodiments, altering gene expression may be provided. In some embodiments, a functional CRISPR-Cas system may comprise additional functional domains. In some embodiments, the present invention provides a method of altering or modifying the expression of a gene product. The method may comprise introducing into a cell comprising or comprising and expressing a target nucleic acid, for example, a DNA molecule; for example, the target nucleic acid may encode a gene product or provide for the expression of a gene product (e.g., a regulatory sequence).

In some embodiments, the DD-CRISPR enzyme is DD-Cas9. In some embodiments, the DD-CRISPR enzyme is a V-A or V-B subtype CRISPR enzyme. In some embodiments, the DD-CRISPR enzyme is Cas9. In some embodiments, the DD-CRISPR enzyme is As DD-Cas9. In some embodiments, the CRISPR enzyme is Lb DD-Cas9. In some embodiments, the DD-CRISPR enzyme cleaves both strands of DNA to form a double-strand break (DSB). In some embodiments, the DD-CRISPR enzyme is a nickase. In some embodiments, the DD-CRISPR enzyme is a dual nickase. In some embodiments, the DD-CRISPR enzyme is a non-functional Cas9, such as a Cas9 that exhibits substantially no nuclease activity, such as exhibiting no more than 5% nuclease activity compared to a wild-type Cas9 or a Cas9 that does not have a mutation. Suitable Cas9 mutations are described elsewhere herein and include, for example, D917A, E1006A, E1028A, D1227A, D1255A, N1257A, D917A, E1006A, E1028A, D1227A, D1255A, and N1257A, corresponding to amino acid positions in the RuvC domain of FnCas9p; or, for example, N580A, N584A, T587A, W609A, D610A, K613A, E614A, D616A, K624A, D625A, K627A, and Y629A, relative to the putative second nuclease domain described elsewhere herein.

In some general embodiments, the DD-CRISPR enzyme is associated with one or more functional domains. In some more specific embodiments, the DD-CRISPR enzyme is a non-functional Cas9 and/or is associated with one or more functional domains. In some embodiments, the DD-CRISPR enzyme comprises a truncation, for example, of an α-helix or a mixed α/β secondary structure. In some embodiments, the truncation involves the removal or replacement of a linker. In some embodiments, the linker is branched or otherwise provides for the attachment of the DD and/or the functional domain. In some embodiments, the CRISPR enzyme is associated with the DD in the form of a fusion protein. In some embodiments, the CRISPR enzyme is fused to the DD. In other words, the DD can be associated with the CRISPR enzyme by fusion to said CRISPR enzyme. In some embodiments, the enzyme can be considered a modified CRISPR enzyme, wherein the CRISPR enzyme is fused to at least one destabilization domain (DD). In some embodiments, the DD may be associated with the CRISPR enzyme via a junction protein, for example, using a system such as a marker system such as the streptavidin-biotin system. Thus, a fusion product of the CRISPR enzyme with a junction protein specific for a high-affinity ligand for such a junction is provided, wherein the DD is linked to said high-affinity ligand. For example, streptavidin may be a junction fused to the CRISPR enzyme, while biotin may be linked to the DD. When co-localized, streptavidin will bind to biotin, thereby linking the CRISPR enzyme to the DD. For simplicity, a fusion product of a CRISPR enzyme and a DD is preferred in some embodiments. In some embodiments, the fusion product comprises a linker between the DD and the CRISPR enzyme. In some embodiments, the fusion may be at the N-terminus of the CRISPR enzyme. In some embodiments, at least one DD is fused to the N-terminus of the CRISPR enzyme. In some embodiments, the fusion may be at the C-terminus of the CRISPR enzyme. In some embodiments, at least one DD is fused to the C-terminus of the CRISPR enzyme. In some embodiments, one DD may be fused to the N-terminus of the CRISPR enzyme and another DD is fused to the C-terminus of the CRISPR enzyme. In some embodiments, the CRISPR enzyme is associated with at least two DDs, and wherein the first DD is fused to the N-terminus of the CRISPR enzyme and the second DD is fused to the C-terminus of the CRISPR enzyme, wherein the first and second DD are the same or different. In some embodiments, the fusion may be at the N-terminus of the DD. In some embodiments, the fusion may be at the C-terminus of the DD. In some embodiments, the fusion may be between the C-terminus of the CRISPR enzyme and the N-terminus of the DD. In some embodiments, the fusion may be between the C-terminus of the DD and the N-terminus of the CRISPR enzyme. Less background was observed with a DD containing at least one N-terminal fusion than with a DD containing at least one C-terminal fusion. The combination of N- and C-terminal fusions had the lowest background, but also the lowest overall activity. Preferably, a DD is provided by at least one N-terminal fusion or at least one N-terminal fusion plus at least one C-terminal fusion. And, of course, a DD may be provided by at least one C-terminal fusion.

In certain embodiments, protein destabilizing domains, such as for induction regulation, can be fused to the N-terminus and/or C-terminus of, for example, Cas9. Additionally, destabilizing domains can be introduced into the primary sequence of, for example, Cas9, in solvent accessible loops. Computer analysis of the primary structure of Cas9 nucleases revealed three distinct regions. The first, a C-terminal RuvC-like domain, which is the only functional domain characterized. The second, an N-terminal alpha-helical region, and the third, a region with mixed alpha and beta structure located between the RuvC-like domain and the alpha-helical region. Several small stretches of unstructured regions are predicted in the primary structure of Cas9. Unstructured regions that are solvent accessible and are not conserved among different Cas9 orthologs represent preferred sites for separations and insertions of small protein sequences. Furthermore, these sides can be used to create chimeric proteins between Cas9 orthologs.

In some embodiments, the DD is ER50. A corresponding stabilizing ligand for such DD is 4HT in some embodiments. Thus, in some embodiments, one of the at least one DD is ER50 and the stabilizing ligand for it is 4HT or CMP8. In some embodiments, the DD is DHFR50. A corresponding stabilizing ligand for such DD is TMP in some embodiments. Thus, in some embodiments, one of the at least one DD is DHFR50 and the stabilizing ligand for it is TMP. In some embodiments, the DD is ER50. A corresponding stabilizing ligand for such DD is CMP8 in some embodiments. Thus, CMP8 can be an alternative stabilizing ligand to 4HT in the ER50 system. While it is possible that CMP8 and 4HT could/should be used competitively, some cell types may be more responsive to one or the other of the two ligands, and based on the present disclosure and prior art information, one skilled in the art will be able to use CMP8 and/or 4HT.

In some embodiments, one or two DDs may be fused to the N-terminus of the CRISPR enzyme, and one or two DDs are fused to the C-terminus of the CRISPR enzyme. In some embodiments, at least two DDs are associated with the CRISPR enzyme, and the DDs are the same DD, i.e., the DDs are homologous. Thus, both (or two or more) of the DDs may be DDs of ER50. This is preferred in some embodiments. Alternatively, both (or two or more) of the DDs may be DDs of DHFR50. This is also preferred in some embodiments. In some embodiments, at least two DDs are associated with the CRISPR enzyme, and the DDs are different DDs, i.e., the DDs are heterologous. Thus, one of the DDs may be ER50, while one or more of the DDs or any other DDs may be DHFR50. The presence of two or more DDs that are heterologous may be advantageous since it may provide a greater level of destruction control. Tandem fusion of more than one DD at the N- or C-terminus may enhance destruction; and such a tandem fusion may be, for example, ER50-ER50-Cas9 or DHFR-DHFR-Cas9. It is envisaged that high levels of destruction may be observed in the absence of both stabilizing ligands, intermediate levels of destruction may be observed in the absence of one stabilizing ligand and in the presence of the other (or different) stabilizing ligand, while low levels of destruction may be observed in the presence of both (or two or more) stabilizing ligands. Control may also be provided by the presence of the N-terminal DD of ER50 and the C-terminal DD of DHFR50.

In some embodiments, the fusion product of a CRISPR enzyme with a DD comprises a linker between the DD and the CRISPR enzyme. In some embodiments, the linker is a GlySer linker. In some embodiments, the DD-CRISPR enzyme further comprises at least one nuclear export signal (NES). In some embodiments, the DD-CRISPR enzyme comprises two or more NESs. In some embodiments, the DD-CRISPR enzyme comprises at least one nuclear localization signal (NLS). This may be present along with the NES. In some embodiments, the CRISPR enzyme comprises, or consists essentially of, or consists of a localization signal (nuclear import or export), as a linker between the CRISPR enzyme and the DD or a portion thereof. HA or Flag tags are also encompassed by the present invention as linkers. Applicants use NLS and/or NES as a linker, and also use glycine-serine linkers as short GS to (GGGGS) ₃ .

In one aspect, the present invention provides a polynucleotide encoding a CRISPR enzyme and an associated DD. In some embodiments, the encoded CRISPR enzyme and the associated DD are operably linked to a first regulatory element. In some embodiments, the DD is also encoded and operably linked to a second regulatory element. Advantageously, the DD herein "stripping" the stabilizing ligand, and thus is advantageously the same DD (i.e., the same type of domain) as that associated with an enzyme, such as those discussed herein (it should be appreciated that the term "stripping" has the meaning discussed herein and can also provide an effect to promote or terminate activity). By stripping the stabilizing ligand with excess DD that is not associated with the CRISPR enzyme, greater degradation of the CRISPR enzyme will be observed. Without being limited by theory, it is contemplated that as additional or excess unassociated DD is added, the equilibrium will shift away from complexing with the stabilizing ligand or binding to the DD associated with the CRISPR enzyme and more toward complexing with the stabilizing ligand or binding to the free DD (i.e., which is not associated with the CRISPR enzyme). Thus, providing excess or additional unassociated (or free) DD is advantageous if CRISPR enzyme activity is to be reduced by enhancing CRISPR enzyme degradation. Excess free DD binds to the residual ligand and also deprives the ligand of the ability to bind to the DD-Cas fusion product. Therefore, it accelerates DD-Cas degradation and enhances temporal control of Cas activity. In some embodiments, the first regulatory element is a promoter and may optionally include an enhancer. In some embodiments, the second regulatory element is a promoter and may optionally include an enhancer. In some embodiments, the first regulatory element is an early promoter. In some embodiments, the second regulatory element is a late promoter. In some embodiments, the second regulatory element is, or comprises, or consists essentially of, an inducible control element, optionally a tet system, or a repressor control element, optionally a tetr system. The inducible promoter may be suitable, for example, rTTA, for inducing tet in the presence of doxycycline.

The attachment or association can be accomplished via a linker, such as a flexible glycine-serine (GlyGlyGlySer) or (GGGS) ₃ , or a rigid alpha-helical linker such as (Ala(GluAlaAlaAlaLys)Ala). Linkers such as (GGGGS) ₃ are preferably used herein to separate protein or peptide domains. (GGGGS) ₃ is preferred because it is a relatively long linker (15 amino acids). Glycine residues are the most flexible, and serine residues increase the likelihood that the linker will be on the outside of the protein. (GGGGS) ₆ , (GGGGS) ₉ or (GGGGS) ₁₂ can preferably be used as alternatives. Other preferred alternatives are (GGGGS) ₁ , (GGGGS) ₂ , (GGGGS) ₄ , (GGGGS) ₅ , (GGGGS) ₇ , (GGGGS) ₈ , (GGGGS) ₁₀ or (GGGGS) ₁₁ . Alternative linkers are available, but highly flexible linkers are thought to better maximize the possibility of combining the two 2 parts of Cas and thus reconstituting Cas activity. One alternative is that the nucleoplasmin NLS can be used as a linker. For example, a linker can also be used between Cas and any functional domain. Again, in this case, the (GGGGS) ₃ linker (or its 6-, 9- or 12-repeat variants) can be used, or the nucleoplasmin NLS can be used as a linker between Cas and the functional domain.

In one aspect of the present invention, there are provided means for delivering the DD-CRISPR-Cas complex of the present invention or the polynucleotides discussed herein, such as a particle(s) delivering the component(s) of the complex, a vector(s) comprising the polynucleotide(s) discussed herein (e.g., encoding the CRISPR enzyme, DD; providing the RNA of the CRISPR-Cas complex). In some embodiments, the vector may be a plasmid or a viral vector, such as AAV or lentivirus. Transient transfection with plasmids, such as HEK cells, may be advantageous, especially given the size limitations of AAV and that, although Cas9 fits into AAV, an upper limit may be reached in the case of additional coding sequence for association with DD.

Also provided is a model that constitutively expresses a CRISPR enzyme and an associated DD. The organism may be transgenic and may be transfected with the vectors of the present invention or may be the progeny of an organism so transfected. In a further aspect, the present invention provides compositions comprising a CRISPR enzyme and an associated DD, or polynucleotides or vectors described herein. Also provided are CRISPR-Cas systems comprising guide RNAs.

Also provided is a method of treating a subject, for example a subject in need thereof, comprising inducing gene editing by transforming the subject with a polynucleotide encoding the system or any of the vectors of the present invention and administering a stabilizing ligand to the subject. Also provided may be a suitable repair template, for example delivered by a vector comprising said repair template. Also provided is a method of treating a subject, for example a subject in need thereof, comprising inducing transcriptional activation or repression by transforming the subject with a polynucleotide encoding the system of the present invention or any of the vectors of the present invention, wherein said polynucleotide or vector encodes or comprises a catalytically inactive CRISPR enzyme and one or more functional domains associated therewith; wherein the method further comprises administering a stabilizing ligand to the subject. These methods may also comprise delivering and/or providing expression of excess DD in the subject. In the event that any processing is carried out ex vivo, for example in cell culture, it should be understood that the term "subject" may be replaced by the phrase "cell or cell culture".

Also provided are compositions comprising the system of the present invention for use in said method of treatment. A single composition may comprise a stabilizing ligand. A kit of parts comprising such compositions may be provided. Also provided is the use of the system of the present invention in the manufacture of a medicament for such methods of treatment. The present invention also provides the use of the system of the present invention in screening, for example, screens for a gain-of-function mutation. Cells in which a gene is artificially overexpressed may decrease gene expression over time (re-equilibration), for example, by negative feedback. By the time the screening begins, the expression level of the unregulated gene may be reduced again. The use of an inducible Cas9 activator allows transcription to be induced immediately before screening, thereby minimizing the likelihood of false negative matches. Accordingly, by using the present invention in screening, for example, screens for a gain-of-function mutation, the likelihood of false negative results can be minimized.

In one aspect, the present invention provides an engineered, non-naturally occurring CRISPR-Cas system comprising a Cas protein and a guide RNA that targets a DNA molecule encoding a gene product in a cell, whereby the guide RNA targets the DNA molecule encoding the gene product, and the Cas protein cleaves the DNA molecule encoding the gene product, whereby expression of the gene product is altered; and wherein the Cas protein and the guide RNA do not occur together in nature. The present invention encompasses a guide RNA that includes a guide sequence fused to a tracr sequence. In one embodiment, the Cas protein is Cas9. The present invention further encompasses encoding 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 a more preferred embodiment, the mammalian cell is a human cell. In a further embodiment of the present invention, expression of the gene product is reduced.

When functional domains and the like are "associated" with one or another part of an enzyme, they are typically fusion products. The term "associated with" is used herein to refer to how one molecule "binds" to another, such as between parts of a CRISPR enzyme and a functional domain. The two can be thought of as being tethered to each other. In the case of such protein-protein interactions, this association can be thought of in terms of recognition, such as an antibody recognizing an epitope. Alternatively, one protein can be associated with another protein by a fusion of the two, such as one subunit being fused to another subunit. Fusion typically occurs by adding one amino acid sequence to another, such as by splicing the nucleotide sequences that encode each protein or subunit. Alternatively, it can be thought of as essentially a linkage between the two molecules, or a direct linkage, such as a fusion protein. In any case, the fusion protein may include a linker between the two subunits of interest (e.g., between the enzyme and the functional domain, or between the adapter protein and the functional domain). Thus, in some embodiments, a portion of the CRISPR enzyme is associated with the functional domain by binding thereto. In other embodiments, the CRISPR enzyme is associated with the functional domain by the two being fused together, optionally through an intervening linker. Examples of linkers include the GlySer linkers discussed herein. While a non-covalently linked DD can initiate the degradation of an associated Cas (e.g., Cas9), degradation by the proteasome involves unwinding the protein chain; and fusion is preferred because it can ensure that the DD remains attached to the Cas during degradation. However, the CRISPR enzyme and DD are brought together in the presence of a stabilizing ligand specific for the DD, forming a stabilization complex. This complex contains a stabilizing ligand bound to the DD. The complex also contains a DD associated with the CRISPR enzyme. In the absence of this stabilizing ligand, the DD and its associated CRISPR enzyme are destroyed.

Destabilizing domains are versatile in conferring instability to a wide range of proteins; see, e.g., Miyazaki, J Am Chem Soc. Mar 7, 2012; 134(9): 3942–3945, incorporated herein by reference. CMP8 or 4-hydroxytamoxifen may represent destabilizing domains. More broadly, a temperature-sensitive mutant of mammalian DHFR (DHFRts) that destabilizes an N-end rule residue was found to be stable at the permissive temperature but unstable at 37°C. Addition of methotrexate, a high-affinity ligand for mammalian DHFR, to cells expressing DHFRts partially inhibited protein degradation. This was important evidence that a small molecule ligand can stabilize a protein otherwise destined for degradation in cells. A rapamycin derivative was used to stabilize an unstable mutant of the FRB domain of mTOR (FRB*) and restore the function of the fusion kinase, GSK-3β.6,7 This system demonstrated that ligand-dependent stability is an attractive strategy for regulating specific protein function in complex biological environments. The system for controlling protein activity could involve a DD that becomes functional upon ubiquitin complementation via rapamycin-induced dimerization of FK506 binding protein and FKBP12. Mutants of human FKBP12 or ecDHFR could be engineered to be metabolically unstable in the absence of their high-affinity ligands, Shield-1 or trimethoprim (TMP), respectively. These mutants represent some of the possible destabilizing domains (DDs) useful in practicing the present invention, and the instability of the DD as a fusion with the CRISPR enzyme ensures that the CRISPR protein as a complete fusion protein is degraded by the proteasome. Shield-1 and TMP bind and stabilize the DD in a dose-dependent manner. The estrogen receptor ligand binding domain (ERLBD, residues 305-549 in ERS1) can also be engineered as a destabilizing domain. Since the estrogen receptor signaling pathway is involved in a number of diseases such as breast cancer, this pathway has been extensively studied and numerous estrogen receptor agonists and antagonists have been developed. Thus, compatible pairs of ERLBD and drugs are known. There are ligands that bind to the mutant form and not to the wild-type form of the ERLBD. By using one of these mutant domains encoding three mutations (L384M, M421G, G521R)12, it is possible to regulate the stability of the ERLBD-derived DD using a ligand that does not disrupt endogenous estrogen-responsive networks. An additional mutation (Y537S) can be introduced to further destabilize the ERLBD and configure it as a potential DD candidate. Such a tetra-mutant is the preferred DD design. The ERLBD mutant can be fused to a CRISPR enzyme and its stability can be regulated or disrupted using a ligand, provided that the CRISPR enzyme has a DD. Another DD could be a 12 kDa (107 amino acids) tag based on the mutated FKBP protein stabilized by the Shield1 ligand; see, e.g., Nature Methods 5, (2008). For example, DD can be a modified FK506 binding protein 12 (FKBP12) that binds to and is reversibly stabilized by the synthetic biologically inert small molecule Shield-1; see, e.g., Banaszynski LA, Chen LC, Maynard-Smith LA, Ooi AG, Wandless TJ. A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules. Cell. 2006;126:995–1004; Banaszynski LA, Sellmyer MA, Contag CH, Wandless TJ, Thorne SH. Chemical control of protein stability and function in living mice. Nat Med. 2008;14:1123–1127; Maynard-Smith LA, Chen LC, Banaszynski LA, Ooi AG, Wandless TJ. A directed approach for engineering conditional protein stability using biologically silent small molecules. The Journal of biological chemistry. 2007;282:24866-24872; and Rodriguez, Chem Biol. Mar 23, 2012; 19(3):391-398, all of which are incorporated herein by reference and can be used in the practice of the present invention in selecting a DD to associate with a CRISPR enzyme to practice the present invention. As can be seen, the prior art includes a variety of DDs, and the DD can be associated with a CRISPR enzyme, for example, fused preferably via a linker, whereby the DD can be stabilized in the presence of a ligand, and in its absence, the DD can become destabilized, resulting in a completely destabilized CRISPR enzyme, or the DD can be stabilized in the absence of a ligand, and when the ligand is present, the DD can become destabilized; wherein the DD enables the regulation and control of the CRISPR enzyme and hence the CRISPR-Cas complex or system - figuratively speaking, turning it on or off, thereby providing a means for regulating or controlling the system, for example, in an in vivo or in vitro environment. For example, if a protein of interest is expressed as a fusion product with a DD tag, it is destabilized and rapidly degraded in the cell, for example, by the proteasome. Thus, the absence of a stabilizing ligand leads to the destruction of Cas associated with the DD. If a new DD is fused to the protein of interest, its instability is transferred to the protein of interest, which leads to the rapid destruction of the entire fused protein. Spikes of Cas activity are sometimes advantageous in reducing off-target effects. Thus, short bursts of high activity are advantageous. The present invention can provide such peaks. In some sense, the system is inducible. In some other sense, the system is repressed in the absence of a stabilizing ligand and reactivated in the presence of a stabilizing ligand. Without going into theory or making any promises, other advantages of the present invention may include the following:

dosability (as opposed to a system that is “switched on” or “switched off”, for example, it is possible to provide variable activity of the CRISPR-Cas system or complex);

orthogonality, e.g. a ligand only affects its cognate DD, so two or more systems can function independently, and/or CRISPR enzymes can be from one or more orthologs;

transportability, for example, can work across different cell types or cell lines;

rapidity;

temporary control;

the ability to reduce background or non-target Cas toxicity or excess Cas accumulation by promoting Cas destruction.

Although the DD may be at the N- and/or C-terminus(es) of the CRISPR enzyme, including DDs at one or more sides of a split (as defined elsewhere herein), for example, Cas9(N)-linker-DD-linker-Cas9(C) is also a way to introduce a DD. In some embodiments, if only one terminal association of the DD is used with the CRISPR enzyme to be used, then it is preferable to use ER50 as the DD. In some embodiments, if both the N-terminus and C-terminus are used, then it is preferable to use ER50 and/or DHFR50. Particularly good results were observed with the N-terminal fusion, which was surprising. Having both an N-terminal and a C-terminal fusion can be synergistic. The size of the destabilization domain varies, but is typically from about 100 to about 300 amino acids. The DD is preferably an engineered protein destabilizing domain. DDs and methods for producing DDs, for example, from a high affinity ligand and its ligand binding domain. The present invention can be considered "orthogonal" because only a specific ligand will stabilize its corresponding (cognate) DD, while it will not affect the stability of non-cognate DDs. A commercially available DD system is the CloneTech, ProteoTuner™ system; the stabilizing ligand is Shield1.

In some embodiments, the stabilizing ligand is a "small molecule". In some embodiments, the stabilizing ligand is cell-permeable. It has a high affinity for its corresponding DD. Suitable DD-stabilizing ligand pairs are known in the art. In general, the stabilizing ligand can be removed by:

natural processing (e.g. degradation by proteasomes), e.g. in vivo;

clearing, for example, ex vivo/in cell culture, by

providing a preferred bonding partner or

providing XS substrate (DD without Cas).

In another aspect of the present invention, there is provided an engineered, non-naturally occurring vector system comprising one or more vectors comprising a first regulatory element operably linked to a guide RNA of the CRISPR-Cas system that targets a DNA molecule encoding a gene product, and a second regulatory element operably linked to a DD-Cas protein. Components (a) and (b) can be located in the same or different vectors of the system. The guide RNA targets a DNA molecule encoding a gene product in a cell, and the DD-Cas protein can cleave the DNA molecule encoding the gene product (it can cleave one or both strands or exhibit virtually no nuclease activity), resulting in altered expression of the gene product; and wherein the DD-Cas protein and the guide RNA do not occur together in nature. In one embodiment of the present invention, the DD-Cas protein is a DD-Cas9 protein. The present invention further encompasses encoding a DD-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 a more preferred embodiment, the mammalian cell is a human cell. In a further embodiment, the expression of the gene product is reduced.

In one aspect of the present invention, a vector system is provided, comprising one or more vectors. In some embodiments, the system comprises: (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 upstream or downstream (as appropriate) of the direct repeat sequence, wherein upon expression, the guide sequence directs sequence-specific binding of a DD-CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a DD-CRISPR enzyme in complex with a guide sequence that hybridizes to the target sequence; and (b) a second regulatory element operably linked to an enzyme-coding sequence that encodes said DD-CRISPR enzyme, comprising at least one nuclear localization sequence and/or at least one NES; wherein components (a) and (b) are in the same or different vectors of the system. If applicable, a tracr sequence may also be provided. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences directs sequence-specific binding of the DD-CRISPR complex to its target sequence in a eukaryotic cell. In some embodiments, the DD-CRISPR complex comprises one or more nuclear localization sequences and/or one or more NESs that are sufficiently efficient to direct accumulation of said CRISPR complex in a detectable amount in or outside the nucleus of a eukaryotic cell. Without being bound by theory, it is believed that the nuclear localization sequence and/or NES are not necessary for the activity of the DD-CRISPR complex in eukaryotes, but the inclusion of such sequences enhances the activity of the system, particularly with respect to targeting nucleic acid molecules in the nucleus and/or molecules outside the nucleus. In some embodiments, the DD-CRISPR enzyme is DD-Cas9. In some embodiments, the DD-CRISPR enzyme is a DD-Cas9 enzyme. In some embodiments, the DD-Cas9 enzyme is derived from Cas9 Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, or Porphyromonas macacae (e.g., Cas9 from one of these organisms modified to have or have the ability to associate with at least one DD) and may include additional mutations or changes or may be a chimeric Cas9. The enzyme may be a homolog or ortholog of DD-Cas9. In some embodiments, the DD-CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the DD-CRISPR enzyme directs cleavage of one or both strands at a specific position in a target sequence. In some embodiments, the DD-CRISPR enzyme does not have DNA strand cleavage 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 length of the guide sequence is at least 16, 17, 18, 19, 20, 25 nucleotides, or 16-30, or 16-25, or 16-20 nucleotides. Generally and throughout this specification, the term "vector" refers to a nucleic acid molecule capable of transferring 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, no free ends (e.g., circular); nucleic acid molecules that contain DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular loop of double-stranded DNA into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally derived DNA or RNA sequences are present in the vector for packaging into the virus (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by the virus for transfection into a host cell. Certain vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal vectors for mammals). Other vectors (e.g., non-episomal mammalian vectors) integrate into the host cell genome upon introduction into the host cell and are thus replicated along with the host genome. Furthermore, certain vectors are capable of directing the expression of genes to which they are functionally linked. Such vectors are referred to herein as "expression vectors." Common expression vectors suitable for recombinant DNA techniques are often in the form of plasmids.

Recombinant expression vectors may comprise a nucleic acid of the present invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors comprise one or more regulatory elements, which may be selected taking into account the host cells that are intended to be used for expression, which are operably linked to the nucleic acid sequence that is intended to be expressed. In the context of a recombinant expression vector, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) such that expression of the nucleotide sequence is allowed (e.g. in an in vitro transcription/translation system or in a host cell upon introduction of the vector into the host cell).

In some embodiments, the host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, the cell is transfected while it is in vivo in the subject. In some embodiments, the cell that is transfected is derived from the subject. In some embodiments, the cell is derived from cells derived from the subject, such as cell lines. A variety of cell lines are known in the art for use in tissue culture. Examples of 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, Bcl-1, BC-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 epithelial cells BS-C-1, mouse embryonic fibroblasts BALB/3T3, 3T3 Swiss, 3T3-L1, human fetal fibroblasts 132-d5; 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 cells 293, BxPC3, 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, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, 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 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic variants thereof. Cell lines are available from a variety of sources known to those skilled in the art (see, e.g., American Type Culture Collection (ATCC) (Manassas, VA)). In some embodiments, a cell transfected with one or more vectors described herein is used to generate a new cell line comprising one or more vector-derived sequences. In some embodiments, a cell transiently transfected with components of the CRISPR system described herein (such as by transient transfection with one or more vectors or transfection using RNA) and modified with the activity of the CRISPR complex is used to generate a new cell line comprising cells with the modification but without any other exogenous sequence. In some embodiments, cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells, are used in the evaluation of one or more test compounds.

The term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., 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 direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct expression preferentially in a target tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a time-dependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, which may or may not also be tissue-specific or cell type-specific. In some embodiments, the vector comprises one or more pol III promoters (e.g., 1, 2, 3, 4, 5 or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5 or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5 or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, the U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase gene promoter, the β-actin gene promoter, the glycerophosphate kinase (PGK) gene promoter, and the EF1α promoter. Also encompassed by the term "regulatory element" are enhancer elements such as the WPRE; CMV; the R-U5' segment of the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); the SV40 enhancer; and the intronic sequence between exons 2 and 3 of the rabbit β-globin gene (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981). Those skilled in the art will appreciate that the configuration of the expression vector may depend on factors such as the choice of host cell to be transformed, the desired expression level, etc. The vector can be introduced into host cells to thereby produce transcripts, proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids described herein (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).

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

In one aspect of the present invention, there is provided a vector comprising a regulatory element operably linked to an enzyme coding sequence that encodes a DD-CRISPR enzyme comprising one or more nuclear localization sequences and/or a NES. In some embodiments, said regulatory element directs transcription of the DD-CRISPR enzyme in a eukaryotic cell such that said DD-CRISPR enzyme accumulates in a detectable amount in the nucleus of the eukaryotic cell and/or is exported from the nucleus. In some embodiments, the regulatory element is a polymerase II promoter. In some embodiments, the DD-CRISPR enzyme is DD-Cas9. In some embodiments, the DD-CRISPR enzyme is DD-Cas9. In some embodiments, the DD-Cas9 enzyme is derived from Cas9 of Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, or Porphyromonas macacae (e.g., modified to have or have the ability to associate with at least one DD), and may include an additional alteration or mutation of Cas9 and may be a chimeric Cas9. In some embodiments, the DD-CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the DD-CRISPR enzyme directs cleavage of one or both strands at a specific position in a target sequence. In some embodiments, the DD-CRISPR enzyme does not have or is substantially free of DNA strand cleavage activity (e.g., has no more than 5% nuclease activity compared to a wild-type enzyme or an enzyme without a mutation or alteration that reduces nuclease activity).

In one aspect of the present invention, there is provided a DD-CRISPR enzyme comprising one or more nuclear localization sequences and/or NESs that are sufficiently efficient to direct the accumulation of said DD-CRISPR enzyme in a detectable amount in and/or outside the nucleus of a eukaryotic cell. In some embodiments, the DD-CRISPR enzyme is DD-Cas9. In some embodiments, the DD-CRISPR enzyme is DD-Cas9. In some embodiments, the DD-Cas9 enzyme is derived from Cas9 of Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, or Porphyromonas macacae (e.g., modified to have or have the ability to associate with at least one DD), and may include an additional alteration or mutation of Cas9 and may be a chimeric Cas9. In some embodiments, the DD-CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the DD-CRISPR enzyme directs cleavage of one or both strands at a specific position in a target sequence. In some embodiments, the DD-CRISPR enzyme does not have or is substantially free of DNA strand cleavage activity (e.g., has no more than 5% nuclease activity compared to a wild-type enzyme or an enzyme without a mutation or alteration that reduces nuclease activity).

In one aspect of the present invention, there is provided a eukaryotic host cell comprising (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for insertion of one or more guide sequences upstream or downstream (as appropriate) of the direct repeat sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a DD-CRISPR complex to a target sequence in a eukaryotic cell, wherein the DD-CRISPR complex comprises a DD-CRISPR enzyme in complex with a guide sequence that hybridizes to the target sequence; and/or (b) a second regulatory element operably linked to an enzyme-coding sequence that encodes said DD-CRISPR enzyme comprising at least one nuclear localization sequence and/or NES. In some embodiments, the host cell comprises components (a) and (b). If applicable, a tracr sequence may also be provided. In some embodiments, component (a), component (b), or components (a) and (b) are stably integrated into the genome of a eukaryotic host cell. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences directs sequence-specific binding of the DD-CRISPR complex to its target sequence in a eukaryotic cell. In some embodiments, the DD-CRISPR enzyme comprises one or more nuclear localization sequences and/or nuclear export sequences or NESs that are sufficiently efficient to direct accumulation of said CRISPR enzyme in a detectable amount within and/or outside the nucleus of a eukaryotic cell. In some embodiments, the DD-CRISPR enzyme is Cas9. In some embodiments, the CRISPR enzyme is Cas9. In some embodiments, the DD-Cas9 enzyme is derived from the Cas9 of Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, or Porphyromonas macacae (e.g., modified to have or have the ability to associate with at least one DD), and may include an additional alteration or mutation of Cas9 and may be a chimeric Cas9. In some embodiments, the DD-CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the DD-CRISPR enzyme directs cleavage of one or both strands at a specific position in a target sequence. In some embodiments, the DD-CRISPR enzyme has no or substantially no DNA strand cleavage activity (e.g., has no more than 5% of the nuclease activity of the wild-type enzyme or the enzyme without a mutation or alteration that reduces nuclease 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 length of the guide sequence is at least 16, 17, 18, 19, 20, 25 nucleotides, or 16-30, or 16-25, or 16-20 nucleotides. In one aspect, the present invention provides a non-human eukaryotic organism; preferably a multicellular eukaryotic organism comprising a eukaryotic host cell according to any of the described embodiments. In other aspects of the present invention, there is provided a eukaryotic organism, preferably a multicellular eukaryotic organism, comprising a eukaryotic host cell according to any of the embodiments described. In some embodiments of these aspects, the organism may be an animal; for example, a mammal. The organism may also be an arthropod, such as an insect. The organism may also be a plant. In addition, the organism may be a fungus.

With respect to the use of the CRISPR-Cas system, reference is generally made to documents, including patent applications, patents, and patent publications, cited throughout this disclosure, since embodiments of the present invention may be practiced as in these documents. The CRISPR-Cas system(s) (e.g., single(s) or multiplex(es)) may be used in conjunction with recent advances in crop genomics. Such CRISPR-Cas system(s) may be used to efficiently and cost-effectively interrogate or edit or manipulate a plant gene or genome, such as to rapidly interrogate and/or select and/or interrogate and/or compare and/or manipulate and/or transform plant genes or genomes; such as to obtain, identify, develop, optimize, or impart trait(s) or characteristic(s) to a plant(s) or to transform a plant genome. Accordingly, the production of plants, new plants with new combinations of traits or characteristics, or new plants with improved traits can be improved. Such a CRISPR-Cas system(s) can be used in plants in site-directed integration (SDI) or gene editing (GE), or in any of the techniques of approximate reverse selection (NRB) or reverse selection (RB). For the use of the CRISPR-Cas system in plants, reference should be made to the website "CRISPR-PLANT" of the University of Arizona (http://www.genome.arizona.edu/crispr/) (supported by Pennsylvania State University and AGI). Embodiments of the present invention can be used in genome editing in plants or in a case where RNAi or similar genome editing techniques have been used previously; 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 Aug 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/sst119. online publication 17 Aug 2013; Xu, "Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice", Rice 2014, 7:5 (2014), Zhou et al., "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 only at www.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, www.nature.com/naturecommunications DOI: 10.1038/ncomms7989; U.S. Patent No. 6,603,061 - Agrobacterium-Mediated Plant Transformation Method; U.S. Patent No. 7,868,149 - Plant Genome Sequences and Uses Thereof and US 2009/0100536 - Transgenic Plants with Enhanced Agronomic Traits, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety. In the practice of the present invention, the contents and disclosure of Morrell et al, "Crop genomics: advances and applications", Nat Rev Genet. 2011 Dec 29;13(2):85-96; which is incorporated herein by reference, including how embodiments herein may be applied to plants. Accordingly, reference herein to animal cells may also be applied, mutatis mutandis, to plant cells, unless it is obvious otherwise.

In one aspect of the present invention, a kit is provided comprising one or more components described herein. In some embodiments, the kit comprises a vector system and instructions for using the kit. In some embodiments, the vector system comprises (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 upstream or downstream (as appropriate) of the direct repeat sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell, wherein the CRISPR complex comprises a CRISPR enzyme in complex with a guide sequence that hybridizes to the target sequence; and/or (b) a second regulatory element operably linked by an enzyme-coding sequence that encodes said CRISPR enzyme comprising a nuclear localization sequence. If applicable, a tracr sequence may also be provided. In some embodiments, the kit comprises components (a) and (b) located in the same or different vectors of the system. In some embodiments, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences directs sequence-specific binding of a CRISPR complex to its target sequence in a eukaryotic cell. In some embodiments, the CRISPR enzyme comprises one or more nuclear localization sequences effective enough to direct accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. In some embodiments, the CRISPR enzyme is Cas9. In some embodiments, the CRISPR enzyme is Cas9. In some embodiments, the Cas9 enzyme is derived from the Cas9 of Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW2011_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, or Porphyromonas macacae (e.g., modified to have or have the ability to associate with at least one DD), and may include an additional alteration or mutation of Cas9 and may be a chimeric Cas9. In some embodiments, the DD-CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In some embodiments, the DD-CRISPR enzyme directs cleavage of one or both strands at a specific position in a target sequence. In some embodiments, the DD-CRISPR enzyme does not have or has substantially no DNA strand cleavage activity (e.g., has no more than 5% of the nuclease activity of the wild-type enzyme or the enzyme without a mutation or alteration that reduces nuclease 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 length of the guide sequence is at least 16, 17, 18, 19, 20, 25 nucleotides, or 16-30, or 16-25, or 16-20 nucleotides.

In one aspect, the present invention provides a method for modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method comprises causing a DD-CRISPR complex to bind to the target polynucleotide, such as to effect cleavage of said target polynucleotide, thereby causing the target polynucleotide to be modified, wherein the DD-CRISPR complex comprises a DD-CRISPR enzyme in complex with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a direct repeat sequence. If applicable, a tracr sequence may also be provided (e.g., to provide a single guide RNA, sgRNA). In some embodiments, said cleavage comprises cleavage of one or both strands at a specific position of the target sequence by said DD-CRISPR enzyme. In some embodiments, said cleavage results in reduced transcription of the target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous polynucleotide template, wherein said repair results in a mutation involving an insertion, deletion or substitution of one or more nucleotides in said target polynucleotide. In some embodiments, said mutation results in a change in one or more amino acids in a protein expressed from a gene comprising 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 direct the expression of one or more of a DD-CRISPR enzyme and a guide sequence associated with a direct repeat sequence. If applicable, a tracr sequence may also be provided. In some embodiments, said vectors are delivered to a eukaryotic cell in a subject. 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 the subject's body prior to performing said modification. In some embodiments, the method further comprises returning said eukaryotic cell and/or cells derived therefrom to said subject.

In one aspect of the present invention, there is provided a method for modifying the expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises causing a DD-CRISPR complex to bind to a polynucleotide, such that said binding results in increased or decreased expression of said polynucleotide; wherein the DD-CRISPR complex comprises a DD-CRISPR enzyme, in complex with a guide sequence hybridizing to a target sequence within said polynucleotide, wherein said guide sequence is linked to a direct repeat sequence. If applicable, a tracr sequence may also be provided. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cells, wherein the one or more vectors direct the expression of one or more of a DD-CRISPR enzyme and a guide sequence linked to a direct repeat sequence. If applicable, a tracr sequence may also be provided.

In one aspect of the present invention, there is provided a method for producing a model eukaryotic cell comprising a mutated gene responsible for the development of a disease. In some embodiments, the gene responsible for the development of a disease is any gene associated with an increased risk of having or developing a disease. In some embodiments, the method comprises (a) introducing one or more vectors into a eukaryotic cell, wherein the one or more vectors direct the expression of one or more of a DD-CRISPR enzyme, a guide sequence associated with a direct repeat sequence (if applicable, a tracr sequence may also be provided); and (b) causing the DD-CRISPR complex to bind to a target polynucleotide, such as to effect cleavage of the target polynucleotide within said disease-causing gene, wherein the DD-CRISPR complex comprises a DD-CRISPR enzyme in a complex with a guide sequence that hybridizes to a target sequence within the target polynucleotide, thereby producing a model eukaryotic cell comprising a mutated disease-causing gene. In some embodiments, said cleavage comprises cleavage of one or both strands at a specific position of the target sequence by said DD-CRISPR enzyme. In some embodiments, said cleavage results in reduced transcription of the target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous polynucleotide template, wherein said repair results in a mutation involving an insertion, deletion or substitution of one or more nucleotides in said target polynucleotide. In some embodiments, said mutation results in a change in one or more amino acids in the expression of a protein from a gene comprising the target sequence.

In one aspect of the present invention, there is provided a method for developing a biologically active agent that modulates a cellular signaling event associated with a disease-promoting gene. In some embodiments, the disease-promoting gene is any gene associated with an increased risk of having or developing a disease. In some embodiments, the method comprises (a) contacting a test compound with a model cell according to any of the described embodiments; and (b) detecting a change in a readout that indicates a decrease or increase in a cellular signaling event associated with said mutation in said disease-promoting gene, thereby obtaining said biologically active agent that modulates said cellular signaling event associated with said disease-promoting gene.

In one aspect of the present invention, there is provided a recombinant polynucleotide comprising a guide sequence upstream or downstream (as appropriate) of a direct repeat sequence, wherein the guide sequence, when expressed, directs sequence-specific binding of a DD-CRISPR complex to a corresponding target sequence present in a eukaryotic cell. In some embodiments, the target sequence is a viral sequence present in a eukaryotic cell. If applicable, a tracr sequence may also be provided. In some embodiments, the target sequence is a proto-oncogene or an oncogene.

In one aspect of the present invention, there is provided a method for selecting one or more cells by introducing one or more mutations into a gene in one or more cells, the method comprising introducing one or more vectors into the cell(s), wherein the one or more vectors direct the expression of one or more of a DD-CRISPR enzyme, a guide sequence associated with a direct repeat sequence (if applicable, a tracr sequence may also be provided), and an editing template; wherein the editing template comprises one or more mutations that provide for the termination of cleavage by the DD-CRISPR enzyme; providing for homologous recombination of the editing template with a target polynucleotide in the cell(s) to be selected; allowing the CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide within said gene, wherein the DD-CRISPR complex comprises a DD-CRISPR enzyme in complex with a guide sequence that hybridizes to a target sequence within the target polynucleotide, wherein the binding of the DD-CRISPR complex to the target polynucleotide induces cell death, thereby allowing selection of one or more cells into which one or more mutations have been introduced. In a preferred embodiment, the DD-CRISPR enzyme is DD-Cas9. In another aspect of the present invention, the cell to be selected may be a eukaryotic cell. Aspects of the present invention provide for the selection of specific cells without the need for a selection marker or a two-step method that may include a negative selection system. The cell(s) may be a prokaryotic or a eukaryotic cell.

In a further aspect, the present invention relates to a computer-implemented method for identifying or designing potential compounds for insertion or binding to a DD-CRISPR-Cas9 system or a functional part thereof, or vice versa (a computer-implemented method for identifying or designing potential DD-CRISPR-Cas9 systems or a functional part thereof for binding to desired compounds) or to a computer-implemented method for identifying or designing potential DD-CRISPR-Cas9 systems (e.g., in terms of predicting regions in a DD-CRISPR-Cas9 system that can be manipulated, e.g., based on crystal structure data or based on Cas9 ortholog data, or taking into account where a functional group such as an activator or a repressor can be attached to a DD-CRISPR-Cas9 system, or with respect to Cas9 truncations, or with respect to the design of nickases), said method comprising using a computer system, e.g. a stored program computer comprising a processor, a data storage system, an input device and an output device, the steps of (a) inputting into a computer with a stored program, via said input device, data comprising three-dimensional coordinates of a subset of atoms from a crystal structure of DD-CRISPR-Cas9 or related thereto, for example in a binding domain of the DD-CRISPR-Cas9 system or alternatively or additionally in domains that vary based on differences among Cas9 orthologs or concerning Cas9 or concerning niccases or concerning functional groups, optionally with information on the structure of the complex(es) of the CRISPR-Cas9 system, thereby obtaining a data set; (b) comparing, using said processor, said data set with a computer database of structures stored in said computer data storage system, such as structures of compounds that bind, or are predicted to bind, or are claimed to bind, to the DD-CRISPR-Cas9 system, or to DD-Cas9 orthologs (e.g., Cas9 or to domains or regions that vary among Cas9 orthologs), or to a crystal structure of DD-CRISPR-Cas9, or to nickases or to functional groups; (c) selecting from said database, using computational methods, a structure(s), such as DD-CRISPR-Cas9 structures, that can bind to desired structures, desired structures that can bind to specific DD-CRISPR-Cas9 structures, parts of the DD-CRISPR-Cas9 system that can be manipulated, such as based on data on other parts of the DD-CRISPR-Cas9 crystal structure and/or on DD-Cas9 orthologs, truncated Cas9, novel nicks or specific functional groups or positions for attaching functional groups or for mutating DD-CRISPR-Cas9 systems; (d) constructing, using computer methods, a model of the selected structure(s), and (e) outputting data on the selected structure(s) to said output device, and optionally synthesizing one or more selected structures, and further optionally testing said synthesized selected structure(s) as or in a CRISPR-Cas9 system; or the method comprises obtaining coordinates of at least two atoms of a DD-CRISPR-Cas9 crystal structure, such as at least two atoms from the materials cited herein, or coordinates of at least a subdomain of a DD-CRISPR-Cas9 crystal structure ("selected coordinates"), obtaining a candidate structure comprising a binding molecule or parts of the DD-CRISPR-Cas9 system that can be manipulated, such as from data on other parts of the DD-CRISPR-Cas9 crystal structure and/or on Cas9 orthologs or on the structure of functional groups, and fitting the candidate structure to the selected coordinates, thereby obtaining product data comprising DD-CRISPR-Cas9 structures that can bind to desired structures, desired structures that can bind to specific DD-CRISPR-Cas9 structures, parts of the CRISPR-Cas9 system that can be manipulated, truncated Cas9, novel niccases, or specific functional groups, or positions for attaching functional groups or for mutating DD-CRISPR-Cas9 systems, with output data thereof; and optionally synthesizing compound(s) based on said product data, and further optionally testing said synthesized compound(s) as a DD-CRISPR-Cas9 system or the like therein. Testing may include analyzing a DD-CRISPR-Cas9 system obtained based on said synthesized selected structure(s), for example, for binding or performing a desired function. Output information in the above-mentioned methods may include data transmission, for example, transmission of information via telecommunication, telephone, video conference, mass media, for example, a presentation, such as a computer presentation (e.g. POWERPOINT), the Internet, e-mail, communication via documents, such as a computer program document (e.g. WORD), etc. Accordingly, the present invention also encompasses computer-readable media comprising atomic coordinate data in accordance with the materials cited herein, wherein said data define a three-dimensional structure of DD-CRISPR-Cas9 or at least one subdomain thereof, or structure factor data for CRISPR-Cas9, wherein said structure factor data can be obtained from the materials cited herein. The computer-readable media may also comprise any of the data from the above-mentioned methods. The present invention further encompasses methods using a computer system for obtaining or performing rational design as in the above-mentioned methods, providing either atomic coordinate data in accordance with the materials cited herein, wherein said data define a three-dimensional structure of DD-CRISPR-Cas9 or at least one subdomain thereof, or structure factor data for CRISPR-Cas9, wherein said structure factor data is obtained from the atomic coordinate data from the materials cited herein. The present invention further encompasses a method of conducting business, including providing a user with a computer system, or a storage medium, or data of a three-dimensional structure of DD-CRISPR-Cas9, or at least one subdomain thereof, or a structure factor for DD-CRISPR-Cas9, wherein said structure is set forth in the materials cited herein, and said structure factor data can be obtained from the atomic coordinate data from the materials cited herein, or by the electronic storage medium provided herein, or by the data transmission provided herein.

"Binding site" or "active site" includes, or consists essentially of, or consists of a site (such as an atom, a functional group of an amino acid residue, or a plurality of such atoms and/or groups) in a binding cavity or region that can bind to a compound, such as a nucleic acid molecule, that is involved in the binding. By "fitting" is meant determining, by automated or semi-automated means, interactions between one or more atoms of a candidate molecule and at least one atom of a structure of the present invention and calculating the degree of stability of such interactions. The interactions include attractions and repulsions due to charge, steric aspects, and the like. Various computer fitting methods are described below. By "root mean square (or rms) deviation" is meant the square root of the arithmetic mean of the squares of the deviations from the mean. By "computer system" is meant hardware, software and data storage means used for analyzing the data by atomic coordinates. The minimum hardware of the computer systems of the present invention typically comprises a central processing unit (CPU), input devices, output devices and data storage means. For visualizing the data by structure, it is desirable to provide a display or monitor. The data storage means can be RAM or means for accessing the machine-readable media of the present invention. Examples of such systems are a computer and tablet devices running Unix, Windows or Apple operating systems. By "machine-readable media" is meant any medium or media that can be read and accessed directly or indirectly by a computer, for example, such that the media are suitable for use in the above-mentioned computer system. Such media include, but are not limited to, magnetic storage devices such as floppy disks, hard disk storage and magnetic tape; optical storage devices such as optical disks or CD-ROMs; electrical storage devices such as RAM and ROM; flash drives; cloud storage devices; and hybrids of these categories such as magnetic/optical storage devices.

In particular embodiments of the present invention, conformational changes in the crystal structures of the DD-CRISPR-Cas9 system or DD-CRISPR-Cas9 components provide important and necessary information about the flexibility or mobility of protein structural regions relative to nucleotide (RNA or DNA) structural regions, which may be important for the function of the DD-CRISPR-Cas system. The structural information provided for Cas9 in the materials cited herein can be used to further design and optimize the DD-CRISPR-Cas system according to this document, and can be extrapolated to a detailed study of the structure-function relationships in another CRISPR enzyme, such as in systems with a DD-CRISPR enzyme, as well as, for example, in other systems with a type V or VI CRISPR enzyme (e.g., in other systems with a type V or VI DD-CRISPR enzyme). The present invention encompasses optimized functional DD-CRISPR-Cas enzyme systems. In particular, the DD-CRISPR enzyme comprises one or more mutations that convert it into a DNA-binding protein to which functional domains that exhibit a function of interest can be recruited or added or inserted or attached. In certain embodiments, the CRISPR enzyme comprises one or more mutations in RuvC1 of the DD-CRISPR enzyme and/or another mutation discussed herein. In some embodiments, the DD-CRISPR enzyme has one or more mutations in the catalytic domain, wherein, when transcribed, the guide sequence directs sequence-specific binding of the DD-CRISPR complex to a target sequence, and wherein the enzyme further comprises a functional domain (e.g., to provide or facilitate a destabilized domain). The structural information provided in the materials cited herein provides a detailed understanding of the interaction of the guide with the target DNA and the CRISPR enzyme (e.g., Cas9; e.g., a DD-CRISPR enzyme, e.g., DD-Cas9)), providing the ability to design or alter the structure of the sgRNA to optimize the functionality of the overall DD-CRISPR-Cas system. For example, the loops in the guide can be enlarged without overlapping with the Cas9 protein by inserting adaptor proteins that can bind to the RNA. Such adaptor proteins can further recruit effector proteins or fusion products that contain one or more functional domains. A functional domain can comprise, consist essentially of, or consist of a transcriptional activation domain, such as VP64. A functional domain can comprise, consist essentially of, or consist of a transcriptional repression domain, such as KRAB. In some embodiments, the transcriptional repression domain is, or comprises, or consists essentially of SIDs or SID concatemers (e.g., SID4X). In some embodiments, the functional domain comprises, or consists essentially of, an epigenetic modification domain, such that an epigenetic modification enzyme is provided. In some embodiments, the functional domain comprises, or consists essentially of, an activation domain, which may be a P65 activation domain.

Aspects of the present invention encompass a non-naturally occurring or engineered composition that can comprise a guide RNA (gRNA) that includes a guide sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell, and a DD-CRISPR enzyme that can comprise at least one or more nuclear localization sequences, wherein the DD-CRISPR enzyme comprises one or two or more mutations that cause the enzyme to have altered or reduced nuclease activity compared to a wild-type enzyme, wherein at least one loop of the gRNA is modified by insertion of different RNA sequence(s) that binds to one or more adaptor proteins, and wherein the adaptor protein further recruits one or more heterologous functional domains. In one embodiment of the present invention, the DD-CRISPR enzyme comprises one or two or more mutations. In another embodiment, the functional domain comprises, consists essentially of, a transcriptional activation domain, such as VP64. In another embodiment, the functional domain comprises, consists essentially of, a transcriptional repression domain, such as a KRAB domain, a SID domain, or a SID4X domain. In embodiments of the present invention, the one or more heterologous functional domains are characterized by one or more activities selected from the group consisting of, consisting essentially of, or consisting of, methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcript release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. In further embodiments of the present invention, the cell is a eukaryotic cell or a mammalian cell or a human cell. In further embodiments, the adapter protein is selected from the group consisting of, consisting essentially of, or consisting of MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, PRR1. In another embodiment, at least one loop of the gRNA is a tetraloop and/or loop 2. One aspect of the present invention encompasses methods of modifying a genomic locus of interest to alter gene expression in a cell by introducing into the cell any of the compositions described herein.

One aspect of the present invention is that the above-mentioned elements are contained in a single composition or are contained in separate compositions. These compositions can advantageously be applied to a host to induce a functional effect at the genome level.

In general, gRNAs are modified in a manner that provides specific binding sites (e.g., aptamers) for adaptor proteins containing one or more functional domains (e.g., by forming a fusion protein) to bind. Modified sgRNAs are modified such that once the gRNA forms a DD-CRISPR complex (i.e., the DD-CRISPR enzyme binds to the gRNA and the target), binding of the adaptor proteins occurs and the functional domain on the adaptor protein is positioned in a spatial orientation that is advantageous for the efficiency of its inherent function. For example, if the functional domain contains, consists essentially of, a transcriptional activator (e.g., VP64 or p65), the transcriptional activator is positioned in a spatial orientation that allows it to affect the transcription of the target. Similarly, a transcriptional repressor will be positioned preferentially to affect transcription of the target, and a nuclease (e.g., Fok1) will be positioned preferentially to cleave or partially cleave the target.

It will be appreciated by those skilled in the art that modifications to a gRNA that allow binding of the adapter + functional domain but do not allow proper placement of the adapter + functional domain (e.g., due to steric hindrance in the three-dimensional structure of the CRISPR complex) are modifications that are not intended. The one or more modified gRNAs may be modified at a tetraloop, stem-loop 1, stem-loop 2, or stem-loop 3 described herein, preferably at either the tetraloop or stem-loop 2, and most preferably at both the teraloop and stem-loop 2.

As explained herein, the functional domains may be, for example, one or more domains from the group comprising, consisting essentially of, or consisting of domains with methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcript release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switches (e.g., light-inducible). In some cases, it is advantageous to further provide at least one NLS and/or NES. In some cases, it is advantageous to place the NLS and/or NES at the N-terminus. When more than one functional domain is included, the functional domains may be the same or different.

The gRNA can be designed to include multiple recognition binding sites (e.g., aptamers) specific for the same or different adaptor proteins. The gRNA can be designed to bind to a promoter region located -1000 to +1 nucleotides upstream of the transcription start site (i.e., TSS), preferably -200 nucleotides. This placement improves functional domains that affect gene activation (e.g., transcriptional activators) or gene inhibition (e.g., transcriptional repressors). The modified gRNA can be one or more modified gRNAs (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) targeting one or more target loci contained in the composition.

In addition, the DD-CRISPR enzyme with reduced nuclease activity is most effective if the nuclease activity is inactivated (e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% nuclease inactivation compared to the wild-type enzyme; or, in other words, the DD-Cas9 enzyme or DD-CRISPR enzyme is characterized by predominantly about 0% nuclease activity relative to the unmutated Cas9 enzyme or CRISPR enzyme or wild-type enzyme, or no more than about 3%, or about 5%, or about 10% nuclease activity relative to the unmutated Cas9 enzyme or CRISPR enzyme or wild-type enzyme). This is possible by introducing mutations into the nuclease domain of RuvC Cas9 and its orthologs. The inactivated CRISPR enzyme may be associated (e.g., by forming a fusion protein) with one or more functional domains, such as at least one destabilizing domain; or, for example, similar to those described herein for modified gRNAs that bind adaptor proteins, including, for example, one or more domains from the group comprising, consisting essentially of, or consisting of domains with methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcript release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switches (e.g., light-inducible). Preferred domains are Fok1, VP64, P65, HSF1, MyoD1. In the case where Fok1 is provided, it is advantageous that multiple functional domains of Fok1 are provided to provide a functional dimer, and that gRNAs are designed to provide the appropriate distance for functional use (Fok1, as specifically described in Tsai et al. Nature Biotechnology, Vol. 32, Number 6, June 2014). Known linkers can be used in the adapter protein to attach such functional domains. In some cases, it is advantageous to additionally provide at least one NLS or NES. In some cases, it is advantageous to place the NLS or NES at the N-terminus. When more than one functional domain is included, the functional domains can be the same or different. In general, the placement of one or more functional domains in the inactivated DD-CRISPR enzyme ensures the correct spatial orientation of the functional domain to act on the target with its inherent functional effect. For example, if the functional domain is a transcriptional activator (e.g., VP64 or p65), the transcriptional activator is positioned in a spatial orientation that allows it to affect transcription of the target. Similarly, a transcriptional repressor would be positioned preferentially to affect transcription of the target, and a nuclease (e.g., Fok1) would be positioned preferentially to cleave or partially cleave the target. Positions other than the N-/C-terminus of the DD-CRISPR enzyme may be envisaged.

The adaptor protein may be any of a number of proteins that bind to an aptamer or recognition site introduced into the modified gRNA and that allows one or more functional domains to be correctly positioned after the gRNA has been incorporated into the CRISPR complex to act on the target within its intended function. As explained in detail in this application, they may be coat proteins, preferably bacteriophage coat proteins. Functional domains associated with such adapter proteins (e.g. in the form of a fusion protein) may comprise, for example, one or more domains from the group comprising, consisting essentially of or consisting of domains with methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcript release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity and molecular switches (e.g. inducible by light). Preferred domains are Fok1, VP64, P65, HSF1, MyoD1. In case the functional domain is a transcriptional activator or a transcriptional repressor, it is advantageous to additionally provide at least an NLS or NES and preferably at the N-terminus. When including more than one functional domain, the functional domains may be the same or different. The adaptor protein can use known linkers to attach such functional domains. Such linkers can be used to associate the DD with the CRISPR enzyme or to provide a CRISPR enzyme containing the DD.

Thus, all of the gRNA, such as the modified gRNA, the inactivated DD-CRISPR enzyme (with or without functional domains) and the binding protein with one or more functional domains may be separately contained in a composition and may be administered to the host separately or together. Alternatively, these components may be provided in a single composition for administration to the host. Administration to the host may be accomplished via viral vectors known to the skilled artisan or described herein for delivery to the host (e.g., a lentiviral vector, an adenoviral vector, an AAV-based vector). As explained herein, the use of different selection markers (e.g., for lentiviral sgRNA selection) and different gRNA concentrations (e.g., depending on whether multiple gRNAs are used) may be advantageous to induce an improved effect. Based on this concept, several options are suitable for inducing transformation at a genomic locus, including DNA cleavage, gene activation or gene inactivation. Using the provided compositions, a person skilled in the art will be able to efficiently and specifically target one or multiple loci using the same or different functional domains to induce one or more transformations at a genomic locus. The compositions can be used in a variety of methods for screening libraries in cells and functional modeling in vivo (e.g., lincRNA gene activation and function identification; gain-of-function modeling; loss-of-function modeling; using the compositions of the present invention to create cell lines and transgenic animals for optimization and screening purposes).

The present invention encompasses the use of the compositions of the present invention to generate and use transgenic cells/animals with conditionally or inducibly expressed DD-CRISPR; see, e.g., Platt et al., Cell (2014), 159(2):440-455, or the PCT patent publications cited therein, such as WO 2014/093622 (PCT/US2013/074667). For example, cells or animals, such as non-human animals, e.g., vertebrates or mammals, such as rodents, e.g., mice, rats, or other laboratory or non-laboratory animals, e.g., cats, dogs, sheep, etc., can be characterized by a "knockin" state, whereby the animal conditionally or inducibly expresses DD-Cas9, as described in Platt et al. Thus, the target cell or animal comprises a conditionally dependent or inducible DD-CRISPR enzyme (e.g., DD-Cas9) (e.g., in the form of Cre-dependent constructs) and/or a conditionally dependent or inducible adapter protein or DD, and when the vector introduced into the target cell is expressed, the vector expresses that which induces or provides a condition for the expression of the DD-CRISPR enzyme (e.g., DD-Cas9) and/or the expression of the adapter or DD in the target cell. By applying the ideas and compositions according to the present invention with a known method for creating a CRISPR complex, inducible genome transformations are also an aspect of the present invention. One example of this is the generation of a CRISPR knockin/conditional expression transgenic animal (e.g., a mouse containing, e.g., a Lox-Stop-polyA-Lox (LSL) cassette) and subsequent delivery of one or more compositions providing one or more modified gRNAs (e.g., -200 nucleotides to the TSS in a target gene of interest for purposes of gene activation, e.g., modified gRNAs with one or more aptamers recognized by coat proteins, e.g., MS2), one or more adaptor proteins described herein (MS2 binding protein linked to one or more VP64), and means for inducing the animal to have conditional expression (e.g., Cre recombinase to provide inducible expression of DD-Cas9). Alternatively, the adaptor protein or DD can be provided as a conditionally dependent or inducible element with a conditionally dependent or inducible CRISPR enzyme to provide an efficient model for screening purposes that advantageously requires only minimal design and introduction of specific gRNAs for a variety of applications.

In some embodiments, the phenotypic change is preferably the result of modification of the genome in targeting a genetic disease, particularly in therapeutic methods, and preferably if a repair template is provided to correct or alter the phenotype.

In some embodiments, diseases that can be targeted include diseases that are caused by pathogenic splicing defects.

In some embodiments, cellular targets include hematopoietic stem/progenitor cells (CD34+); human T cells, and ocular cells (retinal cells), such as photoreceptor progenitor cells.

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

In some embodiments, the target diseases also include cancer; sickle cell anemia (caused by a point mutation); HBV, HIV; beta-thalassemia; and an ophthalmologic or eye disease, such as Leber congenital amaurosis (LCA), caused by a splicing defect.

In some embodiments, delivery methods include cationic lipid-mediated "direct" delivery of the enzyme-targeting (ribonucleoprotein) complex and electroporation of plasmid DNA.

The methods, products, and uses described herein may be used for purposes other than therapy. In addition, any of the methods described herein may be used in vitro and ex vivo.

In one aspect, there is provided a non-naturally occurring or engineered composition comprising:

I. two or more polynucleotide sequences of the CRISPR-Cas system, providing

(a) a first guide sequence capable of hybridizing to a first target sequence at a polynucleotide locus,

(b) a second guide sequence capable of hybridizing to a second target sequence at the polynucleotide locus,

(c) direct repeat sequence,

(d) optionally, if applicable, the tracr sequence; and

II. the Cas9 enzyme or a second polynucleotide sequence encoding it,

wherein the Cas9 enzyme is a modified enzyme comprising one or more DDs described herein,

wherein, when transcribed, the first and second guide sequences direct sequence-specific binding of the first and second CRISPR complexes to the first and second target sequences, respectively,

wherein the first CRISPR complex comprises a Cas9 enzyme in complex with a first guide sequence that can hybridize to a first target sequence,

wherein the second CRISPR complex comprises a Cas9 enzyme in complex with a second guide sequence that can hybridize to a second target sequence, and

wherein the first guide sequence directs cleavage of one strand of a DNA duplex near a first target sequence, and the second guide sequence directs cleavage of the other strand near a second target sequence, thereby inducing a double-strand break, thereby providing modification of an organism, either non-human or non-animal.

In another embodiment, Cas9 is delivered to the cell as a protein. In another and particularly preferred embodiment, Cas9 is delivered to the cell as a protein or as a nucleotide sequence encoding it. Delivery to the cell as a protein may include delivery of a ribonucleoprotein (RNP) complex, where the protein is in a complex with a guide.

In one aspect, host cells and cell lines modified with or containing the compositions, systems or modified enzymes of the present invention are provided, including stem cells and their progeny.

In one aspect, methods of cell therapy are provided in which, for example, a single cell or population of cells is isolated or cultured, wherein such cell or cells are or have been modified ex vivo as described herein, and then returned (selected cells) or introduced (cultured cells) into the body. Stem cells, whether embryonic, induced pluripotent or totipotent stem cells, are also particularly preferred in this regard. But, of course, in vivo embodiments are also contemplated.

The methods according to the present invention may further comprise delivery of templates, such as repair templates, which may be dsODN or ssODN, see below. Delivery of the templates may be carried out simultaneously or separately from delivery of any or all of the CRISPR enzyme or guide and by the same or different delivery mechanisms. In some embodiments, it is preferred that the template is delivered together with the guide and, preferably, also with the CRISPR enzyme. An example is an AAV-based vector, wherein the CRISPR enzyme is AsCas9 or LbCas9.

The methods of the present invention may further comprise: (a) delivering to a cell a double-stranded oligodeoxynucleotide (dsODN) comprising sticky ends complementary to the sticky ends created by said double-strand break, wherein said dsODN integrates into a locus of interest; or (b) delivering to a cell a single-stranded oligodeoxynucleotide (ssODN), wherein said ssODN acts as a template for homologous repair of said double-strand break. The methods of the present invention may be used to prevent or treat a disease in an individual, optionally wherein said disease is caused by a defect in said locus of interest. The methods of the present invention may be performed in vivo in an individual or ex vivo on a cell removed from an individual, optionally wherein said cell is returned to the individual.

The present invention also encompasses products obtained by using a CRISPR enzyme, or a Cas enzyme, or a Cas9 enzyme, or a CRISPR-CRISPR enzyme, or a CRISPR-Cas system, or a CRISPR-Cas9 system of the present invention.

Structural homology, homologues and orthologues

In embodiments, a Cas9 protein referred to herein also encompasses a homolog or ortholog of Cas9, such as SpCas9 or eSpCas9. The terms "orthologous" (also referred to herein as an "ortholog") and "homologous" (also referred to herein as a "homolog") are well known in the art. As a further guide, a "homologous" protein, as used herein, is a protein of the same species that performs the same or similar function as the protein to which it is homologous. Homologs and orthologs can be identified using homology modeling (see, e.g., Greer, Science vol. 228 (1985) 1055, and Blundell et al. Eur J Biochem vol 172 (1988), 513) or "structural BLAST" (Dey F, Cliff Zhang Q, Petrey D, Honig B. Toward a "structural BLAST": using structural relationships to infer function. Protein Sci. 2013 Apr;22(4):359-66. doi: 10.1002/pro.2225.). See also Shmakov et al. (2015) for applications to CRISPR-Cas loci. Homologous proteins may, but do not necessarily, be structurally related, or they are only partially structurally related. An "orthologous" protein, as used herein, is a protein from another species that performs the same or similar function as the protein to which it is orthologous. Orthologous proteins may, but need not, be structurally related, or are only partially structurally related. In particular embodiments, a homolog or ortholog of Cas9 as referred to herein has a homology or sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as at least 95%, to Cas9. In further embodiments, a homolog or ortholog of Cas9 as referred to herein has a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as at least 95%, to wild-type Cas9. In particular embodiments, a homolog or ortholog of Cas9 as referred to herein has a homology or sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as at least 95%, with Cas9. In further embodiments, a homolog or ortholog of Cas9 as referred to herein has a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as at least 95%, with wild-type SpCas9. If Cas9 has one or more mutations (mutated), then the homolog or ortholog of said Cas9 as referred to herein has a sequence identity of at least 80%, more preferably at least 85%, even more preferably at least 90%, such as at least 95%, with the mutated Cas9.

Specific domains of orthologous proteins are similarly related. In certain embodiments, an orthologous domain of Cas9 as referred to herein has at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% homology or sequence identity with Cas9. In particular embodiments, an orthologous domain of Cas9 as referred to herein has at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% homology or sequence identity with SpCas9.

Delivery of the CRISPR-Cas9 complex or its components

By utilizing this disclosure and the prior art, a CRISPR-Cas system, particularly the novel CRISPR systems described herein, or components thereof, or nucleic acid molecules thereof (including, for example, an HDR template), or nucleic acid molecules encoding or comprising components thereof, can be delivered using a delivery system described herein, both generally and in detail.

Vector delivery, e.g., plasmid, viral delivery. A CRISPR enzyme, e.g., Cas9, a Cas9 ortholog or mutant thereof, and/or any of the RNAs of the present invention, e.g., a guide RNA, can be delivered using any suitable vector, e.g., a plasmid or viral vectors such as an adeno-associated virus (AAV), a lentivirus, an adenovirus, or other types of viral vectors, or combinations thereof. Cas9 and one or more guide RNAs can be packaged in one or more vectors, e.g., a plasmid or viral vectors. In some embodiments, a vector, e.g., a plasmid or a viral vector, is delivered to the tissue of interest by, e.g., intramuscular injection, while in other cases, delivery is via intravenous, transdermal, intranasal, oral, transmucosal, or other delivery methods. Such delivery can be in the form of a single dose or multiple doses. One skilled in the art will appreciate that the actual dose to be delivered herein may vary significantly depending on a number of factors such as the choice of vector, the target cell, organism or tissue, the general condition of the subject being treated, the degree of transformation/modification required, the route of administration, the route of administration, the type of transformation/modification required, etc.

Such a dose may further comprise, for example, a carrier (water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, a pharmaceutically acceptable carrier (e.g., phosphate buffered saline), a pharmaceutically acceptable excipient, and/or other compounds known in the art. The dose may further comprise one or more pharmaceutically acceptable salts, such as, for example, a salt of an inorganic acid, such as hydrochloride, hydrobromide, phosphate, sulfate, etc.; and salts of organic acids, such as acetates, propionates, malonates, benzoates, etc. Additionally, it may also contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gels or gelling materials, flavorings, coloring agents, microspheres, polymers, suspending agents, etc. In addition, one or more other conventional pharmaceutical ingredients such as preservatives, humectants, suspending agents, surfactants, antioxidants, anti-caking agents, fillers, chelating agents, coating agents, chemical stabilizers, etc. may also be present, especially if the dosage form is a form to be reconstituted. Suitable illustrative ingredients include microcrystalline cellulose, sodium carboxymethylcellulose, 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 detailed discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991), incorporated herein by reference.

In one embodiment according to this document, delivery is carried out via adenovirus, which can be in a single booster dose containing at least 1 x 10 ⁵ particles (also referred to as particle units, pu) of adenoviral vector. In one embodiment according to this document, the dose is preferably at least about 1 x 10 ⁶ particles (e.g., about 1 x 10 ⁶ - 1 x 10 ¹² particles), more preferably at least about 1 x 10 ⁷ particles, more preferably at least about 1 x 10 ⁸ particles (e.g., about 1 x 10 ⁸ - 1 x 10 ¹¹ particles or about 1 x 10 ⁸ - 1 x 10 ¹² particles) and most preferably at least about 1 x 10 ⁹ particles (e.g., about 1 x 10 ⁹ - 1 x 10 ¹⁰ particles or about 1 x 10 ⁹ - 1 x 10 ¹² particles) or even at least about 1 x 10 ¹⁰ particles (e.g., about 1 x 10 ¹⁰ - 1 x 10 ¹² particles) of adenoviral vector. Alternatively, the dose contains no more than about 1 x 10 ¹⁴ particles, preferably no more than about 1 x 10 ¹³ particles, even more preferably no more than about 1 x 10 ¹² particles, even more preferably no more than about 1 x 10 ¹¹ particles, and most preferably no more than about 1 x 10 ¹⁰ particles (e.g., no more than about 1 x 10 ⁹ particles). Thus, the dose may comprise a single dose of adenoviral vector, for example, with about 1 x 10 ⁶ particle units (pu), about 2 x 10 ⁶ pu, about 4 x 10 ⁶ pu, about 1 x 10 ⁷ pu, about 2 x 10 ⁷ pu, about 4 x 10 ⁷ pu, about 1 x 10 ⁸ pu, about 2 x 10 ⁸ pu, about 4 x 10 ⁸ pu, about 1 x 10 ⁹ pu, about 2 x 10 ⁹ pu, about 4 x 10 ⁹ pu, about 1 x 10 ¹⁰ pu, about 2 x 10 ¹⁰ pu, about 4 x 10 ¹⁰ pu, about 1 x 10 ¹¹ pu, about 2 x 10 ¹¹ pu, about 4 x 10 ¹¹ pu, about 1 x 10 ¹² pu, about 2 x 10 ¹² pu, or about 4 x 10 ¹² pu of an adenovirus vector. See, for example, the adenovirus vectors in U.S. Patent No. 8,454,972 B2 to Nabel, et al., issued June 4, 2013; incorporated herein by reference, and the doses at column 29, lines 36-58 of that patent. In one embodiment according to this document, the adenovirus is delivered via multiple doses.

In one embodiment according to the present document, delivery is carried out via AAV. It is believed that a therapeutically effective dose for in vivo delivery of AAV to a human is in the range of about 20 to about 50 ml of saline solution containing about 1 x 10 ¹⁰ to about 1 x 10 ¹⁰ functional AAV particles/ml of solution. The dose can be adjusted to balance the therapeutic benefit and any side effects. In one embodiment according to the present document, the dose of AAV is typically in the concentration range of about 1 x 10 ⁵ to 1 x 10 ⁵⁰ AAV genomes, about 1 x 10 ⁸ to 1 x 10 ²⁰ AAV genomes, about 1 x 10 ¹⁰ to about 1 x 10 ¹⁶ genomes, or about 1 x 10 ¹¹ to about 1 x 10 ¹⁶ AAV genomes. A human dose may be about 1 x 10 ¹³ AAV genomes. Such concentrations may be delivered in a carrier solution of about 0.001 ml to about 100 ml, about 0.05 ml to about 50 ml, or about 10 ml to about 25 ml. Other effective doses may be readily determined by one of ordinary skill in the art through routine dose-response testing. See, for example, U.S. Patent No. 8,404,658 B2 to Hajjar, et al., issued March 26, 2013, at column 27, lines 45-60.

In one embodiment according to the present document, delivery is carried out via a plasmid. In such plasmid compositions, the dose should be an amount of plasmid sufficient to cause an effect. For example, suitable amounts of plasmid DNA in plasmid compositions can be from about 0.1 to about 2 mg, or from about 1 μg to about 10 μg, based on an individual weighing 70 kg. Plasmids of the present invention will generally comprise (i) a promoter; (ii) a sequence encoding a CRISPR enzyme operably linked to said promoter; (iii) a selectable marker; (iv) an origin of replication; and (v) a downstream transcription terminator operably linked to (ii). The plasmid may also encode RNA components of the CRISPR complex, but along with this, one or more of them may be encoded by another vector.

The doses in this document are based on an average weight of 70 kg. The frequency of administration is within the competence of a medical practitioner or veterinarian (e.g., a doctor, a veterinarian) or a scientist who is a specialist in this field. It is also noted that the weight of the mice used in the experiment is usually approximately 20 g, which is proportional to an individual weighing 70 kg when experiments are carried out with mice.

In some embodiments, the RNA molecules of the present invention are delivered in liposomal or Lipofectin-based formulations and the like, and can be prepared using methods well known to those skilled in the art. Such methods are described, for example, in U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,859, incorporated herein by reference. Delivery systems specifically designed to enhance the efficiency and improve the delivery of siRNA into 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 they can be used in the present invention. Recently, siRNAs have been successfully used to inhibit gene expression in primates (see, e.g., Tolentino et al., Retina 24(4):660), and they can also be used in the present invention.

Indeed, RNA delivery is also a useful method for in vivo delivery. Cas9 and gRNA (and, for example, a template for HR repair) can be delivered to cells using liposomes or particles or nanoparticles. Thus, the delivery of a CRISPR enzyme, such as Cas9, and/or the delivery of RNA according to the present invention can be carried out in the form of RNA and via microvesicles, liposomes or particles or nanoparticles. For example, Cas9 mRNA and gRNA can be packaged in liposomal particles for in vivo delivery. Liposomal transfection reagents, such as Lipofectamine from Life Technologies and other reagents available commercially, can efficiently deliver RNA molecules to the liver.

Also preferred RNA delivery vehicles include RNA delivery via particles or nanoparticles (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 siRNA delivery, a system somewhat similar to the CRISPR 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. electronic publication November 15, 2012) describe how exosomes, which are promising tools for drug delivery across various biological barriers, can be adapted to deliver siRNA in vitro and in vivo. This approach involves the generation of targeted exosomes by transfection with an expression vector containing the exosomal protein fused to a peptide ligand. The exosomes are then purified from the supernatant of transfected cells and characterized, and then the exosomes are loaded with RNA. The delivery or administration according to the present invention can be carried out by means of exosomes, in particular without limitation to the brain. Vitamin E (α-tocopherol) can be conjugated to CRISPR-Cas and delivered to the brain together with high-density lipoprotein (HDL), for example, similar to what was done by Uno et al. (HUMAN GENE THERAPY 22:711-719 (June 2011)) for the delivery of short interfering RNA (siRNA) to the brain. Mice were infused using osmotic minipumps (model 1007D; Alzet, Cupertino, CA) filled with phosphate-buffered saline (PBS) or free Toc-siBACE or Toc-siBACE/HDL and connected to Brain Infusion Kit 3 (Alzet). The brain infusion cannula was placed approximately 0.5 mm posterior to bregma in the midline for infusion into the dorsal third ventricle. Uno et al. found that as little as 3 nmol of Toc-siRNA with HDL in the same ICV infusion route could induce a similar degree of targeting. A similar dose of α-tocopherol-conjugated CRISPR-Cas co-administered with HDL targeting the brain could be provided in the present invention for humans, for example, could be provided in an amount of from about 3 nmol to about 3 μmol of brain-targeting CRISPR-Cas. Zou et al. (HUMAN GENE THERAPY 22:465-475 (April 2011)) describe a method for lentiviral-mediated delivery of short hairpin RNAs targeting PKCγ for in vivo gene silencing in the rat spinal cord. Zou et al. administered approximately 10 μl of recombinant lentivirus at a titer of 1 x 10 ⁹ transducing units (TU)/ml via an intrathecal catheter. A similar dose of expressed CRISPR-Cas in a brain-targeting lentiviral vector may be provided in the present invention for humans, for example, approximately 10-50 ml of brain-targeting CRISPR-Cas in a lentivirus at a titer of 1 x 10 ⁹ transducing units (TU)/ml may be provided.

If local delivery to the brain is intended, this can be achieved in a variety of ways. For example, the material can be delivered intrastriatically, such as by injection. The injection can be performed stereotactically via craniotomy.

Increasing the efficiency of NHEJ or HR also facilitates delivery. Preferably, NHEJ efficiency is increased by co-expression of end-processing enzymes such as Trex2 (Dumitrache et al. Genetics. 2011 August; 188(4): 787-797). Preferably, HR efficiency is increased by transient inhibition of components of the NHEJ machinery such as Ku70 and Ku86. HR efficiency can also be increased by co-expression of prokaryotic or eukaryotic homologous recombination enzymes such as RecBCD, RecA.

Packaging and promoters

The following methods exist for packaging nucleic acid molecules encoding Cas9 of the present invention, such as DNA, into vectors, such as viral vectors, to mediate genome modification in vivo.

To achieve NHEJ-mediated gene knockout, the following is required.

One viral vector

A vector containing two or more expression cassettes:

promoter - a nucleic acid molecule encoding the Cas9 terminator;

promoter-gRNA1-terminator;

promoter-gRNA2-terminator;

promoter-gRNA(N)-terminator (up to the maximum vector size).

Two viral vectors

Vector 1 containing one expression cassette to drive Cas9 expression:

promoter - a nucleic acid molecule encoding the Cas9 terminator;

vector 2 containing one or more expression cassettes for directing the expression of one or more guide RNAs:

promoter-gRNA1-terminator;

promoter-gRNA(N)-terminator (up to the maximum vector size).

To mediate repair involving homologous recombination.

In addition to the single and dual viral vector approaches described above, an additional vector can be used to deliver a template for repair involving homologous recombination.

The promoter used to drive expression of the nucleic acid molecule encoding Cas9 may include the following.

- AAV ITR can serve as a promoter: this is advantageous in that it eliminates the need for an additional promoter element (which may take up space in the vector). The freed up extra space can be used to drive the expression of additional elements (gRNA, etc.). Also, the ITR activity is relatively weaker, so it can be used to reduce the potential toxicity caused by Cas9 overexpression.

- For ubiquitous expression, promoters that can be used include: CMV, CAG, CBh, PGK, SV40, ferritin heavy or light chain genes, etc.

For expression in the brain or another part of the CNS, the following promoters can be used: the synapsin I gene for all neurons, the CaMKII-alpha gene for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. For expression in the liver, the albumin gene promoter can be used. For expression in the lung, SP-B can be used. For endothelial cells, ICAM can be used. For hematopoietic cells, the IFN-beta or CD45 gene promoter can be used. For osteoblasts, OG-2 can be used.

The promoter used to drive the guide RNA may include the following:

- Pol III promoters such as U6 or H1;

- use of the Pol II promoter and intron cassettes for gRNA expression.

Adeno-associated virus (AAV)

Cas9 or a Cas9 mutant or ortholog and one or more guide RNAs can be delivered using an adeno-associated virus (AAV), lentivirus, adenovirus or other types of plasmid or viral vectors, in particular using formulations and dosages according to, for example, U.S. Patent Nos. 8,454,972 (formulations, dosages for adenovirus), 8,404,658 (formulations, dosages for AAV) and 5,846,946 (formulations, dosages for plasmid DNA) and clinical trials and publications of clinical trial results using lentivirus, AAV and adenovirus. For example, for AAV, the route of administration, formulation and dosage can be as defined in U.S. Patent No. 8,454,972 and in clinical trials using AAV. For adenovirus, the route of administration, formulation, and dosage may be as defined in U.S. Patent No. 8,404,658 and in clinical trials using adenovirus. For plasmid delivery, the route of administration, formulation, and dosage may be as defined in U.S. Patent No. 5,846,946 and in clinical trials using plasmids. Doses may be based on or extrapolated to an individual with an average weight of 70 kg (e.g., an adult male), and may be adjusted for patients, subjects, mammals of other weights, and other species. The frequency of administration is within the discretion of the practitioner or veterinarian (e.g., a doctor, a veterinarian) and will depend on conventional factors, including the age, sex, general health, other conditions of the patient or subject, and the particular conditions or symptoms being addressed. Viral vectors may be injected into the tissue of interest. In the case of cell type-specific genome modification, Cas9 expression can be driven by a cell type-specific promoter. For example, liver-specific expression could use the albumin gene promoter, while neuron-specific expression (e.g., to target CNS disorders) could use the synapsin I gene promoter.

With regard to in vivo delivery, AAV is advantageous over other viral vectors for two reasons:

low toxicity (this may be due to the purification method, which does not require ultracentrifugation of cellular particles that can activate the immune response);

low probability of causing insertional mutagenesis since it does not integrate into the host genome.

AAV has a packaging limit of 4.5 or 4.75 kb. This means that all of Cas9, as well as the promoter and transcription terminator, must fit into the same viral vector. Constructs larger than 4.5 or 4.75 kb will result in a significant reduction in virus production. SpCas9 is quite large, the gene itself being larger than 4.1 kb, making it difficult to package into AAV. Therefore, embodiments of the present invention include the use of shorter Cas9 homologues. For example:

View Size Cas9 Corynebacterium diphtheriae 3252 Eubacterium ventriosum 3321 Streptococcus pasteurianus 3390 Lactobacillus farciminis 3378 Sphaerochaeta globus 3537 Azospirillum B510 3504 Gluconacetobacter diazotrophicus 3150 Neisseria cinerea 3246 Roseburia intestinalis 3420 Parvibaculum lavamentivorans 3111 Staphylococcus aureus 3159 Nitratifractor salsuginis DSM 16511 3396 Campylobacter lari CF89-12 3009 Streptococcus thermophilus LMD-9 3396

These species are thus generally preferred species for Cas9 production.

With respect to AAV, the AAV may be AAV1, AAV2, AAV5 or any combination thereof. One may select an AAV from among AAVs based on the cells to be targeted; for example, one may select AAV serotypes 1, 2, 5 or a hybrid capsid of AAV1, AAV2, AAV5 or any combination thereof to target the brain or neurons; and one may select AAV4 to target cardiac tissue. AAV8 is useful for liver delivery. The above promoters and vectors are individually preferred herein. The mapping of specific AAV serotypes to specific cells (see Grimm, D. et al, J. Virol. 82: 5887-5911 (2008)) is shown 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 10 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 1 0.2 0,1 HepG2 3 100 16.7 0.3 1.7 5 0.3 No data Hep1A 20 100 0.2 1.0 0,1 1 0.2 0,0 911 17 100 11 0.2 0,1 17 0,1 No data CHO 100 100 14 1.4 333 50 10 1.0 COS 33 100 33 3.3 5.0 14 2.0 0.5 MeWo 10 100 20 0.3 6.7 10 1.0 0.2 NIH3T3 10 100 2.9 2.9 0.3 10 0.3 No data A549 14 100 20 No data 0.5 10 0.5 0,1 HT1180 20 100 10 0,1 0.3 33 0.5 0,1 Monocytes 1111 100 No data No data 125 1429 No data No data Immature DC 2500 100 No data No data 222 2857 No data No data Mature DC 2222 100 No data No data 333 3333 No data No data

Lentivirus

Lentiviruses are complex retroviruses that have the ability to infect both mitotic and postmitotic cells and express their genes in them. The best-known lentivirus is the human immunodeficiency virus (HIV), which uses the envelope glycoproteins of other viruses to target a wide range of cell types.

Lentiviruses can be generated as follows. After cloning pCasES10 (which contains the lentiviral transfer plasmid backbone), low-passage HEK293FT cells (p=5) were seeded in a T-75 flask to 50% confluency the day before transfection in DMEM with 10% fetal bovine serum and no antibiotics. After 20 h, the medium was changed to OptiMEM (serum-free) and transfection was performed 4 h later. Cells were transfected with 10 μg of the lentiviral transfer plasmid (pCasES10) and the following packaging plasmids: 5 μg of pMD2.G (pseudotype VSV-g) and 7.5 μg of psPAX2 (gag/pol/rev/tat). Transfections were performed in 4 ml OptiMEM with a cationic lipid delivery vehicle (50 µl Lipofectamine 2000 and 100 µl Plus reagent). After 6 h, the medium was replaced with antibiotic-free DMEM with 10% fetal bovine serum. These methods used serum during cell culture, but serum-free methods are preferred.

Lentivirus can be purified as follows. Virus-containing supernatants were collected after 48 h. The supernatants were first cleared of debris and filtered through a 0.45 μm low protein binding filter (PVDF). They were then centrifuged in an ultracentrifuge for 2 h at 24,000 rpm. Virus-containing supernatants were resuspended in 50 μl DMEM overnight at 4°C. They were then aliquoted and immediately frozen at -80°C.

In another embodiment, minimal lentiviral vectors for non-primate organisms based on equine infectious anemia virus (EIAV) are also provided, especially for gene therapy of ocular diseases (see, for example, Balagaan, J Gene Med 2006; 8: 275-285). In another embodiment, RetinoStat®, an equine infectious anemia virus lentiviral vector for gene therapy expressing the angiostatic proteins endostatin and angiostatin, which is delivered by subretinal injection for the treatment of wet age-related macular degeneration (see, for example, Binley et al., HUMAN GENE THERAPY 23:980-991 (September 2012)), is also provided, and this vector can be modified for the CRISPR-Cas system of the present invention.

In another embodiment, self-inactivating lentiviral vectors with siRNA targeting a common exon that has tat/rev HIV, a TAR trap nucleolar localization signal, and a CCR5-specific hammerhead ribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) can be used and/or adapted for the CRISPR-Cas system of the present invention. At least 2.5 × 10 ⁶ CD34+ cells/kg patient weight can be harvested and prestimulated for 16-20 hours in X-VIVO 15 medium (Lonza) containing 2 μmol/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. Prestimulated cells can be transduced with lentivirus at an 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.).

Lentiviral vectors have been disclosed with respect to the treatment of Parkinson's disease, see, e.g., U.S. Patent Application Publication No. 20120295960 and U.S. Patent Nos. 7,303,910 and 7,351,585. Lentiviral vectors have also been disclosed with respect to the treatment of eye diseases, see, e.g., U.S. Patent Application Publication Nos. 20060281180, 20090007284, US20110117189; US20090017543; US20070054961, US20100317109. Lentiviral vectors have also been disclosed with respect to delivery to the brain, see, e.g., U.S. Patent Application Publication Nos. US20110293571; US20110293571, US20040013648, US20070025970, US20090111106 and US Patent No. US7259015.

RNA delivery

Delivery of RNA. The CRISPR enzyme, such as Cas9, and/or any of the RNAs of the present invention, such as guide RNA, can also be delivered in the form of RNA. Cas9 mRNA can be produced by in vitro transcription. For example, Cas9 mRNA can be synthesized using a PCR cassette comprising the following elements: T7 promoter-Kozak sequence (GCCACC)-Cas9-3'-UTR of the beta-globin gene-poly(A) tail (a chain of 120 or more adenine residues). The cassette can be used for transcription by T7 polymerase. Guide RNAs can also be transcribed by in vitro transcription from a cassette comprising the T7 promoter-GG-guide RNA sequence.

To enhance expression and reduce potential toxicity, the CRISPR enzyme coding sequence and/or guide RNA can be modified to include one or more modified nucleosides, such as pseudo-U or 5-methyl-C.

mRNA delivery methods are currently particularly promising for delivery to the liver.

Many clinical studies on RNA delivery have focused on RNAi or antisense RNA, but these systems can be adapted to deliver RNA for the practice of the present invention. Accordingly, referenced literature on RNAi, etc. is also needed below. Indeed, RNA delivery is also a useful method for in vivo delivery. Cas9 and gRNA (and, for example, a template for HR repair) can be delivered to cells using liposomes or nanoparticles. Thus, the delivery of a CRISPR enzyme such as Cas9 and/or the delivery of RNA according to the present invention can be carried out in the form of RNA and via microvesicles, liposomes or nanoparticles. For example, Cas9 mRNA and gRNA can be packaged in liposomal particles for in vivo delivery. Liposomal transfection reagents such as Lipofectamine from Life Technologies and other reagents available commercially can efficiently deliver RNA molecules to the liver.

Particle-mediated RNA delivery

Also preferred RNA delivery vehicles include RNA delivery via nanoparticles (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 siRNA delivery, a system somewhat similar to the CRISPR 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. electronic publication from November 15, 2012) describe how exosomes, which are promising tools for drug delivery across various biological barriers, can be adapted to deliver siRNA in vitro and in vivo. This approach involves the creation of targeted exosomes by transfection with an expression vector containing an exosomal protein fused to a peptide ligand. The exosomes are then purified from the supernatant with transfected cells and characterized, and then RNA is loaded into the exosomes. Delivery or administration according to the present invention can be carried out using exosomes, in particular, but not limited to the brain. Vitamin E (α-tocopherol) can be conjugated to CRISPR-Cas and delivered to the brain along with high-density lipoprotein (HDL), for example, similar to what was done by Uno et al. (HUMAN GENE THERAPY 22:711–719 (June 2011)) to deliver short interfering RNA (siRNA) to the brain. Mice were infused using osmotic minipumps (model 1007D; Alzet, Cupertino, CA) filled with phosphate-buffered saline (PBS) or free Toc-siBACE or Toc-siBACE/HDL and connected to Brain Infusion Set 3 (Alzet). The brain infusion cannula was positioned approximately 0.5 mm posterior to bregma in the midline for infusion into the dorsal third ventricle. Uno et al. found that as little as 3 nmol of Toc-siRNA with HDL in the same ICV infusion route could induce a similar degree of targeted reduction. A similar dose of α-tocopherol-conjugated HDL that targets the brain could be provided in the present invention for humans, for example, could be provided in an amount of from about 3 nmol to about 3 μmol of brain-targeting CRISPR-Cas. Zou et al. (HUMAN GENE THERAPY 22:465-475 (April 2011)) describe a method for lentiviral-mediated delivery of short hairpin RNAs targeting PKCγ for in vivo gene silencing in the rat spinal cord. Zou et al. Approximately 10 μl of the recombinant lentivirus with a titer of 1 x 10 ⁹ transducing units (TU)/ml were administered via an intrathecal catheter. A similar dose of the expressed CRISPR-Cas in the lentiviral vector targeting the brain can be provided in the present invention for humans, for example, approximately 10-50 ml of the CRISPR-Cas targeting the brain in the lentivirus with a titer of 1 x 10 ⁹ transducing units (TU)/ml can be provided.

If local delivery to the brain is intended, this can be achieved in a variety of ways. For example, the material can be delivered intrastriatically, such as by injection. The injection can be performed stereotactically via craniotomy.

Increasing the efficiency of NHEJ or HR also facilitates delivery. Preferably, NHEJ efficiency is increased by co-expression of end-processing enzymes such as Trex2 (Dumitrache et al. Genetics. 2011 August; 188(4): 787-797). Preferably, HR efficiency is increased by transient inhibition of components of the NHEJ machinery such as Ku70 and Ku86. HR efficiency can also be increased by co-expression of prokaryotic or eukaryotic homologous recombination enzymes such as RecBCD, RecA.

Delivery via plasmids

In one embodiment according to the present document, delivery is carried out via a plasmid. In such plasmid compositions, the dose should be an amount of plasmid sufficient to cause an effect. For example, suitable amounts of plasmid DNA in plasmid compositions can be from about 0.1 to about 2 mg, or from about 1 μg to about 10 μg, based on an individual weighing 70 kg. Plasmids of the present invention will generally comprise (i) a promoter; (ii) a sequence encoding a CRISPR enzyme operably linked to said promoter; (iii) a selectable marker; (iv) an origin of replication; and (v) a downstream transcription terminator operably linked to (ii). The plasmid may also encode RNA components of the CRISPR complex, but along with this, one or more of them may be encoded by another vector.

The doses in this document are based on an average weight of 70 kg. The frequency of administration is within the competence of a medical practitioner or veterinarian (e.g., a doctor, a veterinarian) or a scientist who is a specialist in this field. It is also noted that the weight of the mice used in the experiment is usually approximately 20 g, which is proportional to an individual weighing 70 kg when experiments are carried out with mice.

In some embodiments, the RNA molecules of the present invention are delivered in liposomal or Lipofectin-based formulations and the like, and can be prepared using methods well known to those skilled in the art. Such methods are described, for example, in U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,859, incorporated herein by reference. Delivery systems specifically designed to enhance the efficiency and improve the delivery of siRNA into 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 they can be used in the present invention. Recently, siRNAs have been successfully used to inhibit gene expression in primates (see, e.g., Tolentino et al., Retina 24(4):660), and they can also be used in the present invention.

General information regarding particle delivery

Also cited is PCT application PCT/US14/70057, Attorney Docket No. 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 to one or more or all of U.S. Provisional Patent Applications 62/054,490, filed September 24, 2014; 62/010,441, filed June 10, 2014; and 61/915,118, 61/915,215, and 61/915,148, each filed December 12, 2013) ("the Particle Delivery PCT"), incorporated herein by reference, regarding a method of producing a particle comprising an sgRNA and a Cas9 protein, comprising mixing a mixture comprising an sgRNA and a Cas9 protein (and optionally a matrix for HDR) with a mixture comprising, consisting essentially of, or consisting of a surfactant, a phospholipid, a biodegradable polymer, a lipoprotein, and an alcohol; thereby producing particles. For example, 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, such as 20-25°C, such as room temperature, for a suitable period of time, such as 15-45, such as 30 minutes, preferably in a sterile nuclease-free buffer, such as 1X PBS. Individually, the particle components, such as or including a surfactant, such as a cationic lipid, such as 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); a phospholipid, such as dimyristoylphosphatidylcholine (DMPC); a biodegradable polymer such as ethylene glycol polymer or PEG and a lipoprotein such as low density lipoprotein, for example cholesterol, were dissolved in an alcohol, preferably a C1-6 alkyl alcohol such as methanol, ethanol, isopropanol, for example 100% ethanol. The two solutions were mixed together to form particles containing Cas9-sgRNA complexes. Accordingly, it is possible to provide a pre-complex of sgRNA with Cas9 protein before assembling the entire complex in the particle. The formulations can be prepared with different molar ratios of various components known to facilitate the delivery of nucleic acids into cells (e.g. 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), polyethylene glycol (PEG) and cholesterol). For example, the molar ratios of DOTAP: DMPC: PEG: cholesterol can be as follows: 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. This application accordingly encompasses mixing of sgRNA, Cas9 protein and components that form a particle; as well as particles obtained as a result of such addition. Aspects of the present invention can encompass particles; for example, particles using a method similar to that in "Particle Delivery PCT", for example by adding a mixture containing sgRNA and/or Cas9, as in the present invention, and components that form a particle, for example as in "Particle Delivery PCT", to form a particle and particles as a result of such mixing (or, of course, other particles containing sgRNA and/or Cas9, as in the present invention).

Delivery systems and/or particle-based formulations

Several types of particle-based delivery systems and/or formulations are known to be applicable in a wide range of biomedical applications. A particle is generally defined as a small object that behaves as a whole unit with respect to its transport and properties. Particles are further classified according to diameter. Large particles cover the range from 2,500 to 10,000 nanometers. Fine particles have a size of 100 to 2,500 nanometers. Ultrafine particles or nanoparticles typically have a size of 1 to 100 nanometers. The basis for the 100 nm limit is the fact that novel properties that distinguish particles from bulk material typically occur at a critical linear scale of less than 100 nm.

As used herein, a particle-based delivery system and/or formulation is defined as any biological delivery system/formulation comprising particles according to the present invention. A particle according to the present invention is any object having a largest dimension (e.g., diameter) of less than 100 microns (μm). In some embodiments, the particles of the present invention have a largest dimension of less than 10 μm. In some embodiments, the particles of the present invention have a largest dimension of less than 2000 nanometers (nm). In some embodiments, the particles of the present invention have a largest dimension of less than 1000 nanometers (nm). In some embodiments, the particles of the present 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. The particles of the present invention typically have a largest dimension (e.g., diameter) of 500 nm or less. In some embodiments, the particles of the present invention have a largest dimension (e.g., diameter) of 250 nm or less. In some embodiments, the particles of the present invention have a largest dimension (e.g., diameter) of 200 nm or less. In some embodiments, the particles of the present invention have a largest dimension (e.g., diameter) of 150 nm or less. In some embodiments, the particles of the present invention have a largest dimension (e.g., diameter) of 100 nm or less. In some embodiments, smaller particles are used, for example, having a largest dimension of 50 nm or less. In some embodiments, the particles of the present invention have a largest dimension ranging from 25 nm to 200 nm.

Particle characterization (including, for example, characterization of morphology, size, etc.) is performed using a number of different techniques. Standard techniques include electron microscopy (TEM, SEM), atomic force microscopy (AFM), dynamic light scattering (DLS), X-ray photoelectron spectroscopy (XPS), powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF), ultraviolet-visible spectroscopy, dual polarization interferometry, and nuclear magnetic resonance (NMR). Characterization (size measurements) can be performed on the native particles (i.e., prior to loading) or after loading of the cargo molecule (herein, cargo molecule refers to, e.g., one or more components of the CRISPR-Cas system, e.g., a CRISPR enzyme or mRNA, or a guide RNA, or any combination thereof, and may include additional carriers and/or excipients) to produce particles having an optimal size for delivery for any in vitro, ex vivo, and/or in vivo use of the present invention. In certain preferred embodiments, particle size characterization (e.g., diameter) is based on dynamic laser light scattering (DLS) measurements. Reference is made to U.S. Patent No. 8,709,843; U.S. Patent No. 6,007,845; U.S. Patent No. 5,855,913; U.S. Patent No. 5,985,309; Patent No. 5,543,158 and James E. Dahlman and Carmen Barnes et al. Nature Nanotechnology (2014), published online May 11, 2014, doi:10.1038/nnano.2014.84, relate to the particles, methods for producing and using them, and their measurement.

Particle-based delivery systems within the scope of the present invention may be in any form, including but not limited to solid, semi-solid, emulsified or colloidal particles. Thus, any delivery systems described herein, including but not limited to, for example, lipid-based systems, liposomes, micelles, microvesicles, exosomes or gene guns, may be envisaged as particle-based delivery systems within the scope of the present invention.

Particles

CRISPR enzyme mRNA and guide RNA can be delivered simultaneously using particles or lipid shells; for example, the CRISPR enzyme and RNA of the present invention, e.g. as a complex, can be delivered via a particle as in Dahlman et al., WO 2015089419 A2 and documents cited therein, such as 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), e.g., a delivery particle comprising a lipid or lipidoid and a hydrophilic polymer, e.g., a cationic lipid and a hydrophilic polymer, e.g., wherein the cationic lipid comprises 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) or 1,2-diteteradecanoyl-sn-glycero-3-phosphocholine (DMPC), and/or wherein the hydrophilic polymer comprises ethylene glycol or polyethyleneglycol (PEG); and/or wherein the particle further comprises cholesterol (e.g., a particle of Formulation 1 = DOTAP 100, DMPC 0, PEG 0, cholesterol 0; Formulation Number 2 = DOTAP 90, DMPC 0, PEG 10, cholesterol 0; Formulation Number 3 = DOTAP 90, DMPC 0, PEG 5, cholesterol 5), wherein the particles are formed using an efficient multi-step process in which the first effector protein and RNA are mixed together, e.g., at a 1:1 molar ratio, e.g., at room temperature, e.g., for 30 minutes, e.g., in sterile nuclease-free 1X PBS; and separately DOTAP, DMPC, PEG, and cholesterol applicable to the formulation are dissolved in an alcohol, e.g., 100% ethanol; and the two solutions are mixed together to form particles containing the complexes).

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/mp100390w. electronic publication April 1, 2011) describe biodegradable core-shell particles with a poly(β-amino ester) (PBAE) core surrounded by a phospholipid bilayer shell. They were designed for in vivo mRNA delivery. The pH-sensitive component of the PBAE was chosen to promote endosomal disruption, while the surface lipid layer was chosen to minimize the toxicity of the polycationic core. Thus, they are preferred for RNA delivery according to the present invention.

In one embodiment, particles based on self-assembling bioadhesive polymers are provided, which can be used for oral delivery of peptides, intravenous delivery of peptides and intranasal delivery of peptides, in all cases to the brain. Other embodiments are also provided, such as absorption by oral use and intraocular delivery of hydrophobic drugs. Molecular shell technology involves an engineered polymer shell that protects and delivers to the site of disease (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, I.F., et al. Int J Pharm, 2001. 224:185-199). Doses of approximately 5 mg/kg are envisaged, which will be single or multiple doses depending on the target tissue.

In one embodiment, particles that can deliver RNA to cancer cells to stop tumor growth, developed in the laboratory of Dan Anderson at MIT, can be used for and/or adapted to the CRISPR-Cas system of the present invention. In particular, the Anderson laboratory has developed fully automated, combinatorial systems for synthesizing, purifying, characterizing, and formulating novel biomaterials and nanoformulations. See, e.g., 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.

US Patent Application 20110293703 relates to lipid-like compounds that are also particularly useful in the administration of polynucleotides that can be used to deliver the CRISPR-Cas system of the present invention. In one aspect, amino alcohol lipid-like compounds are combined with an agent to be delivered to a cell or subject to form microparticles, nanoparticles, liposomes or micelles. The agent to be delivered by particles, liposomes or micelles can be in the form of a gas, liquid or solid, and the agent can be a polynucleotide, protein, peptide or small molecule. Amino alcohol lipid-like compounds can be combined with other amino alcohol lipid-like compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form particles. These particles may then optionally be combined with a pharmaceutical excipient to form a pharmaceutical composition.

U.S. Patent Application Publication No. 20110293703 also provides methods for preparing amino alcohol lipid-like compounds. One or more equivalents of an amine are allowed to react with one or more equivalents of an epoxide-terminated compound under suitable conditions to form an amino alcohol lipid-like compound of the present invention. In certain embodiments, all of the amino groups of the amine are completely reacted with the epoxide-terminated compound to form tertiary amines. In other embodiments, all of the amino groups of the amine are not completely reacted with the epoxide-terminated compound to form tertiary amine groups, thereby forming primary or secondary amine groups of the amino alcohol lipid-like compound. These primary or secondary amine groups are left as is or may be reacted with another electrophile, such as another epoxide-terminated compound. One skilled in the art will appreciate that reacting an amine with less than an excess of an epoxide-terminated compound will result in a variety of different amino alcohol lipid-like compounds with varying numbers of tails. Certain amines may be fully functionalized with two epoxide-derived tails, while other molecules may be incompletely functionalized with epoxide-derived tails. For example, a diamine or polyamine may contain one, two, three, or four epoxide-derived tails at different amino moieties of the molecule, resulting in primary, secondary, and tertiary amine groups. In certain embodiments, all of the amine groups are incompletely functionalized. In certain embodiments, two epoxide-terminated compounds of the same type are used. In other embodiments, two or more different epoxide-terminated compounds are used. The synthesis of the amino alcohol lipid-like compounds is carried out with or without a solvent, and the synthesis can be carried out at higher temperatures ranging from 30-100°C, preferably at about 50-90°C. The resulting amino alcohol lipid-like compounds can optionally be purified. For example, a mixture of amino alcohol lipid-like compounds can be purified to obtain an amino alcohol lipid-like compound with a certain amount of "tails" of compounds derived from epoxides. Or, the mixture can be purified to obtain a certain stereo- or regioisomer. The amino alcohol lipid-like compounds can also be alkylated with an alkyl halide (e.g., methyl iodide) or other alkylating agent and/or they can be acylated.

U.S. Patent Application Publication No. 20110293703 also discloses libraries of amino alcohol lipid-like compounds produced according to the methods of the present invention. These amino alcohol lipid-like compounds can be produced and/or screened using high-throughput techniques involving the use of liquid dispensers, robotic handlers, microtiter plates, computers, etc. In certain embodiments, the amino alcohol lipid-like compounds are screened for their ability to transfect polynucleotides or other agents (e.g., proteins, peptides, small molecules) into a cell.

U.S. Patent Application Publication No. 20130302401 relates to a class of poly(beta-amino alcohols) (PBAAs) prepared by combinatorial polymerization techniques. The PBAAs of the present invention can be used in biotechnology and biomedical applications as coatings (such as film coatings or multilayer films for medical instruments or implants), additives, materials, fillers, antifouling agents, micropatterning agents, and cell encapsulating agents. When used as surface coatings, these PBAAs induce varying levels of inflammation both in vitro and in vivo depending on their chemical structures. The wide chemical diversity of this class of materials allows the identification of polymer coatings that inhibit macrophage activation in vitro. Moreover, these coatings reduce inflammatory cell recruitment and reduce the severity of fibrosis following subcutaneous implantation of carboxylated polystyrene microparticles. These polymers can be used to form capsules based on polyelectrolyte complexes for encapsulating cells. The present invention can also have many other applications in biology, such as the production of antimicrobial coatings, DNA or siRNA delivery, and tissue engineering using stem cells. The teachings of U.S. Patent Application Publication No. 20130302401 can be applied to the CRISPR-Cas system of the present invention. In some embodiments, sugar-based particles, such as GalNAc, can be used as described herein and with reference to WO 2014118272 (incorporated herein by reference) and Nair, JK et al., 2014, Journal of the American Chemical Society 136 (49), 16958-16961), as well as according to the teachings herein, especially with respect to delivery applications for all particles, unless otherwise obvious.

In another embodiment, lipid particles (LNPs) are provided. In particular, small interfering RNAs targeting transthyretin have been encapsulated in lipid particles and used for delivery in humans (see, for example, Coelho et al., N Engl J Med 2013;369:819-29), and such a system can be adapted and used with the CRISPR-Cas system of the present invention. Doses of about 0.01 to about 1 mg per kg of body weight administered intravenously are provided. Medications to reduce the risk of infusion reactions are provided, such as dexamethasone, acetaminophen, diphenhydramine or cetirizine and ranitidine. Multiple doses of five doses of about 0.3 mg per kilogram taken every 4 weeks are also provided.

LNPs have been shown to be highly efficient in delivering siRNA to the liver (see, e.g., Tabernero et al., Cancer Discovery, April 2013, Vol. 3, No. 4, pages 363-470) and are thus envisaged for liver delivery of RNA encoding CRISPR-Cas. A dosing regimen of approximately four doses of 6 mg/kg LNPs every two weeks may be envisaged. Tabernero et al. demonstrated that tumor regression was observed after the first 2 dosing cycles of LNPs at 0.7 mg/kg and by the end of 6 cycles the patient had achieved a partial response with complete regression of lymph node metastases and significant reduction in liver tumor size. In this patient, who remained in remission and completed treatment after receiving dosing for 26 months, a complete response was achieved after 40 doses. Two patients with RCC and extrahepatic sites of disease including kidney, lung, and lymph nodes that had progressed after prior VEGF pathway inhibitor therapy had stable disease in all sites for approximately 8-12 months, and a patient with PNET and liver metastases continued in the extension study for 18 months (36 doses) with stable disease.

However, the charge of LNPs must be taken into account. Thus, association of cationic lipids with negatively charged lipids induces the formation of non-bilayer structures that facilitate intracellular delivery. Since charged LNPs are rapidly cleared from the circulation following 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 (e.g., pH 4), where ionizable lipids exhibit a positive charge. However, at physiological pH, LNPs exhibit a low surface charge compatible with long residence times in the circulation. The main focus is on four kinds of ionizable cationic lipid molecules, namely 1,2-dilinoleoyl-3-dimethylammoniumpropane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxyketo-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 their activity ranging from DLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP using the factor VII gene silencing model (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011). A dose of 1 μg/mL LNP or CRISPR-Cas RNA in or associated with LNP may be envisaged, particularly for a formulation containing DLinKC2-DMA.

The production of LNPs and encapsulation of CRISPR-Cas can be used and/or adapted according to Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011. Cationic lipids 1,2-dilinoleoyl-3-dimethylammoniumpropane (DLinDAP), 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA), (3-o-[2″-(methoxypolyethyleneglycol 2000)-succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG) and R-3-[(ω-methoxypoly(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 CRISPR-Cas RNA can be encapsulated in LNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:cholesterol: PEG-S-DMG or PEG-C-DOMG in a 40:10:40:10 molar ratio). If desired, 0.2% SP-DiOC18 (Invitrogen, Burlington, Canada) can be included to determine cellular uptake, intracellular delivery, and biodistribution. Encapsulation can be accomplished by dissolving lipid mixtures containing 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 ethanolic lipid solution can be added dropwise to 50 mmol/L citrate, pH 4.0, to form multilamellar vesicles to a final ethanol concentration of 30% v/v. Large unilamellar vesicles can be formed by extruding the multilamellar vesicles through two stacked Nuclepore 80 nm polycarbonate filters using an extruder (Northern Lipids, Vancouver, Canada). Encapsulation can be accomplished by adding RNA dissolved at 2 mg/mL in 50 mmol/L citrate, pH 4.0, containing 30% ethanol. v/v, dropwise into extruded preformed large unilamellar vesicles and incubated at 31°C for 30 min with constant agitation to a final RNA/lipid weight ratio of 0.06/1 w/w. Ethanol removal and neutralization of the formulation buffer were performed by dialysis against phosphate-buffered saline (PBS), pH 7.4, for 16 h using Spectra/Por 2 regenerated cellulose dialysis membranes. Particle size distribution can be determined by dynamic light scattering using a NICOMP 370 Particle Sizer, bubble volume/scattered light intensity modes, and Gaussian fit (Nicomp Particle Sizing, Santa Barbara, CA). Particle size for all three LNP systems can be ~70 nm in diameter. Encapsulation efficiency of siRNA can be determined by removing free RNA from samples collected before or after dialysis using VivaPureD MiniH columns (Sartorius Stedim Biotech). Encapsulated RNA can be extracted from the eluted particles and quantified at 260 nm. The RNA to lipid ratio was determined by measuring the cholesterol content of the vesicles using the Cholesterol E enzymatic assay from Wako Chemicals USA (Richmond, VA). In connection with the discussion of LNPs and PEG-lipids in this paper, PEGylated liposomes or LNPs are also suitable for delivery of the CRISPR-Cas system or its components.

The preparation of large LNPs can be used and/or adapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011. A solution of a pre-mixed lipid (total lipid concentration 20.4 mg/mL) can be prepared in ethanol containing DLinKC2-DMA, DSPC, and cholesterol in a molar ratio of 50:10:38.5. Sodium acetate can be added to the pre-mixed lipid in a molar ratio of 0.75:1 (sodium acetate:DLinKC2-DMA). The lipids can then be hydrogenated by combining the mixture with 1.85 volumes of citrate buffer (10 mmol/L, pH 3.0) with vigorous stirring to induce spontaneous formation of liposomes in aqueous buffer containing 35% ethanol. The liposome solution can be incubated at 37°C to provide a time-dependent increase in particle size. Aliquots can be taken at various time points during the incubation to study liposome size changes using dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK). Once the desired particle size has been achieved, an aqueous solution of PEG-lipid conjugates (stock solution = 10 mg/mL PEG-DMG in 35% (v/v) ethanol) can be added to the liposome mixture to give a final molar concentration of PEG of 3.5% of total lipid. Following addition of the PEG-lipid conjugates, the liposomes should maintain their size with effective inhibition of further growth. RNA can then be added to the empty liposomes at a ratio of RNA to total lipids of approximately 1:10 (w:w) followed by incubation for 30 min at 37°C to form loaded LNPs. The mixture can then be dialyzed overnight in PBS and filtered through a 0.45 μm syringe filter.

Spherical nucleic acid constructs (SNAs™) and other particles (particularly gold particles) are also envisaged as a means of delivering the CRISPR/Cas system to intended targets. Representative data show that AuraSense Spherical Nucleic Acid (SNAs™) constructs of nucleic acid-functionalized gold particles are also useful.

Literature sources that may be useful in conjunction with the ideas presented in this paper include: 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, 209ra152 (2013) and Mirkin, et al., Small, 10:186-192.

Self-assembling RNA particles can be constructed with polyethyleneimine (PEI) that is pegylated with an Arg-Gly-Asp (RGD) peptide ligand attached to the distal end of a polyethyleneglycol (PEG) chain. This system has been used, for example, as a means to target integrin-expressing tumor vasculature and to deliver siRNA that down-regulates vascular endothelial growth factor receptor 2 (VEGF R2), thereby suppressing tumor angiogenesis (see, for example, Schiffelers et al., Nucleic Acids Research, 2004, Vol. 32, No. 19). Nanoplexes can be prepared by mixing equal volumes of aqueous solutions of a cationic polymer and nucleic acid to produce a net molar excess of ionizable nitrogen (polymer) relative to phosphate (nucleic acid) in the range of 2 to 6. Electrostatic interactions between the cationic polymers and nucleic acid result in the formation of polyplexes characterized by a particle size distribution with an average size of approximately 100 nm, hence the name nanoplexes. For delivery in self-assembling particles, a dose of approximately 100 to 200 mg of CRISPR-Cas is suggested by Schiffelers et al.

Nanoplexes according to Bartlett et al. (PNAS, September 25, 2007, vol. 104, no. 39) can also be used in the present invention. Nanoplexes according to Bartlett et al. are prepared by mixing equal volumes of aqueous solutions of a cationic polymer and nucleic acid to obtain a net molar excess of ionizable nitrogen (polymer) relative to phosphate (nucleic acid) in the range of 2 to 6. Electrostatic interactions between the cationic polymers and nucleic acid result in the formation of polyplexes characterized by a particle size distribution with an average size of approximately 100 nm, hence they are called nanoplexes. DOTA-siRNA conjugates according to Bartlett et al. were synthesized as follows. 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid mono(N-hydroxysuccinimide ester) (DOTA-NHS ester) was purchased from Macrocyclics (Dallas, TX). Amine-modified sense strand RNA was added to a microcentrifuge tube with a 100-fold molar excess of DOTA-NHS ester in carbonate buffer (pH 9). The contents were reacted by stirring for 4 h at room temperature. The DOTA-sense strand RNA conjugate was precipitated with ethanol, resuspended in water, and annealed with the unmodified antisense strand to yield the DOTA-siRNA conjugate. All fluids were pretreated with Chelex-100 (Bio-Rad, Hercules, CA) to remove trace amounts of metal impurities. Tf-targeting and non-targeting siRNA particles could be formed using cyclodextrin-containing polycations. Typically, particles were prepared in water at a charge ratio of 3 (+/-) and an siRNA concentration of 0.5 g/liter. One percent of the adamantane-PEG conjugate molecules on the surface of the targeted particles were modified with Tf (adamantane-PEG-Tf). The particles were suspended in 5% (w/v) glucose as the injection vehicle.

Davis et al. (Nature, Vol 464, 15 April 2010) conducted a clinical trial with RNA using a targeted particle delivery system (clinical trial registration number NCT00689065). Patients with solid cancers refractory to standard treatments were given doses of targeted particles on days 1, 3, 8, and 10 of a 21-day cycle via a 30-minute intravenous infusion. The particles consist of a synthetic delivery system containing: (1) a linear cyclodextrin (CDP)-based polymer, (2) a ligand targeting human transferrin protein (TF) displayed on the outer surface of the particles, which engages TF receptors (TFRs) on the surface of cancer cells, (3) a hydrophilic polymer (polyethylene glycol (PEG), used to ensure particle stability in biological fluids), and (4) siRNA designed to downregulate RRM2 expression (the sequence used in clinical practice was previously designated siR2B+5). TFR expression has long been known to be increased in cancer cells, and RRM2 is a well-established target for anti-cancer therapy. These particles (clinical variant designated CALAA-01) have been shown to be well tolerated in repeated-dose studies in non-human primates. Even though a single patient with chronic myeloid leukemia was administered siRNA via liposome delivery, the clinical trial by Davis et al. is the first human trial to systemically deliver siRNA using a targeted delivery system and treat patients with solid cancer. To determine whether a targeted delivery system could efficiently deliver functional siRNA to human tumors, Davis et al. examined biopsies from three patients in three different dosing groups; Patients A, B, and C, all of whom had metastatic melanoma and received CALAA-01 doses of 18, 24, and 30 mg m ^-2 siRNA, respectively. Similar doses can also be envisaged for the CRISPR-Cas system of the present invention. Delivery according to the present invention can be achieved using particles comprising a linear cyclodextrin-based polymer (CDP), a human transferrin protein (TF) targeting ligand presented on the outer surface of the particles that contacts TF receptors (TFRs) on the surface of cancer cells, and/or a hydrophilic polymer (e.g., polyethylene glycol (PEG) used to provide stability of the particles in biological fluids).

In the context of the present invention, it is preferred that one or more components of the CRISPR complex, such as the CRISPR enzyme or mRNA or guide RNA, are delivered using particles or lipid shells. Other delivery systems or vectors may be used together with the particle aspects of the present invention.

In general, a "nanoparticle" refers to any particle having a diameter of less than 1000 nm. In certain preferred embodiments, the nanoparticles of the present invention have a largest dimension (e.g., diameter) of 500 nm or less. In other preferred embodiments, the nanoparticles of the present invention have a largest dimension ranging from 25 nm to 200 nm. In other preferred embodiments, the nanoparticles of the present invention have a largest dimension of 100 nm or less. In other preferred embodiments, the nanoparticles of the present invention have a largest dimension ranging from 35 nm to 60 nm.

The particles encompassed by the present invention may be in various forms, such as solid particles (e.g., metal such as silver, gold, iron, titanium, non-metal, lipid solids, polymers), suspensions of particles, or combinations thereof. Metal, dielectric, and semiconductor particles may be produced, as well as hybrid structures (e.g., core/shell particles). Particles made of a semiconductor material may also be labeled with quantum dots if they are small enough (typically less than 10 nm) to allow quantization of electron energy levels. Such nanosized particles are used in biomedical applications as drug carriers or imaging agents, and may be adapted for similar purposes in the present invention.

Semi-solid and soft particles have been obtained and are within the scope of the present invention. The prototype particle of semi-solid nature is the liposome. Various types of liposome particles are currently used in clinical practice as delivery systems for anti-cancer drugs and vaccines. Particles with one hemisphere hydrophilic and the other hemisphere hydrophobic 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.

U.S. Patent No. 8,709,843, incorporated herein by reference, provides a drug delivery system for targeted delivery of particles containing a therapeutic agent to tissues, cells, and intracellular compartments. The present invention provides targeted particles comprising a polymer conjugated to a surfactant, a hydrophilic polymer, or a lipid. U.S. Patent No. 6,007,845, incorporated herein by reference, provides particles having a core of a multiblock copolymer formed by covalently bonding a compound with multiple functional groups to one or more hydrophobic polymers and one or more hydrophilic polymers, and containing a biologically active material. U.S. Patent No. 5,855,913, incorporated herein by reference, provides a particulate composition comprising aerodynamically lightweight particles having a tapped density of less than 0.4 g/cm3 and an average diameter of 5 μm to 30 μm containing a surfactant on their surface for delivery of drugs to the pulmonary system. U.S. Patent No. 5,985,309, incorporated herein by reference, provides particles comprising a surfactant and/or a hydrophilic or hydrophobic complex of a positively or negatively charged therapeutic or diagnostic agent and a charged molecule having an opposite charge for delivery to the pulmonary system. U.S. Patent No. 5,543,158, incorporated herein by reference, provides biodegradable injectable particles having a biodegradable solid core comprising a biologically active material and poly(alkylene glycol) moieties on the surface. WO2012135025 (also published as US20120251560), incorporated herein by reference, describes conjugated polyethyleneimine (PEI)-based polymers and conjugated azamacrocycles (collectively referred to as "conjugated lipopolymer" or "lipopolymers"). In certain embodiments, it may be envisaged that such conjugated lipopolymers can be used in the case of the CRISPR-Cas system to effect genome editing in vitro, ex vivo and in vivo with modification of gene expression, including modulation of protein expression.

In one embodiment, the particle may be a hybrid of an epoxide-terminated lipid and a 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 epoxide-terminated C15 lipids with PEI600 in a 14:1 molar ratio and formulated with C14PEG2000 to produce particles (35 to 60 nm in diameter) that were stable in PBS solution for at least 40 days.

The hybrid of lipid modified with epoxide groups and polymer can be used to deliver the CRISPR-Cas system of the present invention to cells of the lung, cardiovascular system or kidney, however, one skilled in the art can adapt the system for delivery to other target organs. A dose ranging from about 0.05 to about 0.6 mg/kg is contemplated. Dosing over several days or weeks is also contemplated, with a total dose of about 2 mg/kg.

Exosomes

Exosomes are endogenous RNA and protein-carrying nanovesicles that can deliver RNA to the brain and other target organs. To reduce immunogenicity, Alvarez-Erviti et al. (2011, Nat Biotechnol 29: 341) used autologous dendritic cells to produce exosomes. Brain targeting was achieved by engineering dendritic cells expressing Lamp2b, an exosome membrane protein fused to the neuron-specific peptide RVG. Purified exosomes were loaded with exogenous RNA by electroporation. RVG-labeled targeting exosomes injected intravenously specifically delivered siRNA for GAPDH to neurons, microglia, and oligodendrocytes in the brain, causing knockdown of the specific gene. Pre-exposure to RVG-labeled exosomes did not attenuate the magnitude of knockdown, and non-specific uptake into other tissues was not observed. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated by the potent mRNA (60%) and protein (62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease.

To generate a pool of immunologically inert exosomes, Alvarez-Erviti et al. collected bone marrow from inbred C57BL/6 mice with a homogeneous major histocompatibility complex (MHC) haplotype. Because immature dendritic cells produce large amounts of exosomes lacking T cell activators such as MHC-II and CD86, Alvarez-Erviti et al. selected dendritic cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) for 7 days. Exosomes were purified from the culture supernatant the following day using established ultracentrifugation protocols. The exosomes produced were physically homogeneous and characterized by a size distribution with a peak at 80 nm in diameter, as determined by particle tracking assay (NTA) and electron microscopy. Alvarez-Erviti et al. 6-12 μg of exosomes (measured by protein concentration) were obtained per 10 ⁶ cells.

Alvarez-Erviti et al. then investigated the possibility of loading modified exosomes with exogenous cargo molecules using electroporation protocols tailored for nanoscale applications. Since electroporation for membrane particles at the nanometer scale is not well understood, non-specific Cy5-labeled RNA was used to empirically optimize the electroporation protocol. The amount of encapsulated RNA was analyzed after ultracentrifugation and lysis of exosomes. Electroporation at 400 V and 125 μF resulted in the highest RNA retention and was used for all subsequent experiments.

Alvarez-Erviti et al. injected 150 μg of each BACE1 siRNA encapsulated in 150 μg of RVG-labeled exosomes into normal C57BL/6 mice and compared the knockdown efficiency with that of four control groups: untreated mice, mice injected with RVG-labeled exosomes only, mice injected with BACE1 siRNA complexed with a cationic liposome-based in vivo delivery reagent, and mice injected with BACE1 siRNA complexed with RVG-9R, an RVG peptide conjugated to 9 D-arginine residues that electrostatically binds to the siRNA. Cortical tissue samples were analyzed 3 days after administration and both siRNA-RVG-9R and RVG-labeled exosomes-siRNA-treated mice showed significant protein knockdown (45%, P < 0.05 and 62%, P < 0.01) due to significant reductions in BACE1 mRNA levels (66% [+ or -] 15%, P < 0.001 and 61% [+ or -] 13%, P < 0.01, respectively). Furthermore, we demonstrated a significant reduction (55%, P < 0.05) in total [beta]-amyloid peptide 1-42 levels, a major component of amyloid plaques in the Alzheimer's disease pathology process, in animals treated with RVG-labeled exosomes. The observed reduction was greater than the reduction in β-amyloid peptide 1-40 levels demonstrated in normal mice following intracerebroventricular injection of BACE1 inhibitors. Alvarez-Erviti et al. performed rapid amplification of 5' cDNA ends (RACE) on the BACE1 cleavage product, indicating RNAi-mediated knockdown by siRNA.

Finally, Alvarez-Erviti et al. investigated whether RVG-labeled RNA exosomes induce immune responses in vivo by determining serum concentrations of IL-6, IP-10, TNFα, and IFN-α. All cytokines showed little change after exosome treatment, similar to siRNA transfection reagent treatment and unlike siRNA-RVG-9R, which strongly stimulated IL-6 secretion, confirming the immunological inertness of exosome treatment. Given that exosomes encapsulate only 20% of siRNA, delivery by RVG-labeled exosomes appears to be more efficient than that by RVG-9R, since comparable mRNA knockdown and greater protein knockdown were achieved using fivefold less siRNA without corresponding stimulation of the immune response. This experiment demonstrated the therapeutic potential of RVG-labeled exosome technology, which is potentially suitable for long-term silencing of genes associated with neurodegenerative diseases. The exosome-based delivery system of Alvarez-Erviti et al. can be used to deliver the CRISPR-Cas system of the present invention to therapeutic targets, especially in neurodegenerative diseases. The present invention can provide a dose of about 100-1000 mg of CRISPR-Cas encapsulated in about 100-1000 mg of RVG-labeled exosomes.

El-Andaloussi et al. (Nature Protocols 7, 2112–2126(2012)) describe how exosomes derived from cultured cells can be tailored for RNA delivery in vitro and in vivo. This protocol describes for the first time the generation of targeted exosomes by transfection with an expression vector containing an exosomal protein fused to a peptide ligand. El-Andaloussi et al. then explain how to purify and characterize exosomes from the supernatant of transfected cells. El-Andaloussi et al. then detail the critical steps in loading RNA into exosomes. Finally, El-Andaloussi et al. outline how to use exosomes to efficiently deliver RNA in vitro and in vivo to the mouse brain. Examples of predictive results are also provided, in which exosome-mediated RNA delivery is assessed through functional assays and imaging. The complete protocol takes ~3 weeks to complete. The delivery or administration according to the present invention can be carried out using exosomes obtained from autologous dendritic cells. Among the ideas given herein, this one can be used in the practical application of the present invention.

In another embodiment, plasma exosomes are provided according to Wahlgren et al. (Nucleic Acids Research, 2012, Vol. 40, No. 17 e130). Exosomes are nanosized vesicles (30-90 nm in size) produced by many types of cells, including dendritic cells (DCs), B cells, T cells, mast cells, epithelial cells, and tumor cells. These vesicles are formed by inward budding of late endosomes and are then released into the extracellular environment upon fusion with the plasma membrane. Since exosomes naturally transfer RNA between cells, this property may be useful in gene therapy and, according to this disclosure, may be used in the practice of the present invention.

Exosomes can be prepared from plasma by centrifugation of the buffy coat at 900 g for 20 min to separate the plasma, followed by collection of the cell culture supernatants, centrifugation at 300 g for 10 min to remove cells and at 16,500 g for 30 min followed by filtration through a 0.22 mm filter. Exosomes are pelleted by ultracentrifugation at 120,000 g for 70 min. The introduction of siRNA into exosomes by chemical transfection is carried out according to the manufacturer's instructions in the RNAi Human/Mouse Starter Kit (Quiagen, Hilden, Germany). siRNA is added to 100 ml PBS at a final concentration of 2 mmol/ml. After addition of HiPerFect transfection reagent, the mixture is incubated for 10 min at RT. In order to remove excess micelles, exosomes are re-isolated using latex particles with aldehyde/sulfate groups. The introduction of CRISPR-Cas into exosomes by chemical transfection can be carried out similarly to the introduction of siRNA. Exosomes can be co-cultured with monocytes and lymphocytes isolated from the peripheral blood of healthy donors. Thus, it can be envisaged that exosomes containing CRISPR-Cas can be introduced into monocytes and lymphocytes and autologously reintroduced into the human body. Accordingly, the delivery or administration according to the present invention can be carried out using plasma exosomes.

Liposomes

The delivery or administration according to the present invention can be carried out using liposomes. Liposomes are spherical vesicular structures containing a mono- or multi-lamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes have received considerable attention as drug delivery vehicles because they are biocompatible, non-toxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo molecule from degradation by plasma enzymes, and transport their "cargo" across biological membranes and the blood-brain barrier (BBB) (for review, see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679).

Liposomes can be prepared from several different types of lipids; however, phospholipids are most commonly used to create liposomes as drug carriers. Although liposome formation is spontaneous upon mixing of the lipid film with an aqueous solution, it can also be accelerated by applying force in the form of shaking using a homogenizer, ultrasonic disperser, or extrusion apparatus (for a review, see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679).

Some other additives can be added to liposomes to modify their structure and properties. For example, cholesterol or sphingomyelin can be added to the liposome mixture to help stabilize the liposome structure and prevent leakage of the internal liposome cargo molecules. In addition, liposomes are prepared from hydrogenated egg phosphatidylcholine or egg phosphatidylcholine, cholesterol and diacetyl phosphate, and their average vesicle sizes are adjusted to approximately 50 and 100 nm (for a review, see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679).

The liposomal formulation may contain mainly natural phospholipids and lipids such as 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), sphingomyelin, egg forms of phosphatidylcholine, and monosialoganglioside. Since this formulation consists only of phospholipids, liposomal formulations face many problems, one of which is instability in plasma. Several attempts have been made to overcome these problems, particularly through manipulation of the lipid membrane. One of these attempts is aimed at manipulating cholesterol. The addition of cholesterol to traditional formulations reduces the rapid release of the encapsulated bioactive compound into blood plasma, or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) increases stability (for review, see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679).

In a particularly advantageous embodiment, liposomes "Trojan horses" (also known as "molecular Trojan horses") are desirable and protocols can be found at http://cshprotocols.cshlp.org/content/2010/4/pdb.prot5407.long. These particles provide for delivery of the transgene to the entire brain following intravascular injection. Without limitation, it is believed that neutral lipid particles with specific antibodies conjugated to the surface provide for penetration of the blood-brain barrier via endocytosis. Applicant theoretically envisions the use of liposomes "Trojan horses" to deliver CRISPR family nucleases to the brain via intravascular injection, which would provide animals with transgenes throughout the brain without the need for embryo manipulation. For in vivo administration, approximately 1-5 g of DNA or RNA can be provided in the liposomes.

In another embodiment, the 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 about 1, 3, or 5 mg/kg/day of specific targeting CRISPR-Cas in the SNALP are contemplated. Treatment can be performed daily for about three days and then weekly for about five weeks. In another embodiment, specific CRISPR-Cas encapsulated in the SNALP is also contemplated and administered by intravenous injection at doses of about 1 or 2.5 mg/kg (see, e.g., Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006). The SNALP-based formulation may contain the lipids 3-N-[(ω-methoxypoly(ethylene glycol)2000)-carbamoyl]-1,2-dimyristyloxypropylamine (PEG-C-DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol in a molar percentage ratio of 2:40:10:48 (see, e.g., Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006).

In another embodiment, stable nucleic acid lipid particles (SNALPs) were confirmed to be effective delivery molecules for highly vascularized HepG2-derived liver tumors, but not for poorly vascularized HCT-116-derived liver tumors (see, e.g., Li, Gene Therapy (2012) 19, 775-780). SNALP liposomes can be prepared by formulating D-Lin-DMA and PEG-C-DMA with distearoylphosphatidylcholine (DSPC), cholesterol, and siRNA using a lipid/siRNA ratio of 25:1 and a cholesterol/D-Lin-DMA/DSPC/PEG-C-DMA molar ratio of 48/40/10/2. The resulting SNALP liposomes have a size of approximately 80-100 nm.

In another embodiment, the SNALP may comprise synthetic cholesterol (Sigma-Aldrich, St. Louis, MO, USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, AL, USA), 3-N-[(ω-methoxypoly(ethylene glycol)2000)carbamoyl]-1,2-dimyristyloxypropylamine, and cationic 1,2-dilinoleyloxy-3-N,N-dimethylaminopropane (see, e.g., Geisbert et al., Lancet 2010; 375: 1896-905). A dosing regimen of approximately 2 mg/kg total CRISPR-Cas per dose may be contemplated, administered, e.g., as a bolus intravenous infusion.

In another embodiment, the SNALP may comprise synthetic cholesterol (Sigma-Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids Inc.), PEG-C-DMA, and 1,2-dilinoleyloxy-3-(N,N-dimethyl)aminopropane (DLinDMA) (see, e.g., Judge, J. Clin. Invest. 119:661-673 (2009)). Formulations used for in vivo studies may comprise lipids and RNA in a final weight ratio of about 9:1.

The safety profile of RNAi nanomedicines has been reviewed by Barros and Gollob of Alnylam Pharmaceuticals (see, e.g., Advanced Drug Delivery Reviews 64 (2012) 1730–1737). The stable nucleic acid–lipid particle (SNALP) contains four different lipids—an ionizable lipid (DLinDMA) that is cationic at low pH, a neutral helper lipid, cholesterol, and a diffusible polyethyleneglycol (PEG)–lipid conjugate. The particle is approximately 80 nm in diameter and is electroneutral at physiological pH. During formulation, the ionizable lipid serves to condense the lipid onto the anionic RNA during particle formation. Being positively charged under the increasingly acidic conditions of endosomes, the ionizable lipid also mediates the fusion of the SNALP with the endosomal membrane, allowing the release of RNA into the cytoplasm. The PEG-lipid conjugate stabilizes the particle and reduces aggregation during formulation and subsequently provides a neutral hydrophilic outer surface that improves pharmacokinetic properties.

To date, two clinical trial programs have been initiated using SNALP RNA-based formulations. Tekmira Pharmaceuticals recently completed a Phase I, single-dose study of SNALP-ApoB in adult volunteers with elevated LDL-C. ApoB is primarily expressed in the liver and small intestine and is key for the assembly and secretion of VLDL and LDL. Seventeen subjects received a single dose of SNALP-ApoB (dose escalation spanning 7 dose levels). No evidence of hepatotoxicity (suggested as a possible dose-limiting toxicity based on preclinical studies) was observed. One (or two) subjects at the highest dose experienced flu-like symptoms consistent with immune stimulation and the decision was made to terminate the trial.

Alnylam Pharmaceuticals has similarly successfully conducted the ALN-TTR01 study, which uses the SNALP technology described above and targets hepatocyte production of both mutant and wild-type TTR to treat TTR-mediated amyloidosis (ATTR). Three syndromes have been described in ATTR: familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC), both caused by autosomal dominant mutations in TTR; and senile systemic amyloidosis (SSA), caused by deposition of wild-type TTR. They recently completed a phase I, placebo-controlled, single-dose escalation trial of ALN-TTR01 in patients with ATTR. ALN-TTR01 was administered as a 15-minute IV infusion to 31 patients (study drug in 23 and placebo in 8) over a dose range of 0.01-1.0 mg/kg (based on siRNA). Treatment was well tolerated, with no significant elevations in liver function tests. Infusion reactions were observed in 3 of 23 patients at ≥ 0.4 mg/kg; all responded to slowing the infusion rate and all continued in the study. Minimal and transient increases in serum cytokines IL-6, IP-10, and IL-1ra were observed in two patients at the highest dose of 1 mg/kg (as expected based on preclinical and NHP studies). A decrease in serum TTR, an expected pharmacodynamic effect of ALN-TTR01, was observed at 1 mg/kg.

In another embodiment, SNALP can be prepared by solubilizing the cationic lipid, DSPC, cholesterol, and PEG-lipid conjugate, such as in ethanol, such as at a molar ratio of 40:10:40:10, respectively (see Semple et al., Nature Biotechnology, Volume 28 Number 2 February 2010, pp. 172-177). The lipid mixture was added to aqueous buffer (50 mM citrate, pH 4) with stirring to a final ethanol and lipid concentration of 30% (v/v) and 6.1 mg/mL, respectively, and allowed to equilibrate at 22°C for 2 min prior to extrusion. Hydrogenated lipids were extruded through two stacked 80 nm filters (Nuclepore) at 22°C using a Lipex extruder (Northern Lipids) until vesicle diameters of 70–90 nm were achieved as determined by dynamic light scattering analysis. This typically required 1–3 passes. siRNA (solubilized in aqueous solution containing 30% ethanol with 50 mM citrate, pH 4) was added to pre-equilibrated (35°C) vesicles at ~5 ml/min with mixing. After reaching a final target siRNA/lipid ratio of 0.06 (w/w), the mixture was incubated for an additional 30 min at 35°C to allow vesicle reorganization and siRNA encapsulation. Ethanol was then removed and the external buffer was exchanged with PBS (155 mM NaCl, 3 _{mM Na2HPO4} , 1 mM _KH2PO4 , pH 7.5) by either dialysis or tangential flow diafiltration. SNALPs were encapsulated with siRNA using a controlled step-dilution method. The lipid moieties _of KC2-SNALPs were DLin-KC2-DMA (a cationic lipid), dipalmitoylphosphatidylcholine (DPPC; Avanti Polar Lipids), synthetic cholesterol (Sigma), and PEG-C-DMA used at a molar ratio of 57.1:7.1:34.3:1.4. Following formation of loaded particles, SNALPs were dialyzed against PBS and sterilized by filtration through a 0.2 μm filter prior to use. The average particle size was 75-85 nm, and 90-95% of the siRNA was encapsulated in the lipid particles. The final siRNA/lipid ratio in the formulations used for in vivo testing was ~0.15 (w/w). The LNP-siRNA systems containing siRNA for Factor VII were diluted to the appropriate concentrations in sterile PBS immediately prior to use, and the formulations were administered intravenously via the lateral tail vein in a total volume of 10 ml/kg. This method and delivery system data can be extrapolated to the CRISPR-Cas system of the present invention.

Other lipids

Other cationic lipids, such as the aminolipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), can be used to encapsulate CRISPR-Cas or its components or nucleic acid molecules encoding them, similar to siRNA (see, e.g., Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529-8533), and therefore can be used in the practice of the present invention. A preformed vial with the following lipid composition can be provided: aminolipid, distearoylphosphatidylcholine (DSPC), cholesterol and (R)-2,3-bis(octadecyloxy)propyl-1-(methoxypoly(ethyleneglycol)2000)propylcarbamate (PEG-lipid conjugate) in a molar ratio of 40/10/40/10, respectively, and with a FVII siRNA/total lipid ratio of approximately 0.05 (w/w). To ensure a narrow particle size distribution in the range of 70-90 nm and a low polydispersity index of 0.11+0.04 (n=56), the particles can be extruded up to three times through 80 nm membranes before addition of CRISPR-Cas RNA. Particles containing highly active aminolipid 16 can be used, in which the molar ratio of the four lipid components 16, DSPC, cholesterol, and PEG-lipid conjugate (50/10/38.5/1.5) can be further optimized to enhance in vivo activity.

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 membranes for RNA delivery. The use of lipid membranes is also preferred in the present invention.

In another embodiment, lipids can be formulated with the CRISPR-Cas system of the present invention to form lipid particles (LNPs). Lipids include, but are not limited to, DLin-KC2-DMA4, C12-200, and the co-lipids distearoylphosphatidylcholine, cholesterol, and PEG-DMG, which can be formulated with CRISPR-Cas in place of siRNA (see, e.g., Novobrantseva, Molecular Therapy-Nucleic Acids (2012) 1, e4; doi:10.1038/mtna.2011.3) using a spontaneous vesicle formation procedure. The molar ratio of the components can be approximately 50/10/38.5/1.5 (DLin-KC2-DMA or C12-200/distearoylphosphatidylcholine/cholesterol/PEG-DMG). The final lipid:siRNA weight ratio can be ~12:1 and 9:1 for DLin-KC2-DMA and C12-200 based lipid particles (LNPs), respectively. The formulations can have average particle diameters of ~80 nm with >90% incorporation efficiency. A dose of 3 mg/kg can be envisaged.

Tekmira has a portfolio of approximately 95 patent families issued in the United States and abroad that are directed to various aspects of LNP and LNP-based formulations (see, e.g., U.S. Patent Nos. 7,982,027; 7,799,565; 8,058,069; 8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263; 7,915,399; 8,236,943; and 7,838,658 and European Patent Nos. 1,766,035; 1,519,714; 1,781,593; and 1,664,316), all of which may be applied and/or adapted to the present invention.

The CRISPR-Cas system, or components thereof, or nucleic acid molecules encoding them can be delivered encapsulated in PLGA-based microspheres, such as those further described in published U.S. patent applications 20130252281, and 20130245107, and 20130244279 (assigned to Moderna Therapeutics), which relate to aspects of formulating compositions comprising modified nucleic acid molecules that can encode a protein, a protein precursor, or a partially or fully processed form of a protein or protein precursor. The formulation can be characterized by a molar ratio of 50:10:38.5:1.5-3.0 (cationic lipid:fusogenic lipid:cholesterol:PEG-lipid conjugate). The PEG-lipid conjugate can be selected from, but is not limited to, PEG-C-DOMG, PEG-DMG. The fusogenic lipid may be DSPC. See also Schrum et al., Delivery and Formulation of Engineered Nucleic Acids, U.S. Patent Application Published 20120251618.

Nanomerics technology overcomes bioavailability challenges for a wide range of therapeutics, including small molecule hydrophobic drugs, peptides, and nucleic acid-based therapeutics (plasmids, siRNA, miRNA). Specific routes of administration for which the technology has demonstrated clear advantages include the oral route, blood-brain barrier transport, delivery to solid tumors, and to the eye. See, e.g., Mazza et al., 2013, ACS Nano. 2013 Feb 26;7(2):1016–26; Uchegbu and Siew, 2013, J Pharm Sci. 102(2):305–10; and Lalatsa et al., 2012, J Control Release. 2012 Jul 20;161(2):523–36.

U.S. Patent Application Publication No. 20050019923 describes cationic dendrimers for delivering biologically active molecules, such as polynucleotide molecules, peptides and polypeptides and/or pharmaceuticals, to a mammal. Dendrimers are suitable for providing targeted delivery of biologically active molecules, such as to the liver, spleen, lung, kidney or heart (or even the brain). Dendrimers are synthetic 3-dimensional macromolecules obtained in a stepwise manner from simple branched monomer units, the nature and number of functional groups of which can be easily controlled and changed. Dendrimers are synthesized by repeated addition of building blocks from a multifunctional core (divergent synthesis) or from a multifunctional core (convergent synthesis), each addition of a 3-dimensional shell of building blocks yielding a higher generation of dendrimers. Synthesis of polypropyleneimine dendrimers starts with a diaminobutane core to which double the number of amino groups is added by double Michael addition of acetonitrile to the primary amino groups followed by hydrogenation of the nitrile groups. This results in a doubling of the number of amino groups. Polypropyleneimine dendrimers contain 100% protonatable nitrogen atoms and up to 64 terminal amino groups (generation 5, DAB 64). The protonatable groups are typically amino groups capable of accepting protons at neutral pH. The application of dendrimers as gene delivery vehicles has mainly focused on the use of polyamidoamine and phosphorus-containing compounds with an amine/amide mixture or N--P(O ₂ )S as conjugating units, respectively, while the use of low-generation polypropyleneimine dendrimers for gene delivery has not been reported. Polypropyleneimine dendrimers have also been studied as pH-sensitive, controlled-release systems for drug delivery and for encapsulation of guest molecules in the case of chemical modification of peripheral amino acid groups. The cytotoxicity and interaction of polypropyleneimine dendrimers with DNA, as well as the transfection efficiency using DAB 64, have also been studied.

The publication of U.S. Patent Application No. 20050019923 is based on the observation that, contrary to earlier reports, cationic dendrimers such as polypropyleneimine dendrimers exhibit favorable properties such as specific targeting and low toxicity for use in targeted delivery of biologically active molecules such as genetic material. In addition, cationic dendrimer derivatives also exhibit favorable properties for targeted delivery of biologically active molecules. See also "Bioactive Polymers," U.S. Patent Application Publication No. 20080267903, which discloses the following: "Various polymers, including cationic polyamine polymers and dendrimeric polymers, have been shown to have antiproliferative activity and may thus be useful for the treatment of disorders characterized by unwanted cell proliferation, such as neoplasias and tumors, inflammatory disorders (including autoimmune disorders), psoriasis, and atherosclerosis. The polymers may be used alone as active agents or as delivery vehicles for other therapeutic agents, such as drug molecules or nucleic acids for gene therapy. In such cases, the intrinsic antitumor activity of the polymers may complement the activity of the agent to be delivered. The disclosures of these patent publications may be used in conjunction with the teachings of this document to deliver the CRISPR Cas system(s), or component(s) thereof, or encoding(s) thereof nucleic acid molecule(s).

Overcharged proteins

Overcharged proteins are a class of engineered or naturally occurring proteins that typically have a high net theoretical charge, either positive or negative, and can be used to deliver the CRISPR Cas system(s) or component(s) thereof or the nucleic acid molecule(s) encoding them. Both overcharged negative and overcharged positive proteins exhibit the unique property of being resistant to thermally or chemically induced aggregation. Overcharged positive proteins are also capable of entering mammalian cells. Association of cargo molecules such as plasmid DNA, RNA with these proteins or other proteins can provide functional delivery of these macromolecules to mammalian cells both in vitro and in vivo. In 2007, David Liu's laboratory reported the creation and characterization of proteins with excess charge (Lawrence et al., 2007, Journal of the American Chemical Society 129, 10110-10112).

Non-viral delivery of RNA and plasmid DNA into mammalian cells is of interest for both research and therapeutic applications (Akinc et al., 2010, Nat. Biotech. 26, 561-569). Purified +36 GFP protein (or another positively charged protein) is mixed with RNA in a suitable serum-free medium and allowed to complex before addition to cells. Inclusion of serum at this step inhibits the formation of positively charged protein-RNA complexes and reduces the efficiency of the treatment. The following protocol has been found effective for a range of cell lines (McNaughton et al., 2009, Proc. Natl. Acad. Sci. USA 106, 6111-6116) (however, pilot experiments with varying doses of protein and RNA should be performed to optimize the procedure for specific cell lines). (1) One day before treatment, seed 1 x 10 ⁵ cells/well in a 48-well plate. (2) On the day of treatment, dilute purified +36 GFP protein in serum-free medium to a final concentration of 200 nM. Add RNA to a final concentration of 50 nM. Vortex and incubate at room temperature for 10 min. (3) During incubation, aspirate medium from cells and wash once with PBS. (4) After incubation of +36 GFP and RNA, add protein-RNA complexes to cells. (5) Incubate cells with complexes at 37°C for 4 h. (6) After incubation, aspirate medium and wash three times with 20 U/ml heparin in PBS. Incubate cells in serum-free medium for an additional 48 h or longer depending on the activity assay. (7) Analyze cells by immunoblotting, qPCR, phenotypic analysis, or other appropriate method.

David Liu's laboratory further discovered that +36-charge GFP is an efficient reagent for delivering plasmids into a variety of cells. Because plasmid DNA is a larger cargo molecule than siRNA, proportionally more +36-charge GFP protein is required to form an efficient complex with plasmids. To efficiently deliver plasmids, we engineered a +36-charge GFP variant carrying a C-terminal peptide tag of HA2, a known endosomal disrupting peptide derived from the influenza virus hemagglutinin protein. The following protocol was effective for many cells, but as outlined above, it is recommended that the doses of plasmid DNA and excess-charge protein be optimized for specific cell lines and delivery routes. (1) One day prior to treatment, seed 1 x 10 ⁵ cells per well in a 48-well plate. (2) On the day of treatment, dilute purified þ36-charged GFP protein in serum-free medium to a final concentration of 2 mM. Add 1 mg of plasmid DNA. Vortex and incubate at room temperature for 10 min. (3) During incubation, aspirate medium from cells and wash once with PBS. (4) After incubation of þ36-charged GFP and plasmid DNA, carefully add protein-DNA complexes to cells. (5) Incubate cells with complexes at 37°C for 4 h. (6) After incubation, aspirate medium and wash with PBS. Incubate cells in serum-containing medium and incubate for an additional 24–48 h. (7) If desired, analyze plasmid delivery (e.g., via plasmid-mediated gene expression). See, e.g., 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, DB, et al., Chemistry & Biology 19 (7), 831-843 (2012). Methods using overcharged proteins can be used to deliver and/or adapted to the CRISPR-Cas system of the present invention. These systems, according to Dr. Liu and the publications cited in this document in connection with the ideas of this document, can be used in the delivery of the CRISPR Cas system(s), or its component(s), or the nucleic acid molecule(s) encoding them.

Cell Penetrating Peptides (CPP)

In another embodiment, cell penetrating peptides (CPPs) are provided for delivering a CRISPR-Cas system. CPPs are short peptides that facilitate the uptake of various cargo molecules (ranging from nanosized particles to small chemical molecules and large DNA fragments) by cells. The term "cargo molecule" as used herein includes, but is not limited to, a group consisting of therapeutic agents, diagnostic probes, peptides, nucleic acids, antisense oligonucleotides, plasmids, proteins, particles, liposomes, chromophores, small molecules, and radioactive materials. In aspects of the present invention, a cargo molecule may also comprise any component of a CRISPR-Cas system or an entire functional CRISPR-Cas system. Aspects of the present invention further provide methods of delivering a desired cargo molecule to a subject comprising: (a) providing a complex comprising a cell penetrating peptide of the present invention and the desired cargo molecule, and (b) orally, intra-articularly, intraperitoneally, intrathecally, intra-arterially, intranasally, intraparenchymatously, subcutaneously, intramuscularly, intravenously, cutaneously, rectally or topically administering the complex to the subject. The cargo molecule is chemically linked to the peptides via covalent bonds or via non-covalent interactions.

The function of CPPs is to deliver cargo molecules into cells, a process that typically occurs via endocytosis, resulting in the delivery of cargo molecules into endosomes of living mammalian cells. Cell-penetrating peptides vary in size, amino acid sequence, and charge, but all CPPs share one distinguishing characteristic, which is the ability to translocate across the plasma membrane and facilitate the delivery of various cargo molecules into the cytoplasm or organelle. CPP trafficking can be divided into three main entry mechanisms: direct membrane passage, endocytosis-mediated entry, and trafficking via intermediate structure formation. CPPs have found numerous applications in medicine as drug delivery vehicles for a variety of diseases, including cancer, and virus inhibitors, as well as cell labeling contrast agents. Examples of the latter include acting as a carrier for GFP, MRI contrast agents, or quantum dots. CPPs have great potential as in vitro and in vivo delivery vectors for applications in research and medicine. CPPs typically have an amino acid composition that is characterized by a high relative abundance of positively charged amino acids such as lysine or arginine, or have sequences characterized by alternating polar/charged amino acids and non-polar hydrophobic amino acids. These two types of structures are called polycationic or amphipathic, respectively. A third class of CPPs are hydrophobic peptides that contain only non-polar residues with a low net charge or have hydrophobic amino acid groups that are essential for cellular uptake. One of the first CPPs discovered was the transactivating activator of transcription (Tat) of human immunodeficiency virus 1 (HIV-1), which was found to be efficiently taken up from the environment by numerous cell types in culture. Since then, the number of known CPPs has increased significantly, and small molecule synthetic analogs with more efficient protein transduction properties have been developed. CPPs include, but are not limited to, penetratin, Tat (48-60), transportan, and (R-AhX-R4) (Ahx = aminohexanoyl).

U.S. Patent 8,372,951 discloses a CPP derived from eosinophil cationic protein (ECP) that exhibits high cell penetration efficiency and low toxicity. Aspects of delivering the CPP with its cargo molecule to a vertebrate subject are also disclosed. Additional aspects regarding CPPs and their delivery are disclosed in U.S. Patents 8,575,305; 8,614,194; and 8,044,019. The CPPs can be used to deliver the CRISPR-Cas system or components thereof. These CPPs can be used to deliver the CRISPR-Cas system or components thereof, as also disclosed 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. [Electronic publication preceding print], incorporated by reference in its entirety, showing that treatment with recombinant Cas9 protein conjugated to CPP and guide RNA complexed with CPP results in disruption of endogenous genes in human cell lines. In this paper, Cas9 protein was conjugated to CPP via a thioester linkage, while guide RNA was complexed with CPP to form condensed positively charged particles. It was shown that simultaneous and sequential treatment of human cells, including embryonic stem cells, dermal fibroblasts, HEK293T cells, HeLa cells, and embryonal carcinoma cells, with modified Cas9 and guide RNA resulted in efficient gene disruption with a reduced frequency of off-target mutations compared to plasmid transfections.

Implantable devices

In another embodiment, implantable devices for delivering the CRISPR Cas system, or component(s) thereof, or the nucleic acid molecule(s) encoding them are also contemplated. For example, US Patent Application Publication 20110195123 discloses an implantable medical device that releases a drug locally and over a prolonged period, including several types of such a device, methods of treatment and methods of implantation implemented. The device comprises a polymeric substrate, such as a matrix, for example, used as a body of the device, and drugs, and in some cases additional three-dimensional carrier substrates, such as metals or additional polymers, and materials for improving visibility and visualization. An implantable delivery device may be advantageous in providing local and long-term release, where the drug is released directly into the extracellular matrix (ECM) of a diseased site, such as in the case of a tumor, inflammation, degeneration, or for symptomatic therapy, or into diseased smooth muscle cells, or for prevention. One type of drug is RNA, as disclosed above, and this system can be used for and/or adapted to the CRISPR-Cas system of the present invention. The implantation methods in some embodiments are existing implantation procedures developed and currently used for other treatments, including brachytherapy and needle biopsy. In such cases, the dimensions of the new implant described in the present invention are similar to the dimensions of the original implant. Typically, several devices are implanted during a single treatment.

U.S. Patent Application Publication No. 20110195123 discloses an implantable or insertable drug delivery system, including systems useful for insertion into a cavity such as the peritoneal cavity and/or for any other type of administration, in which the drug delivery system is not fixed or attached, comprising a biostable and/or degradable and/or bioabsorbable polymeric substrate, which may, for example, optionally be a matrix. It should be noted that the term "insert" also includes implantation. The drug delivery system is preferably implemented as a "Loder" described in U.S. Patent Application Publication No. 20110195123.

The polymer or plurality of polymers containing the agent and/or plurality of agents are biocompatible, providing for the release of the agent at a controlled rate, where the total volume of the polymer substrate, such as a matrix, for example, in some embodiments, optionally and preferably does not exceed the maximum volume that allows achieving a therapeutic level of the agent. As a non-limiting example, such a volume is preferably in the range of 0.1 m ³ to 1000 mm ³ , as required by the volume of the loaded agent. The loader may optionally have a larger size, for example, being included in a device, the size of which is determined by the functional purpose, for example, without limiting the knee joint, intrauterine or cervical ring, etc.

The drug delivery system (for delivering the composition) in some embodiments is designed to preferably use degradable polymers, where the main release mechanism is bulk erosion; or in some embodiments, non-degradable or slowly degradable polymers are used, where the main release mechanism is diffusion, rather than bulk erosion, so that their outer part functions as a membrane and their inner part functions as a drug depot, which is substantially not exposed to the environment for an extended period (e.g., from about a week to about several months). Combinations of different polymers with different release mechanisms can also optionally be used. The concentration gradient across the surface is preferably effectively maintained constant for a significant period during the overall period of drug release, and thus the diffusion rate (called "zero-order diffusion") is effectively constant. By "constant" is meant a diffusion rate that is preferably maintained above the lower threshold of therapeutic efficacy, but which may nevertheless optionally exhibit an initial burst and/or fluctuate, such as rising and falling to some extent. The diffusion rate is preferably maintained in this manner over a long period, and up to a certain level it may be considered constant to optimize the therapeutically effective period, such as the effective silencing period.

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

The drug delivery system of U.S. Patent Application Publication No. 20110195123 is optionally related to sensing and/or actuating devices that operate during and/or after implantation of the device via non-invasive and/or minimally invasive actuation and/or acceleration/deceleration methods, such as, but not limited to, methods or devices using thermal heating and cooling, laser beams, and ultrasound, including focused ultrasound, and/or RF (radio frequencies).

According to some embodiments of U.S. Patent Application Publication No. 20110195123, the local delivery site may optionally include target sites characterized by high abnormal cell proliferation and suppression of apoptosis, including tumors, sites of active and/or chronic inflammation and infection, including autoimmune disease states, tissue with degenerative changes, including muscle and nerve tissue, sites of chronic pain, sites with degenerative changes, and bone fracture locations, and other wound locations, to enhance tissue regeneration, as well as damaged cardiac, smooth and striated muscles.

The site for implantation of the composition or the target site is preferably characterized by a radius, area and/or volume small enough for targeted local delivery. For example, the target site optionally has a diameter in the range of about 0.1 mm to about 5 cm.

The location of the target site is preferably selected to achieve maximum therapeutic efficacy. For example, the composition of the drug delivery system (optionally together with the implantation device described above) is optionally and preferably implanted in or near the tumor environment or in the bloodstream associated therewith.

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

The target location is optionally selected from the group consisting of (by way of non-limiting examples only, since not all areas of the body may be suitable for implantation of Loder): 1. areas of the brain such as the basal ganglia, white matter and gray matter, with degenerative changes similar to those in Parkinson's disease or Alzheimer's disease; 2. the spinal cord, as in amyotrophic lateral sclerosis (ALS); 3. the cervix to prevent infection due to HPV; 4. joints with active and chronic inflammation; 5. the dermis, as in psoriasis; 6. areas of sympathetic and sensory nerves for an analgesic effect; 7. areas of intraosseous implantation; 8. foci of acute and chronic infection; 9. intravaginal sites; 10. the inner ear of the auditory system, the labyrinth of the inner ear, the vestibular system; 11. intratracheal sites; 12. Intracardiac sites; Coronary vessel sites, epicardial sites; 13. Urinary bladder; 14. Biliary system; 15. Parenchymatous tissue sites, including but not limited to kidney, liver, spleen; 16. Lymph nodes; 17. Salivary glands; 18. Gum sites around teeth; 19. Intra-articular sites (implantation into joints); 20. Intraocular sites; 21. Brain tissue; 22. Cerebral ventricles; 23. Cavities, including the abdominal cavity (e.g., without limitation for the treatment of ovarian cancer); 24. Intraesophageal sites; and 25. Intrarectal sites.

Insertion of the system (e.g., device containing the composition) optionally involves injection of material into the ECM at the target site and the surrounding area of that site to affect local pH and/or temperature and/or other biological factors that influence drug diffusion and/or drug kinetics into the ECM of the target site and the surrounding area of that site.

According to some embodiments, the release of said agent may optionally be associated with sensing and/or activating devices operating before and/or during and/or after insertion by means of non-invasive and/or minimally invasive and/or other activation and/or acceleration/deceleration methods, including methods or devices using laser beams, ionizing radiation, thermal heating and cooling, and ultrasound, including focused ultrasound, and/or RF (radio frequencies), as well as chemical activators.

According to other embodiments, in U.S. Patent Application Publication No. 20110195123, the medicament preferably comprises RNA, for example, for the treatment of localized breast, pancreatic, brain, kidney, bladder, lung, and prostate cancers described below. Although examples have been given with RNAi, many useful medicaments are amenable to encapsulation in Loder and can be used in the context of the present invention, provided that such medicaments can be encapsulated in a Loder substrate, such as, for example, a matrix, and this system can be used and/or adapted to deliver the CRISPR Cas system of the present invention.

As another example of a specific application, degenerative diseases of the nervous system and muscles develop due to abnormal gene expression. Local delivery of RNA may have therapeutic properties that prevent such abnormal gene expression. Local delivery of anti-apoptotic, anti-inflammatory and anti-degenerative drugs, including small molecule drugs and macromolecules, may also optionally be therapeutic. In such cases, Loder is used for prolonged release at a constant rate and/or via a dedicated device that is implanted separately. All of this can be used for and/or adapted to the CRISPR-Cas system of the present invention.

As another example of a specific application, mental and cognitive disorders are treated with gene modifiers. Gene knockdown is a possible treatment method. The use of Loders for local delivery of agents to areas of the central nervous system is a possible treatment method for mental and cognitive disorders, including but not limited to psychosis, bipolar disorders, neurotic disorders and behavioral disorders. Loders can also provide local delivery of drugs, including small molecule drugs and macromolecules, when implanted in specific areas of the brain. All of this can be used for and/or adapted to the CRISPR-Cas system of the present invention.

As another example of a specific application, silencing of genes mediating the innate and/or adaptive immune response at local sites prevents rejection of a transplanted organ. Local delivery of RNA and immunomodulatory reagents by Loder implanted in a transplanted organ and/or implantation site activates suppression of local immunity to the transplanted organ by diverting immune cells such as CD8. All of this can be used for and/or adapted to the CRISPR-Cas system of the present invention.

As another example of a specific application, vascular growth factors, including VEGF and angiogenin, and others, are essential for neovascularization. Local delivery of factors, peptides, peptidomimetics or suppression of their repressors is an important therapeutic intervention; silencing of repressors and local delivery of factors, peptides, macromolecules and small molecule drugs that stimulate angiogenesis by Loder are therapeutic measures for peripheral vascular disease, systemic vascular disease and cardiac vascular disease.

An insertion method such as implantation may optionally also be used for other types of tissue implantation and/or insertions and/or tissue sampling, optionally without modifications or, alternatively, optionally with only minor modifications of such methods. Such methods optionally include, but are not limited to, brachytherapy methods, biopsy, endoscopy with and/or without ultrasound technologies such as ERCP, stereotactic methods in relation to brain tissue, laparoscopy, including implantation using a laparoscope in joints, abdominal organs, bladder wall and body cavities.

The implantable device technology described herein can be used with the teachings herein and thus, using this disclosure and the knowledge in the art, the CRISPR-Cas system, or its components, or its nucleic acid molecules, or the encoded or provided components can be delivered via an implantable device.

Aerosol delivery

Subjects treated for a lung disease, for example, may receive a pharmaceutically effective amount of an AAV-based vector system in the form of an aerosol per lung, delivered endobronchially during spontaneous breathing. Therefore, delivery in the form of an aerosol is preferred for the delivery of AAV. An adenovirus or an AAV particle can be used for delivery. Suitable constructs with genes, each operably linked to one or more regulatory sequences, can be cloned into the delivery vector. In this case, the following constructs are given as examples: the Cbh or EF1a promoter for Cas (Cas9), the U6 or H1 promoter for the guide RNA. A preferred scheme is to use a guide targeting CFTR with the delta-508 mutation, a template for repair of the delta-F508 mutation and a codon-optimized Cas9 enzyme, optionally with one or more nuclear localization signals or sequences (NLS), for example with two (2) NLS. Designs without NLS are also provided.

CRISPR enzyme mRNA and guide RNA

The guide RNA and CRISPR enzyme mRNA can also be delivered separately. The CRISPR enzyme mRNA can be delivered before the guide RNA to allow time for the CRISPR enzyme to be expressed. The CRISPR enzyme mRNA can be administered 1-12 hours (preferably about 2-6 hours) before the guide RNA is administered.

Alternatively, CRISPR enzyme mRNA and guide RNA can be co-administered. A second booster dose of guide RNA can advantageously be administered 1-12 hours (preferably approximately 2-6 hours) after the first administration of CRISPR enzyme mRNA + guide RNA.

Administration of additional doses of CRISPR enzyme mRNA and/or guide RNA may be useful to achieve the most effective levels of genome modification. In some embodiments, the phenotypic change is preferably the result of genome modification when targeting a genetic disease, especially in therapeutic methods, and preferably provides a repair template to correct or alter the phenotype.

In some embodiments, diseases that can be targeted include diseases that are caused by pathogenic splicing defects.

In some embodiments, cellular targets include hematopoietic stem/progenitor cells (CD34+); human T cells, and ocular cells (retinal cells), such as photoreceptor progenitor cells.

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

In the discussions herein regarding a target being associated with a mutation or a disease state, such mutation or disease state may be, for example, hemophilia B, SCID, SCID-X1, ADA-SCID, hereditary tyrosinemia, sickle cell anemia, β-thalassemia, X-linked CGD, Wiskott-Aldrich syndrome, Fanconi anemia, adrenoleukodystrophy (ALD), metachromatic leukodystrophy (MLD), Usher syndrome, retinitis pigmentosa, Leber congenital amaurosis, cystic fibrosis, HIV/AIDS, HSV-1, HSV-2, or more broadly an immunodeficiency disorder, hematological condition, or lysosomal storage disease. The target may be associated with immunotherapy, such as, for example, cancer immunotherapy.

In some embodiments, delivery methods include cationic lipid-mediated "direct" delivery of the enzyme-targeting (ribonucleoprotein) complex and electroporation of plasmid DNA.

The methods of the present invention may further comprise delivery of templates, such as repair templates, which may be dsODN or ssODN, see below. Delivery of templates may be simultaneous or separate from delivery of any or all of the CRISPR enzyme, guide, tracr-mate or tracr-RNA, and by the same or a different delivery mechanism. In some embodiments, it is preferred that the template is delivered together with the guide, tracr-mate and/or tracr-RNA, and also preferably with the CRISPR enzyme. An example is an AAV-based vector, wherein the CRISPR enzyme is SaCas9 (with the N580 mutation).

The methods of the present invention may further comprise: (a) delivering to a cell a double-stranded oligodeoxynucleotide (dsODN) comprising sticky ends complementary to the sticky ends created by said double-strand break, wherein said dsODN integrates into a locus of interest; or (b) delivering to a cell a single-stranded oligodeoxynucleotide (ssODN), wherein said ssODN acts as a template for homologous repair of said double-strand break. The methods of the present invention may be used to prevent or treat a disease in an individual, optionally wherein said disease is caused by a defect in said locus of interest. The methods of the present invention may be performed in vivo in an individual or ex vivo on a cell removed from an individual, optionally wherein said cell is returned to the individual.

The enzymes according to the present invention can be used in optimized functional CRISPR-Cas systems that are of interest for functional screening; SAM test

In one aspect, the present invention relates to a non-naturally occurring or engineered composition comprising a Type V, more particularly a Cas9 CRISPR guide RNA comprising a guide sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell, wherein the guide RNA is modified by inserting different RNA sequence(s) that bind(s) to two or more adaptor proteins (e.g., aptamers), and wherein each adaptor protein is associated with one or more functional domains; or wherein the guide RNA is modified such that it has at least one non-coding functional loop. In particular embodiments, the guide RNA is modified by inserting different RNA sequence(s) at the 5' end of a direct repeat, in a direct repeat, or at the 3' end of the guide sequence. If there is more than one functional domain, the functional domains may be the same or different, such as two same or two different activators or repressors. In one aspect, the present invention relates to a composition based on a non-naturally occurring or engineered composition of a CRISPR-Cas complex comprising a guide RNA discussed herein and a CRISPR enzyme that is a Cas9 enzyme, optionally wherein the Cas9 enzyme comprises at least one mutation such that the Cas9 enzyme has no more than 5% of the nuclease activity of a Cas9 enzyme lacking the at least one mutation, and optionally one or more comprise at least one or more nuclear localization sequences. In one aspect, the present invention relates to a Cas9 CRISPR guide RNA or Cas9-based CRISPR-Cas complex as discussed herein, including a non-naturally occurring or engineered composition comprising two or more adaptor proteins, wherein each protein associates with one or more functional domains and wherein the adaptor protein binds to a different RNA sequence(s) inserted into the guide RNA. In particular embodiments, the guide RNA is additionally or alternatively modified to still allow binding of the Cas9 CRISPR complex, but prevents cleavage by the Cas9 enzyme (as described in detail elsewhere herein).

In one aspect, the present invention relates to a non-naturally occurring or engineered composition comprising a guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell, a Cas9 enzyme comprising at least one or more nuclear localization sequences, wherein the Cas9 enzyme comprises at least one mutation such that the Cas9 enzyme has no more than 5% of the nuclease activity of a Cas9 enzyme lacking the at least one mutation, wherein the guide RNA is modified by inserting different RNA sequence(s) that bind(s) to one or more adaptor proteins, and wherein the adaptor protein is associated with one or more functional domains; or wherein the guide RNA is modified such that it has at least one non-coding functional loop, and wherein the composition comprises two or more adaptor proteins, wherein each protein is associated with one or more functional domains. In one aspect, the present invention relates to a composition as discussed herein, wherein a Cas9 enzyme has a reduced nuclease activity of at least 97% or 100% compared to a Cas9 enzyme lacking at least one mutation. In one aspect, the present invention relates to a composition as discussed herein, wherein the Cas9 enzyme comprises two or more mutations. In one aspect, the present invention relates to a composition as discussed herein, wherein the Cas9 enzyme is associated with one or more functional domains. In one aspect, the present invention relates to a composition as discussed herein, wherein each of the two or more functional domains associated with the adapter protein is a heterologous functional domain. In one aspect, the present invention relates to a composition as discussed herein, wherein each of the one or more functional domains associated with the Cas9 enzyme is a heterologous functional domain. In one aspect, the present invention relates to a composition as discussed herein, wherein the adapter protein is a fusion protein comprising a functional domain, wherein the fusion protein optionally comprises a linker between the adapter protein and the functional domain, wherein the linker optionally comprises a GlySer linker. In one aspect, the present invention relates to a composition as discussed herein, wherein the gRNA is not modified by insertion of different RNA sequence(s) that bind(s) to two or more adapter proteins. In one aspect, the present invention relates to a composition as discussed herein, wherein one or more functional domains associated with the adapter protein are transcriptional activation domains. In one aspect, the present invention relates to a composition as discussed herein, wherein one or more functional domains associated with the Cas9 enzyme are transcriptional activation domains. In one aspect, the present invention relates to a composition as discussed herein, wherein one or more functional domains associated with an adapter protein are transcriptional activation domains comprising VP64, p65, MyoD1, HSF1, RTA, or SET7/9. In one aspect, the present invention relates to a composition as discussed herein, wherein one or more functional domains associated with a Cas9 enzyme are transcriptional activation domains comprising VP64, p65, MyoD1, HSF1, RTA, or SET7/9. In one aspect, the present invention relates to a composition as discussed herein, wherein one or more functional domains associated with an adapter protein are transcriptional repression domains. In one aspect, the present invention relates to a composition as discussed herein, wherein one or more functional domains associated with a Cas9 enzyme are transcriptional repression domains. In one aspect, the present invention relates to a composition as discussed herein, wherein the transcriptional repression domain is a KRAB domain. In one aspect, the present invention relates to a composition as discussed herein, wherein the transcriptional repression domain is a NuE domain, an NcoR domain, a SID domain, or a SID4X domain. In one aspect, the present invention relates to a composition as discussed herein, wherein at least one of the one or more functional domains associated with the adapter protein is characterized by one or more activities comprising a methylase activity, a demethylase activity, a transcriptional activation activity, a transcriptional repression activity, a transcript release factor activity, a histone modification activity, a DNA integration activity, an RNA cleavage activity, a DNA cleavage activity, or a nucleic acid binding activity. In one aspect, the present invention relates to a composition as discussed herein, wherein one or more functional domains associated with a Cas9 enzyme are characterized by one or more activities comprising methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcript release factor activity, histone modification activity, DNA integration activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, or molecular switch activity, or chemical inducibility, or light inducibility. In one aspect, the present invention relates to a composition as discussed herein, wherein the DNA cleavage activity is mediated by a Fok1 nuclease. In one aspect, the present invention relates to a composition as discussed herein, wherein the one or more functional domains are attached to a Cas9 enzyme such that upon binding to a gRNA and a target, the functional domain is in a spatial orientation that allows the functional domain to function in its intended function; or optionally wherein one or more functional domains are attached to a Cas9 enzyme via a linker, optionally a GlySer linker. In one aspect, the present invention relates to a composition as discussed herein, wherein the gRNA is modified such that upon binding of the gRNA to the adaptor protein and subsequent binding to the Cas9 enzyme and the target, the functional domain is in a spatial orientation that allows the functional domain to function with its intended function. In one aspect, the present invention relates to a composition as discussed herein, wherein one or more functional domains associated with the Cas9 enzyme are attached to the RuvC domain of Cas9. In one aspect, the present invention relates to a composition as discussed herein, wherein the direct repeat of the guide RNA is modified by insertion of different RNA sequence(s). In one aspect, the present invention relates to a composition as discussed herein, wherein the insert of a different RNA sequence(s) that binds to one or more adapter proteins is an aptamer sequence. In one aspect, the present invention relates to a composition as discussed herein, wherein the aptamer sequence is two or more aptamer sequences specific for the same adapter protein. In one aspect, the present invention relates to a composition as discussed herein, wherein the aptamer sequence is two or more aptamer sequences specific for different adapter proteins. In one aspect, the present invention relates to a composition as discussed herein, wherein the adapter protein comprises MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ϕCb5, ϕCb8r, ϕCb12r, ϕCb23r, 7s, PRR1. Therefore, in particular embodiments, the aptamer is selected from a binding protein that specifically binds any of the adapter proteins listed above. In one aspect, the present invention relates to a composition as discussed herein, wherein the cell is a eukaryotic cell. In one aspect, the present invention relates to a composition as discussed herein, wherein the eukaryotic cell is a mammalian cell, a plant cell, or a yeast cell, wherein the mammalian cell is optionally a mouse cell. In one aspect, the present invention relates to a composition as discussed herein, wherein the mammalian cell is a human cell. In one aspect, the present invention relates to a composition as discussed herein, wherein the first adapter protein associates with a p65 domain and the second adapter protein associates with an HSF1 domain. In one aspect, the present invention relates to a composition as discussed herein, wherein the composition comprises a CRISPR-Cas complex having at least three functional domains, at least one of which associates with a Cas9 enzyme and at least two of which associate with a gRNA.

In one aspect, the present invention relates to a composition as discussed herein above, wherein there is more than one gRNA, and the gRNAs target different sequences, whereby multiplexing occurs when the composition is used. In one aspect, the present invention relates to a composition, wherein there is more than one gRNA modified by insertion of different RNA sequence(s) that bind(s) to one or more adaptor proteins.

In one aspect, the present invention relates to a composition discussed herein, wherein one or more adaptor proteins associated with one or more functional domains are present and bind to a different RNA sequence(s) inserted into a guide RNA.

In one aspect, the present invention relates to a composition as discussed herein, wherein the target sequence(s) is(are) non-coding or regulatory sequence(s). The regulatory sequence(s) may be a promoter, enhancer, or silencer sequence(s).

In one aspect, the present invention relates to a composition discussed herein, wherein the guide RNA is modified to comprise at least one non-coding functional loop, for example, wherein the at least one non-coding functional loop is repressor; for example, wherein the at least one non-coding functional loop comprises Alu.

In one aspect, the present invention relates to a method of transformation at a genomic locus comprising administering to a host or expressing in vivo in a host one or more of the compositions discussed herein. In one aspect, the present invention relates to a method discussed herein, wherein transformation at a genomic locus comprises affecting gene activation, gene inhibition, or cleavage at the locus.

In one aspect, the present invention relates to a method as discussed herein, wherein the host is a eukaryotic cell. In one aspect, the present invention relates to a method as discussed herein, wherein the host is a mammalian cell, optionally a mouse cell or a plant cell or a yeast cell. In one aspect, the present invention relates to a method as discussed herein, wherein the host is a non-human eukaryotic organism. In one aspect, the present invention relates to a method as discussed herein, wherein the non-human eukaryotic organism is a non-human mammal. In one aspect, the present invention relates to a method as discussed herein, wherein the non-human mammal is a mouse.

In one aspect, the present invention relates to a method for modifying a genomic locus of interest to alter gene expression in a cell by introducing into the cell or expressing in a cell a composition discussed herein. In one aspect, the present invention relates to a method discussed herein comprising delivering a composition or a nucleic acid molecule(s) encoding the same, wherein said nucleic acid molecule(s) is(are) operably linked to a regulatory sequence(s) and is(are) expressed in vivo. In one aspect, the present invention relates to a method discussed herein, wherein the in vivo expression is accomplished by a lentivirus, an adenovirus, or an AAV.

In one aspect, the present invention relates to a mammalian cell line of the cells discussed herein, wherein the cell line is optionally a human cell line or a mouse cell line. In one aspect, the present invention relates to a transgenic mammalian model, optionally a mouse, wherein the model has been transformed with the composition discussed herein or is a progeny of said transformant.

In one aspect, the present invention relates to a nucleic acid molecule(s) encoding a guide RNA or a Cas9-based CRISPR-Cas complex or composition as discussed herein. In one aspect, the present invention relates to a vector comprising a nucleic acid molecule encoding a guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell, wherein a direct repeat in the gRNA is modified by inserting different RNA sequence(s) that bind(s) to two or more adaptor proteins, and wherein each adaptor protein is associated with one or more functional domains; or wherein the gRNA is modified such that it has at least one non-coding functional loop. In one aspect, the present invention relates to a vector(s) comprising a nucleic acid molecule(s) encoding a non-naturally occurring or engineered composition of a CRISPR-Cas complex comprising a gRNA discussed herein and a Cas9 enzyme, wherein optionally the Cas9 enzyme comprises at least one mutation such that the Cas9 enzyme has no more than 5% of the nuclease activity of a Cas9 enzyme lacking the at least one mutation, and optionally one or more comprise at least one or more nuclear localization sequences. In one aspect, the vector may further comprise a regulatory element(s) that functions in a eukaryotic cell, operably linked to a nucleic acid molecule encoding a guide RNA (gRNA) and/or a nucleic acid molecule encoding a Cas9 enzyme, and/or optionally to a nuclear localization sequence(s).

In one aspect, the present invention relates to a kit comprising one or more components described herein above. In some embodiments, the kit comprises a vector system discussed above and instructions for using the kit.

In one aspect, the present invention relates to a method for screening for a gain-of-function (GOF) or loss-of-function (LOF) mutation, or for screening non-coding RNAs or potential regulatory regions (e.g., enhancers, repressors), comprising a cell line as discussed herein or cells of a model as discussed herein that contain or express Cas9, and administering a composition as discussed herein to cells of the cell line or model, thereby incorporating either an activator or a repressor into the gRNA, and monitoring for GOF or LOF, respectively, in those cells in which the administered gRNA comprises the activator or in those cells in which the administered gRNA comprises the repressor. The screening according to the present invention is referred to as a SAM test.

In one aspect, the present invention relates to a Cas9-based CRISPR-Cas complex comprising a Cas9 enzyme and a guide RNA (gRNA), wherein the Cas9 enzyme comprises at least one mutation such that the Cas9 enzyme has no more than 5% of the nuclease activity of a Cas9 enzyme lacking the at least one mutation, and optionally at least one or more nuclear localization sequences; wherein the guide RNA (gRNA) comprises a guide sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell; and wherein the gRNA is modified by inserting different RNA sequence(s) that binds to one or more adaptor proteins, and wherein the adaptor protein is associated with two or more functional domains, or wherein the gRNA is modified such that it has at least one non-coding functional loop; or the Cas9 enzyme is associated with one or more functional domains and the gRNA is modified by inserting a different RNA sequence(s) that binds to one or more adaptor proteins, and where the adaptor protein is associated with two or more functional domains, or where the gRNA is modified such that it has at least one non-coding functional loop.

In one aspect, the present invention relates to a genome-wide library comprising a plurality of Cas9 guide RNAs (gRNAs) comprising guide sequences each of which is capable of hybridizing to a target sequence at a genomic locus of interest in a cell, and thereby the library is capable of targeting a plurality of target sequences at a plurality of genomic loci in a population of eukaryotic cells, wherein each gRNA is modified by inserting a different RNA sequence(s) that binds to one or more or two or more adaptor proteins, and wherein the adaptor protein is associated with one or more functional domains; or wherein the gRNA is modified such that it has at least one non-coding functional loop. And where there is more than one functional domain, the functional domains may be the same or different, such as two same or two different activators or repressors. In one aspect, the present invention relates to a library of non-naturally occurring or engineered composition(s) with a CRISPR-Cas complex comprising a gRNA according to the present invention and a Cas9 enzyme, wherein optionally the Cas9 enzyme comprises at least one mutation such that the Cas9 enzyme has no more than 5% of the nuclease activity of a Cas9 enzyme lacking the at least one mutation, and optionally one or more comprise at least one or more nuclear localization sequences. In one aspect, the present invention relates to a gRNA or Cas9-based CRISPR-Cas complex(es) according to the present invention, including a non-naturally occurring or engineered composition comprising one or two or more adaptor proteins, wherein each protein is associated with one or more functional domains, and wherein the adaptor protein binds to distinct RNA sequence(s) inserted into at least one loop of the gRNA.

In one aspect, the present invention provides a library of non-naturally occurring or engineered compositions, each comprising a Cas9 CRISPR guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell, a Cas9 enzyme comprising at least one or more nuclear localization sequences, wherein the Cas9 enzyme comprises at least one mutation such that the Cas9 enzyme has no more than 5% of the nuclease activity of a Cas9 enzyme lacking the at least one mutation, wherein at least one loop in the gRNA is modified by insertion of different RNA sequence(s) that bind(s) to one or more adaptor proteins, and wherein the adaptor protein is associated with one or more functional domains, wherein the composition comprises one or more or two or more adaptor proteins, wherein each protein is associated with one or more functional domains, and wherein the gRNAs comprise a genome-wide library, comprising a plurality of Cas9 guide RNAs (gRNAs). In one aspect, the present invention relates to a library as discussed herein, wherein the Cas9 enzyme has a reduced nuclease activity of at least 97% or 100% compared to a Cas9 enzyme lacking at least one mutation. In one aspect, the present invention relates to a library as discussed herein, wherein the Cas9 enzyme comprises two or more mutations. In one aspect, the present invention relates to a library as discussed herein, wherein the Cas9 enzyme comprises two or more mutations. In one aspect, the present invention relates to a library as discussed herein, wherein the Cas9 enzyme is associated with one or more functional domains. In one aspect, the present invention relates to a library as discussed herein, wherein one or two or more functional domains associated with the adaptor protein are a heterologous functional domain. In one aspect, the present invention relates to a library as discussed herein, wherein one or more functional domains associated with a Cas9 enzyme are a heterologous functional domain. In one aspect, the present invention relates to a library as discussed herein, wherein the adapter protein is a fusion protein comprising a functional domain. In one aspect, the present invention relates to a library as discussed herein, wherein the gRNA is not modified by insertion of different RNA sequence(s) that bind(s) to two or more adapter proteins. In one aspect, the present invention relates to a library as discussed herein, wherein one or two or more functional domains associated with an adapter protein are a transcriptional activation domain. In one aspect, the present invention relates to a library as discussed herein, wherein one or two or more functional domains associated with a Cas9 enzyme are a transcriptional activation domain. In one aspect, the present invention relates to a library as discussed herein, wherein one or two or more functional domains associated with an adapter protein are a transcriptional activation domain comprising VP64, p65, MyoD1, or HSF1. In one aspect, the present invention relates to a library as discussed herein, wherein one or more functional domains associated with a Cas9 enzyme are a transcriptional activation domain comprising VP64, p65, MyoD1, or HSF1. In one aspect, the present invention relates to a library as discussed herein, wherein one or two or more functional domains associated with an adapter protein are a transcriptional repression domain. In one aspect, the present invention relates to a library as discussed herein, wherein one or more functional domains associated with a Cas9 enzyme are a transcriptional repression domain. In one aspect, the present invention relates to a library as discussed herein, wherein the transcriptional repression domain is a KRAB domain. In one aspect, the present invention relates to a library as discussed herein, wherein the transcriptional repression domain is a SID domain or a SID4X domain. In one aspect, the present invention relates to a library as discussed herein, wherein at least one or two or more functional domains associated with an adapter protein are characterized by one or more activities comprising methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcript release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, or nucleic acid binding activity. In one aspect, the present invention relates to a library as discussed herein, wherein one or more functional domains associated with a Cas9 enzyme are characterized by one or more activities comprising methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcript release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, or molecular switch activity, or chemical inducibility or light inducibility. In one aspect, the present invention relates to a library as discussed herein, wherein the DNA cleavage activity is mediated by a Fok1 nuclease. In one aspect, the present invention relates to a library as discussed herein, wherein one or more functional domains are linked to a Cas9 enzyme such that upon binding to a gRNA and a target, the functional domain is in a spatial orientation that allows the functional domain to function in its intended function. In one aspect, the present invention relates to a library as discussed herein, wherein the gRNA is modified such that upon binding of the gRNA to an adaptor protein and subsequent binding to a Cas9 enzyme and a target, the functional domain is in a spatial orientation that allows the functional domain to function in its intended function. In one aspect, the present invention relates to a library as discussed herein, wherein one or more functional domains associated with a Cas9 enzyme are fused to the N-terminus of a Cas9 enzyme. In one aspect, the present invention relates to a library as discussed herein, wherein one or more functional domains associated with a Cas9 enzyme are fused to RuvC of a FnCas9 protein or any ortholog corresponding to these domains. In one aspect, the present invention relates to a library as discussed herein, wherein a direct repeat in a gRNA is modified by insertion of different RNA sequence(s). In one aspect, the present invention relates to a library as discussed herein, wherein the insert of a different RNA sequence(s) that binds to one or more adapter proteins is an aptamer sequence. In one aspect, the present invention relates to a library as discussed herein, wherein the aptamer sequence is two or more aptamer sequences specific for the same adapter protein. In one aspect, the present invention relates to a library as discussed herein, wherein the aptamer sequence is two or more aptamer sequences specific for a different adapter protein. In one aspect, the present invention relates to a library as discussed herein, wherein the adapter protein comprises MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s, PRR1. In one aspect, the present invention relates to a library as discussed herein, wherein the cellular population of cells is a population of eukaryotic cells. In one aspect, the present invention relates to a library as discussed herein, wherein the eukaryotic cell is a mammalian cell, a plant cell, or a yeast cell. In one aspect, the present invention relates to a library as discussed herein, wherein the mammalian cell is a human cell. In one aspect, the present invention relates to a library as discussed herein, wherein the population of cells is a population of embryonic stem (ES) cells. In one aspect, the present invention relates to a library as discussed herein, wherein the target sequence at a genomic locus is a non-coding sequence. In one aspect, the present invention relates to a library as discussed herein, wherein the gene function of one or more gene products is altered by said targeting; or wherein a gain-of-function mutation is observed with respect to the gene function; or wherein a change in function is observed with respect to the gene function; or wherein a loss of function is observed with respect to the gene function; or wherein the assay is a assay for non-coding RNAs or potential regulatory regions (e.g., enhancers, repressors). In one aspect, the present invention relates to a library as discussed herein, wherein said targeting results in a knockout of gene function. In one aspect, the present invention relates to a library as discussed herein, wherein the targeting spans about 100 or more sequences. In one aspect, the present invention relates to a library as discussed herein, wherein the targeting spans about 1,000 or more sequences. In one aspect, the present invention relates to a library as discussed herein, wherein the targeting spans about 20,000 or more sequences. In one aspect, the present invention relates to a library as discussed herein, wherein the targeting spans the entire genome. In one aspect, the present invention relates to a library as discussed herein, wherein the targeting spans a panel of target sequences focused on a relevant or desired pathway. In one aspect, the present invention relates to a library as discussed herein, wherein the pathway is an immune pathway. In one aspect, the present invention relates to a library as discussed herein, wherein the pathway is a cell division pathway. In one aspect, the present invention relates to a library as discussed herein, wherein altering gene function comprises introducing into each cell in a population of cells a vector system of one or more vectors comprising an engineered, non-naturally occurring Cas9-based CRISPR-Cas system comprising I. a Cas9 protein and II. one or more types of Cas9 guide RNAs, wherein components I and II may be on the same or on different vectors of the system introducing components I and II into each cell, wherein the guide sequence targets a unique gene in each cell, wherein the Cas9 protein is operably linked to a regulatory element, wherein when transcribed, the guide RNA comprising the guide sequence directs sequence-specific binding of the Cas9-based CRISPR-Cas system to a target sequence in genomic loci of a unique gene to induce cleavage of the genomic locus by the Cas9 protein and confirming different mutations in a plurality of unique genes in each cell of the population of cells, thereby generating a library of mutant cells. In one aspect, the present invention relates to a library as discussed herein, wherein one or more vectors are plasmid vectors. In one aspect, the present invention relates to a library as discussed herein, wherein the regulatory element is an inducible promoter. In one aspect, the present invention relates to a library as discussed herein, wherein the inducible promoter is a doxycycline inducible promoter. In one aspect, the present invention relates to a library as discussed herein, wherein confirmation of various mutations is accomplished by whole exome sequencing. In one aspect, the present invention relates to a library as discussed herein, wherein mutation is achieved in 100 or more unique genes. In one aspect, the present invention relates to a library as discussed herein, wherein mutation is achieved in 1,000 or more unique genes. In one aspect, the present invention relates to a library as discussed herein, wherein mutation is achieved in 20,000 or more unique genes. In one aspect, the present invention relates to a library as discussed herein, wherein mutation is achieved throughout the genome. In one aspect, the present invention relates to a library as discussed herein, wherein alteration of gene function is achieved in a plurality of unique genes that function in a particular physiological pathway or condition. In one aspect, the present invention relates to a library as discussed herein, wherein the pathway or condition is an immune pathway or condition. In one aspect, the present invention relates to a library as discussed herein, wherein the pathway or condition is a cell division pathway or condition. In one aspect, the present invention relates to a library as discussed herein, wherein the first adapter protein associates with a p65 domain and the second adapter protein associates with an HSF1 domain. In one aspect, the present invention relates to a library as discussed herein, wherein each Cas9-based CRISPR-Cas complex has at least three functional domains, at least one of which associates with a Cas9 enzyme and at least two of which associate with a gRNA. In one aspect, the present invention relates to a library as discussed herein, wherein the change in gene function is a knockout mutation.

In one aspect, the present invention relates to a method for functionally screening genes in a genome in a cell pool ex vivo or in vivo comprising administering or expressing a library comprising multiple Cas9-based CRISPR-Cas guide RNAs (gRNAs), and wherein the screening further comprises using a Cas9 enzyme, wherein the CRISPR complex is modified to comprise a heterologous functional domain. In one aspect, the present invention relates to a method for screening a genome comprising administering a library to a host or expressing it in a host in vivo. In one aspect, the present invention relates to a method as discussed herein further comprising an activator administered to the host or expressed in the host. In one aspect, the present invention relates to a method as discussed herein, wherein the activator is attached to a Cas9 enzyme. In one aspect, the present invention relates to a method as discussed herein, wherein the activator is attached to the N-terminus or the C-terminus of the Cas9 enzyme. In one aspect, the present invention provides a method as discussed herein, wherein an activator is attached to a direct repeat in a Cas9 CRISPR gRNA. In one aspect, the present invention provides a method as discussed herein, further comprising a repressor introduced into the host or expressed in the host. In one aspect, the present invention provides a method as discussed herein, wherein screening comprises targeting and detecting gene activation, gene inhibition, or cleavage at a locus. In one aspect, the present invention provides a method as discussed herein, wherein the host is a eukaryotic cell. In one aspect, the present invention provides a method as discussed herein, wherein the host is a mammalian cell, a yeast cell, or a plant cell. In one aspect, the present invention provides a method as discussed herein, wherein the host is a eukaryotic organism other than a human. In one aspect, the present invention relates to a method as discussed herein, wherein the non-human eukaryotic organism is a non-human mammal. In one aspect, the present invention relates to a method as discussed herein, wherein the non-human mammal is a mouse. In one aspect, the present invention relates to a method as discussed herein comprising delivering Cas9-based CRISPR-Cas complexes, or component(s) thereof, or nucleic acid molecule(s) encoding the same, wherein said nucleic acid molecule(s) is(are) operably linked to regulatory sequence(s) and is(are) expressed in vivo. In one aspect, the present invention relates to a method as discussed herein, wherein the in vivo expression is accomplished by a lentivirus, an adenovirus, or an AAV. In one aspect of the present invention, there is provided a method as discussed herein, wherein delivery is via a cell penetrating particle, nanoparticle, lipid or peptide (CPP).

In one aspect, the present invention relates to a pair of Cas9-based CRISPR-Cas complexes, each comprising a Cas9 guide RNA (gRNA) comprising a guide sequence capable of hybridizing to a target sequence at a genomic locus of interest in a cell, wherein the gRNA is modified by insertion of different RNA sequence(s) that bind(s) to one or more adaptor proteins, and wherein the adaptor protein is associated with one or more functional domains, wherein each gRNA of each Cas9-based CRISPR-Cas comprises a functional domain characterized by DNA cleavage activity. In one aspect, the present invention relates to the paired Cas9-based CRISPR-Cas complexes discussed herein, wherein the DNA cleavage activity is mediated by the Fok1 nuclease.

In particular embodiments, the methods and compositions herein employ a nucleotide sequence encoding a Cas9 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, a yeast cell, or a plant cell. Alternatively, the eukaryotic cell is a plant cell. In a further embodiment of the present invention, expression of the gene product is reduced.

In some embodiments of the methods and compositions provided herein, the Cas9 enzyme is obtained from Acidaminococcus sp. BV3L6, Lachnospiraceae MA2020 bacteria, or Francisella tularensis 1 Novicida Cas9 and may include a mutated Cas9 obtained from these organisms. The enzyme may be a homolog or ortholog of Cas9.

In one aspect, the present invention relates to a method for creating a model eukaryotic cell comprising a gene with modified expression. In some embodiments, the gene responsible for the development of a disease is any gene associated with an increased risk of having or developing a disease. In some embodiments, the method comprises (a) introducing one or more vectors described herein above into a eukaryotic cell and (b) causing a CRISPR complex to bind to a target polynucleotide such that the gene locus is modified, thereby producing a model eukaryotic cell comprising the modified gene locus.

In one aspect of the present invention, there is provided a method for developing a biologically active agent that modulates a cellular signaling event associated with a disease-promoting gene. In some embodiments, the disease-promoting gene is any gene associated with an increased risk of having or developing a disease. In some embodiments, the method comprises (a) contacting a test compound with a model cell according to any of the embodiments described above; and (b) detecting a change in a readout that indicates a decrease or increase in a cellular signaling event associated with said mutation in said disease-promoting gene, thereby obtaining said biologically active agent that modulates said cellular signaling event associated with said disease-promoting gene.

The present invention encompasses optimized functional Cas9-based CRISPR-Cas enzyme systems, especially in combination with modified guides according to the present invention, and also if the Cas9 enzyme is also associated with a functional domain. In particular, the Cas9 enzyme comprises one or more mutations that convert it into a DNA binding protein to which functional domains exhibiting a function of interest can be added or attached or inserted or linked. In certain embodiments, the Cas9 enzyme comprises one or more mutations, and/or the one or more mutations are in the RuvC1 domain of the Cas9 enzyme or are a mutation otherwise discussed herein. In some embodiments, the Cas9 enzyme has one or more mutations in the catalytic domain, wherein upon transcription, the guide sequence directs sequence-specific binding of the CRISPR complex to the target sequence, and wherein the enzyme further comprises a functional domain. In some embodiments, a mutation in E1006 corresponding to the FnCas9 protein is preferred.

The structural information presented herein provides a detailed study of the interaction of the guide RNA with the target DNA and the Cas9 enzyme, providing the possibility of designing or modifying the structure of the guide RNA to optimize the functionality of the overall Cas9-based CRISPR-Cas system. For example, loops in the guide RNA can be enlarged without overlapping with the Cas9 protein by inserting adaptor proteins that can bind to the RNA. Such adaptor proteins can further recruit effector proteins or fusion products that contain one or more functional domains.

In some preferred embodiments, the functional domain is a transcriptional activation domain, preferably VP64. In some embodiments, the functional domain is a transcriptional repression domain, preferably KRAB. In some embodiments, the transcriptional repression domain is SID or SID concatemers (e.g., SID4X). In some embodiments, the functional domain is an epigenetic modification domain, such that an epigenetic modification enzyme is provided. In some embodiments, the functional domain is an activation domain, which may be a P65 activation domain.

One aspect of the present invention is that the above-mentioned elements are contained in a single composition or are contained in separate compositions. These compositions can advantageously be applied to a host to induce a functional effect at the genome level.

In general, the guide RNA is modified in a manner that provides specific binding sites (e.g., aptamers) for adaptor proteins containing one or more functional domains (e.g., through formation of a fusion protein) to bind. The modified guide RNA is modified such that once the guide RNA forms a CRISPR complex (i.e., the Cas9 enzyme binds to the guide RNA and the target), the adaptor proteins associate and the functional domain in the adaptor protein is positioned in a spatial orientation that is favorable for the efficiency of the intrinsic function. For example, if the functional domain is a transcriptional activator (e.g., VP64 or p65), the transcriptional activator is positioned in a spatial orientation that allows it to influence the transcription of the target. Similarly, a transcriptional repressor will be positioned preferentially to affect transcription of the target, and a nuclease (e.g., Fok1) will be positioned preferentially to cleave or partially cleave the target.

It will be appreciated by one skilled in the art that modifications to the guide RNA that allow binding of the adaptor + functional domain but do not allow proper placement of the adaptor + functional domain (e.g., due to steric hindrance in the three-dimensional structure of the CRISPR complex) are modifications that are not intended. One or more modified guide RNAs may be modified by introducing different RNA sequence(s) at the 5' end of the direct repeat, in the direct repeat, or at the 3' end of the guide sequence.

As explained herein, the functional domains may be, for example, one or more domains from the group consisting of domains with methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcript release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switches (e.g., light-inducible). In some cases, it is preferable that at least one NLS is additionally provided. In some cases, the position of the NLS at the N-terminus is preferable. When more than one functional domain is included, the functional domains may be the same or different.

The guide RNA can be designed to contain multiple binding recognition sites (e.g., aptamers) specific for the same or different adaptor proteins. The Cas9 guide RNA is characterized by being typically 37-43 nucleotides long and by containing only one stem-loop. The guide RNA can be designed to bind to the promoter region -1000 to +1 nucleic acids upstream of the transcription start site (i.e., TSS), preferably -200 nucleic acids. This placement favors functional domains that affect gene activation (e.g., transcriptional activators) or gene inhibition (e.g., transcriptional repressors). The modified guide RNA may be one or more modified guide RNAs targeting one or more target loci (e.g., at least 1 guide RNA, at least 2 guide RNAs, at least 5 guide RNAs, at least 10 guide RNAs, at least 20 guide RNAs, at least 30 guide RNAs, at least 50 guide RNAs) contained in the composition.

In addition, the Cas9 enzyme with reduced nuclease activity is most efficient at inactivating nuclease activity (e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% nuclease inactivation compared to the wild-type enzyme; or, in other words, the Cas9 enzyme has substantially about 0% of the nuclease activity of the wild-type or unmutated Cas9 enzyme or no more than about 3%, or about 5%, or about 10% of the nuclease activity of the wild-type or unmutated Cas9 enzyme). This is possible by introducing mutations into the nuclease domains of RuvC FnCas9 and its orthologs. For example, mutations are used at a residue selected from the group consisting of D917A, E1006A, E1028A, D1227A, D1255A or N1257, as in FnCas9, and more preferably one or more mutations are introduced selected from the group consisting of locations D917A, E1006A, E1028A, D1227A, D1255A, N1257A, D917A, E1006A, E1028A, D1227A, D1255A and N1257 in FnCas9 or a corresponding ortholog. In specific embodiments, the mutations are D917A with E1006A in FnCas9.

The inactivated Cas9 enzyme may be associated (e.g., by forming a fusion protein) with one or more functional domains, such as those described herein for modified guide RNA adaptor proteins, including, for example, one or more domains from the group consisting of domains with methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcript release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switches (e.g., light-inducible). Preferred domains are Fok1, VP64, P65, HSF1, MyoD1. In the case of providing Fok1, it is preferred that several functional domains of Fok1 are provided for a functional dimer and that the guide RNAs are designed to provide an appropriate interval for the functional use (of Fok1, as specifically described in Tsai et al. Nature Biotechnology, Vol. 32, Number 6, June 2014). Known linkers can be used in the adapter protein to attach such functional domains. In some cases, it is preferred that at least one NLS is additionally provided. In some cases, the position of the NLS at the N-terminus is preferred. When more than one functional domain is included, the functional domains can be the same or different.

In general, the placement of one or more functional domains in an inactivated Cas9 enzyme ensures the correct spatial orientation of the functional domain to act on the target with the intended functional effect. For example, if the functional domain is a transcriptional activator (e.g., VP64 or p65), the transcriptional activator is placed in a spatial orientation that allows it to affect transcription of the target. Similarly, a transcriptional repressor will be preferentially positioned to affect transcription of the target, and a nuclease (e.g., Fok1) will be preferentially positioned to cleave or partially cleave the target. Positions other than the N-/C-terminus of the Cas9 enzyme may be envisaged.

The adaptor protein may be any of a number of proteins that bind to an aptamer or recognition site introduced into a modified guide RNA and that ensures the proper placement of one or more functional domains, once the guide RNA has been inserted into the CRISPR complex, to affect the target with the inherent function. As explained in detail in the present application, they may be coat proteins, preferably bacteriophage coat proteins. The functional domains associated with such adaptor proteins (e.g., in the form of a fusion protein) may include, for example, one or more domains from the group consisting of domains with methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcript release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switches (e.g., light-inducible). Preferred domains are Fok1, VP64, P65, HSF1, MyoD1. In case the functional domain is a transcription activator or transcription repressor, it is advantageous to additionally provide at least an NLS and preferably at the N-terminus. When including more than one functional domain, the functional domains can be the same or different. Known linkers can be used in the adapter protein to attach such functional domains.

Thus, the modified guide RNA, the inactivated Cas9 enzyme (with or without functional domains) and the binding protein with one or more functional domains may be separately contained in a composition and may be administered to the host separately or together. Alternatively, these components may be provided in a single composition for administration to the host. Administration to the host may be accomplished using viral vectors known to the skilled artisan or described herein for delivery to the host (e.g., a lentiviral vector, an adenoviral vector, an AAV-based vector). As explained herein, the use of different selection markers (e.g., for lentiviral gRNA selection) and different gRNA concentrations (e.g., depending on whether multiple gRNAs are used) may be advantageous to induce an improved effect.

Based on this concept, several options are suitable for inducing a transformation at a genomic locus, including DNA cleavage, gene activation, or gene inactivation. Using the provided compositions, one skilled in the art will be able to efficiently and specifically target one or multiple loci using the same or different functional domains to induce one or more transformations at a genomic locus. The compositions can be used in a variety of methods for screening libraries in cells and functional modeling in vivo (e.g., lincRNA gene activation and function identification; gain-of-function mutation modeling; loss-of-function mutation modeling; use of the compositions of the present invention to generate cell lines and transgenic animals for optimization and screening purposes).

The present invention encompasses the use of the compositions of the present invention for the generation and use of conditional or inducible CRISPR transgenic cells/animals. (See, e.g., Platt et al., Cell (2014), http://dx.doi.org/10.1016/j.cell.2014.09.014, or the PCT patent publications cited therein, such as WO 2014/093622 (PCT/US2013/074667), which are not considered prior art to the present invention or application.) For example, the target cell comprises a conditionally dependent or inducible Cas9 CRISPR enzyme (e.g., in the form of Cre-dependent constructs) and/or a conditionally dependent or inducible adapter protein, and when the vector is expressed in the target cell, the vector expresses something that induces or provides a condition for the expression of the Cas9 enzyme and/or the expression of the adapter in the target cell. By using the ideas and compositions of the present invention with a known method for creating a CRISPR complex, inducible genome transformations that are affected by the functional domains are also an aspect of the present invention. One example of this is the generation of a CRISPR knockin/conditionally expressing transgenic animal (e.g., a mouse containing, for example, a Lox-Stop-polyA-Lox (LSL) cassette) and subsequent delivery of one or more compositions providing one or more modified guide RNAs (e.g., -200 nucleotides to the TSS in a target gene of interest for gene activation purposes) described herein (e.g., modified guide RNAs with one or more aptamers recognized by coat proteins, such as MS2), one or more adaptor proteins described herein (MS2 binding protein linked to one or more VP64), and means for inducing the animal to have conditionally expressing (e.g., Cre recombinase to provide inducible expression of Cas9). Alternatively, the adaptor protein can be provided as a conditional or inducible element with a conditional or inducible Cas9 enzyme to provide an efficient screening model that advantageously requires only minimal development and introduction of specific gRNAs for a wide range of applications.

Deactivated/inactivated Cas protein

If the Cas9 protein has nuclease activity, the Cas9 protein may be modified to have reduced nuclease activity, such as at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% nuclease inactivation compared to the wild-type enzyme; or, in other words, the Cas9 enzyme has substantially about 0% of the nuclease activity of the unmutated or wild-type Cas9 enzyme or CRISPR enzyme, or no more than about 3%, or about 5%, or about 10% of the nuclease activity of the unmutated or wild-type Cas9 enzyme, such as the unmutated or wild-type Cas9 enzyme or CRISPR enzyme of S. pyogenes. This is possible by introducing mutations into the nuclease domains of Cas9 and its orthologs.

The inactivated Cas9 CRISPR enzyme may be associated (e.g., by forming a fusion protein) with one or more functional domains, including, for example, one or more domains from the group comprising, consisting essentially of, or consisting of methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcript release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and molecular switches (e.g., light-inducible). Preferred domains are Fok1, VP64, P65, HSF1, MyoD1. In the case of providing Fok1, it is preferred that several functional domains of Fok1 are provided for a functional dimer and that the sgRNAs are designed to provide an appropriate spacing for functional use (Fok1), as specifically described in Tsai et al. Nature Biotechnology, Vol. 32, Number 6, June 2014). Known linkers can be used in the adapter protein to attach such functional domains. In some cases, it is preferable that at least one NLS is additionally provided. In some cases, the position of the NLS at the N-terminus is preferable. When including more than one functional domain, the functional domains can be the same or different.

In general, the placement of one or more functional domains in an inactivated Cas9 enzyme ensures the correct spatial orientation of the functional domain to act on the target with the intended functional effect. For example, if the functional domain is a transcriptional activator (e.g., VP64 or p65), the transcriptional activator is placed in a spatial orientation that allows it to affect transcription of the target. Similarly, a transcriptional repressor will be preferentially placed to affect transcription of the target, and a nuclease (e.g., Fok1) will be preferentially placed to cleave or partially cleave the target. Positions other than the N-/C-terminus of the CRISPR enzyme may be envisaged.

In one embodiment, Cas9 may comprise one or more mutations (and therefore the nucleic acid molecule(s) encoding it may have mutation(s)). The mutations may be artificially introduced mutations and may include, but are not limited to, one or more mutations in the catalytic domain. Examples of catalytic domains with respect to the Cas9 enzyme may include, but are not limited to, the RuvC I, RuvC II, RuvC III, and HNH domains.

In one embodiment, Cas9 may contain one or more mutations. The mutations may be artificially introduced mutations and may include, but are not limited to, one or more mutations in the catalytic domain to provide a nickase, for example. Examples of catalytic domains for the Cas enzyme may include, but are not limited to, the RuvC I, RuvC II, RuvC III, and HNH domains.

In one embodiment, Cas9 can be used as a generic nucleic acid binding protein in fusion or in operative association with a functional domain. Exemplary functional domains can include, but are not limited to, a translation initiator, a translation activator, a translation repressor, nucleases, in particular ribonucleases, a spliceosome, granules, a light-inducible/controlled domain, or a chemically inducible/controlled domain.

In some embodiments, an unmodified nucleic acid targeting effector protein may have a cleavage activity. In some embodiments, an RNA targeting effector protein may direct the cleavage of one or both strands of a nucleic acid (DNA or RNA) at a specific position by or near a target sequence, such as within the target sequence and/or in a sequence complementary to the target sequence or in sequences associated with the target sequence. In some embodiments, a Cas9 targeting effector protein may direct the cleavage of one or both strands of DNA or RNA within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500 or more base pairs of the first or last nucleotide of the target sequence. In some embodiments, the cleavage may be "blunt", i.e., forming "blunt" ends. In some embodiments, the cleavage may be staggered, i.e., forming sticky ends. In some embodiments, the cleavage may be a staggered break with a 5' sticky end, such as a 1-5 nucleotide 5' sticky end. In some embodiments, the cleavage may be a staggered break with a 3' sticky end, such as a 1-5 nucleotide 3' sticky end. In some embodiments, the vector encodes a nucleic acid targeting Cas protein that may be mutated relative to the corresponding wild-type enzyme such that the mutant nucleic acid targeting Cas protein lacks the ability to cleave one or both strands of DNA or RNA of a target polynucleotide containing a target sequence. As a further example, two or more catalytic domains of Cas (RuvC I, RuvC II, and RuvC III, or the HNH domain) can be mutated to produce a mutated Cas that lacks substantially all RNA cleavage activity. The corresponding catalytic domains of the Cas9 effector protein described herein can also be mutated to produce a mutated Cas9 that lacks full DNA cleavage activity or has substantially reduced DNA cleavage activity. In some embodiments, a nucleic acid targeting effector protein can be considered to lack substantially all RNA cleavage activity if the RNA cleavage activity of the mutated enzyme is no more than about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the nucleic acid cleavage activity of the unmutated form of the enzyme; an example would be where the DNA cleavage activity of the mutated form is absent or insignificant compared to the unmutated form. The effector protein can be identified relative to a general class of enzymes that have homology to the largest multiple nuclease domain nuclease of the Type II CRISPR system. Most preferably, the effector protein is a Type II protein such as Cas9. By "derived" the applicants mean that the derived enzyme is essentially based on the wild-type enzyme in the sense that it has a high degree of sequence homology to that enzyme, but has been mutated (modified) in some manner as known in the art or described herein.

Again, it should be appreciated that the terms Cas and CRISPR enzyme and CRISPR protein and Cas protein are generally used interchangeably and, in all references herein, refer by analogy to the novel CRISPR effector proteins further described in the present application, unless otherwise obvious, such as by specific reference to Cas9. As mentioned above, many of the residue numbering orders used herein refer to the effector protein from the type II CRISPR locus. However, it should be appreciated that the present invention includes many more effector proteins from other microbial species.

List of organisms as a possible source of Cas protein

The Cas protein may include a Cas protein from an organism of the genus Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, or Corynebacter.

The Cas9 protein may include a Cas9 protein from an organism of the genus Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, or Corynebacter.

Preferred examples include S. pyogenes, S. aureus.

In one embodiment, the Cas9 protein may be an ortholog from an organism of a genus including, but 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 species of such organism may be other than those discussed herein.

Some methods for identifying orthologs of CRISPR-Cas enzymes may involve identifying tracr sequences in the genomes of interest. Identifying tracr sequences may involve the following steps: searching a database for direct repeats or tracr mate sequences to identify the CRISPR region containing the CRISPR enzyme; searching for homologous sequences in the CRISPR region flanking the CRISPR enzyme in both the sense and antisense directions; searching for transcription terminators and secondary structures; identifying any sequence that is not a direct repeat or tracr mate sequence but has greater than 50% identity to a direct repeat or tracr mate sequence as a potential tracr sequence; obtaining a potential tracr sequence and analyzing it for associated transcription terminator sequences.

It should be appreciated that any of the functional properties described herein may be engineered into CRISPR enzymes from other orthologs, including chimeric enzymes comprising fragments from multiple orthologs. Examples of such orthologs are described elsewhere in this document. Thus, chimeric enzymes may include fragments of orthologs of a CRISPR enzyme from an organism that 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 comprise a first fragment and a second fragment, and wherein the fragments may be orthologs of a CRISPR enzyme from organisms of the genera referred to herein or from species referred to herein; preferably, the fragments are fragments of orthologs of a CRISPR enzyme from different species.

To minimize toxicity and off-target effects, regulation of the concentration of CRISPR enzyme mRNA and guide RNA delivered will be important. Optimal concentrations of CRISPR enzyme mRNA and guide RNA can be determined by testing different concentrations in a cell or animal model and using deep sequencing to analyze the degree of modification at potential off-target loci in the genome. For example, for a guide sequence targeting 5'-GAGTCCGAGCAGAAGAAGAA-3' in the EMX1 gene of the human genome, deep sequencing can be used to determine the level of modification at the following two off-target loci: 1: 5'-GAGTCCTAGCAGGAGAAGAA-3' and 2: 5'-GAGTCTAAGCAGAAGAAGAA-3'. For in vivo delivery, the concentration that results in the highest level of on-target modification while minimizing the level of off-target modification should be selected.

Delivery: options for DNA/RNA, or DNA/DNA, or RNA/RNA, or protein/RNA

In some embodiments, the components of the CRISPR system may be delivered in various forms, such as DNA/RNA, or RNA/RNA, or protein/RNA combinations. For example, Cas9 may be delivered as a DNA-encoding polynucleotide, or an RNA-encoding polynucleotide, or as a protein. The guide may be delivered as a DNA-encoding polynucleotide or RNA. All possible combinations are contemplated, including mixed forms of delivery.

In some embodiments, all such combinations (DNA/RNA, or DNA/DNA, or RNA/RNA, or protein/RNA) are provided.

In a particular embodiment, if Cas9 is delivered in protein form, it may be pre-assembled with one or more guides.

Delivery: nanocones

In addition, the CRISPR system can be delivered using nanococoons, such as described in Sun W et al., Cocoon-like self-degradable DNA nanoclew for anticancer drug 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 Engl. 2015 Oct 5;54(41):12029-33. doi: 10.1002/anie.201506030. Epub 2015 Aug 27.

Delivery - GalNAc

The components of the CRISPR complex can be delivered by conjugation or association with transport moieties (adapted, for example, from the approaches disclosed in U.S. Patent Nos. 8,106,022; 8,313,772, incorporated herein by reference). Nucleic acid delivery strategies, for example, can be used to improve the delivery of guide RNA, or messenger RNA, or coding DNA encoding components of the CRISPR complex, including the CRISPR protein. For example, RNAs may include modified RNA nucleotides to improve 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 constructs have been described that can be used to modify nucleic acids such as gRNA for more efficient delivery, such as reversible charge neutralizing modifications of the phosphotriester backbone that can be adapted to modify gRNA to be more hydrophobic and non-anionic, thereby improving cellular uptake (Meade BR et al., 2014, Nature Biotechnology 32, 1256-1261). In further alternative embodiments, selected RNA motifs can be used to mediate cellular transfection (Magalhães 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 adding aptamers to gRNA (Tan W. et al., 2011, Trends in Biotechnology, December 2011, Vol. 29, No. 12).

In some embodiments, conjugation of tri-branched N-acetylgalactosamine (GalNAc) to oligonucleotide components can be used to improve delivery, such as delivery for selection of cell types, such as hepatocytes (see, WO2014118272, incorporated herein by reference; Nair, JK et al., 2014, Journal of the American Chemical Society 136 (49), 16958-16961). This can be considered a sugar-based particle, and further details on other particulate delivery systems and/or formulations are provided in the relevant section herein. Therefore, GalNAc can be considered a particle in the same sense as the other particles described herein, such that the general applications and other considerations, such as delivery of said particles, also apply to GalNAc particles. A liquid phase conjugation strategy, for example, can be used to attach tri-branched GalNAc clusters (MW ~2000), activated as PFP (pentafluorophenyl) esters, to 5′-hexylamino-modified oligonucleotides (5′-HA ASO, MW ~8000 Da; ∅stergaard et al., Bioconjugate Chem., 2015, 26 (8), pp 1451-1455). Similarly, poly(acrylate) polymers have been described for in vivo nucleic acid delivery (see WO2013158141, incorporated herein by reference). In further alternative embodiments, pre-mixing of CRISPR nanoparticles (or protein complexes) with naturally occurring whey proteins can be used to improve delivery (Akinc A et al, 2010, Molecular Therapy vol. 18 no. 7, 1357-1364).

Screening methodologies are available to identify delivered enhancers, such as by screening chemical libraries (Gilleron J. et al., 2015, Nucl. Acids Res. 43 (16): 7984-8001). Approaches to assess the efficiency of delivery vehicles, such as lipid nanoparticles, have also been described, which can be used to identify efficient delivery vehicles for CRISPR components (see Sahay G. et al., 2013, Nature Biotechnology 31, 653-658).

In some embodiments, delivery of CRISPR protein components can be facilitated by adding functional peptides to the protein, such as peptides that alter the hydrophobicity of the protein, such as to improve in vivo functionality. Protein components of the CRISPR complex can similarly be modified to facilitate subsequent chemical reactions. For example, amino acids that have a group susceptible to click chemistry can be added to the protein (Nikić I. et al., 2015, Nature Protocols 10,780-791). In embodiments, such a click chemistry group can then be used in a wide variety of alternative structures, such as poly(ethylene glycol) for stability, cell penetrating peptides, RNA aptamers, lipids, or carbohydrates such as GalNAc. As further alternatives, the protein component of the CRISPR complex can be modified to tailor the protein for cell entry (see Svensen et al., 2012, Trends in Pharmacological Sciences, Vol. 33, No. 4), such as by adding cell-penetrating 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, patients or subjects can be pre-treated with compounds or formulations that facilitate subsequent delivery of the CRISPR complex components.

Induced systems

In some embodiments, the CRISPR enzyme may form a component of an inducible system. The inducible nature of the system will allow for spatiotemporal control of gene editing or gene expression using a form of 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.), or light-inducible systems (phytochrome, LOV, or cryptochrome domains). In one embodiment, the CRISPR enzyme may be part of a light-inducible transcriptional effector (LITE) to drive changes in transcriptional activity in a sequence-specific manner. Components of a light-inducible system may include a CRISPR enzyme, a light-sensitive cytochrome heterodimer (e.g., from Arabidopsis thaliana), and a transcriptional activation/repression domain. Additional examples of inducible DNA binding proteins and methods of using them are provided in US 61/736,465, US 61/721,283 and WO 2014/018423, which are incorporated herein by reference in their entireties.

Self-inactivating systems

Once all copies of a gene in a cell's genome have been edited, further CRISRP/Cas9 expression in that cell is no longer required. Indeed, maintaining expression would be undesirable in the event of off-target effects at sites in the genome not intended for editing, etc. Thus, time-limited expression would be desirable. Inducible expression provides one solution to this problem, but the applicants have also constructed a self-inactivating CRISPR-Cas9 system that relies on the use of a non-coding targeting guide sequence in the CRISPR vector itself. Thus, once expression has begun, the CRISPR system will cause its own destruction, but before the destruction is complete, it will have enough time to edit the genomic copies of the target gene (which, in terms of a normal point mutation in a diploid cell, would require no more than two edits). Briefly, the self-inactivating CRISPR-Cas system includes an additional RNA (i.e., a guide RNA) that targets a coding sequence for the CRISPR enzyme itself or that targets one or more non-coding guide target sequences complementary to unique sequences present in one or more of the following:

(a) in the promoter that controls the expression of non-coding RNA elements,

(b) in the promoter driving the expression of the Cas9 gene,

(c) in the 100 bp sequence of the translation initiation start codon ATG in the Cas9 coding sequence,

(d) in the inverted terminal repeat (iTR) of a viral delivery vector, such as in the AAV genome.

Moreover, such RNA can be delivered via a vector, such as a separate vector or the same vector that encodes the CRISPR complex. When administered via a separate vector, the CRISPR RNA that targets Cas expression can be administered sequentially or simultaneously. When administered sequentially, the CRISPR RNA that targets Cas expression can be delivered after the CRISPR RNA that is intended, for example, for gene editing or gene recombination. The period can be a period measured in minutes (e.g., 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes). The period can be a period measured in hours (e.g., 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours). The period can be a period measured in days (e.g., 2 days, 3 days, 4 days, 7 days). The period may be a period measured in weeks (e.g., 2 weeks, 3 weeks, 4 weeks). The period may be a period measured in months (e.g., 2 months, 4 months, 8 months, 12 months). The period may be a period measured in years (e.g., 2 years, 3 years, 4 years). In this way, the Cas enzyme binds to a first gRNA/chiRNA capable of hybridizing to a first target, such as a genomic locus or loci of interest, and performs the function(s) required for the CRISPR-Cas system (e.g., gene editing); and subsequently, the Cas enzyme may then associate with a second gRNA/chiRNA capable of hybridizing to a sequence containing at least a portion of the Cas or CRISPR cassette. If the gRNA/chiRNA is targeted to sequences encoding the expression of the Cas protein, the enzyme is blocked and the system becomes self-inactivating. Similarly, CRISPR RNA that targets the expression of Cas delivered via, for example, liposome, lipofection, particles, microvesicles, as explained in this paper, can be delivered sequentially or simultaneously. In simple terms, self-inactivation can be used to inactivate one or more guide RNAs used to target one or more targets.

In some aspects, a single gRNA is provided that is capable of hybridizing to a sequence downstream of a start codon of a CRISPR enzyme, wherein after a certain period of time, there is a loss of expression of the CRISPR enzyme. In some aspects, 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, wherein after a certain period of time, there is inactivation of one or more, or in some cases, all, of the CRISPR-Cas systems. In some aspects of the system and without being limited by theory, a cell can comprise a plurality of CRISPR-Cas complexes, wherein a first subset of the CRISPR complexes comprises a first chiRNA capable of targeting a locus or loci of the genome to be edited, and a second subset of the CRISPR complexes comprises at least one second chiRNA capable of targeting a polynucleotide encoding a CRISPR-Cas system, wherein the first subset of the CRISPR-Cas complexes mediate editing of the target locus or loci of the genome, and the second subset of the CRISPR complexes subsequently inactivate the CRISPR-Cas system, thereby further inactivating CRISPR-Cas expression in the cell.

Thus, the present invention relates to a CRISPR-Cas system comprising one or more vectors for delivery into a eukaryotic cell, wherein the vector(s) encode(s): (i) a CRISPR enzyme; (ii) a first guide RNA capable of hybridizing to a target sequence in the cell; (iii) a second guide RNA capable of hybridizing to one or more target sequences in a vector that encodes the CRISPR enzyme; (iv) at least one tracr mate sequence; and (v) at least one tracr sequence. The first and second complexes may use the same tracr and tracr mate, differing only in the guide sequence, wherein, upon expression in the cell, the first guide RNA directs sequence-specific binding of the first CRISPR complex to a sequence in the cell; the second guide RNA directs sequence-specific binding of the second CRISPR complex to a target sequence in the vector that encodes the CRISPR enzyme; wherein the CRISPR complexes comprise (a) a tracr mate sequence hybridized to a tracr sequence, and (b) a CRISPR enzyme associated with a guide RNA such that the guide RNA can hybridize to its target sequence; and a second CRISPR complex inactivates the CRISPR-Cas system to prevent further expression of the CRISPR enzyme by the cell.

Additional characteristics of the vector(s), encoded enzymes, guide sequences, etc. are disclosed elsewhere in this document. For example, one or both guide sequences may be part of a chiRNA sequence that provides a guide, a tracr mate, and a tracr sequence in a single RNA such that such a system can encode (i) a CRISPR enzyme; (ii) a first chiRNA comprising a sequence capable of hybridizing to a first target sequence in a cell, a first tracr mate sequence, and a first tracr sequence; (iii) a second guide RNA capable of hybridizing to a vector that encodes a CRISPR enzyme, a second tracr mate sequence, and a second tracr sequence. In simple terms, the enzyme may include one or more NLSs, etc.

The different coding sequences (CRISPR enzyme, guide RNAs, tracr, and tracr mate) can be introduced into a single vector or into multiple vectors. For example, it is possible to encode the enzyme in one vector and the different RNA sequences in another vector, or to encode the enzyme and one chiRNA in one vector and the remaining chiRNA in another vector, or any other combination. In general, a system using only one or two different vectors is preferred.

When using multiple vectors, it is possible to deliver them in unequal quantities, and ideally with an excess of vector that encodes the first guide RNA linked to the second guide RNA, thereby delaying the final inactivation of the CRISPR system until genome editing has occurred.

The first guide RNA may target any target sequence of interest in the genome, as described elsewhere herein. The second guide RNA targets any sequence in a vector that encodes the CRISPR Cas9 enzyme, thereby inactivating expression of the enzyme driven by the vector. Thus, the target sequence in the vector should be capable of inactivating expression. Suitable target sequences may be, for example, adjacent to or within the translation initiation start codon of the Cas9 coding sequence, in a non-coding sequence in a promoter driving expression of non-coding RNA elements, within a promoter driving expression of the Cas9 gene, within 100 bp of the translation initiation ATG start codon in the Cas9 coding sequence, and/or within an inverted terminal repeat (iTR) of a viral delivery vector, such as in the AAV genome. A double-strand break near this site could induce a frameshift in the Cas9 coding sequence, causing loss of protein expression. An alternative target sequence for the "self-inactivating" guide RNA would be to target editing/inactivation of regulatory regions/sequences that are essential for expression of the CRISPR-Cas9 system or for vector stability. For example, if the promoter structure for the Cas9 coding sequence is disrupted, then transcription would be repressed or prevented. In simple terms, if the vector includes sequences that ensure replication, maintenance, or stability, then these sequences could be targeted. For example, in an AAV-based vector, a suitable target sequence is within the iTR. Other suitable targeting sequences could be promoter sequences, polyadenylation sites, etc.

Moreover, if the guide RNAs are expressed in an array format, then "self-inactivating" guide RNAs that target both promoters simultaneously will result in excision of intervening nucleotides within the CRISPR-Cas expression construct, causing virtual complete inactivation. Simply put, excision of intervening nucleotides will result from targeting both ITRs with guide RNAs or from targeting two or more CRISPR-Cas components simultaneously. As explained in this paper, self-inactivation is generally applicable to CRISPR-Cas9 systems to mediate CRISPR-Cas9 regulation. For example, as explained in this paper, self-inactivation can be used for CRISPR-mediated repair of mutations, such as expansion-mediated disorders, as explained in this paper. The result of such self-inactivation is transient CRISPR-mediated repair activity.

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 to ensure editing at a target genomic locus prior to CRISPR-Cas9 shutdown.

In one aspect of the self-inactivating AAV-CRISPR-Cas9 system, plasmids that co-express one or more sgRNAs that target sequences of interest in the genome (e.g., 1-2, 1-5, 1-10, 1-15, 1-20, 1-30) can be engineered with "self-inactivating" sgRNAs that target a SpCas9 sequence at or near an engineered ATG start site (e.g., within 5 nucleotides, within 15 nucleotides, within 30 nucleotides, within 50 nucleotides, within 100 nucleotides). A regulatory sequence in the U6 promoter region can also be targeted by sgRNAs. U6-controlled sgRNAs can be engineered in an array format so that multiple sgRNA sequences can be released simultaneously. Upon initial delivery to target tissue/cells (left cell), sgRNAs begin to accumulate while Cas9 levels increase in the nucleus. Cas9 complexes with all sgRNAs to mediate genome editing and self-inactivation of CRISPR-Cas9-bearing plasmids.

One aspect of the self-inactivating CRISPR-Cas9 system is the expression in a single or tandem array 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 guide sequence can specifically target a different target. Such sequences can be processed, for example, from a single chimeric pol3 transcript. Pol3 promoters such as the U6 or H1 promoters can be used. Pol2 promoters are mentioned throughout this document. Inverted terminal repeat (iTR) sequences can flank the Pol3 promoter-sgRNA-Pol2 promoter-Cas9.

In one aspect, the chimeric transcript in tandem format comprises one or more guide sequences that edit one or more targets, while one or more self-inactivating guide sequences inactivate the CRISPR/Cas9 system. Thus, for example, the described CRISPR-Cas9 system for repairing expansion-related disorders can be directly combined with the self-inactivating CRISPR-Cas9 system described herein. Such a system can, for example, have two guide sequences directed to a target site for repair, as well as at least a third guide sequence that directs self-inactivation of CRISPR-Cas9. Reference is made to application Serial No. PCT/US2014/069897, entitled "Compositions and methods for using CRISPR-Cas systems in nucleotide repeat-associated disorders," published December 12, 2014 as WO/2015/089351.

Sets

In one aspect, the present invention relates to kits comprising any one or more of the elements disclosed in the above methods and compositions. The elements may be provided separately or in combinations and may be provided in any suitable container, such as an ampoule, vial or tube. In some embodiments, the kit includes instructions in one or more languages, such as multiple languages.

In some embodiments, the kit comprises one or more reagents for use in a method that uses one or more of the elements described herein. The reagents can be provided in any suitable container. For example, the kit can provide one or more reaction buffers or storage buffers. The reagents can be provided in a form that is useful in a particular assay or in a form that allows for the addition of one or more other components prior to use (e.g., in a concentrate form or a lyophilized form). The buffer can be any buffer, including but not limited to, a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH of about 7 to about 10. In some embodiments, the kit comprises one or more oligonucleotides corresponding to the guide sequence for insertion into the vector to operably link the guide sequence and the regulatory element. In some embodiments, the kit comprises a template polynucleotide for homologous recombination. In some embodiments, the kit comprises one or more vectors and/or one or more polynucleotides described herein. Advantageously, the kit may provide all elements of the systems of the present invention.

Nucleic acid targeting systems and methods

The term "nucleic acid targeting system", wherein the nucleic acid is DNA or RNA, and in some aspects may also be DNA-RNA hybrids or derivatives thereof, collectively refers to transcripts and other elements involved in the expression of or control of the activity of genes associated with DNA- or RNA-targeting CRISPR ("Cas"), which may include sequences encoding a DNA- or RNA-targeting Cas protein and a DNA- or RNA-targeting guide RNA comprising a CRISPR RNA sequence (crRNA) and (in some, but not all systems) a CRISPR/Cas transactivating RNA sequence (tracrRNA), or other sequences and transcripts from a DNA- or RNA-targeting CRISPR locus. Typically, an RNA targeting system is characterized by elements that facilitate the formation of a DNA- or RNA-targeting complex at the site of a DNA or RNA target sequence. In the context of forming a DNA or RNA targeting complex, a "target sequence" refers to a DNA or RNA sequence to which a DNA or RNA targeting guide RNA is designed to be complementary, wherein hybridization between the target sequence and the RNA targeting guide RNA facilitates formation of the RNA targeting complex. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell.

In one aspect of the present invention, novel DNA targeting systems, also referred to as DNA targeting CRISPR/DNA targeting Cas system or CRISPR-Cas according to the present application, are based on the identified Cas9 proteins, which do not require the production of individualized proteins to target specific DNA sequences, but rather one effector protein or enzyme can be programmed by an RNA molecule to recognize a specific DNA target, in other words, the enzyme can bind to a specific DNA target using said RNA molecule. Aspects of the present invention relate in particular to DNA targeting RNA-guided CRISPR SpCas9 systems.

In one aspect, the present invention provides methods of using one or more elements of a nucleic acid targeting system. The nucleic acid targeting complex of the present invention provides an effective means for modifying a target DNA or RNA (single- or double-stranded, linear or supercoiled). The nucleic acid targeting complex of the present invention has a wide utility, including modifying (e.g., causing deletion, insertion, translocation, inactivation, activation) of a target DNA or RNA in a variety of cell types. As such, the nucleic acid targeting complex of the present invention has a wide range of applications, such as in gene therapy, drug screening, diagnosis and prognosis of diseases. A typical nucleic acid targeting complex comprises a DNA or RNA targeting effector protein complexed with a guide RNA hybridized to a target sequence at a target locus of interest.

The nucleic acid targeting systems, vector systems, vectors and compositions described herein can be used in a variety of nucleic acid targeting applications, altering or modifying the synthesis of a gene product such as a protein, cleaving nucleic acids, editing nucleic acids, splicing nucleic acids; turnover of target nucleic acids, tracking of target nucleic acids, isolation of target nucleic acids, visualization of target nucleic acids, etc.

Aspects of the present invention also encompass methods and uses of the compositions and systems described herein in genome engineering, such as 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.

In one embodiment, the present invention relates to a method of cleaving a target DNA. The method may comprise modifying the target DNA using a nucleic acid targeting complex that binds to the target DNA and cleaves the target DNA. In one embodiment, the nucleic acid targeting complex of the present invention, when introduced into a cell, may form a break (e.g., a single- or double-strand break) in an RNA sequence. For example, the method may be used to cleave a disease-causing RNA in a cell. For example, an exogenous RNA template comprising a sequence to be integrated flanked by an upstream sequence and a downstream sequence may be introduced into the cell. The upstream and downstream sequences are characterized by sequence similarity to each side of the integration site in the RNA. Optionally, the donor RNA may be mRNA. The exogenous RNA template comprises a sequence to be integrated (e.g., a mutated RNA). The sequence to be integrated may be a sequence endogenous or exogenous to the cell. Examples of a sequence to be integrated include a protein encoding RNA or a non-coding RNA (e.g., a microRNA). Thus, the sequence to be integrated may be operably linked to a corresponding regulatory sequence or sequences. Alternatively, the sequence to be integrated may provide a regulatory function. The upstream and downstream sequences of the exogenous RNA template are selected to promote recombination between the RNA sequence of interest and the donor RNA. An upstream sequence is an RNA sequence that shares sequence similarity with an RNA sequence upstream of the integration site to be targeted. Similarly, a downstream sequence is an RNA sequence that shares sequence similarity with an RNA sequence downstream of the integration site to be targeted. The upstream and downstream sequences of the exogenous RNA template may have 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the RNA sequence to be targeted. Preferably, the upstream and downstream sequences of the exogenous RNA template have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the RNA sequence to be targeted. In some methods, the upstream and downstream sequences of the exogenous RNA template have about 99% or 100% sequence identity to the RNA sequence to be targeted. The upstream or downstream sequence may comprise from about 20 bp to about 25 bp. to about 2,500 bp, such as 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, the exemplary upstream or downstream sequence is from about 200 bp to about 2,000 bp, from about 600 bp to about 1,000 bp, or more particularly from about 700 bp. to about 1000 bp. In some methods, the exogenous RNA template may further comprise a marker. Such a marker may facilitate screening for targetable integrations. Examples of suitable markers include restriction sites, fluorescent proteins, or selectable markers. The exogenous RNA template of the present invention can be constructed using recombination techniques (see, e.g., Sambrook et al., 2001, and Ausubel et al., 1996). In a method of modifying a target DNA by integrating an exogenous DNA template, a break (e.g., a double- or single-strand break) is introduced into the DNA sequence by a nucleic acid targeting complex, and the break is repaired by homologous recombination involving the exogenous template DNA such that the template integrates into the target DNA. The presence of the double-strand break ensures integration of the template. In other embodiments, the present invention relates to a method of modifying RNA expression in a eukaryotic cell. The method comprises increasing or decreasing the expression of a target polynucleotide using a nucleic acid targeting complex that binds to a DNA-encoding RNA (e.g., mRNA or pre-mRNA). In some methods, the target DNA can be inactivated to effect the modification of expression in the cell. For example, after the DNA targeting complex binds to a target sequence in the cell, the target DNA is inactivated such that the sequence is not transcribed, whereby the encoded protein is not produced, or the sequence does not function as a wild-type sequence. For example, a sequence encoding a protein or microRNA can be inactivated such that the protein or microRNA or pre-microRNA transcript is not produced. The target DNA of the DNA targeting complex can be any DNA, endogenous or exogenous to the eukaryotic cell. For example, the target DNA can be DNA located in the nucleus of the eukaryotic cell. The target DNA may be a sequence encoding a gene product (e.g., mRNA or pre-mRNA), encoding a gene product (e.g., a protein), or a non-coding sequence (e.g., ncRNA, lncRNA, tRNA, or rRNA). Examples of target DNA include a sequence associated with a biochemical signaling pathway, such as DNA associated with a biochemical signaling pathway. Examples of target DNA include disease-associated DNA. "Disease-associated" DNA refers to any DNA that provides transcription products at an abnormal level or in an abnormal form in cells derived from disease-affected tissues compared to disease-free control tissues or cells. This may be DNA transcribed from a gene that begins to be expressed at an abnormally high level; it may be DNA transcribed from a gene that begins to be expressed at an abnormally low level, where the altered expression correlates with the onset and/or progression of the disease. Disease-associated DNA also means DNA transcribed from a gene carrying a mutation(s) or genetic variation that is directly responsible for, or is in linkage disequilibrium with, the gene(s) responsible for the etiology of the disease. The translated products may be known or unknown and may be present at normal or abnormal levels. The target DNA of a DNA-targeting complex may be any DNA, endogenous or exogenous to the eukaryotic cell. For example, the target DNA may be DNA located in the nucleus of a eukaryotic cell. The target DNA may be a sequence encoding a gene product (e.g., mRNA, pre-mRNA, protein) or a non-coding sequence (e.g., ncRNA, lncRNA, tRNA, or rRNA).

In some embodiments, the method may comprise causing a nucleic acid targeting complex to bind to a target DNA to effect cleavage of said target DNA, thereby modifying the target DNA, wherein the nucleic acid targeting complex comprises a nucleic acid targeting effector protein in complex with a guide RNA hybridized to a target sequence within said target DNA. In one aspect, the present invention relates to a method of modifying the expression of DNA or RNA in a eukaryotic cell. In some embodiments, the method comprises causing a nucleic acid targeting complex to bind to the DNA such that said binding results in increased or decreased expression of said DNA; wherein the nucleic acid targeting complex comprises a nucleic acid targeting effector protein in complex with a guide RNA. Similar considerations and conditions apply to the methods of modifying a target DNA set forth above. In fact, these sampling, culturing and reintroduction options are encompassed by aspects of the present invention. In one aspect, the present invention relates to methods for modifying target DNA in a eukaryotic cell, which may occur in vivo, ex vivo or in vitro. In some embodiments, the method comprises selecting a cell or a population of cells from a human or non-human animal and modifying the cell or cells. The culturing may be performed at any stage ex vivo. The cell or cells may even be reintroduced into a non-human animal or into a plant. With respect to the cells reintroduced, it is particularly preferred that the cells are stem cells.

Indeed, in any aspect of the present invention, the nucleic acid targeting complex may comprise a nucleic acid targeting effector protein in complex with a guide RNA hybridized to a target sequence.

The present invention relates to the design and optimization of systems, methods and compositions used to control gene expression, providing for targeting a DNA sequence, which relate to a nucleic acid targeting system and its components. An advantage of the methods of the present invention is that the CRISPR system minimizes or eliminates off-target binding and the resulting side effects. This is achieved by using systems that provide for a high degree of sequence specificity with respect to the target DNA.

With respect to the nucleic acid targeting complex or system, it is preferable that the tracr sequence has one or more hairpins and has a length of 30 or more nucleotides, 40 or more nucleotides, or 50 or more nucleotides; wherein the crRNA sequence has a length of 10 to 30 nucleotides, the effector protein for targeting the nucleic acid is a type II Cas9 effector protein.

Editing and modification

In one aspect, the present invention relates to methods of using one or more elements of a CRISPR system. The CRISPR complex of the present invention provides an efficient means for modifying a target polynucleotide. The CRISPR complex of the present invention has broad applicability, including modifying (e.g., causing deletion, insertion, translocation, inactivation, activation) of a target polynucleotide in a variety of cell types. As such, the CRISPR complex of the present invention has a wide range of applications, such as in gene therapy, drug screening, diagnosis and prognosis of diseases. An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence in a target polynucleotide. In certain embodiments, a direct repeat sequence is linked to the guide sequence.

DNA cleavage and repair

The method includes modifying a target polynucleotide using a CRISPR complex that binds to the target polynucleotide and cleaves the target polynucleotide. Typically, the CRISPR complex of the present invention, when introduced into a cell, creates a break (e.g., a single-strand or double-strand break) in a genomic sequence. For example, the method can be used to cleave a gene responsible for the development of a disease in a cell. The break created by the CRISPR complex can be repaired by a repair process, such as error-prone non-homologous end joining (NHEJ) or high-fidelity repair involving homologous recombination (HDR). During this repair process, an exogenous template polynucleotide can be introduced into the genomic sequence. In some methods, the HDR process is used to modify the genomic sequence. For example, an exogenous template polynucleotide comprising a sequence to be integrated flanked by an upstream sequence and a downstream sequence is introduced into a cell. The upstream and downstream sequences are characterized by sequence similarity to each side of the integration site in the chromosome. If desired, the donor polynucleotide can be DNA, such as plasmid DNA, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear DNA fragment, a PCR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. The exogenous template polynucleotide comprises a sequence to be integrated (e.g., a mutated gene). The sequence to be integrated can be a sequence endogenous or exogenous to the cell. Examples of a sequence to be integrated include polynucleotides encoding a protein or non-coding RNA (e.g., a microRNA). Thus, the sequence to be integrated may be operably linked to a corresponding regulatory sequence or sequences. Alternatively, the sequence to be integrated may provide a regulatory function. The upstream and downstream sequences in the exogenous template polynucleotide are selected to promote recombination between the chromosomal sequence of interest and the donor polynucleotide. The upstream sequence is a nucleic acid sequence that shares sequence similarity with a genomic sequence located upstream of the integration site to be targeted. Similarly, the downstream sequence is a nucleic acid sequence that shares sequence similarity with a chromosomal sequence located downstream of the integration site to be targeted. The upstream and downstream sequences of the exogenous template polynucleotide may have 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the genomic sequence to be targeted. The upstream and downstream sequences of the exogenous template polynucleotide preferably have 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the genomic sequence to be targeted. In some methods, the upstream and downstream sequences of the exogenous template polynucleotide have about 99% or 100% sequence identity to the genomic sequence to be targeted. The upstream or downstream sequence may comprise from about 20 bp to about 25 bp. to about 2,500 bp, such as 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, the exemplary upstream or downstream sequence is from about 200 bp to about 2,000 bp, from about 600 bp to about 1,000 bp, or more particularly from about 700 bp. to about 1000 bp. In some methods, the exogenous template polynucleotide may further comprise a marker. Such a marker may facilitate screening for targetable integrations. Examples of suitable markers include restriction sites, fluorescent proteins, or selectable markers. The exogenous template polynucleotide of the present invention can be constructed using recombinant DNA techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996). In a method for modifying a target polynucleotide by integrating an exogenous template polynucleotide into a genomic sequence, a double-strand break is introduced using a CRISPR complex, the break is repaired by homologous recombination involving the exogenous template polynucleotide, such that the template is integrated into the genome. The presence of the double-strand break ensures integration of the template. In other embodiments, the present invention relates to a method for modifying the expression of a polynucleotide in a eukaryotic cell. The method comprises increasing or decreasing the expression of a target polynucleotide using a CRISPR complex that binds to the polynucleotide. In some methods, the target polynucleotide may be inactivated to effect the modification of expression in the cell. For example, after the CRISPR complex binds to the target sequence in the 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 a wild-type sequence. For example, a sequence encoding a protein or microRNA may be inactivated such that the protein or microRNA or pre-microRNA transcript is not produced. In some methods, a regulatory sequence may be inactivated such that it no longer functions as a regulatory sequence. As used herein, the term "regulatory sequence" refers to any nucleic acid sequence that affects the transcription, translation, or accessibility of a nucleic acid sequence. Examples of a regulatory sequence include a promoter, a transcription terminator, and an enhancer, which are regulatory sequences. A target polynucleotide for a CRISPR complex can be any polynucleotide that is endogenous or exogenous to a eukaryotic cell. For example, a target polynucleotide can be a polynucleotide found in the nucleus of a eukaryotic cell. A target polynucleotide can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA). Examples of target polynucleotides include a sequence associated with a biochemical signaling pathway, such as a gene or polynucleotide associated with a biochemical signaling pathway. Examples of target polynucleotides include a disease-associated gene or polynucleotide. A "disease-associated" gene or polynucleotide means any gene or polynucleotide that provides transcription or translation products at an abnormal level or in an abnormal form in cells derived from diseased tissues compared to disease-free control tissues or cells. This may be a gene that begins to be expressed at an abnormally high level; it may be a gene that begins to be expressed at an abnormally low level, where the altered expression correlates with the onset and/or progression of a disease. A disease-associated gene also means a gene that carries a mutation(s) or genetic variation that is directly responsible for, or is in linkage disequilibrium with, the gene(s) responsible for the etiology of the disease. The transcribed or translated products may be known or unknown and may be present at normal or abnormal levels. The target polynucleotide for a CRISPR complex may be any polynucleotide, endogenous or exogenous to the eukaryotic cell. For example, the target polynucleotide may be a polynucleotide located in the nucleus of a eukaryotic cell. The target polynucleotide may be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA).

Gene editing or target locus modification using Cas9; HDR and arrays

The double-strand break or single-strand break in one of the strands should preferably be close enough to the target position so that correction occurs. In one embodiment, the distance is no more than 50, 100, 200, 300, 350, or 400 nucleotides. Without being limited by theory, it is believed that the break should be close enough to the target position so that the break is in a region that is subject to exonuclease-mediated removal during the final resection. If the distance between the target position and the break is too large, the mutation cannot be included in the final resection and therefore cannot be corrected, since only the template nucleic acid sequence can be used to correct the sequence at the final resection site.

In one embodiment, wherein the guide RNA and a Type II molecule, in particular Cas9 or its ortholog or homolog, preferably Cas9 nuclease, induce a double-strand break to induce HDR-mediated correction, the cleavage site is 0-200 bp away. (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, 75-125, 75-100 bp) from the target position. In one embodiment, the cleavage site is 0-100 bp away. (e.g., 0-75, 0-50, 0-25, 25-100, 25-75, 25-50, 50-100, 50-75, or 75-100 bp) from the target position. In a further embodiment, two or more guide RNAs complexed with Cas9 or an ortholog or homolog thereof can be used to induce multiplex breaks to induce HDR-mediated correction.

The homology arm should extend at least to the site where the final resection can occur, for example, to allow the resected single-stranded "sticky" end to find a complementary site in the donor template. The overall length may be limited by parameters such as plasmid size or virus packaging limits. In one embodiment, the homology arm may not extend to the repeat elements. A typical homology arm length is at least 50, 100, 250, 500, 750, or 1000 nucleotides.

A target position, as used herein, refers to a site in a target nucleic acid or a target gene (e.g., a chromosome) that is modified by a Cas9 type II dependent process, in particular Cas9 or an ortholog or homolog thereof, preferably a Cas9 molecule. For example, a target position can be a modified Cas9 molecule cleavage of a target nucleic acid and a modification directed to a template nucleic acid, such as a correction of a target position. In one embodiment, a target position can be a site between two nucleotides, such as adjacent nucleotides, in a target nucleic acid, to which one or more nucleotides are added. A target position can comprise one or more nucleotides that are altered, such as corrected, by a template nucleic acid. In one embodiment, a target position is in a target sequence (e.g., a sequence to which a guide RNA binds). In one embodiment, the target position is upstream or downstream of a target sequence (e.g., a sequence to which a guide RNA binds).

A template nucleic acid, as used herein, refers to a nucleic acid sequence that can be used in combination with a Type II molecule, in particular Cas9 or an ortholog or homolog thereof, preferably a Cas9 molecule and a guide RNA molecule to alter the structure of a target location. In one embodiment, the target nucleic acid is modified to provide some or all of the template nucleic acid sequence, typically at or near the cleavage site(s). In one embodiment, the template nucleic acid is single-stranded. In an alternative embodiment, the template nucleic acid is double-stranded. In one embodiment, the template nucleic acid is DNA, such as double-stranded DNA. In an alternative embodiment, the template nucleic acid is single-stranded.

In one embodiment, the template nucleic acid alters the structure of the target position by participating in homologous recombination. In one embodiment, the template nucleic acid alters the sequence of the target position. In one embodiment, the template nucleic acid results in the incorporation of a modified or non-naturally occurring base into the target nucleic acid.

The template sequence may undergo cleavage-mediated or catalyzed recombination with the target sequence. In one embodiment, the template nucleic acid may include a sequence that corresponds to a site in the target sequence that is cleaved by Cas9-mediated cleavage conversion. In one embodiment, the template nucleic acid may include a sequence that corresponds to both a first site in the target sequence that is cleaved by a first Cas9-mediated event and a second site in the target sequence that is cleaved by a second Cas9-mediated event.

In certain embodiments, the template nucleic acid may include a sequence that results in a change in the coding sequence of the translated sequence, such as a sequence that results in a substitution of one amino acid for another in the protein product, such as a transformation of a mutant allele into a wild-type allele, a transformation of a wild-type allele into a mutant allele, and/or the introduction of a stop codon, an insertion of an amino acid residue, a deletion of an amino acid residue, or a nonsense mutation. In certain embodiments, the template nucleic acid may include a sequence that results in a change in a non-coding sequence, such as a change in an exon or in a 5' or 3' untranslated or untranscribed region. Such changes include a change in a control element, such as a promoter, an enhancer, and a change in a cis-acting or trans-acting control element.

A template nucleic acid having homology to a target position in a target gene can be used to alter the structure of the target sequence. The template sequence can be used to alter an unwanted structure, such as an unwanted or mutant nucleotide. The template nucleic acid can include a sequence that when integrated results in a decrease in the activity of a positive control element; an increase in the activity of a positive control element; a decrease in the activity of a negative control element; an increase in the activity of a negative control element; a decrease in gene expression; an increase in gene expression; an increase in resistance to a disorder or disease; an increase in resistance to viral entry; a correction of a mutation or an alteration of an unwanted amino acid residue, provision, enhancement, abolition or reduction of a biological property of a gene product, such as an increase in the enzymatic activity of an enzyme, or an increase in the ability of the gene product to interact with another molecule.

The template nucleic acid may include a sequence that results in a change 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 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 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, 110 +/- 20, 120 +/- 20, 130 +/- 20, 140 +/- 20, 150 +/- 20, 160 +/- 20, 170 +/- 20, 180 +/- 20, 190 +/- 20, 200 +/- 20, 210 +/- 20, or 220 +/- 20 nucleotides. In one embodiment, the template nucleic acid has a length of 10-1000, 20-900, 30-800, 40-700, 50-600, 50-500, 50-400, 50-300, 50-200, or 50-100 nucleotides.

The template nucleic acid comprises the following components: [5' homologous arm]-[substitution sequence]-[3' homologous arm]. The homologous arms mediate recombination in the chromosome, thereby replacing an undesired element, such as a mutation or signature, with a substitution sequence. In one embodiment, the homologous arms flank the most distal cleavage sites. In an embodiment, the 3' end of the 5' homologous arm is a position adjacent to the 5' end of the substitution sequence. In one embodiment, the 5' homologous arm may extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5' from the 5' end of the substitution sequence. In an embodiment, the 5' end of the 3' homologous arm is a position adjacent to the 3' end of the substitution sequence. In an embodiment, the 3' homologous arm may extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 3' from the 3' end of the substitution sequence.

In certain embodiments, one or both homologous arms may be shortened to avoid the inclusion of certain repetitive sequence elements. For example, the 5' homologous arm may be shortened to avoid the repetitive sequence element. In other embodiments, the 3' homologous arm may be shortened to avoid the repetitive sequence element. In some embodiments, both the 5' and 3' homologous arms may be shortened to avoid the inclusion of certain repetitive sequence elements.

In certain embodiments, mutation correction template nucleic acids can be designed for use as a single-stranded oligonucleotide. When using a single-stranded oligonucleotide, the length of the 5' and 3' homologous arms can vary up to about 200 base pairs (bp), such as at least 25, 50, 75, 100, 125, 150, 175, or 200 bp.

DNA repair and NHEJ

In certain embodiments, nuclease-induced non-homologous end joining (NHEJ) can be used to target gene-specific knockouts. Nuclease-induced NHEJ can also be used to remove (e.g., delete) a sequence in a gene of interest. Typically, NHEJ repairs a double-strand break in DNA by joining the two ends together; however, typically, the original sequence is restored only if the two compatible ends, just as if they were formed by the double-strand break, are completely ligated. The DNA ends of a double-strand break are often enzymatically processed, resulting in the addition or removal of nucleotides on one or both strands before the ends are rejoined. This results in the presence of insertion and/or deletion (insertional-deletion) mutations in the DNA sequence at the NHEJ repair site. Two-thirds of such mutations typically alter the reading frame and therefore produce a non-functional protein. Additionally, mutations that maintain the reading frame but that insert or delete a significant portion of sequence may disrupt protein functionality. This depends on the locus, since mutations in critical functional domains are likely to be less tolerated than mutations in non-critical regions of the protein. The insertion/deletion mutations created by NHEJ are unpredictable in nature; however, at a given breakpoint, some insertion/deletion sequences are favored and overrepresented in the population, likely due to small regions of microhomology. Deletion lengths can vary widely; most commonly in the range of 1-50 bp, but they can freely exceed 50 bp, for example, they can freely reach more than approximately 100-200 bp. Insertions are typically shorter and often involve short repeats of sequences immediately surrounding the breakpoint. However, large insertions can occur, and in these cases the inserted sequence often extends to other regions of the genome or to plasmid DNA present in the cells.

Because NHEJ is a mutagenic process, it can also be used to remove small sequence motifs, provided that formation of a specific final sequence is not required. If a double-strand break is targeted near a short target sequence, deletion mutations induced by NHEJ repair often span and therefore remove the unwanted nucleotides. For deletion of larger DNA segments, introduction of two double-strand breaks, one on either side of the sequence, can result in NHEJ between the ends, removing the entire insertion sequence. Both of these approaches can be used to remove specific DNA sequences; however, the error-prone nature of NHEJ can still result in insertion/deletion mutations at the repair site.

Both a double-stranded type II cleavage molecule, in particular Cas9 or an ortholog or homolog thereof, preferably Cas9 molecules, and a single-stranded or type II nickase molecule, in particular Cas9 or an ortholog or homolog thereof, preferably Cas9 molecules, can be used in the methods and compositions described herein to create NHEJ-mediated insertions/deletions. NHEJ-mediated insertions/deletions targeting a gene, such as a coding region, such as an early coding region of a gene of interest, can be used to knock out (i.e., eliminate expression of) the gene of interest. For example, an early coding region of a gene of interest includes a sequence immediately after the transcription start site, in the first exon of the coding sequence, or within 500 bp. transcription start site (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp).

In one embodiment, wherein the guide RNA and the Type II molecule, in particular Cas9 or an ortholog or homolog thereof, preferably a Cas9 nuclease, forms a double-strand break to induce NHEJ-mediated insertions/deletions, the guide RNA may be configured to place one double-strand break in close proximity to the nucleotide of the target position. In one embodiment, the cleavage site may be within 0-500 bp 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 bp of the target position).

In one embodiment, wherein two guide RNAs complexed with type II molecules, in particular Cas9 or its ortholog or homolog, preferably Cas9 nickases, induce two single-strand breaks to induce NHEJ-mediated insertions/deletions, the two guide RNAs may be configured to accommodate two single-strand breaks to ensure NHEJ repair of the target nucleotide.

Delivery of functional effectors

Unlike CRISPR-Cas-mediated gene knockout, which permanently abolishes expression by mutating the gene at the DNA level, CRISPR-Cas knockdown allows for a transient reduction in gene expression using artificial transcription factors. Mutating key residues in both DNA cleavage domains of the Cas9 protein results in the formation of a crystal-inactive Cas9. The crystal-inactive Cas9 complexes with a guide RNA and localizes to a DNA sequence defined by the targeting domain of the guide RNA, but does not cleave the target DNA. Fusion of the inactive Cas9 protein to an effector domain, such as a transcriptional silencing domain, facilitates recruitment of the effector to a DNA site defined by the guide RNA. In certain embodiments, Cas9 may be fused to a transcriptional silencing domain and recruited to a promoter region of a gene. In particular, for gene silencing, it is envisaged herein that blocking the binding site of an endogenous transcription factor will promote gene expression silencing. In another embodiment, an inactive Cas9 may be fused to a chromatin modifying protein. The alteration of the chromatin state may result in decreased expression of the target gene.

In one embodiment, a guide RNA molecule may target known transcriptional responsive elements (e.g., promoters, enhancers, etc.), known upstream activating sequences, and/or sequences of unknown or known function that are believed to be capable of controlling the expression of the target DNA.

In some methods, the target polynucleotide may be inactivated to modify expression in the cell. For example, after the CRISPR complex binds to the target sequence in the cell, the target polynucleotide is inactivated so that the sequence is not transcribed, the encoded protein is not produced, or the sequence does not function as the wild-type sequence. For example, a sequence encoding a protein or microRNA may be inactivated so that the protein is not produced.

In certain embodiments, the CRISPR enzyme comprises one or more mutations selected from the group consisting of D917A, E1006A, and D1225A, and/or the one or more mutations are in the RuvC domain of the CRISPR enzyme or are another mutation discussed herein. In some embodiments, the CRISPR enzyme has one or more mutations in the catalytic domain, wherein upon transcription, the direct repeat sequence forms a single stem-loop and the guide sequence directs sequence-specific binding of the CRISPR complex to a target sequence, and wherein the enzyme further comprises a functional domain. In some embodiments, the functional domain is a transcriptional activation 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 concatemers of SIDs (e.g., SID4X). In some embodiments, the functional domain is an epigenetic modification domain, such that an epigenetic modification enzyme is provided. In some embodiments, the functional domain is an activation domain, which may be a P65 activation domain.

Functional effectors (domains)

Gene editing can be performed using Cas9 according to the present invention. Cas9 can be inactivated and fused to one or more functional domains (effectors).

In some embodiments, the functional domain may be selected from the group consisting of a transposase domain, an integrase domain, a recombinase domain, a resolvase domain, an invertase domain, a protease domain, a DNA methyltransferase domain, a DNA hydroxylmethylase domain, a DNA demethylase domain, a histone acetylase domain, a histone deacetylase domain, a nuclease domain, a repressor domain, an activator domain, nuclear localization signal domains, a transcription regulatory protein (or transcription recruitment complex) domain, a cellular uptake activity-associated domain, a nucleic acid binding domain, an antibody presentation domain, a histone modifying enzyme, a histone modifying enzyme recruiter; inhibitor of histone modifying enzymes, histone methyltransferase, histone demethylase, histone kinase, histone phosphatase, histone ribosylase, histone deribosylase, histone ubiquitinase, histone deubiquitinase, histone biotinase and histone tail protease.

In some preferred embodiments, the functional domain is a transcriptional activation domain, preferably VP64. In some embodiments, the functional domain is a transcriptional repression domain, preferably KRAB. In some embodiments, the transcriptional repression domain is SID or SID concatemers (e.g., SID4X). In some embodiments, the functional domain is an epigenetic modification domain, such that an epigenetic modification enzyme is provided. In some embodiments, the functional domain is an activation domain, which may be a P65 activation domain.

Targeting a gene in non-dividing cells (neurons and muscle)

Non-dividing (especially non-dividing, fully differentiated) cell types, including muscle cells and especially neurons, present challenges for gene targeting or genome engineering, for example, because homologous recombination (HR) is typically downregulated in the G1 phase of the cell cycle. However, by investigating how cells control normal repair systems, Orthwein et al. reported a previously unknown switch that keeps HR “off” in non-dividing cells, and they developed a strategy to turn on this switch. Orthwein et al. (Daniel Durocher lab at Mount Sinai Hospital in Ottawa, Canada, in Nature 16142, published online December 9, 2015) showed that HR downregulation could be reversed and gene targeting successful in both kidney (293T) and osteosarcoma (U2OS) cells. Tumor suppressors BRCA1, PALB2, and BRAC2 are known to repair DNA DSBs via HR. They found that the formation of the BRCA1 complex with PALB2-BRAC2 is regulated by a ubiquitin site in PALB2, for example, by the action of an E3 ubiquitin ligase on the site. This E3 ubiquitin ligase consists of KEAP1 (a PALB2-interacting protein) in a complex with cillin-3 (CUL3)-RBX1. Ubiquitination of PALB2 inhibits its interaction with BRCA1 and is neutralized by deubiquitylase USP11, which itself is under cell cycle control. Restoration of the BRCA1-PALB2 interaction in combination with activation of DNA end resection is sufficient to induce homologous recombination in G1, as measured by a number of methods, including a CRISPR-Cas9-based gene targeting assay targeting USP11 or KEAP1 (expressed from the pX459 vector). However, when the BRCA1-PALB2 interaction was restored in G1-resected cells using either KEAP1 depletion or PALB2-KR mutant expression, a significant increase in gene targeting events was detected.

Thus, reactivation of HR in cells, especially non-dividing, fully differentiated cell types, including muscle cells and especially neurons, is preferred in some embodiments. In some embodiments, providing BRCA1-PALB2 interaction is preferred in some embodiments. In some embodiments, the target cell is a non-dividing cell. In some embodiments, the target cell is a neuron or a muscle cell. In some embodiments, the target cell is targeted in vivo. In some embodiments, the cell is in G1 and HR is suppressed.

In some embodiments, it is preferred to use KEAP1 depletion, such as inhibition of KEAP1 expression activity. KEAP1 depletion can be achieved via siRNA, such as shown in Orthwein et al. Alternatively, it is preferred to express a PALB2-KR mutant (lacking all eight Lys residues in the BRCA1 interaction domain), either in combination with KEAP1 depletion or alone.

PALB2-KR interacts with BRCA1 regardless of position in the cell cycle. Thus, ensuring or restoring the BRCA1-PALB2 interaction, especially in G1 cells, is advantageous in some embodiments, especially if the target cells are non-dividing or if removal and recapitulation (ex vivo gene targeting) is problematic, such as in neuronal or muscle cells. KEAP1 siRNA is preferred and is available from ThermoFischer.

In some embodiments, the BRCA1-PALB2 complex can be delivered to a G1 cell, either as a protein complex, a fusion protein, polynucleotides encoding BRCA1 and PALB2, or polynucleotides encoding a BRCA1-PALB2 fusion protein. Such polynucleotides can be under the control of a suitable promoter, for example, and delivered as described herein, either simultaneously and optionally in the same vector or vector system, as a CRISPR protein, or separately. Other options for providing HR in non-dividing, fully differentiated cell types, including muscle cells and especially neurons, can include direct delivery of PALB2 (using Cas9 fused to an affinity molecule for PALB2) and/or direct delivery of BRCA2 (using Cas9 fused to an affinity molecule for BRCA2).

In some embodiments, deubiquitylation of PALB2 can be achieved, for example, by enhanced expression or activity of USP11 deubiquitylase. Thus, in some embodiments, it is envisaged that a construct can be provided to achieve or activate expression or activity of USP11 deubiquitylase.

Knockdown of CRL4 can also be used to make KEAP1 inactive or reduce its activity. For example, CRL4 siRNA can be used. Alternatively, MLN4924 (an inhibitor of all CRL) can also be used to inactivate KEAP1.

It is particularly advantageous that 53BP knockout is also achieved, as it has been suggested that this is necessary for HDR activation (shown in Orthwein et al). 53BP knockout can also be achieved by siRNA.

Activating DNA resection (creating 3' sticky ends on either side of a DNA double-strand break) is also essential for HR activation in G1. This was accomplished by delivering the CtIP (or SAE2) gene ORF with a mutation (T847E) that mimics activating phosphorylation. The authors claim that this requirement can be circumvented by using dual Cas9 nickases to introduce 3' sticky ends (Hsu et al.). Therefore, this use of dual Cas9 nickases to introduce 3' sticky ends is advantageous in targeting non-dividing, fully differentiated cell types, including muscle cells and especially neurons.

In one embodiment, the present invention relates to a method for cleaving a target polynucleotide. The method includes using a CRISPR complex that binds to a target polynucleotide and cleaves said target polynucleotide. Typically, the CRISPR complex of the present invention, when introduced into a cell, creates a break (e.g., a single-strand break or a double-strand break) in a genomic sequence. For example, the method can be used to cleave a disease-causing gene in a cell.

The break created by the CRISPR complex can be repaired by a repair process such as error-prone non-homologous end joining (NHEJ) or high-fidelity repair involving homologous recombination (HDR). During this repair process, an exogenous template polynucleotide can be introduced into the genomic sequence. In some methods, the HDR process is used to modify the genomic sequence. For example, an exogenous template polynucleotide is introduced into the cell that contains the sequence to be integrated, flanked by an upstream sequence and a downstream sequence. The upstream and downstream sequences are characterized by sequence similarity to each side of the integration site in the chromosome.

If desired, the donor polynucleotide may be DNA, such as plasmid DNA, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), viral vector, linear DNA fragment, PCR fragment, naked nucleic acid, or nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.

The exogenous template polynucleotide comprises a sequence to be integrated (e.g., a mutated gene). The sequence to be integrated may be a sequence endogenous or exogenous to the cell. Examples of a sequence to be integrated include polynucleotides encoding a protein or non-coding RNA (e.g., microRNA). Thus, the sequence to be integrated may be operably linked to a corresponding regulatory sequence or corresponding regulatory sequences. Alternatively, the sequence to be integrated may provide a regulatory function.

The upstream and downstream sequences of the exogenous template polynucleotide are selected to promote recombination between the chromosomal sequence of interest and the donor polynucleotide. The upstream sequence is a nucleic acid sequence that has sequence similarity to a genomic sequence located upstream of the integration site to be targeted. Similarly, the downstream sequence is a nucleic acid sequence that has sequence similarity to a chromosomal sequence located downstream of the integration site to be targeted. The upstream and downstream sequences of the exogenous template polynucleotide may have 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the genomic sequence to be targeted. The upstream and downstream sequences of the exogenous template polynucleotide preferably have 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the genomic sequence being targeted. In some methods, the upstream and downstream sequences of the exogenous template polynucleotide have approximately 99% or 100% sequence identity to the genomic sequence being targeted.

The upstream or downstream sequence may be from about 20 bp to about 2500 bp, such as 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, an exemplary upstream or downstream sequence is from about 200 bp to about 2000 bp, from about 600 bp to about 1000 bp, or from about 1000 bp to about 1000 bp. or, more specifically, from about 700 bp to about 1000 bp.

In some methods, the exogenous template polynucleotide may further comprise a marker. Such a marker may facilitate screening for targetable integrations. Examples of suitable markers include restriction sites, fluorescent proteins, or selectable markers. The exogenous template polynucleotide of the present invention may be constructed using recombinant DNA techniques (see, e.g., Sambrook et al., 2001 and Ausubel et al., 1996).

In an exemplary method for modifying a target polynucleotide by integrating an exogenous template polynucleotide into a genomic sequence, a double-strand break is introduced using a CRISPR complex, the break is repaired by homologous recombination involving the exogenous template polynucleotide, so that the template is integrated into the genome. The presence of the double-strand break ensures the integration of the template.

In other embodiments, the present invention relates to a method for modifying the expression of a polynucleotide in a eukaryotic cell. The method comprises increasing or decreasing the expression of a target polynucleotide using a CRISPR complex that binds to the polynucleotide.

In some methods, the target polynucleotide may be inactivated to modify expression in the cell. For example, after the CRISPR complex binds to the target sequence in the cell, the target polynucleotide is inactivated so that the sequence is not transcribed, the encoded protein is not produced, or the sequence does not function as the wild-type sequence. For example, a sequence encoding a protein or microRNA may be inactivated so that the protein is not produced.

In some methods, a regulatory sequence can be inactivated such that it no longer functions as a regulatory sequence. As used herein, the term "regulatory sequence" refers to any nucleic acid sequence that affects the transcription, translation, or accessibility of a nucleic acid sequence. Examples of a regulatory sequence include a promoter, a transcription terminator, and an enhancer, which are regulatory sequences. The inactivated target sequence may comprise a deletion mutation (i.e., a deletion of one or more nucleotides), an insertion mutation (i.e., an insertion of one or more nucleotides), or a nonsense mutation (i.e., a substitution of one nucleotide for another nucleotide such that a stop codon is introduced). In some methods, inactivation of a target sequence results in a "knockout" of the target sequence.

Illustrative applications of the CRISPR Cas system

The present invention relates to a non-naturally occurring or engineered composition, or one or more polynucleotides encoding components of said composition, or a vector or delivery systems comprising one or more polynucleotides encoding components of said composition, for use in modifying a target cell in vivo, ex vivo or in vitro, and they can be made using a method that alters the cell such that after modification, the progeny or cell line of the cell modified by CRISPR retains the altered phenotype. The modified cells and progeny can be part of a multicellular organism, such as a plant or animal, using the CRISPR system ex vivo or in vivo with respect to the desired cell types. The CRISPR invention can be a therapeutic method of treatment. The therapeutic method of treatment can include gene or genome editing or gene therapy.

Modification of a target using the CRISPR-Cas system or complex

In one aspect, the present invention relates to methods for modifying a target polynucleotide in a eukaryotic cell, which may occur in vivo, ex vivo or in vitro. In some embodiments, the method comprises selecting a cell or a population of cells from a human or non-human animal and modifying the cell or cells. The culturing may be performed at any stage ex vivo. The cell or cells may even be reintroduced into a non-human animal or into a plant. With respect to the cells reintroduced, it is particularly preferred that these cells are stem cells.

In some embodiments, the method includes providing binding of a CRISPR complex to a target polynucleotide to effect cleavage of said target polynucleotide, thereby providing modification of the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence that hybridizes or is capable of hybridizing with a target sequence in said target polynucleotide, wherein said guide sequence is associated with a mate tracr sequence that, in turn, hybridizes with the tracr sequence.

In one aspect of the present invention, there is provided a method for modifying the expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises causing a CRISPR complex to bind to the polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence that hybridizes or is capable of hybridizing to a target sequence in said polynucleotide, wherein said guide sequence is linked to a tracr mate sequence that in turn hybridizes to the tracr sequence. Similar factors and conditions apply to the methods for modifying a target polynucleotide set forth above. Indeed, these sampling, culturing, and reintroduction options are encompassed by aspects of the present invention.

Indeed, in any aspect of the present invention, the CRISPR complex may comprise a CRISPR enzyme complexed with a guide sequence that hybridizes or is capable of hybridizing to a target sequence, wherein said guide sequence may be associated with a mate tracr sequence that in turn can hybridize to the tracr sequence. Similar factors and conditions apply to the methods for modifying a target polynucleotide set forth above.

Thus, each of the non-naturally occurring CRISPR enzymes described herein comprises at least one modification, and thus the enzyme is characterized by certain improved properties. In particular, each of the enzymes is capable of forming a CRISPR complex with a guide RNA. When such a complex is formed, the guide RNA is capable of binding to a target polynucleotide sequence, and the enzyme is capable of modifying the target locus. In addition, the enzyme in the CRISPR complex is characterized by a reduced ability to modify one or more off-target loci compared to the unmodified enzyme.

In addition, the modified CRISPR enzymes described herein include enzymes wherein in a CRISPR complex the enzyme is characterized by an increased ability to modify one or more target loci compared to an unmodified enzyme. Such functionality may be provided alone or provided in combination with the above-described functionality of reduced ability to modify one or more off-target loci. Any such enzymes may be provided with one of the additional modifications of the CRISPR enzyme as described herein, such as in combination with the activity provided by one or more associated heterologous functional domains, any additional mutations to reduce nuclease activity, etc.

In preferred embodiments of the present invention, a modified CRISPR enzyme is provided with a reduced ability to modify one or more off-target loci compared to an unmodified enzyme and an increased ability to modify one or more target loci compared to an unmodified enzyme. In combination with additional modifications of the enzyme, significantly increased specificity can be achieved. For example, a combination of such preferred embodiments with one or more additional mutations is provided, wherein the one or more additional mutations are in one or more catalytically active domains. Such additional catalytic mutations can confer nickase functionality, as described in detail elsewhere herein. In such enzymes, increased specificity can be achieved by increased specificity in terms of enzymatic activity.

Modifications to reduce off-target effects and/or enhance on-target effects as described above can be performed on amino acid residues located in the positively charged region/groove located between the RuvC-III and HNH domains. It is expected that any of the functional effects described above can be achieved by modifying amino acids in the aforementioned groove, but also by modifying amino acids near or outside the groove.

Additional functional properties that can be engineered into modified CRISPR enzymes as described herein may include the following. 1. Modified CRISPR enzymes that disrupt DNA-protein interactions without disrupting the tertiary or secondary structure of the proteins. This includes residues that contact any part of the RNA:DNA duplex. 2. Modified CRISPR enzymes that weaken interactions between proteins that hold Cas9 in a conformation required for nuclease cleavage in response to DNA binding (on-target or off-target). For example, a modification that slightly inhibits but still allows the nuclease conformation of the HNH domain (located at the cleavable phosphate). 3. Modified CRISPR enzymes that strengthen interactions between proteins that hold Cas9 in a conformation that inhibits activity in response to DNA binding (on-target or off-target). For example: a modification that stabilizes the HNH domain in a conformation beyond the scissile phosphate. Any such additional functional enhancement may be provided in combination with any other modification of the CRISPR enzyme, as described in detail elsewhere in this document.

Any of the improved functional properties described herein may be performed with respect to any CRISPR enzyme, such as a Cas9 enzyme. The Cas9 enzymes described herein are derived from the Cas9 enzymes from S. pyogenes and S. aureus. However, it is contemplated that any of the functional properties described herein may be engineered into Cas9 enzymes from other orthologs, including chimeric enzymes containing fragments from multiple orthologs. Examples of such orthologs can be found, for example, in Figures 8 and 9 described herein.

In the present invention, nucleic acids are used to bind target DNA sequences. This is advantageous because nucleic acids are much easier and cheaper to produce than proteins, and the specificity can vary with fragment length if homology is desired. For example, complex 3-D determination of the positions of multiple domains is not required. The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably. They denote a polymeric form of nucleotides of any length, both deoxyribonucleotides and ribonucleotides or their analogs. Polynucleotides can have any spatial structure and can perform any function, known or unknown. Non-limiting examples of polynucleotides are: coding or non-coding regions of a gene or gene fragment, loci(s) determined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNAs, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. The term also encompasses nucleic acid-like structures with synthetic backbones, see, for example, Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO 96/39154; 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 be introduced before or after assembly of the polymer. The nucleotide sequence may be interrupted by components other than nucleotides. The polynucleotide may be further modified after polymerization, such as by coupling with a labeling component. As used herein, the term "wild type" is a term of art understood by one skilled in the art and means the typical form of an organism, strain, gene or characteristic that occurs in nature, as distinguished from mutant or variant forms. "Wild type" represents the backbone. As used herein, the term "variant" is to be understood to mean the expression of properties that are characterized by a pattern that differs from that found in nature. The expressions "non-naturally occurring" and "engineered" are used interchangeably and indicate human intervention. The expressions, when applied 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 and found in nature. "Complementarity" means the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence, either by conventional Watson-Crick pairing or other non-conventional types. Percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 would be 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfect complementarity" means that all contiguous residues in the nucleic acid sequence will hydrogen bond with the same number of contiguous residues in the second nucleic acid sequence. As used herein, the term "substantially complementary" means a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% within a region of 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" in relation to hybridization means conditions under which a nucleic acid with complementarity to a target sequence preferentially hybridizes to the target sequence and substantially does not hybridize to non-targeted sequences. Stringent conditions are generally 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 described in detail 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 If a polynucleotide sequence is envisaged, complementary or partially complementary sequences are also envisaged. These sequences are preferably capable of hybridizing to the reference sequence under conditions of high stringency. Typically, to maximize the hybridization rate, hybridization conditions of relatively low stringency are selected: a temperature of approximately 20-25°C below the melting point temperature ( _Tm ). _Tm is the temperature at which 50% of a specific target sequence hybridizes to a perfectly complementary probe in solution at a given ionic strength and pH. Typically, if at least about 85% nucleotide complementarity of the hybridized sequences is desired, very stringent wash conditions are selected with a temperature of approximately 5-15°C lower than _Tm . If at least about 70% nucleotide complementarity of the hybridized sequences is desired, moderately stringent wash conditions are selected with a temperature of approximately 15-30°C lower than _Tm . Highly permissive (very low stringency) wash conditions may have a lowest temperature of 50°C below _Tm , which allows a high level of mismatches between the hybridized sequences. Those skilled in the art will appreciate that other physical and chemical parameters during the hybridization and wash steps may also be varied to influence the resulting detectable hybridization signal based on the particular level of homology between the target and probe sequences. Preferred high stringency conditions include incubation in 50% formamide, 5x SSC, and 1% SDS at 42°C, or incubation in 5x SSC and 1% SDS at 65°C with a wash of 0.2x 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 may occur via Watson-Crick base pairing, Hoogsteen bonding, or any other sequence-specific method. The complex may comprise two strands forming a duplex structure, three or more strands forming a multistrand complex, a single self-hybridizing strand, or any combination thereof. The hybridization reaction may be a step in a larger process, such as the initial step of PCR or the cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing to a given sequence is called a "complementary sequence" for the sequence. As used herein, the term "genomic locus" or "locus" (plural loci) is a specific position of a gene or DNA sequence on a chromosome. "Gene" refers to fragments of DNA or RNA that encode a polypeptide chain or RNA that plays a functional role in an organism, and therefore it is the molecular unit of heredity in living organisms. For the purpose of the present invention, genes may be considered to contain regions that regulate the formation of a gene product, whether the regulatory sequences are adjacent to coding and/or transcribed sequences or not. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences, such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus regulatory regions. As used herein, the term "expression of a genomic locus" or "gene expression" refers to the process by which the information of a gene is used in the synthesis of a functional gene product. Gene expression products are often proteins, but for non-protein-coding 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 necessary for survival. As used herein, "expression" of a gene or nucleic acid encompasses not only the expression of genes in cells, but also the transcription and translation of nucleic acid(s) in cloning systems and in any other context. As used herein, the term "expression" also refers to the process by which a polynucleotide is transcribed from a DNA template (such as to form an mRNA or other RNA transcript) and/or the process by which transcribed mRNA is further translated to form peptides, polypeptides, or proteins. The transcripts and encoded polypeptides may be referred to collectively as the "gene product." If the polynucleotide is derived from genomic DNA, expression may involve splicing of mRNA 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, it may contain modified amino acids, and its structure may be interrupted by non-amino acid components. The terms also encompass a polymer of amino acids that has been modified; for example, by disulfide bonding, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation such as coupling to a labeling component. As used herein, the term "amino acid" includes natural and/or non-natural or synthetic amino acids, including glycine and both D- and L-optical isomers, and 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 in aspects of the present invention, sequence identity refers to sequence homology. Homology comparisons can be made by eye or, more commonly, using readily available sequence comparison programs. These commercially available computer programs can calculate the percentage (%) homology between two or more sequences, and can also calculate the sequence identity between two or more amino acid sequences or nucleic acid sequences. In some preferred embodiments, the dTALE capping region described herein has sequences that are at least 95% identical or have sequence identity to the capping region of the amino acid sequences provided herein. Sequence homology values can be obtained using any of a number of computer programs known in the art, such as BLAST or FASTA, etc. A suitable computer program for performing such alignments is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid - Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410), and the GENEWORKS package as comparison tools. Offline and online searching is available in both BLAST and FASTA (see Ausubel et al., 1999 ibid, pages 7-58 - 7-60). However, the use of the GCG Bestfit program is preferred. Percentage (%) sequence homology can be calculated for contiguous sequences, i.e. one sequence is aligned with another sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called a "gapless" alignment. Typically, such gapless alignments are performed for only a relatively small number of residues. Although this method is very simple and consistent, it does not take into account the fact that, for example, in a pair of otherwise identical sequences, a single insertion or deletion may cause the following amino acid residues to be ignored in the alignment, thus potentially resulting in a significant decrease in % homology when the global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that account for possible insertions and deletions without unduly penalizing the overall homology or identity score. This is achieved by inserting "gaps" into the sequence alignment in an attempt to maximize local homology or identity. However, these more sophisticated methods assign "gap penalties" to each gap that occurs in the alignment, so that for the same number of identical amino acids, an alignment of sequences with the smallest possible number of gaps, reflecting a higher degree of relatedness between the two sequences being compared, may result in a higher score than an alignment with a larger number of gaps. Typically, "affine gap penalty values for related sequences" are used, which assign a relatively high value for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. Of course, high gap penalties can lead to optimized alignments with fewer gaps. Most alignment programs allow the gap penalties to be changed. However, it is preferable to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit software package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each residue beyond it. Calculating the maximum % homology therefore initially requires obtaining an optimal alignment that takes the gap penalties into account. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit software package (Devereux et al., 1984 Nuc. Acids Research 12 p387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 Short Protocols in Molecular Biology, ^4th Ed. - Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol. 403-410), and the GENEWORKS software package as comparison tools. Both BLAST and FASTA provide offline and online searching (see Ausubel et al., 1999, Short Protocols in Molecular Biology, pages 7-58 - 7-60). However, for some tasks the GCG Bestfit program is preferred. A new tool called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol Lett. 1999 174(2): 247-50; FEMS Microbiol Lett. 1999 177(1): 187-8 and the National Center for Biotechnology Information web site at the National Institutes of Health web site). Although the final % homology can be measured in units of identity, the alignment method itself is generally not based on an all-or-none comparison of pairs. Instead, a similarity-scoring substitution matrix is typically used, assigning scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix most commonly used is the BLOSUM62 matrix, the default matrix for the BLAST suite of programs. The GCG Wisconsin programs typically use either publicly available defaults or custom character comparison tables if provided (see the user manual for details). For some purposes, it is preferable to use the publicly available defaults for the GCG suite of programs or, in the case of other software, a default matrix such as BLOSUM62. Alternatively, percent homology can be calculated using the multiple alignment function in DNASIS™ (Hitachi Software) using an algorithm similar to CLUSTAL (Higgins DG & Sharp PM (1988) Gene 73(1), 237–244). Once the software has provided an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this by comparing the sequences and provides a numerical result. Sequences may also have deletions, insertions or substitutions of amino acid residues that result in a silent change and result in a functionally equivalent substance. Intentional amino acid substitutions may be made based on similarities in amino acid properties (e.g. 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 also more useful to include mutation data. The amino acid groups obtained in this way are likely to be conserved for structural reasons. These groups can be described in the form of a Venn diagram (Livingstone CD and Barton GJ (1993) "Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation" Comput. Appl. Biosci. 9: 745-756) (Taylor WR (1986) "The classification of amino acid conservation" J. Theor. Biol. 119; 205-218). Conservative substitutions can be made, for example, according to the table below, which describes the generally accepted 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 Small A G S

Embodiments of the present invention encompass sequences (both polynucleotide and polypeptide) that may comprise a homologous substitution (as used herein, both substitution and replacement mean the exchange of an existing amino acid residue or nucleotide for an alternative residue or nucleotide), which may occur, i.e., in the case of amino acids, a like-for-like substitution, such as basic for basic, acidic for acidic, polar for polar, etc. Non-homologous substitution may also occur, i.e., a residue from one class for a residue from another, or, alternatively, involving the inclusion of amino acids other than naturally occurring, such as ornithine (hereinafter referred to as Z), ornithinediaminobutyric acid (hereinafter referred to as B), norleucineornithine (hereinafter referred to as O), pyridylalanine, thienylalanine, naphthylalanine, and phenylglycine. Variant amino acid sequences may contain suitable spacer groups that can be inserted between any two amino acid residues of the sequence, including alkyl groups, for example, methyl, ethyl or propyl groups, in addition to amino acid spacers such as glycine or β-alanine residues. Another form of variation, which involves the presence of one or more amino acid residues in peptoid form, may be well understood by those skilled in the art. For the avoidance of doubt, "peptoid form" is used to refer to variant amino acid residues wherein the substituent group for the α-carbon is located on the nitrogen atom of the residue rather than on the α-carbon. Methods for producing 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.

Homology modeling. Corresponding residues in other Cas9 orthologs 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)—the protein–protein interaction (PPI) computational method for predicting interactions mediated by domain–motif boundaries. PrePPI (predicted PPI), a framework based on the PPI prediction method, combines structural evidence with non-structural evidence using the concept of Bayesian statistics. The method involves taking a pair of proteins and applying a structural alignment to identify structural elements that match either their experimentally determined structures or homology models. Structural alignment is further used to identify both nearby and distant structural neighbors through global and local geometric relationships. Whenever two neighboring structural elements form a complex described in the Protein Data Bank, it defines a matrix for modeling the interaction between the two proteins of interest. Models of the complex are created by superimposing the element structures onto their corresponding structural neighbor in the matrix. This approach is further described in Dey et al., 2013 (Prot Sci; 22: 359-66).

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 accomplished using natural or recombinant DNA polymerases, such as TaqGold™, T7 DNA polymerase, the Klenow fragment of E. coli DNA polymerase, and reverse transcriptase. The preferred amplification method is PCR.

In certain aspects, the present invention encompasses vectors. As used herein, a "vector" is a vehicle that enables or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another segment of DNA can be inserted to thereby effect replication of the inserted segment. Typically, a vector is capable of replication if associated with appropriate control elements. In general, the term "vector" refers to a nucleic acid molecule capable of transferring 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, no free ends (e.g., circular); nucleic acid molecules that contain DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular loop of double-stranded DNA into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, where virally derived DNA or RNA sequences are present in the vector for packaging into the virus (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses (AAV)). Viral vectors also include polynucleotides carried by the virus for transfection into a host cell. Certain vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., mammalian vectors other than episomal vectors) integrate into the host cell genome upon introduction into the host cell and are thus replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are functionally linked. Such vectors are referred to herein as "expression vectors". Common expression vectors suitable for recombinant DNA techniques are often in the form of plasmids.

Recombinant expression vectors may comprise a nucleic acid of the present invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors comprise one or more regulatory elements, which may be selected taking into account the host cells that are intended to be used for expression, which are operably linked to the nucleic acid sequence that is intended to be expressed. In the context of a recombinant expression vector, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) such that expression of the nucleotide sequence is allowed (e.g. in an in vitro transcription/translation system or in a host cell upon introduction of the vector into the host cell). With respect to recombination and cloning methods, reference should be made to U.S. Patent Application No. 10/815,730, published September 2, 2004 as US 2004-0171156 A1, the contents of which are incorporated herein by reference in their entirety.

Aspects of the present invention relate to bicistronic vectors for chimeric RNA and modified or mutated CRISPR enzymes (e.g. Cas9). Bicistronic expression vectors for chimeric RNA and modified or mutated CRISPR enzymes are preferred. In general and in particular, in this embodiment, the modified or mutated CRISPR enzymes are preferably driven by the CBh promoter. The chimeric RNA may preferably be driven by a Pol III promoter, such as the U6 promoter. Optimally, a combination of both. The chimeric guide RNA typically consists of a 20 bp guide sequence (Ns), which may be linked to a tracr sequence (extending from the first "U" in the downstream strand to the end of the transcript). The tracr sequence may be truncated at various specified positions. The guide and tracr sequences are separated by a tracr pair sequence, which may be GUUUUAGAGCUA. It may be followed by the shown GAAA loop sequence. Both of these are preferred examples. The present inventors have demonstrated Cas9-mediated insertions/deletions at the human EMX1 and PVALB loci using SURVEYOR nuclease assays. ChiRNAs are shown by their "+n" designation, and crRNA refers to a hybrid RNA in which the guide and tracr sequences are expressed as separate transcripts. Throughout this application, the chimeric RNA may also be referred to as a single guide or synthetic guide RNA (sgRNA). The loop is preferably GAAA, but is not limited to this sequence, or indeed only 4 bp in length. Indeed, preferred loop-forming sequences for use in hairpin structures are four nucleotides long, and most preferably have the sequence GAAA. However, longer or shorter loop sequences, as well as alternative sequences, may be used. The sequences preferably include a nucleotide triplet (e.g., AAA) and an additional nucleotide (e.g., C or G). Examples of loop-forming sequences include CAAA and AAAG. In practicing any of the methods disclosed herein, a suitable vector can be introduced into a cell or embryo via one or more methods known in the art, including, but not limited to, microinjection, electroporation, sonoporation, ballistic transfection, calcium phosphate-mediated transfection, cationic lipid particle transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impalefection, optical transfection, proprietary agent-stimulated nucleic acid uptake, and liposome, immunoliposome, virosome, or artificial virion delivery. In some methods, the vector is introduced into the embryo via microinjection. The vector or vectors can be microinjected into the nucleus or cytoplasm of the embryo. In some methods, the vector or vectors can be introduced into the cell by nucleofection.

The term "regulatory element" is intended to encompass promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., 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 direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct expression preferentially in a target tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a time-dependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, which may or may not also be tissue-specific or cell type-specific. In some embodiments, the vector comprises one or more pol III promoters (e.g., 1, 2, 3, 4, 5 or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5 or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5 or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, the U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase gene promoter, the β-actin gene promoter, the glycerophosphate kinase (PGK) gene promoter, and the EF1α promoter. Also encompassed by the term "regulatory element" are enhancer elements such as the WPRE; CMV; the R-U5' segment of the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); the SV40 enhancer; and the intronic sequence between exons 2 and 3 of the rabbit β-globin gene (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981). Those skilled in the art will appreciate that the configuration of the expression vector may depend on factors such as the choice of host cell to be transformed, the desired expression level, etc. The vector can be introduced into host cells to thereby produce transcripts, proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids described herein (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.). With respect to regulatory sequences, reference is made to U.S. Patent Application No. 10/491,026, 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 U.S. Patent Application No. 12/511,940, the contents of which are incorporated herein by reference in their entireties.

Vectors can be designed to express CRISPR transcripts (e.g., 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 further discussed 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 regulatory sequences of the T7 promoter and T7 polymerase.

Vectors can be introduced and propagated in a prokaryote or a prokaryotic cell. In some embodiments, the prokaryote is used to amplify copies of a vector that is to be introduced into a eukaryotic cell, or as an intermediate vector in the preparation of a vector that is to be introduced into a eukaryotic cell (e.g., by amplifying a plasmid as part of a viral vector packaging system). In some embodiments, the prokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism. Expression of proteins in prokaryotes is most often accomplished in Escherichia coli using vectors containing constitutive or inducible promoters that direct the expression of either fusion proteins or proteins other than fusion proteins. Fusion vectors add a number of amino acids to the protein encoded therein, such as to the amino terminus of a recombinant protein. Such fusion vectors may serve one or more purposes, such as: (i) to enhance expression of the recombinant protein; (ii) to enhance the solubility of the recombinant protein; and (iii) to aid in purification of the recombinant protein by functioning as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced into fusion expression vectors at the junction of the fusion fragment and the recombinant protein to facilitate separation of the recombinant protein from the fusion fragment after purification of the fusion protein. Such enzymes and their cognate recognition sequences include factor Xa, thrombin, and enterokinase. Illustrative fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA), and pRIT5 (Pharmacia, Piscataway, NJ), in which glutathione S-transferase (GST), maltose-binding protein E, or protein A, respectively, are fused to the target recombinant protein. Examples of suitable inducible non-fusion expression vectors for E. coli include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). In some embodiments, the vector is a yeast expression vector. Examples of vectors for expression in the yeast Saccharomyces cerivisae include pYepSec1 (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). In some embodiments, the vector directs protein expression in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc group (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL group (Lucklow and Summers, 1989. Virology 170: 31-39).

In some embodiments, the vector is capable of directing the 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 the expression vector are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma virus, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, see, for example, Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In some embodiments, recombinant mammalian expression vectors are capable of directing expression of a nucleic acid preferentially in a particular cell type (e.g., 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 gene promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid tissue-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), particularly the 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), neuron-specific promoters (e.g., the neurofilament gene promoter; Byrne and Ruddle, 1989). Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreatic cell-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary cell-specific promoters (e.g., the whey promoter; U.S. Patent No. 4,873,316 and European Patent Application Publication No. 264,166). Developmental stage-regulated promoters are also included, e.g., the mouse hox gene promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the alpha-fetoprotein gene promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546). With respect to these prokaryotic and eukaryotic vectors, reference should be made to U.S. Patent No. 6,750,059, the contents of which are incorporated herein by reference in their entirety. Other embodiments of the present invention may relate to viral vectors, which are mentioned in U.S. Patent Application Ser. No. 13/092,085, the contents of which are incorporated herein by reference in their entirety. Tissue-specific regulatory elements are known in the art and, in this regard, reference should be made to U.S. Patent No. 7,776,321, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the regulatory element is operably linked to one or more elements of the CRISPR system so as to direct the expression of one or more elements of the CRISPR system. In general, CRISPR (clustered regularly interspaced short palindromic repeats), also known as SPIDR (spacer-interrupted direct repeats), constitute a family of DNA loci that are typically specific to a particular bacterial species. The CRISPR locus includes a particular class of interspersed short sequence repeats (SSRs) that were discovered 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 alternating SSRs 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]; and Mojica et al., Mol. Microbiol., 17:85–93 [1995]). CRISPR loci are typically distinguished from other SSRs by the structure of repeats, which have been termed regularly interspaced short 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 elements that occur in groups that are regularly separated by unique intervening sequences of nearly constant length (Mojica et al., [2000], supra). Although the repeat sequences are highly conserved between strains, a number of interspersed repeats and spacer region sequences typically differ between strains (van Embden et al., J. Bacteriol., 182:2393–2401 [2000]). CRISPR loci have been identified in over 40 prokaryotes (see, e.g., Jansen et al., Mol. Microbiol., 43:1565–1575 [2002]; and Mojica et al., [2005]), including but not limited to Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema and Thermotoga.

In some embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more domains of a heterologous protein (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains in addition to the CRISPR enzyme). The fusion protein comprising the CRISPR enzyme may comprise any additional protein sequence and, optionally, a linker sequence between any two domains. Examples of protein domains that may be fused to the CRISPR enzyme include, but are not limited to, epitope tags, sequences from reporter genes, and protein domains with one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcript release 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 acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). The CRISPR enzyme may be fused to a gene sequence encoding a protein or protein fragment that binds to DNA molecules or binds to other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA-binding domain (DBD) fusions, GAL4 DNA-binding domain fusions, and herpes simplex virus (HSV) BP16 fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US20110059502, incorporated herein by reference. In some embodiments, a labeled CRISPR enzyme is used to identify the location of a target sequence.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA technology, all of which are within the skill of 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)); METHODS IN ENZYMOLOGY series (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)).

Models of genetic and epigenetic conditions

The method of the present invention can be used to create a plant, animal or cell that can be used as a model of a disease. As used herein, the term "disease" refers to a disease, disorder or symptom in a subject. For example, the method of the present invention can be used to create an animal or cell that contains a modification of one or more nucleic acid sequences associated with a disease, or a plant, animal or cell in which the expression of one or more nucleic acid sequences associated with a disease is altered. Such a nucleic acid sequence can encode a protein sequence associated with a disease, or can be a regulatory sequence associated with a disease. Accordingly, it is understood that in embodiments of the present invention, the plant, subject, patient, organism or cell can be a non-human subject, patient, organism or cell. Accordingly, the present invention relates to a plant, animal or cell produced by the method of the present invention, or a progeny thereof. The progeny may be a clone of the obtained plant or animal, or it may be obtained by sexual reproduction by crossing with other individuals of the same species to impart additional desired characteristics to their offspring. The cell may be in vivo or ex vivo in the case of multicellular organisms, in particular animals or plants. In the case of a cell in culture, a cell line may be obtained by performing appropriate culture conditions, and preferably if the cell is suitably adapted for this purpose (e.g. a stem cell). Also contemplated are bacterial cell lines obtained according to the present invention. Therefore, cell lines are also contemplated.

In some methods, the disease model can be used to study the effect of mutations on an animal or cell and the development and/or progression of the disease using measures commonly used in disease studies. Alternatively, such a disease model is useful for studying the effect of a pharmaceutically active compound on a disease.

In some methods, a disease model can be used to evaluate the effectiveness of a potential gene therapy strategy. Thus, a disease-associated gene or polynucleotide can be modified so that the development and/or progression of the disease is slowed or reduced. In particular, the method includes modifying a disease-associated gene or polynucleotide so that an altered protein is produced and, as a result, an altered response is observed in the animal or cell. Accordingly, in some methods, a genetically modified animal can be compared with an animal predisposed to developing a disease so that the effect of performing gene therapy can be assessed.

In another embodiment, the present invention relates to a method for producing a biologically active agent that modulates a signaling process in a cell associated with a gene responsible for the development of a disease. The method involves contacting a test compound with a cell containing one or more vectors that direct the expression of one or more of: a CRISPR enzyme, a guide sequence associated with a tracr pair sequence and a tracr sequence; and detecting a change in reading that indicates a decrease or increase in the signaling process in the cell associated, for example, with a mutation in a gene responsible for the development of a disease contained in the cell.

A cellular model or an animal model can be constructed in combination with the method of the present invention to screen for a change in cellular function. Such a model can be used to study the effect of a genomic sequence modified by a CRISPR complex of the present invention on a cellular function of interest. For example, a cellular function model can be used to study the effect of a modified genomic sequence on intracellular signaling or extracellular signaling. Alternatively, a cellular function model can be used to study the effects of a modified genomic sequence on sensory sensitivity. In some such models, one or more genomic sequences associated with a biochemical signaling pathway in the model are modified.

Several disease models were specifically examined. These include the genes CHD8, KATNAL2, and SCN2A, which are associated with risk for de novo autism, and the gene UBE3A, which is associated with syndromic autism (Angelman syndrome). These genes and the resulting autism models are, of course, preferred, but serve to demonstrate the broad applicability of the present invention to genes and corresponding models.

Altered expression of one or more genomic sequences associated with a biochemical signaling pathway can be determined by analyzing the difference in mRNA levels of the corresponding genes between a test model cell and a control cell when they are contacted with a candidate agent. Alternatively, differential expression of sequences associated with a biochemical signaling pathway is determined by detecting a difference in the level of the encoded polypeptide or gene product.

To analyze the change in the level of mRNA transcripts or corresponding polynucleotides induced by a certain agent, the nucleic acid contained in the sample is first extracted according to standard methods in the art. For example, messenger RNA can be isolated using various lytic enzymes or chemical solutions according to the procedures outlined in Sambrook et al. (1989), or extracted using nucleic acid binding resins according to the accompanying instructions provided by the manufacturers. The mRNA contained in the extracted nucleic acid sample is then detected using amplification techniques or conventional hybridization assays (e.g., Northern blot analysis) according to methods commonly known in the art or based on the methods illustrated herein.

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 using natural or recombinant DNA polymerases such as TaqGold™, T7 DNA polymerase, Klenow fragment of E. coli DNA polymerase and reverse transcriptase. A preferred amplification method is PCR. In particular, the isolated RNA can be subjected to reverse transcription analysis, which is combined with quantitative polymerase chain reaction (RT-PCR), to quantify the expression level of a sequence associated with a biochemical signal transduction pathway.

The detection of gene expression levels can be performed in a real-time amplification assay. In one aspect, the amplified products can be directly visualized using fluorescent DNA binding agents, including but not limited to DNA intercalators and DNA groove binding agents. Since the number of intercalators incorporated into double-stranded DNA molecules is generally proportional to the number of amplified DNA products, the amount of amplified products can be easily determined by quantifying the fluorescence of the intercalating dye using conventional optical systems from the prior art. DNA-binding dye suitable for this task includes SYBR green, SYBR blue, DAPI, propidium iodide, Hoechst, SYBR gold, ethidium bromide, acridines, proflavine, acridine orange, acriflavine, fluorocoumarin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium-polypyridyl complexes, anthramycin, etc.

In another aspect, other fluorescent labels, such as sequence-specific probes, can be used in the amplification reaction to provide detection and quantification of the amplified products. Probe-based quantitative amplification relies on sequence-specific detection of the desired amplified product. Target-specific fluorescent probes (e.g., TaqMan® probes) are used, resulting in increased specificity and sensitivity. Methods for performing probe-based quantitative amplification are well known in the art and are described in U.S. Patent No. 5,210,015.

In another aspect, conventional hybridization assays can be performed using hybridization probes that are characterized by sequence homology to sequences associated with a biochemical signaling pathway. Typically, in a hybridization reaction, the probes are allowed to form stable complexes with sequences associated with a biochemical signaling pathway contained in a biological sample obtained from a subject under study. One skilled in the art will appreciate that if an antisense nucleic acid is used as a probe, the target polynucleotides present in the sample are selected to be complementary to the antisense nucleic acid sequences. Conversely, if the nucleotide probe is a sense nucleic acid, the target polynucleotide is selected to be complementary to the sense nucleic acid sequences.

Hybridization can be performed under conditions of varying stringency. Suitable hybridization conditions for practicing the present invention are those such that the recognition interaction of the probe with sequences associated with the biochemical signaling pathway is both sufficiently specific and sufficiently stable. Conditions that increase the stringency of the hybridization reaction are well known in the art and are published. 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, silicon, and a variety of DNA chips. A preferred hybridization assay is performed on the high-density gene chips described in U.S. Patent No. 5,445,934.

To conveniently detect probe-target complexes formed during the hybridization assay, the nucleotide probes are conjugated to a detectable label. Detectable labels suitable for use in the present invention include any composition detectable by photochemical, biochemical, spectroscopic, immunochemical, electrical, optical or chemical means. A wide range of suitable detectable labels are known in the art, including fluorescent or chemiluminescent labels, radioactive isotope labels, enzymatic or other ligands. In preferred embodiments, a fluorescent label or an enzymatic label, such as digoxigenin, β-galactosidase, urease, alkaline phosphatase or peroxidase, avidin/biotin complex, is likely to be preferred.

The detection methods used to detect or quantify the intensity of hybridization will generally depend on the label selected above. For example, radioactive labels may be detected using photographic film or a phosphovisualizer. Fluorescent markers may be detected and quantified using a photodetector to detect the emitted light. Enzyme labels are typically detected by supplying the enzyme with a substrate and measuring the amount of reaction product formed upon exposure of the enzyme to the substrate; and finally, colorimetric labels are detected by simply visualizing the colored label.

An agent-induced change in the expression of sequences associated with a biochemical signaling pathway can also be determined by examining the corresponding gene products. Determining the level of a protein typically involves a) contacting a protein contained in a biological sample with an agent that specifically binds to a protein associated with a biochemical signaling pathway; and (b) identifying any agent:protein complex formed thereby. In one aspect of this embodiment, the agent that specifically binds a protein associated with a biochemical signaling pathway is an antibody, preferably a monoclonal antibody.

The reaction is carried out by contacting the agent with a sample of proteins associated with the biochemical signal transduction pathway obtained from the test samples under conditions that allow the formation of a complex between the agent and the proteins associated with the biochemical signal transduction pathway. The formation of the complex can be detected directly or indirectly according to standard procedures in the art. In a direct detection method, the agents are provided with a detectable label and unreacted agents can be removed from the complex; the amount of remaining label thus reflects the amount of complex formed. For such a method, it is preferable to select labels that remain attached to the agents even under stringent washing conditions. Preferably, the label does not interfere with the binding reaction. Alternatively, for an indirect detection procedure, an agent can be used that contains a label introduced either chemically or enzymatically. The desired label, as a rule, does not interfere with the binding or stability of the resulting agent:polypeptide complex. However, the label is typically designed to be accessible for efficient binding by the antibody and hence the production of a detectable signal.

A wide range of labels suitable for detecting protein levels are known in the art. Non-limiting examples include radioactive isotopes, enzymes, colloidal metals, fluorescent compounds, bioluminescent compounds, and chemiluminescent compounds.

The amount of agent:polypeptide complexes formed during the binding reaction can be quantified using standard quantitative assays. As illustrated above, agent:polypeptide complex formation can be measured directly by the amount of label remaining at the binding site. Alternatively, the pathway-associated protein is assayed for its ability to compete with a labeled analogue for binding sites on a specific agent. In this competition assay, the amount of label captured is inversely proportional to the amount of pathway-associated protein sequences present in the sample being tested.

A number of protein analysis techniques based on the general principles outlined above are available in the art. These include, but are not limited to, radioimmunoassays, ELISA (enzyme linked immunoradiometric assays), sandwich immunoassays, immunoradiometric assays, in situ immunoassays (using, for example, colloidal gold, enzyme or radioisotope labels), Western blot analysis, immunoprecipitation assays, immunofluorescence assays and SDS-PAGE.

Antibodies that specifically recognize or bind to proteins associated with a biochemical signaling pathway are preferred for performing the above-mentioned protein assays. If desired, antibodies that recognize a specific type of post-translational modification (e.g., modifications induced by a biochemical signaling pathway) can be used. Post-translational modifications include, but are not limited to, glycosylation, lipidation, acetylation, and phosphorylation. These antibodies are available from commercial suppliers. For example, anti-phosphotyrosine antibodies, which specifically recognize tyrosine-phosphorylated proteins, are available from a number of suppliers, including Invitrogen and Perkin Elmer. Anti-phosphotyrosine antibodies are particularly useful in detecting proteins that are differentially phosphorylated on their tyrosine residues in response to ER (endoplasmic reticulum) stress. Such proteins include, but are not limited to, eukaryotic translation initiation factor 2 alpha (eIF-2α). Alternatively, these antibodies can be produced using conventional polyclonal or monoclonal antibody production technologies by immunizing a host animal or antibody-producing cell with a target protein that has undergone the desired post-translational modification.

In the practical implementation of the claimed method, it may be necessary to determine the expression profile of a protein associated with a biochemical signal transduction pathway in various tissues of the body, in various cell types and/or in various subcellular structures. These studies can be carried out using tissue-specific, cell-specific or subcellular structure-specific antibodies capable of binding to protein markers that are predominantly expressed in certain tissues, cell types or subcellular structures.

Altered expression of a gene associated with a biochemical signaling pathway can also be determined by examining the change in activity of the gene product compared to a control cell. The assay for a specific agent-induced change in activity of a protein associated with a biochemical signaling pathway will depend on the biological activity and/or signaling pathway being examined. For example, if the protein is a kinase, a change in its ability to phosphorylate a substrate(s) at subsequent steps can be determined by a number of assays known in the art. Exemplary assays include, but are not limited to, immunoblotting and immunoprecipitation using antibodies, such as anti-phosphotyrosine antibodies, that recognize phosphorylated proteins. In addition, kinase activity can be detected using high-throughput chemiluminescent assays such as the AlphaScreen™ (available from Perkin Elmer) and eTag™ assays (Chan-Hui, et al. (2003) Clinical Immunology 111: 162-174).

If the protein associated with a biochemical signal transduction pathway is part of a signaling cascade that results in fluctuations in intracellular pH conditions, pH-sensitive molecules, such as fluorescent pH-sensitive dyes, can be used as reporter molecules. In another example, if the protein associated with a biochemical signal transduction pathway is an ion channel, fluctuations in membrane potential and/or intracellular ion concentration can be monitored. A number of commercial kits and high-throughput devices are particularly suitable for the rapid and reliable screening of ion channel modulators. Illustrative instruments include FLIPRTM (Molecular Devices, Inc.) and VIPR (Aurora Biosciences). These instruments are capable of simultaneously detecting reactions in more than 1000 sample wells in a microplate and providing real-time measurements and functional data within a second or even millisecond.

In practicing any of the methods disclosed 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, ballistic transfection, calcium phosphate-mediated transfection, cationic lipid particle transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impalefection, optical transfection, proprietary agent-stimulated nucleic acid uptake, and liposome, immunoliposome, virosome, or artificial virion delivery. In some methods, the vector is introduced into the embryo by microinjection. The vector or vectors may be microinjected into the nucleus or cytoplasm of the embryo. In some methods, the vector or vectors can be introduced into the cell via nucleofection.

Target locus, target polynucleotide; PAM sequence

A target polynucleotide for a CRISPR complex may be any polynucleotide that is endogenous or exogenous to a eukaryotic cell. For example, a target polynucleotide may be a polynucleotide located in the nucleus of a eukaryotic cell. A target polynucleotide may be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA). The target may be a control element or a regulatory element or a promoter or an enhancer or a silencer. In some embodiments, a promoter may be located at +200 bp or even +1000 bp from a TTS. In some embodiments, a regulatory region may be an enhancer. An enhancer typically extends more than +1000 bp from a TTS. More specifically, expression of eukaryotic protein-coding genes is typically regulated by multiple cis-acting transcriptional control regions. Some control elements are located close to the start site (promoter-proximal elements), whereas others are located further away (enhancers and silencers). Promoters define the site of transcription initiation and direct the binding of RNA polymerase II. Three types of promoter sequences have been identified in eukaryotic DNA. The TATA box is most often prevalent in rapidly transcribed genes. Initiation promoters are rare in some genes, and CpG islands are characteristic of transcribed genes. Promoter-proximal elements occur within ≈200 bp of the start site. Some such elements, up to ≈20 bp long, may help regulate a particular gene. Enhancers, which are typically ≈100–200 bp long, contain multiple control elements of 8–20 bp. They may be located anywhere from 200 base pairs to tens of thousands of base pairs upstream or downstream of a promoter, in an intron, or downstream of a final exon in a gene. Promoter-proximal elements and enhancers may be cell-type specific, functioning only in certain types of differentiated cells. However, any of these regions may be a target sequence and are encompassed by the concept that a target may be a control element, or a regulatory element, or a promoter, or an enhancer, or a silencer.

Typically, 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 and complexed with one or more nucleic acid targeting effector proteins) results in cleavage of one or both strands of DNA or RNA at or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs) the target sequence. As used herein, the term "sequence(s) associated with a target locus of interest" 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 of the target sequence, wherein the target sequence is contained within the target locus of interest).

Without being bound by theory, it is believed that the target sequence must be associated with a PAM (protospacer adjacent motif); i.e., a short sequence recognized by the CRISPR complex. The exact sequence and length requirements for a PAM vary depending on the CRISPR enzyme used, but PAMs are typically 2-5 bp long sequences located adjacent to the protospacer (i.e., the target sequence). Examples of PAM sequences are provided in the Examples section below, and one skilled in the art will be able to identify additional PAM sequences for use with a given CRISPR enzyme. In addition, engineering of a PAM interacting (PI) domain can provide programmability of PAM specificity, improve target site recognition accuracy, and increase the versatility of a Cas genome engineering platform such as Cas9. Cas proteins such as Cas9 proteins can be engineered to alter their PAM specificity, such as described by Kleinstiver BP et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015 Jul 23;523(7561):481-5. doi: 10.1038/nature14592. In some embodiments, the method comprises causing a CRISPR complex to bind to a target polynucleotide to cleave said target polynucleotide, thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence in said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence that in turn hybridizes to the tracr sequence. In one aspect, the present invention provides a method of modifying the expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises causing a CRISPR complex to bind to a polynucleotide, whereby said binding results in increased or decreased expression of said polynucleotide; wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence in said polynucleotide, wherein said guide sequence is linked to a tracr mate sequence that in turn hybridizes to the tracr sequence. Similar factors and conditions apply to the methods of modifying a target polynucleotide set forth above. Indeed, these sampling, culturing, and reintroduction options are encompassed by aspects of the present invention. In one aspect, the present invention relates to methods of modifying a target polynucleotide in a eukaryotic cell, which may occur in vivo, ex vivo, or in vitro. In some embodiments, the method comprises selecting a cell or population of cells from a human or non-human animal and modifying the cell or cells. The culturing may be performed at any stage ex vivo. The cell or cells can even be reintroduced into a non-human animal or plant. As for the cells to be reintroduced, it is particularly preferred that these cells are stem cells.

Indeed, in any aspect of the present invention, the CRISPR complex may comprise a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence, wherein said guide sequence may be associated with a mate tracr sequence, which in turn may hybridize to the tracr sequence.

The present invention relates to the design and optimization of systems, methods and compositions used to control gene expression, including targeted sequence manipulation, such as genome modification or gene editing, associated with the CRISPR-Cas system and its components. The Cas enzyme is Cas9. An advantage of the methods of the present invention is that the CRISPR system minimizes or eliminates off-target binding and the resulting side effects. This is achieved by using systems that provide for a high degree of sequence specificity with respect to the target DNA.

With respect to the CRISPR-Cas complex or system, it is preferable that the tracr sequence has one or more hairpins and has a length of 30 or more nucleotides, 40 or more nucleotides, or 50 or more nucleotides; wherein the guide sequence has a length of 10 to 30 nucleotides, and the CRISPR/Cas enzyme is a Cas9 type II enzyme.

The target polynucleotide for the CRISPR complex may include a number of disease-associated genes and polynucleotides, as well as genes and polynucleotides associated with biochemical signaling pathways, which are listed in U.S. Provisional Patent Applications 61/736,527 and 61/748,427, collectively referenced BI-2011/008/WSGR, Serial Number 44063-701.101, and BI-2011/008/WSGR, Serial Number 44063-701.102, respectively, both entitled "SYSTEMS METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION," filed December 12, 2012 and January 2, 2013. accordingly, each of the contents of which is incorporated herein by reference in its entirety.

Examples of target polynucleotides include a sequence associated with a biochemical signaling pathway, such as a gene or polynucleotide associated with a biochemical signaling pathway. Examples of target polynucleotides include a disease-associated gene or polynucleotide. A "disease-associated" gene or polynucleotide means any gene or polynucleotide that provides transcription or translation products at an abnormal level or in an abnormal form in cells derived from disease-affected tissues compared to disease-free control tissues or cells. This may be a gene that begins to be expressed at an abnormally high level; it may be a gene that begins to be expressed at an abnormally low level, where the altered expression correlates with the onset and/or progression of the disease. A disease-associated gene also means a gene that carries a mutation(s) or genetic variation that is directly responsible for, or is in linkage disequilibrium with, the gene(s) responsible for the etiology of the disease. The products being transcribed or translated may be known or unknown and may be present at normal or abnormal levels.

Genome-wide knockout screening

The CRISPR-Cas proteins and systems described herein can be used to perform functional genomic tests in an efficient and cost-effective manner. Whole genome CRISPR-Cas libraries can be used for such tests. Such tests and libraries can provide information on gene function, the involvement of genes in cellular pathways, 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 eliminates off-target binding and the resulting side effects. This is achieved by using systems that provide a high degree of sequence specificity for the target DNA.

A genome-wide library may comprise a plurality of CRISPR-Cas guide RNAs described herein comprising guide sequences that are capable of targeting a plurality of target sequences at a plurality of genomic loci in a population of eukaryotic cells. The population of cells 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 gene function of one or more gene products may be altered by the targeting. The targeting may result in a knockout of gene function. Targeting a gene product may involve more than one guide RNA. The gene product can be targeted with 2, 3, 4, 5, 6, 7, 8, 9 or 10 guide RNAs, preferably 3-4 per gene. Off-target modifications can be minimized (see, e.g., 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 Biotechnol doi:10.1038/nbt.2647 (2013)), incorporated herein by reference. Targeting can involve about 100 or more sequences. The targeting may involve approximately 1,000 or more sequences. The targeting may involve approximately 20,000 or more sequences. The targeting may involve the entire genome. The targeting may involve a panel of target sequences directed toward a relevant or desired pathway. The pathway may be an immune pathway. The pathway may be a cell division pathway.

One aspect of the present invention encompasses a genome-wide library that may contain a plurality of CRISPR-Cas system guide RNAs that may contain guide sequences capable of targeting a plurality of target sequences at a plurality of genomic loci, wherein said targeting results in a knockout of gene function. This library may potentially contain guide RNAs that target each and every gene in the genome of an organism.

In some embodiments of the present invention, the organism or subject is a eukaryote (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 subject is a non-human animal and may be an arthropod, such as an insect, or may be a nematode. In some methods of the present invention, the organism or subject is a plant. In some methods of the present invention, the organism or subject is a mammal or a non-human mammal. The non-human mammal may be, for example, a rodent (preferably a mouse or rat), an ungulate, or a primate. In some methods of the present invention, the organism or subject is an alga, including a microalga, or is a fungus.

Knockout of gene function may comprise introducing into each cell in a population of cells a vector system of one or more vectors containing an engineered, non-naturally occurring CRISPR-Cas system comprising I. a Cas protein and II. one or more guide RNAs, wherein components I and II may be on the same or on different vectors of the system introducing components I and II into each cell, wherein the guide sequence targets a unique gene in each cell, wherein the Cas protein is operably linked to a regulatory element, wherein upon transcribing the guide RNA containing the guide sequence directs sequence-specific binding of the Cas CRISPR-Cas system to a target sequence at genomic loci of a unique gene to induce cleavage of the genomic locus by the Cas protein and confirming different knockout mutations in a plurality of unique genes in each cell of the population of cells, thereby forming a library of knockout gene cells. The present invention provides that the cell population is a population of eukaryotic cells, and in a preferred embodiment, the cell population is a population of embryonic stem (ES) cells.

The one or more vectors may be plasmid vectors. The vector may be a single vector containing Cas9, sgRNA, and optionally a selectable marker in target cells. Without wishing to be bound by theory, the ability to simultaneously deliver Cas9 and sgRNA using a single vector allows for application to any cell type of interest without the need to first generate cell lines that express Cas9. The regulatory element may be an inducible promoter. The inducible promoter may be a doxycycline inducible promoter. In some methods of the present invention, expression of the guide sequence is under the control of a T7 promoter and is driven by expression of T7 polymerase. Confirmation of various knockout 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 1,000 or more unique genes. A knockout mutation can be achieved in 20,000 or more unique genes. A knockout mutation can be achieved in an entire genome. A knockout of gene function can be achieved in multiple unique genes that function in a specific physiological pathway or condition. The pathway or condition can be an immune pathway or condition. The pathway or condition can be a cell division pathway or condition.

The present invention also relates to a kit that contains the whole genome libraries mentioned herein. The kit may comprise one container containing vectors or plasmids containing a library according to the present invention. The kit may also comprise a panel that provides for the selection of unique CRISPR-Cas guide RNAs containing guide sequences from a library according to the present invention, wherein the selection is indicative of a particular physiological condition. The present invention contemplates that the targeting is about 100 or more sequences, about 1000 or more sequences, or about 20,000 or more sequences, or the entire genome. In addition, the panel of target sequences may be oriented to a relevant or desired pathway, such as an immune pathway or cell division.

In a further aspect of the present invention, the Cas9 enzyme may comprise one or more mutations and may be used as a standard DNA binding protein, fused or not fused to a functional domain. The mutations may be artificially introduced mutations or gain-of-function or loss-of-function mutations. The mutations may include, but are not limited to, mutations in one of the catalytic domains (D10 and H840) among the catalytic domains of RuvC and HNH, respectively. Additional mutations have been characterized. In one aspect of the present invention, the functional domain may be a transcriptional activation domain, which may be VP64. In other aspects of the present invention, the functional domain may be a transcriptional repression domain, which may be KRAB or SID4X. Other aspects of the present invention relate to a mutated Cas9 enzyme fused to domains that include, but are not limited to, a transcriptional activator, a transcriptional repressor, a recombinase, a transposase, a histone remodeling factor, a demethylase, a DNA methyltransferase, a cryptochrome, a light-inducible/regulated domain, or a chemical-inducible/regulated domain. Some methods according to the present invention may involve inducing expression of target genes. In one embodiment, inducing expression by targeting multiple target sequences at multiple loci in a population of eukaryotic cells is accomplished by using a functional domain.

In carrying out the present invention, it is useful to consider the following references:

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]; published in final edited form as: Science. 2014 Jan 3; 343(6166): 84–87.

Shalem et al. describe a new way to probe gene function at a genome-wide scale. Their studies showed that delivery of a genome-wide CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique targeting sequences enabled screening by both positive and negative selection in human cells. First, the authors demonstrated the use of the GeCKO library to identify genes essential for cell survival in cancer and pluripotent stem cells. Next, in a melanoma model, the authors screened for genes whose loss of function is involved in resistance to vemurafenib, a therapeutic that inhibits mutant protein kinase BRAF. Their studies showed that the top-ranking candidates included previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. The authors observed a high level of concordance between independent guide RNAs targeting the same gene and a high hit confirmation rate, thus demonstrating the promise of Cas9-based genome-scale screening.

Also, reference may be made to US Patent Application No. US20140357530 and PCT Patent Publication No. WO2014093701, which are hereby incorporated by reference.

Functional change and screening

In some embodiments, one or more functional domains are associated with a CRISPR enzyme, such as a type II Cas9 enzyme.

In some embodiments, one or more functional domains are associated with an adaptor protein, such as used with the modified guides of Konnerman et al. (Nature 517, 583-588, 29 January 2015).

In some embodiments, one or more functional domains are associated with a dead-sgRNA (dRNA). In some embodiments, a dRNA complex with active Cas9 directs gene regulation by the functional domain at one gene locus, while the sgRNA directs DNA cleavage by active Cas9 at another locus, e.g., as in Dahlman et al., 'Orthogonal gene control with a catalytically active Cas9 nuclease' (in press). In some embodiments, the dRNA is selected to provide maximum selectivity of regulation for a gene locus of interest over off-target regulation. In some embodiments, the dRNA is selected to provide maximum regulation of the target gene and minimal on-target cleavage.

For the purposes of the following discussion, the reference functional domain may be the functional domain associated with a CRISPR enzyme or the functional domain associated with an adaptor protein.

In practicing the present invention, loops in sgRNA can be increased without overlapping with the Cas9 protein by inserting other RNA loop(s) or other sequence(s) that can recruit adaptor proteins that can bind to the other RNA loop(s) or other sequence(s). Adaptor proteins can include, but are not limited to, orthogonal RNA binding protein/aptamer combinations that are found in a variety of bacteriophage coat proteins. The list of such envelope proteins includes, but is not limited to, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, fCb5, fCb8r, fCb12r, fCb23r, 7s, and PRR1. Such adapter proteins or orthogonal RNA binding proteins may further recruit effector proteins or fusion products that contain one or more functional domains. In some embodiments, the functional domain may be selected from the group consisting of a transposase domain, an integrase domain, a recombinase domain, a resolvase domain, an invertase domain, a protease domain, a DNA methyltransferase domain, a DNA hydroxylmethylase domain, a DNA demethylase domain, a histone acetylase domain, a histone deacetylase domain, a nuclease domain, a repressor domain, an activator domain, nuclear localization signal domains, a transcription regulatory protein (or transcription recruitment complex) domain, a cellular uptake activity-associated domain, a nucleic acid binding domain, an antibody presentation domain, a histone modifying enzyme, a histone modifying enzyme recruiter; an inhibitor of histone modifying enzymes, histone methyltransferase, histone demethylase, histone kinase, histone phosphatase, histone ribosylase, histone deribosylase, histone ubiquitinase, histone deubiquitinase, histone biotinase and histone tail protease. In some preferred embodiments, the functional domain is a transcriptional activation domain, such as, but not limited to, VP64, p65, MyoD1, HSF1, RTA, SET7/9 or histone acetyltransferase. In some embodiments, the functional domain is a transcriptional repression domain, preferably KRAB. In some embodiments, the transcriptional repression domain is SID or concatemers of SID (e.g., SID4X). In some embodiments, the functional domain is an epigenetic modification domain, such that an epigenetic modification enzyme is provided. In some embodiments, the functional domain is an activation domain, which may be a P65 activation domain.

In some embodiments, one or more functional domains are an NLS (nuclear localization sequence) or an NES (nuclear export signal). In some embodiments, one or more functional domains are a transcriptional activation domain that includes VP64, p65, MyoD1, HSF1, RTA, SET7/9, and histone acetyltransferase. Other references herein to activation (or activator) domains with respect to CRISPR enzyme associated domains include any known transcriptional activation domain, and in particular VP64, p65, MyoD1, HSF1, RTA, SET7/9, or histone acetyltransferase.

In some embodiments, one or more functional domains are a transcriptional repression domain. In some embodiments, the transcriptional repression domain is a KRAB domain. In certain embodiments, the transcriptional repression domain is a NuE domain, a NcoR domain, a SID domain, or a SID4X domain.

In some embodiments, one or more functional domains are characterized by one or more activities comprising methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcript release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, DNA integration activity, or nucleic acid binding activity.

Histone modifying domains are also preferred in some embodiments. Exemplary histone modifying domains are discussed below. Transposase domains, HR (homologous recombination) machinery domains, recombinase domains, and/or integrase domains are also preferred as functional domains of the present invention. In some embodiments, HR machinery domains, integrase domains, recombinase domains, and/or transposase domains have DNA integration activity. Histone acetyltransferase is preferred in some embodiments.

In certain embodiments, the DNA cleavage activity is mediated by a nuclease. In some embodiments, the nuclease comprises a Fok1 nuclease. See "Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing" by 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) for dimeric RNA-guided FokI nucleases that recognize extended sequences and can edit endogenous genes with high efficiency in human cells.

In some embodiments, one or more functional domains are attached to a CRISPR enzyme such that, upon binding to the sgRNA and the target, the functional domain is in a spatial orientation that allows the functional domain to function in its intended manner.

In some embodiments, one or more functional domains are attached to an adaptor protein such that upon binding of the CRISPR enzyme to the sgRNA and target, the functional domain is in a spatial orientation that allows the functional domain to function in its intended manner.

In one aspect, the present invention provides a composition as discussed herein, wherein one or more functional domains are attached to a CRISPR enzyme or adapter protein via a linker, optionally a GlySer linker as discussed herein.

Endogenous transcriptional repression is often mediated by chromatin modifying enzymes such as histone methyltransferases (HMTs) and deacetylases (HDACs). Typical histone effector domains are known and an illustrative list is provided below. The illustrative table mentions proteins and functional truncations of small sizes to facilitate efficient viral packaging (e.g., by AAV). In general, however, the domains may include HDACs, histone methyltransferases (HMTs), and histone acetyltransferase (HAT) inhibitors, as well as HDAC and HMT recruiting proteins. The functional domain may be or include, in some embodiments, HDAC effector domains, HDAC recruiter effector domains, histone methyltransferase (HMT) effector domains, histone methyltransferase (HMT) recruiter effector domains, or histone acetyltransferase inhibitory effector domains.

HDAC effector domains

Subtype/
Complex Name Substrate (if known) Modification (if known) Organism Full size (aa) Selected truncation (aa) Final size (aa) 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 IV 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. coli (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

Therefore, the repressor domains according to the present invention can be selected from histone methyltransferases (HMT), histone deacetylases (HDAC), histone acetyltransferase (HAT) inhibitors, as well as HDAC and HMT recruiting proteins.

The HDAC domain can be any of the domains in the table above, namely HDAC8, RPD3, MesoLo4, HDAC11, HDT1, SIRT3, HST2, CobB, HST2, SIRT5, Sir2A, or SIRT6.

In some embodiment, the functional domain may be a HDAC recruiter effector domain. Preferred examples include the domains in the table below, namely MeCP2, MBD2b, Sin3a, NcoR, SALL1, RCOR1. NcoR is typical in the examples of the present invention, and although preferred, it is envisaged that other domains in the class will also be useful.

Table of HDAC Recruiter Effector Domains

Subtype/
Complex Name Substrate (if known) Modification (if known) Organism Full size (aa) Selected truncation (aa) Final size (aa) Catalytic
domain Sin3a MeCP2 - - R. norvegicus 492 207-492 (Nan) 286 - Sin3a MBD2b - - H. sapiens 262 45-262 (Boeke) 218 - Sin3a Sin3a - - H. sapiens 1273 524-851 (Laherty) 328 627-829: HDAC1 interaction 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 -

In some embodiment, the functional domain may be a methyltransferase (HMT) effector domain. Preferred examples include the domains in the table below, namely NUE, vSET, EHMT2/G9A, SUV39H1, dim-5, KYP, SUVR4, SET4, SET1, SETD8, and TgSET8. NUE is typical in the examples of the present invention, and while preferred, it is envisaged that other domains in the class will also be useful.

Histone methyltransferase (HMT) effector domain table

Subtype/
Complex Name Substrate (if known) Modification (if known) Organism Full size (aa) Selected truncation (aa) Final size (aa) Catalytic domain SET NUE H2B, H3, H4 - C. trachomatis 219° 1-219 (Pennini) 219 - SET vSET - H3K27me3 P. bursaria chlorella virus 119 1-119 (Mujtaba) 119 4-112: SET2 Family
SUV39 EHMT2/
G9A H1.4K2, H3K9, H3K27 H3K9me1/2, H1K25me1 M. musculus 1263 969-1263 (Tachibana) 295 1025-1233: preSET, SET, postSET SUV39 SUV39H1 - H3K9me2/3 H. sapiens 412 79-412 (Snowden) 334 172-412: preSET, SET, postSET Suvar3-9 dim-5 - H3K9me3 N. crassa 331 1-331 (Rathert) 331 77-331: preSET, SET, postSET Suvar3-9 (SUVH subfamily) KYP - H3K9me1/2 A. thaliana 624 335-601 267 (Jackson) - Suvar3-9 (SUVR subfamily) SUVR4 H3K9me1 H3K9me2/3 A. thaliana 492 180-492 313 (Thorstensen) 192-462: preSET, SET, postSET Suvar4-20 SET4 - H4K20me3 C. elegans 288 1-288 (Vielle) 288 - SET8 SET1 - H4K20me1 C. elegans 242 1-242 (Vielle) 242 - SET8 SETD8 - H4K20me1 H. sapiens 393° 185-393 209 (Couture) 256-382: SET SET8 TgSET8 - H4K20me1/2/3 T. gondii 1893° 1590-1893 (Sautel) 304 1749-1884: SET

In some embodiment, the functional domain may be a histone methyltransferase (HMT) recruiter effector domain. Preferred examples include the domains in the table below, namely Hp1a, PHF19, and NIPP1.

Table of histone methyltransferase (HMT) recruiter effector domains

Subtype/
Complex Name Substrate (if known) Modification (if known) Organism Full size (aa) Selected truncation (aa) Final size (aa) Catalytic domain - Hp1a - H3K9me3 M. musculus 191 73-191 119 (Hathaway) 121-179: chromoshadow - PHF19 - H3K27me3 H. sapiens 580 (1-250) + GGSG linker + (500-580) 335 (Ballaré) 163-250: PHD2 - NIPP1 - H3K27me3 H. sapiens 351 1-329 (Jin) 329 310-329: EED

In some embodiment, the functional domain may be an inhibitory effector domain of a histone acetyltransferase. Preferred examples include SET/TAF-1β, shown in the table below.

Histone acetyltransferase inhibitory effector domains table

Subtype/
Complex Name Substrate (if known) Modification (if known) Organism Full size (aa) Selected truncation (aa) Final size
(aa) Catalytic domain - SET/TAF-1β - - M. musculus 289 1-289 (Cervoni) 289 -

Also preferred is targeting endogenous (regulatory) control elements (such as enhancers and silencers) in addition to promoter or promoter-proximal elements. Thus, the present invention can also be used to target endogenous control elements (including enhancers and silencers) in addition to targeting a promoter. Such control elements can be located upstream and downstream of a transcription start site (TSS) starting from 200 kb from the TSS to 100 kb from the TSS. Targeting known control elements can be used to activate or repress a gene of interest. In some cases, a single control element can affect the transcription of several target genes. Therefore, targeting a single control element can be used to control the transcription of several genes simultaneously.

On the other hand, targeting putative control elements (e.g., by overlapping the region of a putative control element as well as 200 bp to 100 kb around the element) can be used as a means to confirm such elements (by measuring transcription of the gene of interest) or to identify new control elements (e.g., by overlapping 100 kb upstream and downstream of the TSS of the gene of interest). Additionally, targeting putative control elements can be useful in the context of understanding the genetic causes of disease. Many mutations and common SNP variants associated with disease phenotypes are located outside of coding regions. Targeting such regions with either the upregulation or downregulation systems described herein can be followed by transcriptional readout of either a) a set of putative targets (e.g., a set of genes located in close proximity to a control element) or b) a whole transcriptome readout, such as by RNAseq or a microarray. This would allow identification of likely candidate genes involved in the disease phenotype. Such candidate genes may be useful as new drug targets.

Inhibitors of histone acetyltransferase (HAT) are mentioned herein. However, an alternative in some embodiments is that one or more functional domains comprise an acetyltransferase, preferably a histone acetyltransferase. They are useful in the field of epigenomics, for example in methods for detailed study of the epigenome. Methods for detailed study of the epigenome may include, for example, targeting epigenomic sequences. Targeting epigenomic sequences may include a guide directed to an epigenomic target sequence. The epigenomic target sequence may include, in some embodiments, a promoter, a silencer, or an enhancer sequence.

The use of a functional domain associated with a CRISPR-Cas enzyme described herein, preferably a non-functional Cas, more preferably a non-functional Cas9, to target epigenomic sequences can be used to activate or repress promoters, silencers or enhancers.

Examples of acetyltransferases are known and may include, in some embodiments, histone acetyltransferases. In some embodiments, the histone acetyltransferase may comprise the catalytic core of human acetyltransferase p300 (Gerbasch & Reddy, Nature Biotech 6th April 2015).

In some preferred embodiments, the functional domain binds to a non-functional Cas9 enzyme to target and activate epigenomic sequences such as promoters or enhancers. One or more guides targeting such promoters or enhancers may also be provided to direct the binding of the CRISPR enzyme to such promoters or enhancers.

The term "associated with" is used herein to refer to the association of a functional domain with a CRISPR enzyme or an adaptor protein. It is used in relation to how one molecule "binds" to another, such as between an adaptor protein and a functional domain or between a CRISPR enzyme and a functional domain. In the case of such protein-protein interactions, this association can be thought of in terms of recognition, such as an antibody recognizing an epitope. Alternatively, one protein can be associated with another protein by a fusion of the two, such as one subunit being fused to another subunit. Fusion typically occurs by the addition of one amino acid sequence to another, such as by splicing the nucleotide sequences that encode each protein or subunit. Alternatively, it can be thought of in essence as a binding of two molecules or a direct link, such as a fusion protein. In any case, the fusion protein may include a linker between the two subunits of interest (i.e., between the enzyme and the functional domain or between the adapter protein and the functional domain). Thus, in some embodiments, the CRISPR enzyme or adapter protein is associated with the functional domain by binding thereto. In other embodiments, the CRISPR enzyme or adapter protein is associated with the functional domain because the two are fused together, optionally through an intervening linker.

Attachment of the functional domain or fusion protein can be accomplished via a linker, such as a flexible glycine-serine (GlyGlyGlySer), or (GGGS) ₃ , or a rigid alpha-helical linker such as (Ala(GluAlaAlaAlaLys)Ala). Linkers such as (GGGGS) 3 are preferably used herein to separate protein or peptide domains. (GGGGS) ₃ is preferred because it is a relatively long linker (15 amino acids). Glycine residues are the most flexible, and serine residues increase the likelihood that the linker will be on the outside of the protein. (GGGGS) ₆ (GGGGS) ₉ or (GGGGS) ₁₂ may preferably be used as alternatives. Other preferred alternatives are (GGGGS) ₁ , (GGGGS) ₂ , (GGGGS) ₄ , (GGGGS) ₅ , (GGGGS) ₇ , (GGGGS) ₈ , (GGGGS) ₁₀ or (GGGGS) ₁₁ . Alternative linkers are available, but highly flexible linkers are thought to better maximize the ability to combine the two 2 parts of Cas9 and thus restore Cas9 activity. One alternative is that the nucleoplasmin NLS can be used as a linker. For example, a linker can also be used between Cas9 and some functional domain. Again, in this case, the (GGGGS) ₃ linker (or its 6-, 9- or 12-repeat variants) can be used, or the nucleoplasmin NLS can be used as a linker between Cas9 and a functional domain.

Saturation mutagenesis

With respect to the use of the CRISPR-Cas system, reference is generally made to documents, including patent applications, patents, and patent publications, cited throughout this disclosure, since embodiments of the present invention may be used as in these documents. The CRISPR-Cas system(s) can be used to perform saturation or deep-scan mutagenesis of genomic loci along with the cellular phenotype, for example, to determine critical minimal features and discrete lesions of functional elements required for gene expression, drug resistance, and disease reversibility. By saturation or deep-scan mutagenesis is meant that every or substantially every DNA base is cut at the genomic loci. A library of CRISPR-Cas guide RNAs can be introduced into a population of cells. The library can be introduced such that each cell receives a single guide RNA (sgRNA). When the library is introduced by transduction of the viral vector described herein, a low multiplicity of infection (MOI) is used. The library may include sgRNAs that target each sequence upstream of a PAM (protospacer adjacent motif) sequence in a genomic locus. The library may include at least 100 non-overlapping genomic sequences upstream of a PAM sequence for every 1000 base pairs in a genomic locus. The library may include sgRNA targeting sequences upstream of at least one other PAM sequence. The CRISPR-Cas system(s) may include more than one Cas protein. Any Cas protein described herein may be used, including orthologs or engineered Cas proteins that recognize other PAM sequences. The frequency of off-target sites for an sgRNA can be less than 500. Off-target event estimates can be obtained to select sgRNAs with the lowest number of off-target sites. Any phenotype determined to be associated with cleavage at an sgRNA target site can be confirmed using sgRNA targeting to the same site in a single experiment. Target site confirmation can also be accomplished using the Cas9 nickase described herein and two sgRNAs targeting the genomic site of interest. Without being bound by theory, a target site is a true hit if a change in phenotype is observed in confirmatory experiments.

The loci of the genome may comprise at least one continuous region of the genome. The at least one continuous region of the genome may even comprise the entire genome. The at least one continuous region of the genome may comprise a functional element of the genome. The functional element may be in a non-coding region, a coding region, an intronic region, a promoter or an enhancer. The at least one continuous region of the genome may comprise at least 1 kb, preferably at least 50 kb, of genomic DNA. The at least one continuous region of the genome may comprise a transcription factor binding site. The at least one continuous region of the genome may comprise a DNase I hypersensitivity region. The at least one continuous region of the genome may comprise a transcriptional enhancer or repressor element. The at least one continuous region of the genome may comprise a site enriched in an epigenetic signature. At least one contiguous region of genomic DNA may comprise an epigenetic insulator. The at least one contiguous region of the genome may comprise two or more contiguous genomic regions that physically interact. The regions of the genome that interact may be identified using ‘4C technology’. 4C technology enables the entire genome to be screened in an objective manner for segments of DNA that physically interact with a selected DNA fragment, as described in Zhao et al. ((2006) Nat Genet 38, 1341-7) and in U.S. Patent No. 8,642,295, both of which are incorporated herein by reference in their entireties. The epigenetic signature may be histone acetylation, histone methylation, histone ubiquitination, histone phosphorylation, DNA methylation, or the absence thereof.

The CRISPR-Cas system(s) for saturation or deep scanning mutagenesis can be applied to a population of cells. The CRISPR-Cas system(s) can be applied to eukaryotic cells, including, but not limited to, mammalian and plant cells. The population of cells can be prokaryotic cells. The population of eukaryotic cells can be a population of embryonic stem (ES) cells, neural cells, epithelial cells, immune cells, endocrine cells, muscle cells, red blood cells, lymphocytes, plant cells, or yeast cells.

In one aspect, the present invention relates to a method of screening for functional elements associated with a change in phenotype. A library can be introduced into a population of cells that are adapted to contain a Cas protein. The cells can be sorted into at least two groups based on phenotype. The phenotype can be gene expression, cell growth or cell viability. The relative representation of guide RNAs present in each group is determined, thereby determining the sites of the genome associated with a change in phenotype using the representation of guide RNAs present in each group. The change in phenotype can be a change in the expression of a gene of interest. The gene of interest can be activated, repressed or knocked out. The cells can be sorted into a high expression group and a low expression group. The population of cells can contain a reporter construct that is used to determine the phenotype. The reporter construct can include a detectable marker. The cells can be sorted using the detectable marker.

In another aspect, the present invention relates to a method of screening for genomic sites associated with resistance to a chemical compound. The chemical compound may be a drug or a pesticide. The library may be introduced into a population of cells that are adapted to contain a Cas protein, wherein each cell of the population contains no more than one guide RNA; wherein the population of cells is treated with a chemical compound and the presentation of guide RNAs is determined after treatment with the chemical compound at a later time point compared to an early time point, thereby determining genomic sites associated with resistance to the chemical compound by enrichment of guide RNAs. The presentation of sgRNA may be determined by deep sequencing methods.

In connection with the practice of the present invention, reference may be made to the article entitled "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., Orkin, S.H., & Bauer, D.E. DOI:10.1038/nature15521, published online September 16, 2015, which article is hereby incorporated by reference and is briefly discussed below.

Canver et al. describe a novel library of pooled CRISPR-Cas9 guide RNAs to perform in situ saturation mutagenesis of human and mouse erythroid BCL11A enhancers, previously identified as an enhancer associated with fetal hemoglobin (HbF) levels and whose mouse ortholog is required for erythroid BCL11A expression. This approach identifies critical minimal features and discrete lesions of these enhancers. By editing human primary progenitor cells and mouse transgenes, the authors validated the erythroid BCL11A enhancer as a target for HbF reinduction. The authors generated a detailed enhancer map that provides insights into therapeutic genome editing.

A method for using CRISPR-Cas systems to modify a cell or organism

The present invention in some embodiments encompasses a method for modifying a cell or organism. The cell may be a prokaryotic cell or a eukaryotic cell. The cell may be a mammalian cell. The mammalian cell may be a cell of a non-human primate, bovine, porcine, rodent or mouse. The cell may be a eukaryotic cell from an organism other than a mammal, such as a bird, fish or shrimp. The cell may also be a plant cell. The plant cell may be from a crop plant such as cassava, corn, sorghum, wheat or rice. The plant cell may also be from an alga, tree or vegetable crop. The modification introduced into the cell by the present invention may be such that the cell and the progeny of the cell are altered to improve the production of biological products such as an antibody, starch, alcohol or other desired cellular product. A modification introduced into a cell by the present invention may be such that the cell and progeny of the cell will include a change that alters the biological product produced.

The system may comprise one or more different vectors. In an aspect of the present invention, the Cas protein is codon-optimized for expression in a eukaryotic cell, preferably a mammalian cell or a human cell.

CRISPR systems can be used in plants

The CRISPR-Cas system(s) (e.g., single(s) or multiplex(es)) can be used in combination with recent advances in crop genomics. Such CRISPR-Cas system(s) can be used to efficiently and cost-effectively mine, edit, or manipulate a plant gene or genome, such as to rapidly screen and/or select and/or mine and/or compare and/or manipulate and/or transform plant genes or genomes; such as to obtain, identify, develop, optimize, or impart trait(s) or characteristic(s) to a plant(s) or to transform a plant genome. Accordingly, the production of plants, new plants with new combinations of traits or characteristics, or new plants with improved traits can be improved. Such a CRISPR-Cas system(s) can be used in plants in site-directed integration (SDI) or gene editing (GE) or in any of the near reverse selection (NRB) or reverse selection (RB) techniques. For the use of the CRISPR-Cas system in plants, reference should be made to the University of Arizona's "CRISPR-PLANT" website (http://www.genome.arizona.edu/crispr/) (supported by Pennsylvania State University and AGI). Embodiments of the present invention can be used in genome editing in plants or in cases where RNAi or similar genome editing techniques have been used previously; 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 Aug 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/sst119. online published 17 Aug 2013; Xu, "Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice", Rice 2014, 7:5 (2014), Zhou et al., "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 only at www.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, www.nature.com/naturecommunications DOI: 10.1038/ncomms7989; U.S. Patent No. 6,603,061 - Agrobacterium-Mediated Plant Transformation Method; U.S. Patent No. 7,868,149 - Plant Genome Sequences and Uses Thereof and US 2009/0100536 - Transgenic Plants with Enhanced Agronomic Traits, the entire contents and disclosure of each of which are incorporated herein by reference in their entirety. In the practice of the present invention, the contents and disclosure of Morrell et al, "Crop genomics: advances and applications", Nat Rev Genet. 2011 Dec 29;13(2):85-96; which is incorporated herein by reference, including how embodiments herein may be used with respect to plants. Accordingly, reference herein to animal cells may also apply, mutatis mutandis, to plant cells unless otherwise apparent; and enzymes herein having reduced off-target effects, and systems utilizing such enzymes, may be used in plant applications, including those mentioned herein.

Sugano et al. (Plant Cell Physiol. 2014 Mar;55(3):475-81. doi: 10.1093/pcp/pcu014. Epub 2014 Jan 18) describe the use of CRISPR/Cas9 for site-directed mutagenesis in the liverwort Marchantia polymorpha L., which has become a model species for studying land plant evolution. The M. polymorpha U6 promoter was identified and cloned to express a gRNA. The gRNA target sequence was designed to disrupt the gene encoding auxin response factor 1 (ARF1) in M. polymorpha. Using Agrobacterium-mediated transformation, Sugano et al. isolated stable mutants in the gametophyte generation of M. polymorpha. In vivo CRISPR/Cas9-based site-directed mutagenesis was achieved using cauliflower mosaic virus 35S or the M. polymorpha EF1α promoter to express Cas9. The isolated mutants exhibiting an auxin-resistant phenotype were not chimeric. In addition, stable mutants were obtained by asexual propagation of T1 plants. Several arf1 alleles were readily identified by CRIPSR/Cas9-based site-directed mutagenesis. The methods of Sugano et al. can be applied to the CRISPR Cas system of the present invention.

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 expressing a Cas9 variant, a reporter gene, and up to four sgRNAs from independent RNA polymerase III promoters incorporated into a vector using the convenient Golden Gate cloning method. Each sgRNA was efficiently expressed and could mediate multiplex gene editing and long-term transcriptional activation in immortalized and primary human cells. The methods of Kabadi et al. may be applicable to the CRISPR Cas system of the present invention.

Ling et al. (BMC Plant Biology 2014, 14:327) developed a set of binary CRISPR/Cas9 vectors based on the pGreen or pCAMBIA backbone and gRNA. This toolkit does not require restriction enzymes other than BsaI to generate final constructs carrying maize codon-optimized Cas9 and one or more gRNAs with high efficiency in only a single cloning step. The toolkit was validated using maize protoplasts, transgenic maize lines, and transgenic Arabidopsis lines and was shown to have high efficiency and specificity. More importantly, using this toolkit, targeted mutations in three Arabidopsis genes were detected in T1-generation transgenic seedlings. In addition, several gene mutations could be inherited by the next generation. (Guide RNA) set of modular vectors, as well as a toolkit for multiplexed genome editing in plants. The Lin et al. toolkit can be applied to the CRISPR Cas system of the present invention.

Protocols for targeted plant genome editing via CRISPR/Cas9 are also available in volume 1284 of the series in Methods in Molecular Biology pp 239-255 dated February 10, 2015. A detailed procedure is included for the design, construction, and evaluation of dual gRNAs for plant codon-optimized Cas9-mediated (pcoCas9) genome editing using Arabidopsis thaliana and Nicotiana benthamiana protoplast model cell systems. Strategies for using the CRISPR/Cas9 system to produce targeted genome modifications in whole plants are also described. The protocols in this chapter can be applied to the CRISPR Cas system of the present invention.

Ma et al. (Mol Plant. 2015 Aug 3;8(8):1274-84. doi: 10.1016/j.molp.2015.04.007) describe a robust CRISPR/Cas9 vector system using a plant codon-optimized Cas9 gene for convenient and highly efficient multiplexed genome editing in monocots and dicots. Ma et al. developed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes that can be assembled into binary CRISPR/Cas9 vectors in a single cloning round using Golden Gate ligation or Gibson assembly. Using this system, Ma et al. edited 46 target sites in rice with an average mutation rate of 85.4%, mostly in biallelic and homozygous status. Ma et al. provide examples of loss-of-function gene mutations in T0 rice plants and T1 Arabidopsis 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. may be applicable to the CRISPR Cas system of the present invention.

Lowder et al. (Plant Physiol. 2015 Aug 21. pii: pp.00636.2015) also developed a CRISPR/Cas9 toolkit that enables multiplexed genome editing and transcriptional regulation of expressed, silenced, or noncoding genes in plants. This toolkit provides researchers with a protocol and reagents for the rapid and efficient assembly of functional T-DNA CRISPR/Cas9 constructs in monocots and dicots via the Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of endogenous plant genes. T-DNA-based transformation technology is fundamental to modern biotechnology, genetics, molecular biology, and plant physiology. In this regard, we have developed a method for assembling Cas9 (WT, nickase, or dCas9) and gRNA into a T-DNA destination vector of interest. The assembly method is based on Golden Gate assembly and MultiSite Gateway recombination. Three modules are required for assembly. The first module is a Cas9 entry vector that contains promoterless Cas9 or its derivative genes flanked by attL1 and attR5 sites. The second module is a gRNA entry vector that contains gRNA-based entry expression cassettes flanked by attL5 and attL2 sites. The third module includes attR1-attR2-containing T-DNA destination vectors that provide preferred promoters for Cas9 expression. The Lowder et al. toolbox can be applied to the CRISPR Cas9 system of the present invention.

Petersen ("Towards precisely glycol engineered plants," Plant Biotech Denmark Annual meeting 2015, Copenhagen, Denmark) developed a method for using CRISPR/Cas9 to engineer genomic changes in Arabidopsis, such as glyco-engineering Arabidopsis to produce proteins and products having desired post-translational modifications. Hebelstrup et al. (Front Plant Sci. 2015 Apr 23; 6:247) describe starch bioengineering in plants, involving crops that express starch-modifying enzymes and directly yield products that are typically made using industrial chemical and/or physical starch processing methods. The methods of Petersen and Hebelstrup may be applicable to the CRISPR-Cas9 system of the present invention.

In a preferred embodiment, the plant may be a tree. The present invention may also employ the CRISPR Cas system disclosed herein for herbaceous plant systems (see, e.g., 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 may target a single nucleotide polymorphism (SNP) in trees (see, e.g., Zhou et al., New Phytologist, Volume 208, Issue 2, pages 298-301, October 2015). In the study by Zhou et al. The authors applied the CRISPR Cas system to the tree-like perennial Populus with the 4-coumarate:CoA ligase (4CL) gene family as an example application and achieved 100% mutation efficiency for two 4CL target genes, with each transformant tested having biallelic modifications. In the study by Zhou et al., the CRISPR/Cas9 system was highly sensitive to single nucleotide polymorphisms (SNPs), as segregation for a third 4CL gene was abolished by a SNP in the target sequence.

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 4CL genes, 4CL1 and 4CL2, associated with lignin and flavonoid biosynthesis, respectively, are targets for editing using CRISPR/Cas9. The Populus tremula × alba clone 717-1B4, commonly used for transformation, is different from Populus trichocarpa with a sequenced genome. Therefore, the gRNAs 4CL1 and 4CL2, designed from the reference genome, were screened in detail according to the internal 717 RNA sequencing data to ensure the absence of SNPs that could limit the efficiency of Cas. A third gRNA designed for 4CL5, a genomic duplication of 4CL1, is also included. The corresponding 717 sequence contains one SNP in each allele near/in the PAM, both of which are predicted to abrogate targeting by the 4CL5 gRNA. All three gRNA target sites are located in the first exon. For transformation, the 717 gRNA sequence is expressed from the Medicago U6.6 promoter together with codon-optimized human Cas under the control of the CaMV 35S promoter in a binary vector. Transformation with a Cas-only vector can serve as a control. Randomly selected 4CL1 and 4CL2 lines are subjected to amplicon sequencing. The data are then processed and biallelic mutations are confirmed in all cases.

In plants, pathogens are often host specific. For example, Fusarium oxysporum f. sp. lycopersici causes fusarium wilt of tomato but attacks only tomato, and F. oxysporum f. dianthii and Puccinia graminis f. sp. tritici attack only wheat. Plants have intrinsic and induced defense responses that provide resistance to most pathogens. Mutations and recombination events over generations of plants result in genetic variation that determines susceptibility, especially since pathogens replicate at a higher frequency than plants. Plants may have resistance to non-host species, for example, the host and pathogen are incompatible. There may also be horizontal resistance, for example, partial resistance to all races of a pathogen, usually controlled by many genes, and vertical resistance, for example, complete resistance to some races of a pathogen but not to other races, usually controlled by a few genes. At the level of gene interaction, plants and pathogens evolve together, and genetic changes in one balance changes in the other. Accordingly, using natural variability, breeders combine genes that are most useful for yield, quality, uniformity, endurance, and resistance. Sources of resistance genes include native or alien varieties, ancient varieties, related wild plants, and induced mutations, such as when treating plant material with mutagenic agents. Using the present invention, plant breeders receive a new tool for inducing mutations. Accordingly, a person skilled in the art can analyze the genome of resistance gene sources, and with respect to varieties that have the desired characteristics or traits, use the present invention to induce the appearance of resistance genes with greater accuracy than in the case of previously used mutagenic agents, and, therefore, to accelerate and improve plant breeding programs.

Applications for plants and yeast; applications for biofuels

Application of the Cas9-CRISPR system to plants and yeast

Definitions

In general, the term "plant" refers to any distinct photosynthetic, eukaryotic, unicellular or multicellular organism of the kingdom Plantae that characteristically grows by cell division, contains chloroplasts, and has cell walls composed of cellulose. The term "plant" includes monocots and dicots. In particular, the plants include, without limitation, angiosperms and gymnosperms such as acacia, alfalfa, amaranth, apple, apricot, artichoke, ash, asparagus, avocado, banana, barley, beans, beets, birch, beech, blackberry, blueberry, broccoli, Brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava, cauliflower, cedar, cereal, celery, chestnut, cherry, Chinese cabbage, citrus, clementine, clover, coffee, corn, cotton, cowpea, cucumber, cypress, eggplant, elm, chicory, eucalyptus, fennel, fig, fir, geranium, grape, grapefruit, peanut, cherry shrub, eucalyptus, hemlock, hickory, kale, kiwi, kohlrabi, larch, lettuce, leek, lemon, lime, robinia, maidenhair, corn, mango, maple, melon, millet, mushroom, mustard, nuts, oak, oats, oil palm, okra, onion, orange, ornamental, flowering plant or tree, papaya, palm, parsley, parsnip, pea, peach, peanut, pear, peat, pepper, persimmon, pigeon nut, pine, pineapple, plantain, plum, pomegranate, potato, pumpkin, radicchio, radish, rapeseed, raspberry, rice, rye, sorghum, safflower, willow, soybean, spinach, spruce, pumpkin giant, strawberry, sugar beet, sugar cane, sunflower, sweet potato, sweet corn, mandarin, tea, tobacco, tomato, trees, triticale, moss, turnip, creeper, walnut, watercress, watermelon, wheat, yam, yew, and pumpkin. The term "plant" also includes algae, which are mainly photoautotrophs, grouped together primarily because they lack roots, leaves, and other organs that characterize higher plants.

The genome editing methods using the CRISPR/Cas9 system as described herein can be used to impart desired traits to virtually any plant. A wide variety of plants and plant cell systems can be engineered to have the desired physiological and agronomic characteristics described herein using the nucleic acid constructs of the present disclosure and the various transformation methods mentioned above. In preferred embodiments, target plants and plant cells for engineering include, but are not limited to, monocots and dicots such as cereal crops (e.g., wheat, maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear, strawberry, orange), forage crops (e.g., alfalfa), root vegetables (e.g., carrot, potato, sugar beet, yam), leafy vegetables (e.g., lettuce, spinach); flowering plants (e.g. petunia, rose, chrysanthemum), conifers and pine trees (e.g. pine, fir, spruce); plants used in phytoremediation (e.g. heavy metal-absorbing plants); oil crops (e.g. sunflower, rapeseed) and plants used for experimental purposes (e.g. Arabidopsis). Thus, the CRISPR-Cas methods and systems can be applied to a wide range of plants, such as, for example, dicotyledonous plants belonging to the orders Magnoliales, Illitiaceae, Lauraceae, Piperiales, Aristolochiaceae, Nymphales, Ranunculales, Papiliales, Sarraceniaceae, Trochodendraceae, Hamamelidaceae, Eucomisaceae, Leutneriaceae, Myricaceae, Fagales, Casuarinaceae, Caryophyllales, Batalaceae, Polygonaceae, Plumbagoaceae, Dilleniaceae, Tea, Malvales, Urticaceae, Lecythiaceae, Violaceae, Salixaceae, Caperaceae, Ericales, Diapensiaceae, Ebenaceae, Primulales, Rosaceae, Fabaceae, Podostemaceae, Slanophytale, Myrtales, Cornus, Proteales, Santalales, Rafflesiaceae, Celastrales, Euphorbiales, Rhamnaceae, Sapindoales, Nuciferae, Geraniales, Esthales, Araliales, Gentianales, Polemonium, Lamiaceae, Plantaginaceae, Scrophulariaceae, Campanulales, Rubiaceae, Chamomillaceae and Asterales; The CRISPR-Cas methods and systems can be applied to monocotyledonous plants, such as those belonging to the orders Alistales, Pandanales, Naiadaceae, Triuridaceae, Commelinales, Eriocaulonaceae, Restiaceae, Tenulipediaceae, Rumpleaf, Cyperaceae, Typhaceae, Bromeliadiales, Zingiberales, Palmales, Cyclanthus, Pandanales, Araceae, Liliales, and Orchidales, or to plants belonging to the gymnosperms, such as those belonging to the orders Pinaceae, Ginkgoceae, Cycads, Araucariaceae, Cupressaceae, and Gnetoides.

The CRISPR/Cas9 systems and methods described herein may be applicable to a broad range of plant species included in the non-limiting list of dicotyledonous, monocotyledonous, or gymnospermous genera listed below: 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, Vilis, and Vigna; and genera Allium, Andropogon, Aragrostis, Asparagus, Avena, Cynodon, Elaeis, Festuca, Festulolium, Heterocallis, Hordeum, Lemna, Lolium, Musa, Oryza, Panicum, Pannesetum, Phleum, Poa, Secale, Sorghum, Triticum, Zea, Abies, Cunninghamia, Ephedra, Picea, Pinus, and Pseudotsuga.

The CRISPR/Cas9 systems and methods may also be applicable to a wide range of "algae" or "algal cells", including, for example, algae selected from several eukaryotic phyla, including Rhodophyta (red algae), Chlorophyta (green algae), Phaeophyta (brown algae), Bacillariophyta (diatoms), Eustigmatophyta, and dinoflagellates, as well as the prokaryotic phylum Cyanobacteria (blue-green algae). The term "algae" includes, for example, algae selected from Amphora, Anabaena, Anikstrodesmis, Botryococcus, Chaetoceros, Chlamydomonas, Chlorella, Chlorococcum, Cyclotella, Cylindrotheca, Dunaliella, Emiliana, Euglena, Hematococcus, Isochrysis, Monochrysis, Monoraphidium, Nannochloris, Nannnochloropsis, Navicula, Nephrochloris, Nephroselmis, Nitzschia, Nodularia, Nostoc, Oochromonas, Oocystis, Oscillartoria, Pavlova, Phaeodactylum, Playtmonas, Pleurochrysis, Porhyra, Pseudoanabaena, Pyramimonas, Stichococcus, Synechococcus, Synechocystis, Tetraselmis, Thalassiosira and Trichodesmium.

A part of a plant, i.e., "plant tissue", can be processed according to the methods of the present invention to obtain an improved plant. Plant tissue also includes plant cells. The term "plant cell" as used herein refers to individual units of a living plant, whether in the intact whole plant or in isolated form grown in in vitro tissue cultures, on media or agar, in suspension in a growth medium or buffer, or as part of more highly organized units such as, for example, plant tissue, a plant organ, or a whole plant.

"Protoplast" refers to a plant cell from which the protective cell wall has been completely or partially removed by, for example, mechanical or enzymatic means, resulting in an intact biochemically competent living plant unit that can reform its cell wall, proliferate, and regenerate into a whole plant under appropriate growth conditions.

The term "transformation" refers broadly to the process by which a host plant is genetically modified by the introduction of DNA by Agrobacteria or by a variety of chemical or physical means. As used herein, the term "host plant" refers to any plant cells, tissues, organs, or progeny. Many suitable plant tissues or plant cells can be transformed and include, but are not limited to, protoplasts, somatic embryos, pollen, leaves, seedlings, stems, callus, stolons, microtubers, and shoots. Plant tissue also refers to any clone of such a plant, seeds, progeny, propagules, whether sexually or asexually produced, and the descendants of any of these, such as cuttings or seeds.

The term "transformed" as used herein refers to a cell, tissue, organ or organism into which a foreign DNA molecule, such as a construct, has been introduced. The introduced DNA molecule may be integrated into the genomic DNA of the recipient cell, tissue, organ or organism such that the introduced DNA molecule is passed on to subsequent streams. In these embodiments, a "transformed" or "transgenic" cell or plant may also include progeny of the cell or plant and progeny resulting from a breeding program using such a transformed cell as a parent in a cross and characterized by an altered phenotype resulting from the presence of the introduced DNA molecule. Preferably, the transgenic plant is fertile and capable of passing on the introduced DNA to progeny through sexual reproduction.

The term "progeny," such as progeny of a transgenic plant, is progeny born, produced, or obtained from a plant or transgenic plant. The introduced DNA molecule may also be temporarily introduced into a recipient cell in such a way that the introduced DNA molecule is not inherited by subsequent progeny and is thus not considered "transgenic." Accordingly, as used herein, a "non-transgenic plant" or plant cell is a plant that does not contain foreign DNA stably integrated into its genome.

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. Exemplary suitable plant promoters include, but are not limited to, those derived from plants, plant viruses, and bacteria such as Agrobacterium or Rhizobacterium that contain genes expressed in plant cells.

As used herein, the term "fungal cell" refers to any type of eukaryotic cell in the kingdom Fungi. Divisions in the kingdom Fungi include Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Glomeromycota, Microsporidia, and Neocallimastigomycota. Fungal cells can include yeasts, molds, and filamentous fungi. In some embodiments, the fungal cell is a yeast cell.

As used herein, the term "yeast cell" refers to any fungal cell within the phyla Ascomycota and Basidiomycota. Yeast cells may include budding yeast cells, dividing yeast cells, and mold cells. Without being limited to these organisms, many types of yeast used in laboratory and industrial settings are part of the phylum Ascomycota. In some embodiments, the yeast cell is a S. cerervisiae, Kluyveromyces marxianus, or Issatchenkia orientalis cell. Other types of 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 known as 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 by filaments, i.e., hyphae or mycelium. Examples of filamentous fungal cells include, but are not limited to, Aspergillus spp. (e.g., Aspergillus niger), Trichoderma spp. (e.g., Trichoderma reesei), Rhizopus spp. (e.g., Rhizopus oryzae), and Mortierella spp. (e.g., Mortierella isabellina).

In some embodiments, the fungal cell is an industrial strain. As used herein, the term "industrial strain" refers to any strain of a fungal cell used in or isolated from an industrial process, such as producing a product on a commercial or industrial scale. An industrial strain may refer to a fungal species that is commonly used in an industrial process, or it may refer to an isolate of a fungal species that may also be used for non-commercial purposes (e.g., laboratory testing). Examples of industrial processes may include fermentation (e.g., in the production of food and beverage products), distillation, biofuel production, compound production, and polypeptide production. Examples of industrial strains may include, but are not limited to, JAY270 and ATCC4124.

In some embodiments, the fungal cell is a polyploid cell. As used herein, the term "polyploid" cell may refer to any cell whose genome is present in more than one copy. A polyploid cell may refer to a cell type that occurs naturally in a polyploid state or may refer to a cell that has been induced to exist in a polyploid state (e.g., by specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). A polyploid cell may refer to a cell whose entire genome is polyploid, or may refer to a cell that is polyploid at a particular genomic locus of interest. Without wishing to be bound by theory, it is believed that excess guide RNA may more often be a rate-limiting component when engineering the genomes of polyploid cells than haploid cells, and thus the CRISPR/Cas9 system applications described herein may benefit from using a particular type of fungal cell.

In some embodiments, the fungal cell is a diploid cell. As used herein, a "diploid" cell may refer to any cell whose genome is present in two copies. A diploid cell may refer to a cell type that occurs naturally in the diploid state, or may refer to a cell that has been induced to exist in a diploid state (e.g., by specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). For example, S. cerevisiae strain S228C may be maintained in a haploid or diploid state. A diploid cell may refer to a cell whose entire genome is diploid, or may refer to a cell that is diploid at a particular genomic locus of interest. 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 a single copy. A haploid cell may refer to a cell type that occurs naturally in the haploid state or may refer to a cell that has been induced to exist in a haploid state (e.g., by specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). For example, S. cerevisiae strain S228C may be maintained in a haploid or diploid state. A haploid cell may refer to a cell whose entire genome is haploid or may refer to a cell that is haploid at a particular genomic locus of interest.

As used herein, the term "yeast expression vector" refers to a nucleic acid that contains one or more sequences encoding an RNA and/or a polypeptide, and may further contain any desired elements that control expression of the nucleic acid(s), as well as any elements that ensure replication and maintenance of the expression vector in a yeast cell. Many suitable yeast expression vectors and their characteristics are known in the art; for example, various vectors and techniques 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 comprise, but are not limited to, a centromeric (CEN) sequence, an autonomous replication sequence (ARS), a promoter such as the RNA polymerase III promoter operably linked to a sequence or gene of interest, a terminator such as the RNA polymerase III terminator, an origin of replication, and a marker gene (e.g., selectable auxotroph markers, selectable antibiotic markers, or other selectable markers). Examples of expression vectors for use in yeast may include plasmids, yeast artificial chromosomes, 2 μm plasmids, yeast integrative plasmids, yeast replicative plasmids, shuttle vectors, and episomal plasmids.

Stable integration of CRISPR/Cas9 system components into the genome of plants and plant cells

In certain embodiments, it is envisaged that polynucleotides encoding components of the CRISPR/Cas9 system are introduced for the purpose of stable integration into the genome of a plant cell. In these embodiments, the design of the transformation vector or expression system can be adjusted depending on when, where, and under what conditions the chi/sgRNA and/or Cas9 gene are expressed.

In specific embodiments, stable introduction of components of the CRISPR/Cas9 system into the genomic DNA of a plant cell is provided. Additionally or alternatively, introduction of components of the CRISPR/Cas9 system for the purpose of stable integration into the DNA of a plant organelle, such as, but not limited to, a plastid, mitochondrion or chloroplast, is provided.

An expression system for stable integration into a 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 Cas9 enzyme in the plant cell; a 5' untranslated region to enhance expression; an intron element to further enhance expression in certain cells, such as monocot cells; a multiple cloning site to provide convenient restriction sites for insertion of chi/sgRNA sequences and/or the Cas9 gene and other desired elements; and a 3' untranslated region to provide efficient termination of the expressed transcript.

The elements of the expression system may be in one or more expression constructs, which are circular, such as a plasmid or transformation vector, or non-circular, such as linear double-stranded DNA.

In particular embodiments, the CRISPR-Cas9 expression system comprises at least

a nucleotide sequence encoding a guide sequence or chi/sgRNA that hybridizes to a target sequence in a plant, and wherein the guide sequence or chi/sgRNA comprises a guide sequence and a direct repeat sequence, and

the nucleotide sequence encoding the Cas9 protein,

where components (a) or (b) are located in the same or different constructs, and where different nucleotide sequences may be under the control of the same or different regulatory elements functioning in the cell.

The DNA construct(s) containing the components of the CRISPR/Cas9 system and, if necessary, the template sequence, can be introduced into the genome of a plant, plant part, or plant cell using a variety of standard techniques. This process typically involves the steps of selecting a suitable host cell or host tissue, introducing the construct(s) into the host cell or host tissue, and reconstituting them into plant cells or plants.

In particular embodiments, the DNA construct can be introduced into the plant cell by techniques such as, but not limited to, electroporation, microinjection, aerosol beam injector injection of plant cell protoplasts, or the DNA constructs can be introduced directly into the plant tissue by biolistic methods such as DNA particle bombardment (see also Fu et al., Transgenic Res. 2000 Feb;9(1):11-9). Particle bombardment is based on the acceleration of particles coated with the gene(s) of interest into cells, resulting in penetration of the particles into the cytoplasm and, typically, stable integration into the genome. (See, e.g., Klein et al, Nature (1987), Klein et ah, Bio/Technology (1992), Casas et ah, Proc. Natl. Acad. Sci. USA (1993).).

In particular embodiments, DNA constructs containing components of the CRISPR/Cas9 system can be introduced into a plant by Agrobacterium-mediated transformation. The DNA constructs can be combined with suitable flanking regions of T-DNA and introduced into a standard Agrobacterium tumefaciens host vector. Foreign DNA can be incorporated into the plant genome by infecting the plants or incubating plant protoplasts with Agrobacterium bacteria containing one or more Ti (tumor-inducing) plasmids. (See, e.g., Fraley et al., (1985), Rogers et al., (1987), and U.S. Patent No. 5,563,055).

Plant promoters

In order to ensure appropriate expression in a plant cell, the components of the CRISPR/Cas9 system described herein are typically placed under the control of a plant promoter, i.e., a promoter that functions in plant cells. The use of different types of promoters is envisaged.

A constitutive plant promoter is one that is capable of expressing the open reading frame (ORF) that controls it in all or nearly all plant tissues during all or nearly all stages of plant development (called "constitutive expression"). One non-limiting example of a constitutive promoter is the cauliflower mosaic virus 35S promoter. A "regulatory promoter" refers to promoters that drive gene expression not constitutively but by temporal and/or spatial regulation, and includes tissue-specific, tissue-preferred, and inducible promoters. Different promoters may drive gene expression in different tissues or cell types, or at different developmental stages, or in response to different environmental factors. In particular embodiments, one or more of the CRISPR/Cas9 components are expressed under the control of a constitutive promoter, such as the cauliflower mosaic virus 35S promoter. Tissue-specific promoters can be used to target enhanced expression in specific cell types in a particular plant tissue, such as vascular tissues or specific seed cells. Examples of specific promoters for use in the CRISPR/Cas9 system are 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.

Examples of promoters that are inducible and that provide spatiotemporal control of gene editing or gene expression may utilize some form of energy. The form of energy may include, but is not limited to, sound energy, electromagnetic energy, chemical energy, and/or thermal energy. Examples of inducible systems include tetracycline-inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcriptional activation systems (FKBP, ABA, etc.), or light-inducible systems (phytochrome, LOV, or cryptochrome domains) such as the light-inducible transcriptional effector (LITE), which drives changes in transcriptional activity in a sequence-specific manner. Components of a light-inducible system may include a CRISPR/Cas9 enzyme, a light-sensitive cytochrome heterodimer (e.g., from Arabidopsis thaliana), and a transcriptional activation/repression domain. Additional examples of inducible DNA binding proteins and methods of using them are provided in US 61/736,465 and US 61/721,283, which are incorporated herein by reference in their entireties.

In particular embodiments, transient or inducible expression can be achieved, for example, by chemically regulated promoters, i.e., in the case where the application of an exogenous chemical compound induces gene expression. Modulation of gene expression can also be obtained by a chemically repressible promoter, where the application of a chemical compound represses gene expression. Chemically induced promoters include, but are not limited to, the maize ln2-2 promoter, which is activated by benzenesulfonamide herbicide safeners (De Veylder et al., (1997) Plant Cell Physiol 38:568-77), the maize GST promoter (GST-ll-27, WO93/01294), which is activated by hydrophobic electrophilic compounds used as pre-emergence herbicides, and the tobacco PR-1 promoter (Ono et al., (2004) Biosci Biotechnol Biochem 68:803-7), which is activated by salicylic acid. Promoters that are regulated by antibiotics, such as tetracycline-inducible and tetracycline-repressible promoters (Gatz et al., (1991) Mol Gen Genet 227:229-37; U.S. Patent Nos. 5,814,618 and 5,789,156), may also be used herein.

Translocation and/or expression in specific plant organelles

The expression system may contain elements for translocation and/or expression in a specific plant organelle.

Targeting chloroplasts

In particular embodiments, it is envisaged that the CRISPR/Cas9 system is used to specifically modify chloroplast genes or to ensure expression in a chloroplast. For this purpose, methods of transforming chloroplasts or compartmentalizing components of the CRISPR/Cas9 system in a chloroplast are used. For example, introducing genetic modifications into the plastid genome can reduce biosecurity issues such as gene flow through pollen.

Methods for transforming chloroplasts are known in the art and include particle bombardment, PEG treatment and microinjection. In addition, methods involving translocation of transformation cassettes from the nuclear genome to the plastid can be used as described in WO2010061186.

Alternatively, it is envisaged to target one or more components of the CRISPR/Cas9 system to the plant chloroplast. This is achieved by including in the expression construct a sequence encoding a chloroplast transit peptide (CTP) or a plastid transit peptide operably linked to the 5' region of the Cas9 protein encoding sequence. The CTP is removed in a processing step during translocation into the chloroplast. Targeting of expressed proteins to chloroplasts is well known to those skilled in the art (see, e.g., Protein Transport into Chloroplasts, 2010, Annual Review of Plant Biology, Vol. 61: 157-180). In such embodiments, targeting of chi/sgRNA to the plant chloroplast is also desirable. Methods and constructs that can be used to translocate chi/sgRNA into the chloroplast using a chloroplast localization sequence are described, for example, in US 20040142476, incorporated herein by reference. Such variations of constructs can be incorporated into the expression systems of the present invention for efficient translocation of Cas9-chi/sgRNA.

Introduction of polynucleotides encoding the CRISPR-Cas9 system into algal cells

Transgenic algae (or other plants such as rapeseed) may be particularly useful in the production of vegetable oils or biofuels such as alcohols (especially methanol and ethanol) or other products. They may be engineered to synthesize or oversynthesize oils or alcohols at high levels for use in the oil and fat or biofuel industries.

US 8,945,839 describes a method for engineering microalgae (Chlamydomonas reinhardtii cell species) using Cas9. Likewise, the CRISPR/Cas9 system described herein may be applicable to Chlamydomonas species and other algae. In particular embodiments, Cas9 and chi/sgRNA are introduced into synthesizing algae using a vector that expresses Cas9 under the control of a constitutive promoter, such as Hsp70A-Rbc S2 or the beta 2-tubulin promoter. Chi/sgRNA is optionally delivered using a vector containing a T7 promoter. Alternatively, Cas9 mRNA and in vitro transcribed chi/sgRNA may be delivered into algal cells. Electroporation protocols are available to those skilled in the art, such as the standard recommended protocol from the GeneArt Chlamydomonas Engineering kit.

In particular embodiments, the endonuclease used herein is a Split Cas9 enzyme. Split Cas9 enzymes are preferably used in algae for targeted genomic modification, as described in WO 2015086795. The use of the Cas9 split system is particularly suitable for an inducible method of genome manipulation, and as a result, the potential toxic effect of overexpressing Cas9 in an algal cell is avoided. In particular embodiments, said Cas9 split domains (RuvC and HNH domains) can be simultaneously or sequentially introduced into a cell such that said split Cas9 domain(s) processes a target nucleic acid sequence in an algal cell. The reduced size of split Cas9 compared to wild-type Cas9 facilitates other methods of delivering the CRISPR system to cells, such as the use of penetrating peptides, as described herein. This method is of particular interest for the production of genetically modified algae.

Introduction of polynucleotides encoding Cas9 components into yeast cells

In particular embodiments, the present invention relates to the use of a CRISPR/Cas9 system for editing the genomes of yeast cells. Methods for transforming yeast cells that can be used to introduce polynucleotides encoding components of the CRISPR/Cas9 system are well known to those skilled in the art and are described in Kawai et al., 2010, Bioeng Bugs. 2010 Nov-Dec; 1(6): 395-403). Non-limiting examples include transforming yeast cells by lithium acetate treatment (which may further include treatment with a carrier DNA and PEG), bombardment, or by electroporation.

Transient expression of Cas9 CRISP system components in plants and plant cells

In particular embodiments, it is envisaged that the chi/sgRNA and/or the Cas9 gene are transiently expressed in the plant cell. In these embodiments, the CRISPR/Cas9 system can provide for modification of the target gene only when both the chi/sgRNA and the Cas9 protein are present in the cell, so that the genomic modification can be further controlled. Since the expression of the Cas9 enzyme is transient, plants regenerated from such plant cells typically do not contain foreign DNA. In particular embodiments, the Cas9 enzyme is stably expressed by the plant cell and the guide sequence is expressed transiently.

In specific embodiments, components of the CRISPR/Cas9 system can be introduced into plant cells using a plant virus vector (Scholthof et al. 1996, Annu Rev Phytopathol. 1996;34:299-323). In further specific embodiments, the viral vector is a DNA virus vector. For example, a geminivirus (e.g., cabbage leaf curl virus, bean yellow dwarf virus, wheat dwarf virus, tomato leaf curl virus, maize streak virus, tobacco leaf curl virus, or tomato golden mosaic virus) or a nanovirus (e.g., faba bean yellow necrosis virus). In other specific embodiments, the viral vector is an RNA virus vector. For example, tobravirus (e.g., tobacco rattle virus, tobacco mosaic virus), potexvirus (e.g., potato virus X) or hordeivirus (e.g., barley streak mosaic virus). The replicating genomes of plant viruses are non-integrative vectors.

In particular embodiments, the vector used for transient expression of the CRISPR/Cas9 constructs is, for example, the pEAQ vector, which is suitable for Agrobacterium-mediated transient expression (Sainsbury F. et al., Plant Biotechnol J. 2009 Sep;7(7):682-93) in protoplasts. Precise targeting of genomic localization has been demonstrated using a modified cabbage leaf curl virus (CaLCuV) vector for the expression of gRNA in stable transgenic plants expressing the CRISPR enzyme (Scientific Reports 5, article number: 14926 (2015), doi:10.1038/srep14926).

In particular embodiments, double-stranded DNA fragments encoding chi/sgRNA and/or a Cas9 gene may be transiently introduced into a plant cell. In such embodiments, the introduced double-stranded DNA fragments are provided in sufficient quantity to modify the cell, but are not retained after a predetermined period of time or after one or more cell divisions. Methods for directly transferring DNA into plants are known to those skilled in the art (see, e.g., Davey et al. Plant Mol Biol. 1989 Sep;13(3):273-85.)

In other embodiments, an RNA polynucleotide encoding a Cas9 protein is introduced into a plant cell, which is then translated and processed by the host cell to produce protein in sufficient quantity to modify the cell (in the presence of at least one chi/sgRNA), but which is not retained after a predetermined period of time or after one or more cell divisions. Methods for introducing mRNA into plant protoplasts for transient expression are known to those skilled in the art (see, e.g., Gallie, Plant Cell Reports (1993), 13;119-122).

Combinations of the various methods described above are also provided.

Delivery of CRISPR/Cas9 components into plant cells

In particular embodiments, it is of interest to deliver one or more components of the CRISPR/Cas9 system directly into a plant cell. This is of interest, among other things, for producing non-transgenic plants (see below). In particular embodiments, one or more Cas9 components are produced outside of the plant or plant cell and delivered into the cell. For example, in particular embodiments, the Cas9 protein is produced in vitro prior to introduction into the plant cell. The Cas9 protein can be produced by a variety of methods known to those skilled in the art, including recombinant production. Once expressed, the Cas9 protein is isolated, optionally refolded, purified, and optionally processed to remove any tags, such as a His-tag. Once the crude, partially purified, or more completely purified Cas9 protein is produced, the protein can be introduced into the plant cell.

In specific embodiments, the Cas9 protein is mixed with chi/sgRNA targeting a gene of interest to produce a preassembled ribonucleoprotein.

Individual components or pre-assembled ribonucleoprotein can be introduced into plant cells by electroporation, by bombardment with particles coated with the product of a Cas9-associated gene, by chemical transfection, or by other means of transport across the cell membrane. For example, transfection of plant protoplasts with pre-assembled CRISPR ribonucleoprotein has been demonstrated to mediate targeted modification of the plant genome (as described by Woo et al. Nature Biotechnology, 2015; DOI: 10.1038/nbt.3389).

In particular embodiments, components of the CRISPR/Cas9 system are introduced into plant cells using nanoparticles. The components, either in the form of protein or nucleic acid or a combination thereof, can be loaded or packaged into nanoparticles and applied to plants (such as, for example, those described in WO2008042156 and US20130185823). In particular, embodiments of the present invention provide nanoparticles loaded or packaged with DNA molecule(s) encoding a Cas9 protein, DNA molecules encoding chi/sgRNA and/or isolated chi/sgRNA, as described in WO2015089419.

Additional means for introducing one or more components of the CRISPR/Cas9 system into a plant cell include cell penetrating peptides (CPPs). Accordingly, in particular, embodiments of the present invention provide a cell penetrating peptide linked to a Cas9 protein. In particular embodiments of the present invention, the Cas9 protein and/or chi/sgRNA are linked to one or more CPPs for efficient transport into cell protoplasts (as described by Ramakrishna (2014 Genome Res. 2014 Jun;24(6):1020-7 for Cas9 in human cells). In other embodiments, the Cas9 gene and/or chi/sgRNA are encoded by one or more circular or non-circular DNA molecule(s) that are linked to one or more CPPs for delivery into plant protoplasts. The plant protoplasts are then regenerated to plant cells and then to plants. CPPs are typically described as short peptides of less than 35 amino acids, derived from both proteins and chimeric sequences, that are capable of transporting biomolecules across the cell membrane in a receptor-dependent manner. CPPs can be cationic peptides, peptides having hydrophobic sequences, amphipathic peptides, peptides having a proline-rich sequence and an antimicrobial sequence, and chimeric or bipartite peptides (Pooga and Langel 2005). CPPs are able to penetrate biological membranes and thus induce the movement of various biomolecules across cellular membranes into the cytoplasm, and improve intracellular movement, and thus facilitate the interaction of a biomolecule with a target. Examples of CPPs include, among others, Tat, a nuclear protein transcriptional activator for HIV type 1 viral replication, penetratin, a Kaposi's fibroblast growth factor (FGF)-based signal peptide, a β3 integrin-based signal peptide; Arg-based polyarginine peptide, guanine-rich molecular transporters, the "sweet arrow" peptide, etc.

Application of the CRISPR/Cas9 system to obtain genetically modified non-transgenic plants

In particular embodiments, the methods described herein are used to modify endogenous genes or to modify their expression without permanently introducing any foreign gene, including CRISPR coding components, into the plant genome, so as to avoid the presence of foreign DNA in the plant genome. This may be of interest because regulatory requirements for non-transgenic plants are less stringent.

In particular embodiments, this is achieved by transient expression of CRISPR/Cas9 components. In particular embodiments, one or more of the CRISPR components are expressed by one or more viral vectors that produce sufficient Cas9 protein and chi/sgRNA to stably achieve modification of the gene of interest in accordance with the method described herein.

In particular embodiments, transient expression of the CRISPR/Cas9 constructs is provided in plant protoplasts and is thus not integrated into the genome. The limited expression window may be sufficient to ensure that the CRISPR/Cas9 system provides modification of the target gene as described herein.

In specific embodiments, various components of the CRISPR/Cas9 system are introduced into a plant cell, protoplast, or plant tissue, either separately or in a mixture, to separately deliver molecules, such as nanoparticles or CPP molecules, as described herein above.

Expression of CRISPR/Cas9 components can induce targeted genome modification either through direct Cas9 nuclease activity and optional introduction of template DNA or through modification of target genes by the CRISPR/Cas9 system as described herein. The various strategies described herein above achieve Cas9-mediated targeted genome editing that require the introduction of CRISPR/Cas9 components into the plant genome. Components that are transiently introduced into the plant cell are typically removed by selection.

Detection of modifications in plant genome-selectable markers

In particular embodiments where the method involves modifying an endogenous target gene of the plant genome, any suitable method can be used to determine whether or not gene targeting or site-directed mutagenesis has occurred after the plant, plant part, or plant cell is infected or transfected with the CRISPR/Cas9 system. If the method involves introducing a transgene, the transformed plant cell, callus, tissue, or plant can be identified and isolated by selecting or screening the engineered plant material for the presence of the transgene or traits encoded by the transgene. Physical and biochemical methods can be used to detect transformants of plants or plant cells containing inserted gene constructs or modification of endogenous DNA. These methods include, but are not limited to: 1) Southern analysis or PCR amplification to detect and determine the structure of the recombinant DNA insert or modified endogenous genes; 2) Northern blotting, S1 RNase protection, primer extension, or reverse transcriptase PCR amplification to detect and analyze RNA transcripts of the gene constructs; 3) enzymatic assays to detect enzyme or ribozyme activity, where such gene products are encoded by the gene construct or expression is impaired by genetic modification; 4) protein gel electrophoresis, Western blotting techniques, immunoprecipitation, or enzyme-linked immunoassays, where the gene construct or endogenous gene products are proteins. Additional techniques such as in situ hybridization, enzymatic staining, and immunostaining may also be used to detect the presence or expression of a recombinant construct or to detect endogenous gene modification in specific plant organs or tissues. Methods for performing all of these assays are well known to those skilled in the art.

Additionally (or alternatively), the expression system encoding the CRISPR/Cas9 components is typically engineered to contain one or more selectable or detectable markers that provide a means for isolating or efficiently selecting cells that contain and/or have been modified by the CRISPR/Cas9 system early and extensively.

In the case of Agrobacterium-mediated transformation, the marker cassette may be located near or between the boundaries of the flanking T-DNA and contained in a binary vector. In another embodiment, the marker cassette may be located outside the T-DNA. The selectable marker cassette may also be located at or near the boundaries of the same T-DNA as the expression cassette and may be located elsewhere in the second T-DNA in the binary vector (e.g., a 2 T-DNA system).

In the case of particle bombardment or protoplast transformation, the expression system may comprise one or more isolated linear fragments or may be part of a larger construct that must contain bacterial replication elements, bacterial selectable markers, or other detectable elements. The expression cassette(s) containing the polynucleotides encoding the guide sequence and/or Cas9 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 comprises the necessary elements for expression of a detectable or selectable marker that provides efficient selection of transformed cells.

The selection procedure in the case of cells based on a selectable marker will depend on the nature of the marker gene. In particular embodiments, a selectable marker is used, i.e. a marker that provides direct selection of cells based on the expression of the marker. The selectable marker may provide positive or negative selection and may or may not be dependent on the presence of external substrates (Miki et al. 2004, 107(3): 193-232). Typically, antibiotic or herbicide resistance genes are used as markers, and selection should be performed based on the growth of the engineered plant material on media containing an inhibitory amount of the antibiotic or herbicide to which the marker gene confers resistance. Examples of such genes are genes that confers resistance to antibiotics such as hygromycin (hpt) and kanamycin (nptII), and genes that confers resistance to herbicides such as phosphinothricin (bar) and chlorsulfuron (als).

Transformed plants and plant cells can also be identified by screening for the activity of a visible marker, typically an enzyme capable of processing a colored substrate (e.g., β-glucuronidase, luciferase, B or C1 genes). Such selection and screening techniques are well known to those skilled in the art.

Crops and plant regeneration

In specific embodiments, plant cells have a modified genome, and those generated or obtained by any of the methods described herein can be cultured to regenerate a whole plant that has the transformed or modified genotype and thus the desired phenotype. Standard regeneration techniques are well known to those skilled in the art. Specific examples of such regeneration techniques rely on the action of certain phytohormones in the tissue culture growth medium, and typically rely on a biocidal and/or herbicidal marker that has been co-introduced with the desired nucleotide sequences. In further specific embodiments, plant regeneration is performed from cultured protoplasts, callus, explants, plant organs, pollen, embryos, or parts thereof (see, e.g., Evans et al. (1983), Handbook of Plant Cell Culture, Klee et al (1987) Ann. Rev. of Plant Phys.).

In particular embodiments, the transformed or improved plants as described herein can be self-pollinated to produce seed for homositic improved plants of the present invention (homozygous for the DNA modification) or crossed with non-transgenic plants or different improved plants to produce seed for heterozygous plants. If the recombinant DNA has been introduced into a plant cell, the resulting plant from such selection is a plant that is heterozygous for the recombinant DNA molecule. Both such homozygous and heterozygous plants obtained from the crossed improved plants and containing the genetic modification (which may be recombinant DNA) are referred to herein as "progeny". Daughter plants are plants derived from the original parent plant and containing the genomic modification or recombinant DNA molecule introduced by the methods provided herein. Alternatively, genetically modified plants can be produced by one of the methods described above using Cas9, where foreign DNA is not introduced into the genome. The progeny of such plants, obtained by additional selection, can also contain the genetic modification. Crosses are performed by any of the selection methods that are widely used for various agricultural crops (e.g., Allard, Principles of Plant Breeding, John Wiley & Sons, NY, U. of CA, Davis, CA, 50-98 (1960).

Obtaining plants with improved agronomic characteristics

The Cas9-based CRISPR systems provided herein can be used to introduce targeted double-strand or single-strand breaks and/or to introduce gene activator and/or repressor systems and can be used, without limitation, for gene targeting, gene replacement, site-directed mutagenesis, targeted deletions or insertions, targeted inversions, and/or targeted translocations. Multiplexed modification of genomes can be achieved by co-expressing multiple targeting RNAs directed to produce multiple modifications in a single cell. This technology can be used to precisely engineer plants with improved characteristics, including increased nutritional value, increased resistance to biotic and abiotic stress, and increased production of commercially valuable plant products or heterologous compounds.

In particular embodiments, the CRISPR/Cas9 system as described herein is used to introduce targeted double-strand breaks (DSBs) into an endogenous DNA sequence. The DSB activates cellular DNA repair pathways that can be used to achieve desired modifications to the DNA sequence near the site of the break. This is of interest if inactivation of endogenous genes can confer or promote a desired trait. In particular embodiments, homologous recombination by a template sequence is activated at the site of the DSB to introduce a gene of interest.

In particular embodiments, the CRISPR/Cas9 system can be used as a generic nucleic acid binding protein in fusion or in operably linked to a functional domain for activating and/or repressing endogenous plant genes. Exemplary functional domains can include, but are not limited to, a translation initiator, a translation activator, a translation repressor, nucleases, in particular ribonucleases, a spliceosome, granules, a light-inducible/controlled domain, or a chemically inducible/controlled domain. Typically, in these embodiments, the Cas9 protein comprises at least one mutation, such as having no more than 5% of the activity of a Cas9 protein lacking at least one mutation; the chi/sgRNA comprises a guide sequence capable of hybridizing to a target sequence.

The methods described herein generally result in "improved plants" in that they have one or more desirable traits compared to a wild-type plant. In particular embodiments, the resulting plants, plant cells, or plant parts are transgenic plants that contain an exogenous DNA sequence incorporated into the genome of all or a portion of the cells of the plant. In particular embodiments, non-transgenic genetically modified plants, plant parts, or plant cells are produced such that no exogenous DNA sequence is incorporated into the genome of any of the plant cells of the plant. In such embodiments, the improved plants are non-transgenic. In the event that only an exogenous gene modification is provided and no foreign genes are introduced or maintained in the genome of the plant, the resulting genetically modified crop plants do not contain foreign genes and may thus be considered non-transgenic as such. Various uses of the CRISPR/Cas9 system for editing plant genomes are described in more detail below.

a) Introduction of one or more foreign genes to impart an agricultural characteristic of interest

The present invention relates to methods of editing genomes or modifying sequences associated with or located at a target locus of interest, wherein the method comprises introducing a Cas9 effector protein complex into a plant cell, wherein the Cas9 effector protein complex functions effectively to integrate a DNA insert, for example encoding a foreign gene of interest, into the genome of the plant cell. In preferred embodiments, integration of the DNA insert is facilitated by HR with an exogenously introduced DNA template or repair template. Typically, the exogenously introduced DNA template or repair template is delivered together with the Cas9 effector protein complex or one component or a polynucleotide vector for expressing a component of the complex.

The CRISPR/Cas9 systems provided herein provide targeted gene delivery. It has become increasingly clear that expression of a gene of interest is largely determined by the location of integration into the genome. The methods of the present invention provide targeted integration of a foreign gene into a desired location in the genome. The location may be selected based on information about previously obtained events or may be selected using methods disclosed elsewhere herein.

In particular embodiments, the methods provided herein comprise (a) introducing into a cell a CRISPR/Cas9 complex comprising a chi/sgRNA comprising a direct repeat and a guide sequence, wherein the guide sequence hybridizes to a target sequence that is endogenous to the plant cell; (b) introducing into the plant cell a Cas9 effector molecule that complexes with the chi/sgRNA when the guide sequence hybridizes to the target sequence and induces a double-strand break at or near a sequence targeted by the guide sequence; and (c) introducing into the cell a nucleotide sequence encoding an HDR repair template that encodes a gene of interest and that is introduced at the DS position of the HDR break. In particular embodiments, the introducing step may comprise delivering into the plant cell one or more polynucleotides encoding a Cas9 effector protein, a chi/sgRNA, and a repair template. In particular embodiments, the polynucleotides are delivered to the cell using a DNA virus (e.g., a gemnivirus) or an RNA virus (e.g., a tobravirus). In particular embodiments, the introducing steps comprise introducing into the plant cell a T-DNA comprising one or more polynucleotide sequences encoding a Cas9 effector protein, a chi/sgRNA, and a repair template, wherein the delivery is accomplished by Agrobacterium. The nucleic acid sequence encoding the Cas9 effector protein may be operably linked to a promoter, such as a constitutive promoter (e.g., the cauliflower mosaic virus 35S promoter) or a cell-specific or inducible promoter. In particular embodiments, the polynucleotide is introduced using microparticle bombardment. In particular embodiments, the method further comprises screening the plant cell after the introducing steps to determine whether the repair template, i.e., the gene of interest, has been introduced. In particular embodiments, the methods comprise the step of regenerating a plant from a plant cell. In further embodiments, the methods comprise crossbreeding the plant to produce a genetically desired plant line. Examples of foreign genes encoding a trait of interest are provided below.

b) Editing endogenous genes to impart an agricultural characteristic of interest

The present invention relates to methods of editing genomes or modifying sequences associated with a target locus of interest, the method comprising introducing a Cas9 effector protein complex into a plant cell, wherein the Cas9 complex modifies the expression of an endogenous gene of the plant. This can be achieved in a variety of ways. In particular embodiments, abolishing the expression of the endogenous gene is desired, and a CRISPR/Cas9 complex is used to target the endogenous gene to modify gene expression and cleave it. In these embodiments, the methods provided herein comprise (a) introducing into a plant cell a CRISPR/Cas9 complex comprising a chi/sgRNA comprising a direct repeat and a guide sequence, wherein the guide sequence hybridizes to a target sequence in a gene of interest in the genome of the plant cell; and (b) introducing into the cell a Cas9 effector protein that, when bound to a chi/sgRNA comprising a guide sequence that hybridizes to a target sequence, creates a double-strand break at or near the sequence that the guide sequence targets. In particular embodiments, the introducing step may comprise delivering into the plant cell one or more polynucleotides encoding a Cas9 effector protein and a chi/sgRNA.

In particular embodiments, the polynucleotides are delivered to the cell using a DNA virus (e.g., a gemnivirus) or an RNA virus (e.g., a tobravirus). In particular embodiments, the introducing steps comprise introducing into the plant cell a T-DNA comprising one or more polynucleotide sequences encoding a Cas9 effector protein and a chi/sgRNA, wherein the delivery is accomplished by Agrobacterium. The polynucleotide sequence encoding components of the CRISPR/Cas9 system may be operably linked to a promoter, such as a constitutive promoter (e.g., the cauliflower mosaic virus 35S promoter) or a cell-specific or inducible promoter. In particular embodiments, the polynucleotide is introduced using microprojectile bombardment. In particular embodiments, the method further comprises screening the plant cell after the introducing steps to determine whether expression of the gene of interest has been modified. In particular embodiments, the methods comprise the step of regenerating a plant from the plant cell. In additional embodiments, the methods include cross-breeding the plant to produce a genetically desired plant line.

In particular embodiments of the methods described above, disease-resistant crop plants are produced by targeted mutation of disease susceptibility genes or genes encoding negative regulators (e.g., the Mlo gene) from plant defense genes. In a particular embodiment, herbicide-resistant crop plants are produced by targeted substitution of specific nucleotides in plant genes, such as those encoding acetolactate synthase (ALS) and protoporphyrinogen oxidase (PPO). In particular embodiments, drought-resistant and salt-tolerant crop plants are produced by targeted mutation of genes encoding negative regulators of abiotic stress tolerance, low-amylose grains by mutation of the Waxy gene, rice or other grains with reduced rancidity by targeted mutation of essential aleurone lipase genes, etc. A more detailed list of endogenous genes encoding traits of interest is provided below.

c) Modulation of endogenous genes using the CRISPR/Cas9 system to confer an agricultural trait of interest

The present document also provides methods for modulating (i.e., activating or repressing) the expression of an endogenous gene using the Cas9 protein provided herein. Such methods employ distinct RNA sequence(s) that are targeted to the plant genome using the Cas9 complex. In particular, the distinct RNA sequence(s) bind(s) to two or more adaptor proteins (e.g., aptamers), wherein each adaptor protein is associated with one or more functional domains and wherein at least one or more functional domains associated with the adaptor protein have one or more activities comprising a methylase activity, a demethylase activity, a transcriptional activation activity, a transcriptional repression activity, a transcript release factor activity, a histone modification activity, a DNA integration activity, an RNA cleavage activity, a DNA cleavage activity, or a nucleic acid binding activity. The functional domains are used to modulate the expression of an endogenous plant gene to obtain a desired trait. Typically, in these embodiments, the Cas9 effector protein has one or more mutations such that it has no more than 5% of the nuclease activity of a Cas9 effector protein that does not have at least one mutation.

In particular embodiments, the methods provided herein comprise the steps of (a) introducing into a cell a CRISPR/Cas9 complex comprising a chi/sgRNA comprising a direct repeat and a guide sequence, wherein the guide sequence hybridizes to a target sequence that is endogenous to the plant cell; (b) introducing into the plant cell a Cas9 effector molecule that complexes with the chi/sgRNA when the guide sequence hybridizes to the target sequence; and wherein the chi/sgRNA is modified to comprise a distinct RNA sequence (aptamer) that binds to a functional domain and/or the Cas9 effector protein is modified such that it binds to a functional domain. In particular embodiments, the introducing step may comprise delivering into the plant cell one or more polynucleotides encoding the (modified) Cas9 effector protein and the (modified) chi/sgRNA. Details of the components of the CRISPR/Cas9 system for use in these methods are described elsewhere in this document.

In particular embodiments, the polynucleotides are delivered to the cell using a DNA virus (e.g., a gemnivirus) or an RNA virus (e.g., a tobravirus). In particular embodiments, the introducing steps comprise introducing into the plant cell a T-DNA comprising one or more polynucleotide sequences encoding a Cas9 effector protein and a chi/sgRNA, wherein the delivery is accomplished by Agrobacterium. The nucleic acid sequence encoding one or more components of the CRISPR/Cas9 system may be operably linked to a promoter, such as a constitutive promoter (e.g., the cauliflower mosaic virus 35S promoter) or a cell-specific or inducible promoter. In particular embodiments, the polynucleotide is introduced using microprojectile bombardment. In particular embodiments, the method further comprises screening the plant cell after the introducing steps to determine whether the expression of the gene of interest has been modified. In particular embodiments, the methods comprise the step of regenerating a plant from the plant cell. In additional embodiments, the methods involve crossbreeding the plant to obtain a genetically desired plant line. A more detailed list of endogenous genes encoding the trait of interest is provided below.

Application of Cas9 to modify polyploid plants

Many plants are polyploid, meaning that they carry double copies of their genomes - sometimes up to six, as in wheat. The methods of the present invention that use a CRISPR/Cas9 effector protein can be "multiplexed" to affect all copies of a gene or to target multiple genes at once. For example, in particular embodiments, the methods of the present invention are used to simultaneously introduce a loss-of-function mutation in different genes responsible for suppressing defense against a disease. In particular embodiments, the methods of the present invention are used to simultaneously suppress the expression of a TaMLO-Al, TaMLO-Bl, and TaMLO-Dl nucleic acid sequence in a wheat plant cell and regenerate a wheat plant therefrom to provide powdery mildew resistance to the wheat plant (see also WO2015109752).

Illustrative genes conferring agronomic traits

As described herein above, in specific embodiments, the present invention encompasses the use of a CRISPR/Cas9 system as described herein to insert a DNA of interest, including one or more expressed plant genes. In further specific embodiments, the present invention encompasses methods and means that use a Cas9 system as described herein to partially or completely remove one or more expressed plant genes. In still further embodiments, the present invention encompasses methods and means that use a Cas9 system as described herein to provide for the modification of one or more plant expressed genes by mutation, substitution, insertion of one or more nucleotides. In other embodiments, the present invention encompasses the use of a CRISPR/Cas9 system as described herein to provide for the modification of the expression of one or more plant expressed genes by specifically modifying one or more of the regulatory elements that control the expression of said genes.

In particular embodiments, the present invention encompasses methods that involve introducing exogenous genes and/or manipulating endogenous genes and their regulatory elements, such as the following:

1. Genes that confer resistance to pests or diseases

Genes conferring resistance to plant diseases. Plants can be transformed with cloned resistance genes to engineer plants that are resistant to specific pathogenic strains. See, for example, Jones et al., Science 266:789 (1994) (cloning of the tomato Cf-9 resistance gene to Cladosporium fulvum); Martin et al., Science 262:1432 (1993) (the tomato Pto resistance gene to Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinos et al., Cell 78:1089 (1994) (Arabidopsis may have the RSP2 resistance gene to Pseudomonas syringae).

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.

For proteins of Bacillus thuringiensis, see, for example, Geiser et al., Gene 48:109 (1986).

Lectins, see, for example, Van Damme et al., Plant Molec. Biol. 24:25 (1994).

For a vitamin binding protein such as avidin, see PCT application US93/06487, which describes the use of avidin and avidin homologues as larvacides against insect pests.

Enzyme inhibitors such as protease or proteinase inhibitors or amylase inhibitors. See, e.g., 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 U.S. Patent No. 5,494,813.

Insect-specific hormones or pheromones, such as ecdysteroid or juvenile hormone, its variant, its mimetic, or its antagonist or agonist. See, e.g., Hammock et al., Nature 344:458 (1990).

Insect-specific peptides or neuropeptides that, 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.

A venom specific to insects, produced in nature by a snake, wasp, 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; for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase, and a glucanase, whether 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).

Viral invasive proteins or complex toxin derived from them. See Beachy et al., Ann. rev. Phytopathol. 28:451 (1990).

Developmental arrest proteins produced in nature by a pathogen or parasite. See Lamb et al., Bio/Technology 10:1436 (1992) and Toubart et al., Plant J. 2:367 (1992).

A growth arrest protein produced naturally by plants. For example, Logemann et al., Bio/Technology 10:305 (1992).

In plants, pathogens are often host-specific. For example, some Fusarium species will cause tomato wilt but only attack tomato, while other Fusarium species attack only wheat. Plants have intrinsic and induced defense responses that provide resistance to most pathogens. Mutations and recombination events over generations of plants result in genetic variation that determines susceptibility, especially since pathogens replicate at a higher frequency than plants. Plants may have non-host resistance, e.g., the host and pathogen are incompatible, or they may have partial resistance to all races of the pathogen, usually controlled by many genes, and/or also complete resistance to some races of the pathogen but not to other races. Such resistance is usually controlled by a few genes. Using the methods and components of the CRISP-Cas9 system, a new tool now exists for inducing putative mutations. Accordingly, the genome of resistance gene sources can be analyzed, and the method and components of the CRISPR/Cas9 system can be used in plants having the desired characteristics or traits to induce the formation of resistance genes. The systems of the present invention can do this with greater precision than previously used mutagenic agents, and therefore accelerate and improve plant breeding programs.

2. Genes involved in plant diseases, such as those disclosed 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 tree diseases: Monilinia mali, Valsa ceratosperma, 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;

bottle gourd 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;

cabbage vegetable diseases: Alternaria japonica, Cercosporella brassicae, Plasmodiophora brassicae, Peronospora parasitica;

diseases of chives: 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;

diseases of Turkish beans: Colletrichum lindemthianum;

peanut diseases: Cercospora personata, Cercospora arachidicola, Sclerotium rolfsii;

Pea diseases: Erysiphe pisi;

potato diseases: Alternaria solani, Phytophthora infestans, Phytophthora erythroseptica, Spongospora subterranean, f. sp. Subterranean;

strawberry diseases: Sphaerotheca humuli, Glomerella cingulata;

tea diseases: Exobasidium reticulatum, Elsinoe leucospila, Pestalotiopsis sp., Colletotrichum theaesinensis;

tobacco diseases: Alternaria longipes, Erysiphe cichoracearum, Colletotrichum tabacum, Peronospora tabacina, Phytophthora nicotianae;

rape diseases: Sclerotinia sclerotiorum, Rhizoctonia solani;

cotton diseases: Rhizoctonia solani;

beet diseases: Cercospora beticola, Thanatephorus cucumeris, Thanatephorus cucumeris, Aphanomyces cochlioides;

rose diseases: Diplocarpon rosae, Sphaerotheca pannosa, Peronospora sparsa;

diseases of chrysanthemums and asters: 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;

diseases of zoysia: Sclerotinia homeocarpa, Rhizoctonia solani;

banana diseases: Mycosphaerella fijiensis, Mycosphaerella musicola;

sunflower diseases: Plasmopara halstedii;

seed diseases and diseases at the initial stages of growth 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.

3. Examples of genes that confer herbicide resistance

Resistance to herbicides that inhibit the growing point or meristem, such as imidazolinone or sulfonylurea, e.g., Lee et al., EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449 (1990), respectively.

Tolerance to glyphosate (resistance conferred, for example, by mutant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) genes, aroA genes, and glyphosate acetyltransferase (GAT) genes, respectively), or resistance to other phosphonic compounds, for example by glufosinate genes (phosphinothricin acetyltransferase (PAT) from Streptomyces species, including Streptomyces hygroscopicus and Streptomyces viridichromogenes), and to pyridineoxy- or phenoxypropionic acids and cyclohexons by genes encoding ACCase inhibitors. See, for example, U.S. Patent No. 4,940,835 and U.S. Patent No. 6,248,876, U.S. Patent No. 4,769,061, EP No. 0333033, and U.S. Patent No. 4,975,374. See also EP No. 0242246, DeGreef et al., Bio/Technology 7:61 (1989), Marshall et al., Theor. Appl. Genet. 83:435 (1992), WO 2005012515 of Castle et. al., and WO 2005107437.

Resistance to herbicides that inhibit photosynthesis, such as triazine (psbA and gs+ genes) or benzonitrile (nitrilase gene), and glutathione S-transferase, in Przibila et al., Plant Cell 3:169 (1991), U.S. Patent No. 4,810,648, and Hayes et al., Biochem. J. 285: 173 (1992).

Genes encoding herbicide detoxifying enzymes or a mutant glutamine synthase enzyme that is resistant to inhibition are, for example, in U.S. Patent Application Serial No. 11/760,602. Or 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 U.S. Patent Nos. 5,561,236; 5,648,477; 5,646,024; 5,273,894; 5,637,489; 5,276,268; 5,739,082; 5,908,810; and 7,112,665.

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 U.S. Patent No. 6,768,044.

4. Examples of genes involved in abiotic stress tolerance

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 WO 00/04173 or WO/2006/045633.

Transgenes capable of reducing the expression and/or activity of PARG-encoding genes in plants or plant cells as described in WO 2004/090140.

Transgenes encoding a functional enzyme in plants of the nicotinamide adenine dinucleotide utilization and synthesis pathway, including nicotinamidase, nicotinate phosphoribosyltransferase, nicotinic acid mononucleotide adenyltransferase, nicotinamide adenine dinucleotide synthetase or nicotinamide phosphoribosyltransferase, as described in EP 04077624.7, WO 2006/133827, PCT/EP07/002,433, EP 1999263 or WO 2007/107326.

Enzymes involved in carbohydrate biosynthesis include those described, for example, in EP 0 571 427, WO 95/04826, EP 0 719 338, WO 96/15248, WO 96/19581, WO 96/27674, WO 97/11188, WO 97/26362, 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 2005/030942, 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 07090007.1, EP 07090009.7, WO 01/14569, WO 02/79410, WO 03/33540, 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 Patent No. 6,734,341, WO 00/11192, WO 98/22604, WO 98/32326, WO 01/98509, WO 01/98509, WO 2005/002359, US Patent No. 5,824,790, US Patent No. 6,013,861, WO 94/04693, WO 94/09144, WO 94/11520, WO 95/35026 or WO 97/20936, or enzymes involved in the formation of polyfructose, in particular from inulin or levans as disclosed in EP 0 663 956, WO 96/01904, WO 96/21023, WO 98/39460 and WO 99/24593, the formation of alpha-1,4-glucans as disclosed in WO 95/31553, US 2002 031 826, US Patent No. 6,284,479, US Patent No. 5,712,107, WO 97/47806, WO 97/47807, WO 97/47808 and WO 00/14249, the formation of alpha-1,6 branched alpha-1,4-glucans as disclosed in WO 00/73422, the formation of alternan as disclosed, for example, in WO 00/47727, WO 00/73422, EP 06077301.7, US Patent No. 5908975 and EP 0728213, the formation of hyaluronan, for example as disclosed in WO 2006/032538, WO 2007/039314, WO 2007/039315, WO 2007/039316, JP 2006304779 and WO 2005/012529.

Genes that enhance drought tolerance. For example, WO2013122472 discloses that the absence or reduced level of a functional ubiquitin protein ligase (UPL) protein, in particular UPL3, results in a reduced water requirement or increased drought tolerance in said plant. Other examples of transgenic plants with increased drought tolerance are disclosed, for example, in US2009/0144850, US2007/0266453 and WO2002/083911. US2009/0144850 describes a plant exhibiting a drought tolerance phenotype as a result of altered expression of a DR02 nucleic acid. US 2007/0266453 describes a plant exhibiting a drought tolerance phenotype as a result of altered expression of the DR03 nucleic acid, and WO 2002/08391 1 describes a plant having increased tolerance to drought stress as a result of impaired activity of an ABC transporter expressed in guard cells. Another example is the study of Kasuga et al. (1999), who described that overexpression of a cDNA encoding DREB1 A in transgenic plants activated the expression of many stress tolerance genes under normal growth conditions and resulted in increased tolerance to drought, salt stress, and freezing. However, expression of DREB1A also resulted in severe growth retardation under normal growth conditions (Kasuga (1999) Nat Biotechnol 17(3) 287-291).

In additional specific embodiments, agricultural plants can be improved by influencing certain plant traits. For example, in developing pesticide-resistant plants, increasing disease resistance in plants, increasing resistance to plant-harmful insects and nematodes, increasing plant resistance to parasitic weeds, increasing drought resistance in plants, increasing plant nutritional value, increasing plant stress tolerance, avoiding self-pollination, increasing forage digestibility, biomass, grain yield, etc. Several specific non-limiting examples are provided herein below.

In addition to targeted mutation of single genes, Cas9CRISPR complexes can be designed to provide targeted mutation of multiple genes, deletion of a chromosomal fragment, site-specific transgene integration, site-directed mutagenesis in vivo, and precise gene replacement or allelic replacement in plants. Thus, the methods described herein have broad applications in gene discovery and validation, mutation and cisgenic breeding, and hybrid breeding. These applications facilitate the production of a new generation of genetically modified crops with various improved agronomic traits, such as herbicide tolerance, disease resistance, abiotic stress tolerance, high yield, and excellent quality.

Using Cas9 gene to produce male sterile plants

Hybrid plants generally have favorable agronomic traits compared to inbred plants. However, for self-pollinating plants, producing hybrids can be problematic. Genes that are important for plant fertility, particularly male fertility, have been identified in various plant types. For example, in maize, at least two genes have been identified that are 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;169(2):931-45; Djukanovic et al. Plant J. 2013 Dec;76(5):888-99). The methods provided herein can be used to target genes required for male fertility in order to produce male sterile plants that can be easily crossed to produce hybrids. In particular embodiments, the CRISPR/Cas9 system provided herein is used to site-directed mutagenesis of a cytochrome P450-like gene (MS26) or a meganuclease gene (MS45), thereby rendering a maize plant male sterile. Maize plants that are genetically altered for this reason can be used in hybrid breeding programs.

Increasing the fertility stage in plants

In particular embodiments, the methods provided herein are used to extend the fertile stage of a plant, such as a rice plant. For example, a rice fertile stage gene, such as Ehd3, can be targeted to produce a mutation in the gene, and seedlings can be selected for an extended fertile stage in plant regeneration (as described in CN 104004782)

Using Cas9 to Genetically Genetically Adapt a Crop of Interest

The availability of wild-type germplasm and genetic variation in crop plants is a key point for crop improvement programs, however, the available germplasm variability from crops is limited. The present invention provides methods for obtaining a diversity of genetic variations of a germplasm of interest. In the present application, a CRISPR/Cas9 system is provided with a library of chi/sgRNA targeting various loci in the plant genome and is introduced into plant cells in conjunction with a Cas9 effector protein. In this regard, a collection of genomic point mutations and gene knockouts can be obtained. In particular embodiments, the methods include obtaining a plant part or plant from the cells thus obtained and screening the cells for the presence of a trait of interest. The target genes can include coding and non-coding regions. In particular embodiments, the trait is stress tolerance and the method is a method for producing crop varieties with stress tolerance.

Application of Cas9 to influence fruit ripening

Ripening is a normal phase in the ripening process of fruits and vegetables. Only a few days after its onset, it renders the fruit or vegetable inedible. This process causes significant losses to both farmers and consumers. In particular embodiments, the methods of the present invention are used to reduce ethylene formation. This is achieved by providing one or more of the following. a. Suppressing the expression of the ACC synthase gene. ACC (1-aminocyclopropane-1-carboxylic acid) synthase is an enzyme responsible for the conversion of S-adenosylmethionine (SAM) to ACC, which occurs from the second to the last step in ethylene biosynthesis. Enzyme expression is impaired when an antisense ("mirror image") or truncated copy of the synthase gene is inserted into the plant genome; b. insertion of the ACC deaminase gene. The gene encoding the enzyme is obtained from Pseudomonas chlororaphis, a common non-pathogenic soil bacterium. It converts ACC into another compound, thereby reducing the amount of ACC available for ethylene formation; c. insertion of the SAM hydrolase gene. This approach is similar to the case of ACC deaminase, where ethylene formation is impaired when the amount of its precursor metabolite is reduced; in this case, SAM is converted to homoserine. The gene encoding the enzyme is obtained from the E. coli T3 bacteriophage, and d. suppression of ACC oxidase gene expression. ACC oxidase is an enzyme that catalyzes the oxidation of ACC to ethylene, which is the last step in the ethylene biosynthetic pathway. By the methods described herein, reduction of ACC oxidase gene expression results in suppression of ethylene formation, thereby delaying fruit ripening. In particular embodiments, in addition to or as an alternative to the modifications described herein, the methods described herein are used to modify ethylene receptors to disrupt ethylene signals received by the fruit. In particular embodiments, the expression of the ETR1 gene, which encodes an ethylene binding protein, is modified, in particular suppressed. In particular embodiments, in addition to or as an alternative to the modifications described herein, the methods described herein are used to modify the expression of a gene encoding polygalacturonase (PG), which is an enzyme responsible for the degradation of pectin, a compound that maintains the integrity of plant cell walls. Pectin degradation occurs early in the ripening process, resulting in softening of the fruit. Accordingly, in particular embodiments, the methods described herein are used to introduce a mutation into the PG gene or to suppress the activation of the PG gene to reduce the amount of PG enzyme produced, thereby delaying pectin degradation.

Thus, in particular embodiments, the methods include using a CRISPR/Cas9 system to provide one or more modifications to the genome of a plant cell, such as those described above, and regenerating a plant therefrom. In particular embodiments, the plant is a tomato plant.

Increasing the shelf life of plants

In particular embodiments, the methods of the present invention are used to modify genes involved in the formation of compounds that affect the shelf life of a plant or plant part. In particular, the modification is made in a gene that prevents the accumulation of reducing sugars in potato tubers. When treated with high heat, these reducing sugars react with free amino acids, resulting in the formation of brown, bitter-tasting products and elevated levels of acrylamide, which is a potential carcinogen. In particular embodiments, the methods provided herein are used to reduce or inhibit the expression of the vacuolar invertase (VInv) gene, which encodes a protein that breaks down sucrose into glucose and fructose (Clasen et al. DOI: 10.1111/pbi.12370).

Using the CRISPR/Cas9 system to provide a trait with an additional effect

In particular embodiments, the CRISPR/Cas9 system is used to produce crops with improved nutritional properties. In particular embodiments, the methods provided herein are adapted to produce "functional foods," i.e., a modified food or food component that can provide health benefits beyond the traditional nutrients it contains, or "nutraceuticals," i.e., substances that can be considered a food or part of a food and provide health benefits, including the prevention and treatment of disease. In particular embodiments, the nutraceutical is useful for the prevention and/or treatment of one or more of cancer, diabetes, cardiovascular disease, or hypertension.

Examples of crops with improved nutritional properties include (Newell-McGloughlin, Plant Physiology, July 2008, Vol. 147, pp. 939-953):

modified protein quality, amino acid content and/or composition, such as described for buckwheat (Luciani et al. 2005, Florida Genetics Conference Poster), canola (Roesler et al., 1997, Plant Physiol 113 75-81), maize (Cromwell et al, 1967, 1969 J Anim Sci 26 1325-1331, O'Quin et al. 2000 J Anim 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 1063-1074), 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).

Essential amino acid content, for example, as described for canola (Falco et al. 1995, Bio/Technology 13 577–582), lupin (White et al. 2001, J Sci Food Agric 81 147–154), maize (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), soybean (Falco et al. 1995 Bio/Technology 13 577–582; Galili 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, above); cotton (Chapman et al. (2001) J Am Oil Chem Soc 78 941–947; Liu et al. (2002) J Am Coll Nutr 21 205S-211S [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), maize (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; Kinney and Kwolton, 1998, Blackie Academic and Professional, London, pp 193-213), sunflower (Arcadia, Biosciences 2008).

Carbohydrates such as fructans, described for example 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, for example as described for potato (Hellewege et al. 2000, Proc Natl Acad Sci USA 97 8699-8704), starch, for example 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, such as those 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, Díaz 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), maize and soybean (flavonoids, Yu et al. (2000) Plant Physiol 124 781–794), potato (anthocyanin, alkaloid, and glycoside, 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, stilbene; 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

availability of mineral components, such as described for alfalfa (phytase, Austin-Phillips et al. (1999) http://www.molecularfarming.com/nonmedical.html), lettuce (iron, Goto et al. (2000) Theor Appl Genet 100 658-664), rice (iron, Lucca et al. (2002) J Am Coll Nutr 21 184S-190S), maize, soybean 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).

In particular embodiments, the additional effect trait refers to the intended health benefit of compounds present in the plant. For example, in particular embodiments, the additional effect crop is obtained by the methods of the present invention to provide modification and/or induction/increase in the synthesis of one or more of the following compounds.

Carotenoids such as α-carotene present in carrots neutralize free radicals that can cause cell damage, or β-carotene present in various fruits and vegetables that neutralizes free radicals.

Lutein, present in green vegetables, which helps maintain normal vision.

Lycopene, present in tomatoes and tomato products, which is believed to reduce the risk of prostate cancer.

Zeaxanthin, found in citrus fruits and maize, helps maintain normal vision.

Dietary fibres such as insoluble fibre present in wheat bran which may reduce the risk of breast cancer and/or bowel cancer, and β-glucan present in oats, soluble fibre present in psyllium and whole grains which may reduce the risk of cardiovascular disease (CVD).

Fatty acids such as omega-3 fatty acids, which may reduce the risk of CVD and improve mental and visual function, conjugated linoleic acid, which may improve body composition, may reduce the risk of certain cancers, and GLA, which may reduce the risk of inflammation, cancer, and CVD, may improve body composition.

Flavonoids such as hydroxycinnamates present in wheat, which have similar antioxidant activity and may reduce the risk of degenerative diseases, flavonols, catechins and tannins present in fruits and vegetables, which neutralize free radicals and may reduce the risk of cancer.

Glucosinolates, indoles, isothiocyanates such as sulforaphane, present in cruciferous vegetables (broccoli, kale), radishes, which neutralize free radicals, can reduce the risk of cancer.

Phenolics such as stilbenes present in grapes which may reduce the risk of degenerative diseases, heart disease and cancer may affect longevity, caffeic acid and ferulic acid present in vegetables and citrus fruits which have similar antioxidant activity may reduce the risk of degenerative diseases, heart disease and eye disease, and epicatechin present in cocoa which has similar antioxidant activity may reduce the risk of degenerative diseases and heart disease.

Plant stanols/sterols present in maize, soy, wheat, and tree resins may reduce the risk of coronary heart disease by lowering blood cholesterol levels.

Fructans, inulins, fructooligosaccharides present in Jerusalem artichoke, shallots, onion powder, which can improve the condition of the gastrointestinal tract.

Saponins present in soybeans which can lower LDL cholesterol levels.

Soy protein, present in soybeans, which may reduce the risk of heart disease.

Phytoestrogens such as isoflavones present in soy may reduce menopausal symptoms such as hot flashes, may ease osteoporosis and CVD, and lignans present in flax, rye and vegetables which may protect against heart disease and some cancers may reduce LDL cholesterol, total cholesterol.

Sulfides and thiols such as diallyl sulfide, present in onions, garlic, olives, leeks and green onions, and allyl methyl trisulfide, dithiothiones, present in cruciferous vegetables, which can lower LDL cholesterol levels, help support the immune system.

Tannins such as proanthocyanidins present in cranberries, cocoa, which can improve urinary tract health, may reduce the risk of CVD and high blood pressure, etc.

In addition, the methods of the present invention also provide for modification of protein/starch functional properties, shelf life, taste/aesthetic characteristics, fiber quality, and attributes associated with reduction of allergens, antinutrients, and toxins.

In one embodiment, the plant can be a legume. The present invention can use the CRISP-Cas9 system disclosed herein to study and modify, for example, but not limited to, soybean, pea, and peanut. Curtin et al. provide a toolbox for legume functional genomics. (See Curtin et al., "A genome engineering toolbox for legume Functional genomics," International Plant and Animal Genome Conference XXII 2014). Curtin used CRISPR genetic transformation to knockout/knockdown single copy and duplicate legume genes in root hair and whole plant systems. Some of the target genes were selected for the purpose of exploring and optimizing the performance of the knockout/knockdown systems (e.g., phytoene desaturase), while others were identified using soybean homology to Arabidopsis Dicer-like genes or genome-wide association studies in nodulation in Medicago.

Peanut allergies and legume allergies are generally a real and serious health problem. The CRISPR-Cas9 effector protein system of the present invention can be used to identify and subsequently edit or silence genes encoding allergenic proteins from such legumes. Without limiting such genes and proteins, Nicolaou et al. identified allergenic proteins in peanuts, soybeans, lentils, peas, lupines, green beans, and mung beans. See Nicolaou et al., Current Opinion in Allergy and Clinical Immunology 2011;11(3):222).

Accordingly, the present invention encompasses methods for producing plants with additional nutritional effect, said methods comprising introducing into a plant cell a gene encoding an enzyme involved in the formation of a component with additional nutritional effect using a CRISPR/Cas9 system as described herein, and regenerating a plant from said plant cell, said plant having an increased expression of said component with additional nutritional effect. In particular embodiments, a CRISPR/Cas9 system is used to modify the endogenous synthesis of these compounds indirectly, for example, by 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 a CRISPR/Cas9 system are described herein above.

Some specific examples of modifications in plants that have been engineered to impart traits with additive effects are plants with modified fatty acid metabolism, such as by transforming a plant with an antisense stearyl ACP desaturase gene to increase the stearic acid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci. U.S.A. 89:2624 (1992). Another example involves reducing phytate content, such as by cloning and then introducing DNA linked to one allele that can produce mutants of maize characterized by low phytic acid content. See Raboy et al, Maydica 35:383 (1990).

Similarly, expression of Tfs C1 and R from maize (Zea mays), which regulate flavonoid formation in maize aleurone layers under the control of a strong promoter, resulted in high rates of anthocyanin accumulation in Arabidopsis (Arabidopsis thaliana), presumably resulting from activation of the entire pathway (Bruce et al., 2000, Plant Cell 12:65-80). DellaPenna (Welsch et al., 2007 Annu Rev Plant Biol 57: 711-738) found that Tf RAP2.2 and its interactor SINAT2 increased carotenogenesis in Arabidopsis leaves. Expression of Tf Dof1 induced increased expression of genes encoding enzymes for carbon skeleton formation, a marked increase in amino acid content, and a decrease in Glc levels in transgenic Arabidopsis (Yanagisawa, 2004 Plant Cell Physiol 45: 386–391), and DOF Tf AtDof1.1 (OBP2) activated all steps in the glucosinolate biosynthetic pathway in Arabidopsis (Skirycz et al., 2006 Plant J 47: 10–24).

Reducing allergens in plants

In particular embodiments, the methods provided herein are used to produce plants with reduced levels of allergens, making them safer for the consumer. In particular embodiments, the methods comprise modifying the expression of one or more genes responsible for the production of plant allergens. For example, in particular embodiments, the methods comprise reducing the expression of the Lol p5 gene in a plant cell, such as a ryegrass plant cell, and regenerating a plant therefrom, to reduce the allergenicity of the pollen of the plant (Bhalla et al. 1999, Proc. Natl. Acad. Sci. USA Vol. 96: 11676-11680).

Methods for screening endogenous genes of interest

The methods provided herein further enable the identification of valuable genes encoding enzymes involved in the formation of a component with additional nutritional effect, or in general genes influencing agronomic traits of interest, within a species, type and plant kingdom. By selectively targeting, for example, genes encoding enzymes of metabolic pathways in plants using the CRISPR/Cas9 system as described herein, genes responsible for certain nutritional aspects of the plant can be identified. Similarly, by selectively targeting genes that can influence a desired agronomic trait, corresponding genes can be identified. Accordingly, the present invention encompasses methods for screening genes encoding enzymes involved in the formation of compounds with certain nutritional value and/or agronomic traits.

Additional applications of the CRISPR/Cas9 system in plants and yeast

Application of the CRISPR/Cas9 system in biofuel production

The term "biofuel" as used herein is an alternative fuel derived from plant or plant-derived resources. Renewable biofuels may be extracted from organic matter whose energy has been obtained through the process of carbon fixation or obtained through the use or conversion of biomass. This biomass may be used directly for biofuels or may be converted into useful energy-containing substances through thermal conversion, chemical conversion or biochemical conversion. This conversion of biomass may result in the formation of fuel in solid, liquid or gaseous form. There are two types of biofuels: bioethanol and biodiesel. Bioethanol is formed primarily through the fermentation of sugars from cellulose (starch), which is primarily obtained from maize and sugar cane. Biodiesel, on the other hand, is formed primarily from oil crops such as canola, palm and soybeans. Biofuels are used primarily for transportation.

Improving the properties of plants to produce biofuels

In particular embodiments, methods using the CRISPR/Cas9 system as described herein are used to alter the properties of the cell wall to facilitate access by primary hydrolyzing agents to more efficiently release sugars for fermentation. In particular embodiments, the biosynthesis of cellulose and/or lignin is modified. Cellulose is a major component of the cell wall. The biosynthesis of cellulose and lignin is coregulated. By decreasing the proportion of lignin in a plant, the proportion of cellulose can be increased. In particular embodiments, the methods described herein are used to decrease lignin biosynthesis in a plant to increase the content of fermentable carbohydrates. In particular, the methods described herein are used to reduce the expression 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), hydroxycinnamoyltransferase (HCT), caffeic acid O-methyltransferase (COMT), caffeol CoA 3-O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), cinnamyl alcohol dehydrogenase (CAD), cinnamoyl CoA reductase (CCR), 4-coumarate-CoA ligase (4CL), monolignol-lignin-specific glycosyltransferase and aldehyde dehydrogenase (ALDH), as disclosed in WO 2008064289 A2.

In particular embodiments, the methods described herein are used to produce plant material that produces lower levels of acetic acid during fermentation (see also WO2010096488). In particular, the methods disclosed herein are used to produce mutations in CaslL homologues to reduce acetylation of polysaccharides.

Modifying yeast to produce biofuels

In particular embodiments, the Cas9 enzyme provided herein is used to produce biofuels using recombinant microorganisms. For example, Cas9 can be used to engineer microorganisms, such as yeast, to produce biofuels or biopolymers from fermentable sugars and, optionally, the ability to degrade plant-derived lignocellulose derived from agricultural residues as a source of fermentable sugars. In particular, the present invention relates to methods that use a CRISPR/Cas9 complex to introduce foreign genes required for biofuel production into microorganisms and/or to modify endogenous genes that may interfere with biofuel synthesis. In particular, the 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 another product of interest. In particular embodiments, the methods comprise introducing one or more enzymes that facilitate the breakdown of cellulose by the microorganism, such as cellulase. In yet further embodiments, the CRISPR/Cas9 complex is used to modify endogenous metabolic pathways that compete with the biofuel pathway.

Accordingly, in more specific embodiments, the methods described herein are used to modify a microorganism as follows:

- introducing at least one heterologous nucleic acid or increasing the expression of at least one endogenous nucleic acid encoding an enzyme for degrading a plant cell wall, such that said microorganism is capable of expressing said nucleic acid and forming and secreting said enzyme for degrading a plant cell wall;

- introducing at least one heterologous nucleic acid or increasing the expression of at least one endogenous nucleic acid encoding an enzyme that converts pyruvate into acetaldehyde, optionally in combination with at least one heterologous nucleic acid encoding an enzyme that converts acetaldehyde into ethanol, such that said host cell is capable of expressing said nucleic acid; and/or

- modifying at least one nucleic acid encoding an enzyme in a metabolic pathway in said host cell, wherein said pathway results in the formation of a metabolite other than acetaldehyde from pyruvate or ethanol from acetaldehyde, and wherein said modification results in reduced formation of said metabolite, or introducing at least one nucleic acid encoding an inhibitor of said enzyme.

Modification of algae and plants to produce vegetable oils or biofuels

Transgenic algae or other plants such as rapeseed may be particularly useful in the production of vegetable oils or biofuels such as alcohols (especially methanol and ethanol). They can be engineered to synthesize or oversynthesize oils or alcohols at high levels for use in the oil and fat or biofuel industries.

US 8,945,839 describes a method for engineering microalgae (Chlamydomonas reinhardtii cell species) with Cas9. By similar means, the methods of the CRISPR/Cas9 system described herein may be applied to Chlamydomonas species and other algae. In particular embodiments, Cas9 and chi/sgRNA are introduced into the synthesizing algae using a vector that expresses Cas9 under the control of a constitutive promoter, such as Hsp70A-Rbc S2 or the beta 2-tubulin promoter. Chi/sgRNA will be delivered using a vector containing the T7 promoter. Alternatively, Cas9 mRNA and in vitro transcribed chi/sgRNA may be delivered into the algae cells. The electroporation protocol follows the standard recommended protocol for the GeneArt Chlamydomonas Engineering kit.

Application of Cas9 in obtaining microorganisms capable of producing fatty acids

In particular embodiments, the methods of the present invention are used to produce genetically modified microorganisms capable of producing fatty esters, such as fatty acid methyl esters ("FAME") and fatty acid ethyl esters ("FAEE").

In specific embodiments, it is envisaged to specifically modify genes that are involved in modifying the amount of lipids and/or the quality of lipids formed by an algal cell. Examples of genes encoding enzymes involved in fatty acid synthesis pathways may encode proteins having, for example, acetyl-CoA carboxylase, fatty acid synthase, 3-ketoacyl synthase III acyl protein carrier, glycerol-3-phosphate dehydrogenase (G3PDH), enoyl-acyl carrier protein reductase (enoyl-ACP reductase), glycerol-3-phosphate acyltransferase, lysophosphatidine acyltransferase or diacylglycerol acyltransferase, phospholipid:diacylglycerol acyltransferase, phosphatidine phosphatase, fatty acid thioesterase such as palmitoyl protein thioesterase, or malate dehydrogenase activity. In additional embodiments, it is provided to obtain diatoms that are characterized by increased lipid accumulation. 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 triglycerol and free fatty acids, as well as genes directly involved in fatty acid β-oxidation, such as acyl-CoA synthetase, 3-ketoacyl-CoA thiolase, acyl-CoA oxidase and phosphoglucomutase. The Cas9 system and methods described herein can be used to specifically activate such genes in diatoms to increase their lipid content.

Typically, host cells can be engineered to form fatty esters from a carbon source, such as an alcohol, present in the medium by expressing or overexpressing a gene encoding a thioesterase, a gene encoding an acyl-CoA synthase, and a gene encoding an ester synthase. Accordingly, the methods provided herein are used to modify microorganisms to overexpress or introduce a thioesterase gene, an acyl-CoA synthase gene, and an ester synthase gene. In particular embodiments, the thioesterase gene is selected from tesA, 'tesA, tesB, fatB, fatB2, fatB3, fatAl, or fatA. In particular embodiments, the gene encoding the acyl-CoA synthase is selected from fadDJadK, BH3103, pfl-4354, EAV15023, fadDl, 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 a gene encoding the synthase/acyl-CoA:diacylglyceryl acyltransferase from Simmondsia chinensis, Acinetobacter sp. ADP, Alcanivorax borkumensis, Pseudomonas aeruginosa, Fundibacter jadensis, Arabidopsis thaliana, or Alkaligenes eutrophus, or variants thereof.

Additionally or alternatively, the methods provided herein are used to reduce the expression in said microorganism of at least one 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 particular embodiments, one or more genes are inactivated, such as by introducing a mutation. In particular embodiments, the gene encoding the acyl-CoA dehydrogenase is fadE. In particular embodiments, the gene encoding the transcriptional regulator of fatty acid biosynthesis encodes a DNA transcriptional repressor, such as fabR.

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 particular embodiments, the gene encoding pyruvate formate lyase is pflB. In particular embodiments, the gene encoding lactate dehydrogenase is IdhA. In particular embodiments, one or more genes are inactivated, such as by introducing a mutation therein.

In specific embodiments, the microorganism is selected from the genus Escherichia, Bacillus, Lactobacillus, Rhodococcus, Synechococcus, Synechoystis, Pseudomonas, Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor, Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete, Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophamonas, Schizosaccharomyces, Yarrowia or Streptomyces.

Application of Cas9 in obtaining microorganisms capable of producing organic acids

The methods provided herein are further used to engineer microorganisms capable of producing organic acids, particularly from pentose or hexose sugars. In particular embodiments, the methods comprise introducing an endogenous LDH gene into the microorganism. In particular embodiments, the production of organic acids in said microorganisms is additionally or alternatively increased by inactivating endogenous genes encoding proteins involved in an endogenous metabolic pathway that results in the formation of a metabolite other than the organic acid of interest and/or in the case where the endogenous metabolic pathway consumes an organic acid. In particular embodiments, the modification ensures that the formation of a metabolite other than the organic acid of interest is reduced. According to specific embodiments, methods are used for introducing at least one engineered gene deletion and/or inactivation of an endogenous pathway in which an organic acid is consumed, or a gene encoding a product involved in an endogenous pathway that results in the formation of 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 additional embodiments, at least one engineered deletion and/or gene inactivation is in an endogenous gene encoding pyruvate decarboxylase (pdc).

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 comprises at least one engineered gene deletion or inactivation of an endogenous gene encoding a cytochrome-dependent lactate dehydrogenase, such as cytochrome B2-dependent L-lactate dehydrogenase.

Application of Cas9 in obtaining improved yeast strains that utilize xylose or cellobiose

In particular embodiments, the CRISPR/Cas9 system may be useful for selecting improved xylose or cellobiose utilizing yeast strains. Reduced fidelity PCR may be used to amplify one or more genes involved in xylose or cellobiose utilizing pathways. Examples of genes involved in xylose utilizing pathways and cellobiose utilizing pathways may include, but are not limited to, those described in Ha, S. J., et al. (2011) Proc. Natl. Acad. Sci. USA 108(2):504-9 and Galazka, J. M., et al. (2010) Science 330(6000):84-6. The resulting libraries of double-stranded DNA molecules, each containing a random mutation in such a specific gene, could be co-transformed with components of the CRISPR/Cas9 system into a yeast strain (e.g. S288C) and strains with increased xylose or cellobiose utilization capacity could be selected, as described in WO2015138855.

Application of Cas9 in the production of improved yeast strains for use in isoprenoid biosynthesis

Tadas Jakočiūnas et al. described the successful use of a multiplex CRISPR/Cas9 system to engineer a genome from up to 5 different genomic loci in a single transformation step in the baker's yeast Saccharomyces cerevisiae (Metabolic Engineering Volume 28, March 2015, Pages 213-222), yielding strains with high production of mevalonate, a key intermediate in the industrially important isoprenoid biosynthetic pathway. In particular embodiments, the CRISPR/Cas9 system can be used in the multiplex genome engineering method described herein to identify additional high-yielding yeast strains for use in isoprenoid synthesis.

Application of Cas9 in the production of lactic acid-producing yeast strains

In another embodiment, the successful use of a multiplex CRISPR/Cas9 system is encompassed. In 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 obtained in a single transformation event. In a specific embodiment, the CRISPR/Cas9 system is used to simultaneously insert a heterologous lactate dehydrogenase gene and disrupt two endogenous genes, PDC1 and PDC5.

Additional Applications of the CRISPR/Cas9 System in Plants

In particular embodiments, the CRISPR system and preferably the CRISPR/Cas9 system described herein can be used to visualize the dynamics of genetic elements. For example, CRISPR imaging can visualize both repetitive and non-repetitive genomic sequences, characterize telomere length changes and telomere movement, and monitor the dynamics of gene loci during the cell cycle (Chen et al., Cell, 2013). These methods can be applied to plants.

Other applications of the CRISPR system, and preferably the CRISPR/Cas9 system described herein, include screening for positive selection of target gene dysfunction in vitro and in vivo (Malina et al., Genes and Development, 2013). These methods may be applicable to plants.

In specific embodiments, fusion of inactive Cas9 endonucleases to histone modifying enzymes can introduce specific changes in the complex epigenome (Rusk et al., Nature Methods, 2014). These methods may be applicable to plants.

In particular embodiments, the CRISPR system, and preferably the CRISPR/Cas9 system described herein, can be used to purify a specific region of chromatin and identify associated proteins, thereby establishing their regulatory role in transcription (Waldrip et al., Epigenetics, 2014). These methods can be applied to plants.

In particular embodiments, the present invention can be used as a therapy for virus removal in plant systems because both viral DNA and RNA can be cleaved. Previous studies in human systems have shown success using CRISPR in targeting single-stranded RNA hepatitis C virus (A. Price, et al., Proc. Natl. Acad. Sci, 2015) as well as double-stranded DNA hepatitis B virus (V. Ramanan, et al., Sci. Rep, 2015). These methods can be adapted for use of the CRISPR/Cas9 system in plants.

In particular embodiments, the present invention could be used to alter genomic variability. In further particular embodiments, the CRISPR system, and preferably the CRISPR/Cas9 system described herein, can be used to disrupt or alter chromosome numbers and produce haploid plants that contain chromosomes from only one parent. Such plants can be induced to undergo chromosomal duplication and converted into diploid plants containing only homozygous alleles (Karimi-Ashtiyani et al., PNAS, 2015; Anton et al., Nucleus, 2014). These methods can be applied to plants.

In specific embodiments, the CRISPR/Cas9 system described herein can be used for self-cleavage. The described Cas9 enzyme and sgRNA promoter is a constitutive promoter, and a second sgRNA is introduced into the same transformation cassette, but is controlled by an inducible promoter. This second sgRNA can be engineered to induce site-specific cleavage in the Cas9 gene to produce a non-functional Cas9. In a further specific embodiment, the second sgRNA induces cleavage at both ends of the transformation cassette, resulting in the removal of the cassette from the host genome. This system provides a controlled duration of exposure of the cell to the Cas enzyme and further minimizes off-target editing. In addition, cleavage of both ends of the CRISPR/Cas cassette can be used to generate transgene-free T ₀ plants with biallelic mutations (e.g., Moore et al., Nucleic Acids Research, 2014; Schaeffer et al., Plant Science, 2015). The methods of Moore et al. can be applied to the CRISPR/Cas9 systems described herein.

Improved Plants

The present invention also provides plants and yeast cells obtainable and obtainable by the methods provided in this document. Improved plants obtained by the methods described in this document can be useful in the production of food and feed products by expressing genes that, for example, provide tolerance to plant pests, herbicides, drought, low or high temperatures, excess water, etc.

Improved plants obtained by the methods described herein, in particular crops and algae, may be useful in the production of food and feed by synthesizing, for example, higher levels of protein, carbohydrates, nutrients or vitamins than would normally be observed in the wild type. In this regard, improved plants, in particular legumes and tubers, are preferred.

Enhanced algae or other plants such as rapeseed may be particularly useful in the production of vegetable oils or biofuels such as alcohols (especially methanol and ethanol). They can be engineered to synthesize or oversynthesize oils or alcohols at high levels for use in the oil and fat or biofuel industries.

The present invention also provides improved plant parts. Plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovules, and pollen. Plant parts, as provided herein, may be viable, non-viable, regenerable, and/or non-regenerable.

Plant cells and plants according to the methods of the present invention are also encompassed by this document. Gametes, seeds, embryos, both zygotic and somatic, progeny or hybrids of plants containing a genetic modification that are obtained using traditional breeding methods are also included within the scope of the present invention. Such plants may contain a heterologous sequence or a foreign DNA sequence inserted into or in place of the target sequence. Alternatively, such plants may contain only a change (mutation, deletion, insertion, substitution) in one or more nucleotides. For example, such plants will differ from their progenitor plants only by the presence of a certain modification.

Agricultural and productive animals

The present invention thus relates to a plant, animal or cell obtained by the method of the present invention, or their progeny. The progeny may be a clone of the obtained plant or animal, or it may be obtained by sexual reproduction by crossing with other individuals of the same species to impart additional desired characteristics to their progeny. The cell may be in vivo or ex vivo in the case of multicellular organisms, in particular animals or plants.

Organisms and animals; methods

This application may also extend to other agricultural applications, such as, for example, agricultural and production animals. For example, pigs have many characteristics that make them attractive as biomedical models, particularly in regenerative medicine. In particular, severe combined immunodeficiency (SCID) pigs may provide useful models for regenerative medicine, xenotransplantation, and tumorigenesis, and will facilitate the development of therapeutics for human patients with SCID. Lee et al. (Proc Natl Acad Sci U S A. 2014 May 20;111(20):7260-5) used a reporter-directed transcription activator-like effector nuclease (TALEN) system to produce targeted modifications of the recombination activating gene (RAG) 2 in somatic cells with high efficiency, including some that affected both alleles. The CRISPR Cas system may be applicable to a similar system.

The methods of Lee et al., (Proc Natl Acad Sci U S A. 2014 May 20;111(20):7260-5) can be applied to the present invention as follows. Mutated pigs are generated by targeted modification of RAG2 in fetal fibroblasts, followed by SCNT and embryo transfer. Constructs encoding CRISPR Cas and a reporter are electroporated into fetal fibroblasts. After 48 hours, transfected cells expressing green fluorescent protein are sorted into individual wells of a 96-well plate at an expected dilution of one cell per well. Targeted modifications of RAG2 are screened by amplifying a fragment of genomic DNA flanking any CRISPR Cas restriction sites, followed by sequencing of the PCR products. After screening and ensuring the absence of off-target mutations, cells harboring the targeted modification of RAG2 are used for SCNT. The polar body along with a portion of the adjacent cytoplasm of the oocyte presumed to contain metaphase plate II are removed and the donor cell is placed in perivitelline. The reconstructed embryos are then electroporated to fuse the donor cell with the oocyte and then chemically activated. The activated embryos are incubated in porcine zygote development medium 3 (PZM3) with 0.5 μM scriptaid (S7817; Sigma-Aldrich) for 14–16 h. The embryos are then washed to remove scriptaid and cultured in PZM3 until they are transferred into the fallopian tubes of surrogate pigs.

The present invention is also applicable to SNP modification in other animals, such as cows. Tan et al. (Proc Natl Acad Sci U S A. 2013 Oct 8; 110(41): 16526-16531) expanded the bovine gene editing kit to include transcription activator-like effector nuclease (TAL)-stimulated homologous recombination repair (HDR) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 using plasmid, rAAV, and oligonucleotide templates. The gene-specific gRNA sequences were cloned into Church lab gRNA-based 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). Cas9 nuclease was generated by co-transfection of hCas9 plasmid (Addgene ID: 41815) or synthesized using mRNA from RCIScript-hCas9. This RCIScript-hCas9 system was constructed by subcloning the XbaI-AgeI fragment from hCas9 plasmid (covering the hCas9 cDNA) into RCIScript plasmid.

Heo et al. (Stem Cells Dev. 2015 Feb 1;24(3):393-402. doi: 10.1089/scd.2014.0278. Epub 2014 Nov 3) described highly efficient gene targeting in the bovine genome using bovine pluripotent cells and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease. Heo et al. first generated induced pluripotent stem cells (iPSCs) from bovine somatic fibroblasts by ectopically expressing Yamanaka factors and treating with a GSK3β and MEK inhibitor (2i). Heo et al. observed that these bovine iPSCs were highly similar to naïve pluripotent stem cells with respect to gene expression and teratoma development potential. Furthermore, the CRISPR/Cas9 nuclease, which was specific for the bovine NANOG locus, was characterized by highly efficient editing of the bovine genome in bovine iPSCs and embryos.

Igenity® involves profiling animals, such as cows, to express and transmit genes for economically important traits such as carcass composition, carcass quality, maternal and reproductive traits, and average daily gain. Analysis of a complete Igenity® profile begins with the discovery of DNA markers (most often single nucleotide polymorphisms, or SNPs). All markers within an Igenity® profile were discovered by independent scientists at research institutions, including universities, research organizations, and government agencies such as the USDA. The markers were then analyzed using Igenity® in validation populations. Igenity® utilizes populations from multiple resources that reflect a variety of production environments and biological types, often working with industry partners in the breeding, finishing, finishing, and/or packing segments of the cattle industry to capture phenotypes that are not typically available. Databases of bovine genomes are widely available, see, for example, the NAGRP Cattle Genome Coordination Program (http://www.animalgenome.org/cattle/maps/db.html). Thus, the present invention can be applied to target bovine SNPs. One skilled in the art can use the above-mentioned protocols for targeting SNPs and apply them to bovine SNPs, such as Tan et al. or Heo et al.

Qingjian Zou et al. (Journal of Molecular Cell Biology Advance Access, published October 12, 2015) showed increased muscle mass in dogs by targeting the first exon of the canine myostatin (MSTN) gene (a negative regulator of skeletal muscle mass). First, the efficacy of sgRNA was validated by cotransfecting MSTN-targeting sgRNA with a Cas9 vector in canine embryonic fibroblasts (CEFs). MSTN KO dogs were then generated by microinjecting embryos with a mixture of Cas9 mRNA and MSTN sgRNA and autotransplanting the zygotes into the fallopian tube of the same female. The KO puppies exhibited a pronounced muscular phenotype in the thigh region compared to their wild-type littermate.

Livestock - pigs

Viral targets in livestock may include, in some embodiments, porcine CD163, such as on porcine macrophages. CD163 is associated with infection (presumably as a result of viral entry into the cell) by PRRSv (porcine reproductive and respiratory syndrome virus, an arterivirus). Infection by PRRSv, particularly of porcine alveolar macrophages (found in the lungs), results in a previously incurable porcine syndrome ("mystery pig disease" or "blue ear disease"), which causes disease including reproductive failure, weight loss, and high mortality in domestic pigs. Opportunistic infections such as enzootic pneumonia, meningitis, and ear swelling are often observed as a result of immunodeficiency due to loss of macrophage activity. It also has significant economic and environmental consequences due to increased antibiotic use and financial losses (estimated at $660 million per year).

As described 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 Plc, CD163 was targeted with CRISPR-Cas9 and the offspring of the edited pigs were resistant to PRRSv. One founder boar and one founder sow, both of which had mutations in exon 7 of CD163, were mated to produce offspring. The founder boar had an 11 bp deletion in exon 7 on one allele, resulting in a frameshift and missense translation mutation at amino acid 45 in domain 5 and a subsequent premature stop codon at amino acid 64. The other allele had a 2 bp addition to the gene for CD163. in exon 7 and a 377 bp deletion in the preceding intron that were predicted to result in expression of the first 49 amino acids of domain 5 followed by a premature stop codon at amino acid 85. The sow had a 7 bp addition in one allele that, when translated, was predicted to express the first 48 amino acids of domain 5 followed by a premature stop codon at amino acid 70. The sow's other allele was unamplified. Some offspring were predicted to be null animals (CD163-/-), i.e., CD163 knockouts.

Accordingly, in some embodiments, porcine alveolar macrophages can be targeted with a CRISPR protein. In some embodiments, porcine CD163 can be targeted with a CRISPR protein. In some embodiments, porcine CD163 can be knocked out by inducing a DSB or by insertions or deletions, such as targeting a 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.

An "edited" pig and its offspring, such as a CD163 knockout pig, are also envisaged. This may be envisaged for the purposes of livestock breeding, selection and modeling (i.e., a pig model). Semen containing the gene knockout is also envisaged.

CD163 is a member of the cysteine-rich receptor (SRCR) superfamily. Based on in vitro studies, SRCR domain 5 is the domain responsible for unpacking and releasing the viral genome. For example, other members of the SRCR superfamily can also be targeted to confer resistance to other viruses. PRRSV is also a member of the mammalian arterivirus group, which also includes murine lactate dehydrogenase-increasing virus, simian hemorrhagic fever virus, and equine arteritis virus. Arteriviruses share important pathogenesis properties, including macrophage tropism and the ability to cause both severe disease and chronic infection. Accordingly, arteriviruses, and in particular mouse lactate dehydrogenase-inducing virus, simian hemorrhagic fever virus, and equine arteritis virus, can be targeted, for example, by porcine CD163 or its homologues in other species, and mouse, monkey, and equine models and knockout are also envisaged.

Indeed, this approach could be extended to viruses or bacteria that cause other livestock diseases that can be transmitted to humans, such as the swine influenza virus (SIV) strains that include influenza C and the influenza A subtypes known as H1N1, H1N2, H2N1, H3N1, H3N2 and H2N3, as well as the pneumonia, meningitis and oedema mentioned above.

Xenotransplantation, xenografts

The present invention also provides the use of the CRISPR-Cas system described herein, such as the Cas9 effector protein systems, to produce RNA-guided DNA nucleases adapted for use in producing modified tissues for transplantation. For example, RNA-guided DNA nucleases can be used to knock out, knock down, or disrupt specific genes in an animal, such as a transgenic pig (such as a human heme oxygenase-1 transgenic pig strain), such as to disrupt the expression of genes that encode epitopes recognized by the human immune system, i.e., xenoantigen genes. Candidate porcine genes for disruption can, for example, include the α(1,3)-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydrolase genes (see PCT Patent Application Publication No. WO 2014/066505). In addition, genes encoding endogenous retroviruses can be disrupted, such as the genes encoding all porcine endogenous retroviruses (see Yang et al., 2015, Genome-wide inactivation of porcine endogenous retroviruses (PERVs), Science 27 November 2015: Vol. 350 no. 6264 pp. 1101-1104). Furthermore, RNA-guided DNA nucleases can be used to target the site for integration of additional genes in xenograft donor animals, such as the human CD55 gene, to enhance protection against hyperacute rejection.

Gene drives and application to mosquitoes and malaria

The present invention also provides for the use of the CRISPR-Cas system described herein, such as Cas9 effector protein systems, to provide RNA-guided gene drives, such as in systems similar to the gene drives described in PCT Patent Application Publication No. WO 2015/105928. Systems of this type may, for example, provide methods for altering eukaryotic germline cells by introducing into a germline cell a nucleic acid sequence encoding an RNA-guided DNA nuclease and one or more guide RNAs. The guide RNAs may be designed to be complementary to one or more target loci in the genomic DNA of the germline cell. The nucleic acid sequence encoding the RNA-guided DNA nuclease and the nucleic acid sequence encoding the guide RNAs may be provided in constructs between flanking sequences, with promoters positioned such that the germline cell can express the RNA-guided DNA nuclease and the guide RNAs, together with any desired cargo molecule encoding sequences that are also positioned between the flanking sequences. The flanking sequences will typically include a sequence that is identical to a corresponding sequence on a particular chromosome, such that the flanking sequences function with the components encoded by the construct to facilitate the insertion of the foreign sequences of the nucleic acid constructs into genomic DNA at the target cleavage site by mechanisms such as homologous recombination to produce a germline cell homozygous for the foreign nucleic acid sequence. Thus, gene drive systems are capable of introgression of desired genes throughout the breeder population (Gantz et al., 2015, Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi, PNAS 2015, online publication ahead of print, November 23, 2015, doi:10.1073/pnas.1521077112; Esvelt et al., 2014, Concerning RNA-guided gene drives for the alteration of wild populations eLife 2014;3:e03401). In certain embodiments, target sequences that have few potential off-target sites in the genome can be selected. Targeting multiple sites within a target locus with multiple guide RNAs can increase cleavage frequency and slow the evolution of drive-resistant genes. Truncated guide RNAs can reduce off-target cleavage. Paired nicksases can be used instead of a single nuclease to further increase specificity. Gene drive constructs can include cargo molecule sequences encoding transcriptional regulators, for example, to activate homologous recombinant genes and/or repress non-homologous end joining. Target sites can be selected in an essential gene such that non-homologous end joining events cause lethality rather than the formation of a drive-resistant allele. Gene drive constructs can be designed to function in a range of hosts and 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).

FISH and illustrative applications of inactivated CRISPR Cas9 enzymes

In one aspect, the present invention relates to an engineered non-naturally occurring CRISPR-Cas system comprising a catalytically inactive Cas protein as described herein, preferably inactivated Cas9 (dCas9), and the use of fluorescence in situ hybridization (FISH) in the system. dCas9, which lacks the ability to make double-strand DNA breaks, can be fused to a fluorescent protein, such as enhanced green fluorescent protein (eEGFP), and co-expressed with small guide RNAs to target pericentric, centric, and telocentric repeats in vivo. The dCas9 system can be used to visualize repetitive sequences and individual genes in the human genome. Such novel uses of labeled dCas9 CRISPR-cas systems may be important in cell imaging and the study of functional nuclear architecture, particularly in cases with small nuclear volumes or complex 3-D 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 system. Cell 155(7):1479-91. doi: 10.1016/j.cell.2013.12.001.)

Therapeutic targeting using RNA-guided effector protein complex

As will be appreciated, it is envisaged that the present system 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 a vector or delivery systems comprising one or more polynucleotides encoding components of said composition, for use in modifying a target cell in vivo, ex vivo or in vitro, and can be made using a method that alters the cell such that after modification, the progeny or cell line of 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 an animal, using an ex vivo or in vivo CRISPR system against desired cell types. The CRISPR invention can be a therapeutic method of treatment. The therapeutic method of treatment can include gene or genome editing or gene therapy.

Treatment of pathogens such as bacterial, fungal and parasitic pathogens

The present invention may also be applicable to the treatment of bacterial, fungal and parasitic pathogens. Most research efforts have focused on creating new antibiotics, which, once created, would still be subject to similar problems associated with drug resistance. The present invention relates to new CRISPR-based alternatives that overcome these challenges. In addition, unlike existing antibiotics, CRISPR-based treatments can be pathogen-specific, inducing cell death of the target pathogen while sparing 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. The study, which introduced precise mutations into the genomes, relied on dual RNA:Cas9-guided cleavage at a target site in the genome to kill unmutated cells and eliminated the need for selectable markers or negative selection systems. CRISPR systems have been used to reverse antibiotic resistance and eliminate the transfer of resistance between strains. Bickard et al. showed that Cas9 reprogrammed to target virulence genes kills virulent, but not avirulent, S. aureus. Reprogramming the nuclease to target antibiotic resistance genes destroyed staphylococcal plasmids that harbored antibiotic resistance genes and immunized against the spread of plasmid-borne resistance genes. (See, Bikard et al., "Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials," Nature Biotechnology vol. 32, 1146–1150, doi:10.1038/nbt.3043, published online October 5, 2014.) Bikard showed that CRISPR-Cas9-based antimicrobials function in vivo to kill S. aureus in a mouse model of skin colonization. Similarly, Yosef et al. used the CRISPR system to target genes encoding enzymes that confer resistance to β-lactam antibiotics (see Yousef et al., "Temperate and lytic bacteriophages programmed to sensitize and kill antibiotic-resistant bacteria," Proc. Natl. Acad. Sci. USA, vol. 112, p. 7267–7272, doi: 10.1073/pnas.1500107112, published online May 18, 2015).

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 double-strand breaks 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). Ghorbal 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 1 June 2014) modified the sequences of two genes, orc1 and kelch13, which have putative functions in gene silencing and artemisinin resistance, respectively. Parasites that were modified at the appropriate sites were recovered with very high efficiency despite the lack of direct selection for the modification, indicating that neutral or even deleterious mutations can be generated using this system. The CRISPR-Cas9 system has also been used to modify 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 Sidik et al., "Efficient Genome Engineering of Toxoplasma gondii Using CRISPR/Cas9," PLoS One vol. 9, e100450, doi: 10.1371/journal.pone.0100450, published online June 27, 2014).

Vyas et al. ("A Candida albicans CRISPR system permits genetic engineering of essential genes and gene families," Science Advances, vol. 1, e1500248, DOI: 10.1126/sciadv.1500248, April 3, 2015) used the CRISPR system to overcome long-standing obstacles to genetic engineering in C. albicans and efficiently mutate both copies of several different genes in a single experiment. In an organism where multiple mechanisms contribute to drug resistance, Vyas generated homozygous double mutants that no longer exhibited the hyperresistance to fluconazole or cycloheximide exhibited by the parental clinical isolate Can90. Vyas also generated homozygous loss-of-function mutations in essential 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 repair template that introduced a nonsense mutation and isolated dcr1/dcr1 mutants that could not grow at 16°C.

The CRISPR system of the present invention for use in P. falciparum by disrupting chromosomal loci. Ghorbal et al. ("Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system", Nature Biotechnology, 32, 819-821 (2014), DOI: 10.1038/nbt.2925, from June 1, 2014) used the CRISPR system to introduce specific gene knockouts and single nucleotide substitutions into the genome of the malaria parasite. To adapt the CRISPR-Cas9 system to P. falciparum, Ghorbal et al. generated expression vectors to control Plasmodium regulatory elements in the pUF1-Cas9 episome, which also carries the drug-selectable marker ydhodh, which confers resistance to DSM1, a P. falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitor, and used P. falciparum U6 small nuclear (sn)RNA regulatory elements for sgRNA transcription by placing the guide RNA and the donor DNA template for homologous recombinational repair on the same plasmid, pL7. See also Zhang C. et al. ("Efficient editing of malaria parasite genome using the CRISPR/Cas9 system", MBio, 2014 Jul 1; 5(4):E01414-14, doi: 10.1128/MbIO.01414-14) 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 of pathogens similar to viral pathogens such as HIV

Cas-mediated genome editing can be used to introduce protective mutations into somatic tissues to treat non-genetic or complex diseases. For example, NHEJ-mediated inactivation of the CCR5 receptor in lymphocytes (Lombardo et al., Nat Biotechnol. 2007 Nov; 25(11):1298-306) may represent an effective strategy to eliminate HIV infection, while deletion of PCSK9 (Cohen et al., Nat Genet. 2005 Feb; 37(2):161-5) or angiopoietin (Musunuru et al., N Engl J Med. 2010 Dec 2; 363(23):2220-7) may provide therapeutic effects against statin-resistant hypercholesterolemia or hyperlipidemia. Although these targets can also be targeted by siRNA-mediated protein knockdown, the unique advantage of NHEJ-mediated gene inactivation is the ability to achieve a long-term therapeutic effect without the need for ongoing treatment. As with all gene therapies, it will certainly be important to establish that each proposed therapeutic application has an effective risk-benefit ratio.

Hydrodynamic delivery of plasmid DNA encoding Cas9 and guide RNA together with a repair template to the liver in an adult mouse model of tyrosinemia was shown to correct the mutant Fah gene and restore wild-type Fah protein expression in ~1 in 250 cells (Nat Biotechnol. 2014 Jun; 32(6):551-3). Furthermore, ZF nucleases have been successfully used in clinical studies to treat HIV infection by ex vivo knockout of the CCR5 receptor. In all patients, HIV DNA levels were reduced, and in one out of four patients, HIV RNA became undetectable (Tebas et al., N Engl J Med. 2014 Mar 6; 370(10):901-10). Both of these results highlight the potential of programmable nucleases as a novel therapeutic platform.

In another embodiment, self-inactivating lentiviral vectors with siRNA targeting a common exon that has tat/rev HIV, a TAR trap nucleolar localization signal, and a CCR5-specific hammerhead ribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) can be used and/or adapted for the CRISPR-Cas9 system of the present invention. At least 2.5 × 10 ⁶ CD34+ cells/kg patient weight can be harvested and prestimulated for 16-20 hours in X-VIVO 15 medium (Lonza) containing 2 μmol/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. Prestimulated cells can be transduced with lentivirus at an 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.).

One skilled in the art, using the knowledge in the art and the teachings of the present invention, can correct HSCs with respect to an immunodeficiency state such as HIV/AIDS, including contacting HSCs with a CRISPR-Cas9 system that targets and knocks down CCR5. A guide RNA (and preferably a dual guide approach, such as a pair of different RNAs; for example, guide RNAs targeting two clinically relevant genes, B2M and CCR5, in primary human CD4+ T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs)) that targets and knocks down a particle containing CCR5 and a Cas9 protein is contacted with the HSCs. The cells so contacted can be administered and optionally processed and expanded; see Cartier. See also Kiem, "Hematopoietic stem cell-based gene therapy for HIV disease," Cell Stem Cell. Feb 3, 2012; 10(2): 137-147; incorporated herein by reference, along with the documents listed 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 listed therein. Also cited is Ebina, "CRISPR/Cas9 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 listed therein, as other means of combating HIV/AIDS using the CRISPR-Cas9 system.

The rationale for genome editing to treat HIV comes from the observation that individuals homozygous for loss-of-function mutations in CCR5, a cellular coreceptor for the virus, are highly resistant to infection and otherwise healthy, suggesting that mimicking this mutation with genome editing might be a safe and effective therapeutic strategy [Liu, R., et al. Cell 86, 367–377 (1996)]. This idea was confirmed clinically when an HIV-infected patient received an allogeneic bone marrow transplant from a donor homozygous for a loss-of-function mutation in CCR5, resulting in undetectable levels of HIV and restoration of normal CD4 T cell counts [Hutter, G., et al. The New England journal of medicine 360, 692–698 (2009)]. Although bone marrow transplantation is not an acceptable treatment strategy for most HIV patients due to cost and potential graft-versus-host disease, HIV therapies that transform the patient's own T cells into CCR5 are desirable.

Early studies using ZFN and NHEJ to knock out CCR5 in humanized mouse models of HIV showed that transplantation of CCR5-edited CD4 T cells improved viral load and CD4 T cell counts [Perez, E.E., et al. Nature biotechnology 26, 808–816 (2008)]. Importantly, these models also showed that HIV infection resulted in selection of CCR5 null cells, suggesting that editing confers a fitness advantage and potentially allows a small number of edited cells to exert therapeutic effect.

As a result of this and other promising preclinical studies, a genome-editing therapy that knocks out CCR5 in patients’ T cells is now being 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 were isolated from HIV patients, edited with ZFNs engineered 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)].

In another study (Mandal et al., Cell Stem Cell, Volume 15, Issue 5, p643-652, November 6, 2014), the CRISPR-Cas9 system targeted two clinically relevant genes, B2M and CCR5, in CD4+ T cells and CD34+ human hematopoietic stem and progenitor cells (HSPCs). Use of single guide RNAs resulted in highly efficient mutagenesis in HSPCs but not in T cells. A dual guide approach increased the efficiency of gene ablation in both cell types. HSPCs that underwent genome editing with CRISPR-Cas9 retained multilineage capacity. Putative on- and off-target mutations were examined by targeted capture sequencing in HSPCs, and low levels of off-target mutagenesis were observed at only one site. These results demonstrate that the CRISPR-Cas9 system can efficiently delete genes in HSPCs with minimal off-target mutagenesis, which has broad applicability to hematopoietic cell-based therapies.

Wang et al. (PLoS One. 2014 Dec 26;9(12):e115987. doi: 10.1371/journal.pone.0115987) silenced CCR5 via CRISPR-associated protein 9 (Cas9) and single guide RNAs (gRNAs) with lentiviral vectors expressing Cas9 and CCR5 gRNAs. Wang et al. showed that single-cycle transduction of lentiviral vectors expressing Cas9 and CCR5 gRNAs into HIV-1-susceptible human CD4+ cells resulted in high frequencies of CCR5 gene disruption. Not only are CCR5 disrupted cells resistant to R5-tropic HIV-1, including transmissible/founder (T/F) HIV-1 isolates, but they also have a selective advantage over intact CCR5 cells during R5-tropic HIV-1 infection. Genomic mutations at potential off-target sites that are highly homologous to this CCR5 guide RNA were not detected in stably transduced cells even 84 days post-transduction using the T7 endonuclease I assay.

Fine et al. (Sci Rep. 2015 Jul 1;5:10777. doi: 10.1038/srep10777) identified a dual-cassette system expressing portions of the S. pyogenes Cas9 protein (SpCas9) that were spliced in the cell to form a functional protein capable of site-specific DNA cleavage. Using specific CRISPR guide strands, Fine et al. showed the efficiency of this system in cleaving the HBB and CCR5 genes in human HEK-293T cells as a single Cas9 and a pair of nicase Cas9s. Trans-spliced SpCas9 (tsSpCas9) had ~35% of the nuclease activity of wild-type SpCas9 (wtSpCas9) at standard transfection doses, but had significantly reduced activity at lower dose levels. The significantly reduced length of the tsSpCas9 open reading frame relative to wtSpCas9 potentially facilitates packaging of more complex and longer genetic elements into the AAV-based vector, including tissue-specific promoters, expression of multiplex guide RNA, and fusion of effector domains to SpCas9.

Li et al. (J Gen Virol. 2015 Aug;96(8):2381-93. doi: 10.1099/vir.0.000139. Epub 2015 Apr 8) showed that the CRISPR-Cas9 system can efficiently mediate editing of the CCR5 locus in cell lines, resulting in knockout of CCR5 cell surface expression. By next-generation sequencing, various mutations were found to be introduced near the putative CCR5 cleavage site. For each of the three top-scoring guide RNAs that were analyzed, no significant off-target effects were detected at the 15 potential sites with the highest scores. By constructing chimeric adenoviruses Ad5F35 carrying CRISPR-Cas9 components, Li et al. effectively transduced primary CD4+ T lymphocytes and disrupted CCR5 expression, and positively transduced cells were rendered resistant to HIV-1.

Reference should be made to WO 2015/148670 and in the teachings set forth herein, the present invention pertains to the methods and materials of this document used in combination with the teachings set forth herein. In one aspect of gene therapy, methods and compositions for editing a target sequence associated with or in association with the human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) are pertained. In a related aspect, the present invention described herein pertains to 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. The CCR5 gene is also known as CKR5, CCR-5, CD195, CKR-5, CCCKR5, CMKBR5, IDDM22 and CC-CKR-5. In a further aspect, the present invention described herein involves use for preventing or reducing infection with HIV and/or preventing or reducing the ability of HIV to enter host cells, such as in subjects who are already infected. Exemplary host cells for HIV include, but are not limited to, CD4 cells, T cells, gut-associated lymphoid tissue (GALT), macrophages, dendritic cells, myeloid progenitor cells, and microglia. Entry of the virus into host cells requires interaction of the viral glycoproteins gp41 and gp120 with the CD4 receptor and a coreceptor, such as CCR5. If the coreceptor, such as CCR5, is not present on the surface of the host cells, the virus cannot bind to and enter the host cells. Thus, disease progression is impeded. By knocking out or knocking down CCR5 in host cells, for example by introducing a protective mutation (such as the CCR5 delta 32 mutation), the HIV virus is prevented from entering the host cells.

One skilled in the art can refer to the above-mentioned studies, such as 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, p643-652, 6 November 2014, Wang et al. (PLoS One. 2014 Dec 26;9(12):e115987. doi: 10.1371/journal.pone.0115987), Fine et al. (Sci Rep. 2015 Jul 1;5:10777. doi: 10.1038/srep10777) and Li et al. (J Gen Virol. 2015 Aug;96(8):2381-93. doi: 10.1099/vir.0.000139. Epub 2015 Apr 8), for targeting CCR5 using the CRISPR Cas9 system of the present invention.

Treatment of pathogens similar to viral pathogens such as HBV

Chronic hepatitis B virus (HBV) infection is common, fatal, and rarely curable due to the persistence of viral episomal DNA (cccDNA) in infected cells. Ramanan et al. (Ramanan V, Shlomai A, Cox DB, Schwartz RE, Michailidis E, Bhatta A, Scott DA, Zhang F, Rice CM, Bhatia SN, Sci Rep. 2015 Jun 2;5:10833. doi: 10.1038/srep10833, published online June 2, 2015) showed that the CRISPR/Cas9 system can specifically target and cleave conserved regions in the HBV genome, resulting in persistent suppression of gene expression and replication. With long-term expression of Cas9 and appropriately selected guide RNAs, they showed cleavage of cccDNA by Cas9 and a significant reduction in cccDNA and other parameters of viral gene expression and replication. They thus showed that direct targeting of episomal DNA is a new therapeutic approach to control the virus and possibly cure patients. This is also described in WO2015089465 A1, on behalf of The Broad Institute et al., the contents of which are hereby incorporated by reference.

The present invention can also be used to treat hepatitis B virus (HBV). However, the CRISPR-Cas system should be adapted to avoid the disadvantages of RNAi, such as the risk of oversaturation of endogenous small RNA pathways, by, for example, dose and sequence optimization (see, for example, Grimm et al., Nature vol. 441, 26 May 2006). For example, low doses such as about 1-10 x 10 ¹⁴ particles per person are envisaged. In another embodiment, the CRISPR-Cas system directed against HBV can be administered in liposomes, such as a stable nucleic acid lipid particle (SNALP) (see, for example, Morrissey et al., Nature Biotechnology, Vol. 23, No. 8, August 2005). Daily intravenous injections of about 1, 3, or 5 mg/kg/day of CRISPR Cas targeting HBV RNA at SNALPs are contemplated. Treatment may be daily for about three days, and then weekly for about five weeks. In other embodiments, the system of Chen et al. (Gene Therapy (2007) 14, 11-19) may be applied to and/or adapted from the CRISPR-Cas system of the present invention. Chen et al. used a double-stranded pseudotyped adeno-associated virus 8 (dsAAV2/8) vector to deliver shRNA. A single administration of the dsAAV2/8 vector (1 x 10 ¹² vector genomes per mouse) carrying the HBV-specific shRNA effectively suppressed the steady-state level of HBV protein, mRNA and replicative DNA in the liver of HBV transgenic mice, resulting in a reduction in the circulating HBV load by up to 2-3 log ₁₀ . Significant HBV suppression lasted for at least 120 days after vector administration. The therapeutic effect of the shRNA was dependent on the target sequence and did not involve interferon activation. According to the present invention, the HBV-targeted CRISPR-Cas system can be cloned into an AAV-based vector, such as a dsAAV2/8-based vector, and administered to a human, such as at a dose of from about 1 x 10 ¹⁵ vector genomes to about 1 x 10 ¹⁶ vector genomes per human. In another embodiment, the method of Wooddell et al. (Molecular Therapy vol. 21 no. 5, 973-985 May 2013) can be applied to and/or adapted to the CRISPR-Cas system of the present invention. Woodell et al. demonstrated that simple co-injection of a hepatocyte-targeting N-acetylgalactosamine-conjugated melittin-like peptide (NAG-MLP) with a liver-tropic cholesterol-conjugated siRNA (chol-siRNA) targeting coagulation factor VII (F7) results in efficient knockdown of F7 in mice and non-human primates, without changes in clinical chemistry or cytokine induction. Using transient and transgenic mouse models of HBV infection, Wooddell et al. demonstrated that a single co-injection of NAG-MLP with active chol-siRNA targeting conserved sequences of HBV resulted in multiple repression of viral RNA, proteins and viral DNA with a long duration of effect. Intravenous co-injections of, for example, about 6 mg/kg NAG-MLP and 6 mg/kg HBV-specific CRISPR-Cas can be provided by the present invention. Alternatively, about 3 mg/kg NAG-MLP and 3 mg/kg HBV-specific CRISPR-Cas can be delivered on the first day, followed by about 2-3 mg/kg NAG-MLP and 2-3 mg/kg HBV-specific CRISPR-Cas two weeks later.

Lin et al. (Mol Ther Nucleic Acids. 2014 Aug 19;3:e186. doi: 10.1038/mtna.2014.38) designed eight gRNAs against HBV genotype A. 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-based expression vector. Among the eight gRNAs screened, two were identified as effective. One gRNA targeting a conserved sequence of HBV was active against multiple genotypes. Using a hydrodynamic mouse model of HBV resistance, Lin et al. further demonstrated that this system could cleave the plasmid containing the HBV genome in the liver and facilitate its clearance in vivo, resulting in reduced serum surface antigen levels. These data indicate that the CRISPR-Cas9 system could disrupt HBV-expressing templates both in vitro and in vivo, indicating potential in eliminating chronic HBV infection.

Dong et al. (Antiviral Res. 2015 Jun;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 (gRNAs) targeting conserved regions of HBV. Expression of these gRNAs with Cas9 reduced virus production in Huh7 cells as well as in HepG2.2.15 cells replicating HBV. Dong et al. further demonstrated that direct cleavage by the CRISPR-Cas9 system and cleavage-mediated mutagenesis occurred in cccDNA of HBV transfected cells. In a cccDNA-bearing HBV mouse model, injection of guide RNA- and Cas9-based plasmids into the tail vein resulted in low levels of cccDNA and HBV protein.

Liu et al. (J Gen Virol. 2015 Aug;96(8):2252-61. doi: 10.1099/vir.0.000159. Epub 2015 Apr 22) designed eight guide RNAs (gRNAs) targeting conserved regions of different HBV genotypes that could significantly inhibit HBV replication both in vitro and in vivo to investigate the feasibility of using the CRISPR-Cas9 system to disrupt HBV DNA templates. The HBV-specific gRNA/Cas9 system could inhibit HBV replication of different genotypes in cells, and the viral DNA level was significantly reduced by the single gRNA/Cas9 system and cleared by the combination of different gRNA/Cas9 systems.

Wang et al. (World J Gastroenterol. 2015 Aug 28;21(32):9554-65. doi: 10.3748/wjg.v21.i32.9554) designed 15 gRNAs to HBV genotypes A-D. Eleven combinations of the above two gRNAs (dual gRNAs) covering the HBV regulatory region were selected. The efficacy of each gRNA and the 11 dual gRNAs in suppressing HBV replication (genotypes A-D) was examined by measuring HBV surface antigen (HBsAg) or e antigen (HBeAg) in the culture supernatant. The degradation of HBV-expressing vectors was examined in HuH7 cells co-transfected with dual gRNA and HBV expressing vector by polymerase chain reaction (PCR) and sequencing, and the degradation of cccDNA was examined in HepAD38 cells by KCl precipitation, plasmid-safe ATP-dependent DNase (PSAD) digestion, combined rolling circle amplification method and quantitative PCR. The cytotoxicity of these gRNAs was determined by mitochondrial tetrazolium assay. All of the gRNAs could significantly reduce the formation of HBsAg or HBeAg in the culture supernatant, which depended on the targeting region of the gRNA. All of the dual gRNAs could effectively suppress the formation of HBsAg and/or HBeAg in the case of HBV genotypes A-D, and the efficiency of the dual gRNAs in suppressing the formation of HBsAg and/or HBeAg was significantly increased when compared with the use of single gRNAs alone. In addition, the present inventors confirmed by direct PCR sequencing that these dual gRNAs could specifically degrade HBV-expressing templates by removing the fragment between the cleavage sites of the two gRNAs used. More importantly, the combination of gRNA-5 and gRNA-12 not only could effectively suppress the formation of HBsAg and/or HBeAg but also degrade the cccDNA pool in HepAD38 cells.

Karimova et al. (Sci Rep. 2015 Sep 3;5:13734. doi: 10.1038/srep13734) identified conserved HBV sequences between genotypes in the S and X regions of the HBV genome that were targeted for specific and efficient cleavage by Cas9 nickase. This approach disrupted not only episomal cccDNA and chromosomally integrated HBV target sites in reporter cell lines but also HBV replication in chronically and de novo infected hepatoma cell lines.

One skilled in the art can refer to the above studies, such as 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;118:110-7. 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/wjg.v21.i32.9554) and Karimova et al. (Sci Rep. 2015 Sep 3;5:13734. doi: 10.1038/srep13734) for targeting HBV using the CRISPR Cas system of the present invention.

Methods of personalized screening of patients

A CRISPR-Cas system that specifically targets nucleotides, such as trinucleotide repeats, can be used to screen patients or patient samples for the presence of such repeats. The repeats can be targeted by the RNA of the CRISPR-Cas system, and if they bind to the CRISPR-Cas system, the binding can be detected, thereby indicating the presence of such a repeat. Thus, a CRISPR-Cas system can be used to screen patients or patient samples for the presence of repeats. The patient can then be administered a suitable compound(s) that targets the condition; or the CRISPR-Cas system can be administered to bind to the nucleotide and cause an insertion, deletion, or mutation and alleviate the severity of the condition.

Treatment of diseases with genetic and epigenetic aspects

The CRISPR-Cas9 systems of the present disclosure can be used to correct genetic mutations that have previously been attempted with limited success using TALENs and ZFNs and that have been identified as potential targets for Cas9 systems, including as in published patent applications by Editas Medicine describing methods of using Cas9 systems to target loci for the purpose of therapeutically targeting diseases using gene therapy, including WO 2015/048577 CRISPR-RELATED METHODS AND COMPOSITIONS Gluckmann et al.; WO 2015/070083 CRISPR-RELATED METHODS AND COMPOSITIONS WITH GOVERNING gRNAS Glucksmann et al.

Mention should be made of WO 2015/134812 CRISPR/CAS-RELATED METHODS AND COMPOSITIONS FOR TREATING USHER SYNDROME AND RETINITIS PIGMENTOSA, Maeder et al. In the teachings set forth herein, the present invention contemplates the methods and materials of these documents used in combination with the teachings set forth herein. In one aspect, gene therapy for vision and hearing disorders, methods and compositions for treating Usher syndrome and retinitis pigmentosa can be adapted to the CRISPR-Cas system of the present invention (see, for example, WO 2015/134812). In one embodiment, WO 2015/134812 provides for treating or delaying the onset or progression of Usher syndrome type IIA (USH2A, USH11A) and retinitis pigmentosa type 39 (RP39) by gene editing, such as by CRISPR-Cas9 mediated methods, to correct a guanine deletion at position 2299 in the USH2A gene (e.g., replacing a deleted guanine residue at position 2299 in the USH2A gene). In a related aspect, the mutation is targeted by cleavage with one or more nucleases, one or more nickases, or a combination thereof, such as to induce HDR with a donor template that corrects a point mutation (e.g., a single nucleotide, such as a guanine, deletion). The alteration or correction of the mutant USH2A gene may be mediated by any mechanism. Exemplary mechanisms that may be associated with alteration (e.g., correction) of a mutant HSH2A gene include, but are not limited to, non-homologous end joining, microhomology-mediated end joining (MMEJ), homologous recombination repair (e.g., endogenous donor template mediated), SDSA (synthesis-dependent strand annealing), single-strand annealing, and single-strand invasion. In an embodiment, a method used to treat Usher syndrome and retinitis pigmentosa may include obtaining information about a mutation carried by a subject, such as by sequencing the relevant region of the USH2A gene.

In some embodiments, treatment, prevention and diagnosis of primary open-angle glaucoma (POAG) is provided. The target is preferably the MYOC gene. This is described in WO 2015/153780, the disclosure of which is incorporated herein by reference.

WO 2015/138510 should be mentioned and in the teachings set out in this document, the present invention (using the CRISPR-Cas9 system) involves providing a treatment or delaying the onset or progression of Leber congenital amaurosis type 10 (LCA 10). LCA 10 is caused by a mutation in the CEP290 gene, for example c.2991+1655, an adenine to guanine mutation in the CEP290 gene that results in the formation of a cryptic splice site in intron 26. This is a mutation at nucleotide 1655 of intron 26 of CEP290, for example an A to G mutation. CEP290 is also known as CT87; MKS4; POC3; rd16; BBS14; JBTS5; LCAJO; NPHP6; SLSN6 and 3H11Ag (see, for example, WO 2015/138510). In one aspect of gene therapy, the present invention provides for the introduction of one or more breaks near the LCA target site (e.g., c.2991 + 1655; A to G) in at least one allele of the CEP290 gene. Alteration of the LCA10 target position refers to (1) a break-induced insertion/deletion (also referred to herein as NHEJ-mediated insertion/deletion) in close proximity to or including the LCA10 target position (e.g., c.2991+1655 A to G), or (2) a break-induced deletion (also referred to as NHEJ-mediated deletion) of the genomic sequence, including a mutation at the LCA10 target position (e.g., c.2991+1655 A to G). Both approaches result in loss of function or disruption of the cryptic splice site resulting from the mutation at the LCA 10 target position.

In one aspect, the present invention (using the CRISPR-Cas9 system) involves treating or delaying the onset or progression of Leber congenital amaurosis type 10 (LCA 10). LCA 10 is caused by a mutation in the CEP290 gene, such as c.2991+1655, an adenine to guanine mutation in the CEP290 gene that results in a cryptic splice site in intron 26. This is a mutation at nucleotide 1655 of intron 26 of CEP290, such as an A to G mutation. CEP290 is also known as CT87; MKS4; POC3; rd16; BBS14; JBTS5; LCAJO; NPHP6; SLSN6 and 3H11Ag (see, such as WO 2015/138510). In one aspect of gene therapy, the present invention provides for the introduction of one or more breaks near the site of an LCA target position (e.g., c.2991+1655; A to G) in at least one allele of the CEP290 gene. An alteration of the LCA10 target position refers to (1) a break-induced insertion/deletion introduction (also referred to herein as NHEJ-mediated insertion/deletion introduction) in close proximity to or including the LCA10 target position (e.g., c.2991+1655 A to G), or (2) a break-induced deletion (also referred to as NHEJ-mediated deletion) of the genomic sequence, including a mutation at the LCA10 target position (e.g., c.2991+1655 A to G). Both approaches result in loss of function or disruption of the cryptic splice site resulting from mutation at the target position of LCA 10.

Researchers are considering whether gene therapy can be used to treat a wide range of diseases. The Cas9 effector protein-based CRISPR systems of the present invention are contemplated for such therapeutic applications, including, but not limited to, additional exemplified regions and delivery methods provided below. Some examples of conditions or diseases that can be effectively treated using the system of the present invention, included in the exemplified genes and references incorporated herein and currently associated with such conditions, are also provided herein. The exemplified genes and conditions are not exhaustive.

Treatment of cardiovascular diseases

The present invention also provides for the delivery of a CRISPR-Cas9 system, in particular the novel CRISPR effector protein systems described herein, into blood or hematopoietic stem cells. The plasma exosomes of Wahlgren et al. (Nucleic Acids Research, 2012, Vol. 40, No. 17 e130) have been previously described and can be used to deliver the CRISPR Cas9 system into blood. The nucleic acid targeting system of the present invention is also provided for the treatment of hemoglobinopathies, such as forms of thalassemia and sickle cell anemia. See, for example, International Patent Application Publication No. WO 2013/126794 for potential targets that can be targeted by the CRISPR Cas9 system of the present invention.

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, incorporated herein by reference along with the documents that would be listed therein if they were cited in full, discusses the modification of HSC using a lentivirus that delivers the β-globin or γ-globin gene. In contrast to the use of a lentivirus, a person skilled in the art, using the knowledge in the art and the teachings of the present invention, can correct HSCs for β-thalassemia using a CRISPR-Cas9 system that targets and corrects a mutation (e.g., using a suitable template for HDR that delivers a coding sequence for β-globin or γ-globin, preferably β-globin or γ-globin of non-sickled red blood cells); in particular, the guide RNA can target a mutation that leads to β-thalassemia, and HDR can provide coding that leads to proper expression of β-globin or γ-globin. The guide RNA that targets the mutation and the particle containing the Cas9 protein contacts the HSCs carrying the mutation. The particle may also contain a suitable HDR template for correcting the mutation to properly express β-globin or γ-globin; or the HSC may be contacted with a second particle or vector that contains or delivers the HDR template. The cells so contacted can be administered and optionally processed and expanded; see Cartier. In this regard, mention should be made of Cavazzana's publication "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-thalassaemia", Nature 467, 318-322 (16 September 2010) doi:10.1038/nature09328; Nienhuis "Development of Gene Therapy for Thalassemia, Cold Spring Harbor Perspectives in Medicine, doi: 10.1101/cshperspect.a011833 (2012), LentiGlobin BB305, a lentiviral vector containing an engineered β-globin gene (β ^A-T87Q ); and Xie et al. "Seamless gene correction of β-thalassaemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyback" Genome Research gr.173427.114 (2014) http://www.genome.org/cgi/doi/10.1101/gr.173427.114 (Cold Spring Harbor Laboratory Press); which is the subject of Cavazzana's study, including human β-thalassemia, and the subject of Xie's study, all included in this document by reference, together with all documents listed or linked thereto. In the present invention, the HDR matrix may provide for the expression of an engineered β-globin gene (e.g., β ^A-T87Q ) or a β-globin as disclosed in Xie.

Xu et al. (Sci Rep. 2015 Jul 9;5:12065. doi: 10.1038/srep12065) designed TALENs and CRISPR-Cas9 to directly target the IVS2-654 mutation site of intron 2 in the globin gene. Xu et al. observed different double-strand break (DSB) frequencies at the IVS2-654 loci between TALENs and CRISPR-Cas9, and TALENs mediated higher homologous gene targeting efficiency compared to CRISPR-Cas9 when combined with the piggyBac transposon donor. Furthermore, more obvious off-target events were observed with CRISPR-Cas9 compared to TALENs. Finally, TALEN-corrected iPSC clones were selected for erythroblast differentiation using the OP9 coculture system and revealed relatively high HBB transcription compared to uncorrected cells.

Song et al. (Stem Cells Dev. 2015 May 1;24(9):1053-65. doi: 10.1089/scd.2014.0347. Epub 2015 Feb 5) used the CRISPR/Cas9 system to correct β-Thal gene-corrected iPSCs; the gene-corrected cells had normal karyotypes and were fully pluripotent, as human embryonic stem cells (hESCs) showed no off-target effects. Subsequently, Song et al. assessed the differentiation efficiency of β-Thal gene-corrected iPSCs. Song et al. found that during hematopoietic cell differentiation, β-Thal gene-corrected iPSCs had increased embryoid body ratios and variable percentages of hematopoietic progenitor cells. More importantly, β-Thal gene-corrected iPSC lines restored HBB expression and reduced reactive oxygen species generation compared to the uncorrected group. The study by Song et al. showed that the hematopoietic differentiation efficiency of β-Thal iPSCs was significantly increased immediately after correction with the CRISPR-Cas9 system. Similar approaches can be implemented using the CRISPR-Cas9 systems described herein, such as those containing Cas9 effector proteins.

Reference should be made to WO 2015/148860, in the teachings set forth therein, the present invention contemplates the methods and materials of these documents used in combination with the teachings set forth herein. In one aspect, gene therapy for diseases associated with the blood system, methods and compositions for the treatment of beta-thalassemia can be adapted to the CRISPR-Cas system of the present invention (see, for example, WO 2015/148860). In one embodiment, WO 2015/148860 provides for the treatment or prevention of beta-thalassemia, or its symptoms, for example, by altering the B-cell CLL/lymphoma 11A (BCL11A) gene. The BCL11A gene is also known as B-cell CLL/lymphoma gene 11A, BCL11A-L, BCL11A-S, BCL11AXL, CTIP 1, HBFQTL5, and ZNF. BCL11A encodes a zinc finger protein that is involved in regulating globin gene expression. When the BCL11A gene is altered (e.g., one or both alleles of the BCL11A gene), gamma globin levels may increase. Gamma globin can replace beta globin in the hemoglobin complex and efficiently deliver oxygen to tissues, thereby normalizing the disease phenotypes of beta thalassemia.

Sickle cell anemia is an autosomal recessive inherited disorder in which the red blood cells become sickle-shaped. It is caused by a single base substitution in the β-globin gene, which is located on the short arm of chromosome 11. As a result, valine is produced instead of glutamic acid, which causes the production of sickle cell hemoglobin (HbS). This results in the formation of a curved shape of red blood cells. Due to the abnormal shape, small blood vessels can become blocked, causing severe damage to the bone, spleen, and skin tissue. This can lead to bouts of pain, frequent infections, hand-foot syndrome, or even multiple organ failure. The curved red blood cells are also more susceptible to hemolysis, which leads to severe anemia. As in the case of β-thalassemia, sickle cell anemia can be corrected by modifying HSCs using the CRISPR-Cas9 system. This system allows for specific editing of the cell's genome by cutting its DNA and then ensuring its self-repair. The Cas9 protein is inserted and directed by a guide RNA to the point of mutation, and then it cuts the DNA at this point. At the same time, a normal variant of the sequence is inserted. This system is used by the intrinsic repair system to correct the induced cut. In this regard, the CRISPR-Cas9 system allows for the correction of a mutation in previously obtained stem cells. A person skilled in the art, using the knowledge in the art and the teachings of the present invention, can correct HSCs for sickle cell anemia using the CRISPR-Cas9 system, which targets the mutation and corrects it (e.g., using a suitable template for HDR that delivers a coding sequence for β-globin, preferably β-globin of non-sickled red blood cells); in particular, the guide RNA can target a mutation that causes sickle cell anemia, and HDR can provide encoding that results in proper expression of β-globin. The guide RNA that targets the mutation and the particle containing the Cas9 protein are contacted with HSCs bearing the mutation. The particle can also contain a suitable HDR template for correcting the mutation to properly express β-globin; or the HSCs can be contacted with a second particle or vector that contains or delivers the HDR template. The cells so contacted can be administered and optionally processed and expanded; see Cartier. The HDR template can provide for the expression by the HSCs of an engineered β-globin gene (e.g., βA-T87Q) or of the β-globin described in Xie.

WO 2015/148863 should be mentioned, and in the teachings set forth in this document, the present invention contemplates the methods and materials of these documents, which can be adapted to the CRISPR-Cas system of the present invention. In an aspect of treating or preventing sickle cell anemia, which is an inherited hematological blood disorder, WO 2015/148863 provides for altering the BCL11A gene. By altering the BCL11A gene (for example, one or both alleles of the BCL11A gene), gamma globin levels can be increased. Gamma globin can replace beta globin in the hemoglobin complex and effectively deliver oxygen to tissues, thereby normalizing the phenotypes of sickle cell anemia.

Williams, "Broadening the Indications for Hematopoietic Stem Cell Genetic Therapies," Cell Stem Cell 13:263-264 (2013), incorporated herein by reference along with the papers that would be cited therein if reported in full, reports lentiviral-mediated gene transfer into HSC/P cells from patients with the lysosomal storage disease metachromatic leukodystrophy (MLD), an inherited disorder caused by arylsulfatase A deficiency (ARSA) that results in nerve demyelination; and lentivirus-mediated gene transfer into HSCs of patients with Wiskott-Aldrich syndrome (WAS) (patients with a defective WAS protein, an effector of the small GTPase CDC42 that regulates cytoskeletal function in blood cell lines and thus suffer from immunodeficiency with recurrent infections, symptoms of autoimmune diseases and thrombocytopenia with abnormally small and functionally ineffective platelets, which leads to excessive bleeding and an increased risk of leukemia and lymphoma). In contrast to the use of lentivirus, a person skilled in the art, using the knowledge in the art and the teachings of the present invention, can correct HSCs for MLD (arylsulfatase A deficiency (ARSA)) using the CRISPR-Cas9 system that targets and corrects the mutation (arylsulfatase A deficiency (ARSA)) (e.g., using a suitable template for HDR that delivers the coding sequence for ARSA); in particular, the guide RNA may target a mutation that results in MLD (ARSA deficiency) and HDR may provide coding that results in proper expression of ARSA. The guide RNA that targets the mutation and the particle containing the Cas9 protein are contacted with HSCs bearing the mutation. The particle may also contain a suitable template for HDR to correct the mutation to result in proper expression of ARSA; or the HSCs may be contacted with a second particle or vector that contains or delivers a template for HDR. The cells thus contacted may be administered and optionally processed and expanded; see Cartier. In contrast to the use of a lentivirus, a person skilled in the art, using the knowledge in the art and the teachings of the present invention, can correct HSCs for WAS using a CRISPR-Cas9 system that targets and corrects a mutation (deficiency of the WAS protein) (e.g., using a suitable template for HDR that delivers a coding sequence for the WAS protein); in particular, the guide RNA can target a mutation that leads to WAS (defective WAS protein), and HDR can provide coding that leads to proper expression of the WAS protein. The guide RNA that targets the mutation and the particle containing the Cas9 protein are contacted with HSCs carrying the mutation. The particle can also contain a suitable template for HDR to correct the mutation in order to properly express the WAS protein; or the HSC can be contacted with a second particle or vector that contains or delivers a template for HDR. The cells thus brought into contact can be introduced and optionally processed and multiplied; see Cartier.

In one aspect of the present invention, methods and compositions that involve editing a target nucleic acid sequence or modulating the expression of a target nucleic acid sequence and their uses in connection with cancer immunotherapy are understood by adapting the CRISPR-Cas system of the present invention. Reference is made to the use of gene therapy in WO 2015/161276, which provides methods and compositions that can be used to disrupt the proliferation, survival and/or function of T cells by altering one or more genes expressed by T cells, such as one or more of the FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, TRAC and/or TRBC genes. In related aspects, T cell proliferation may be impaired by alteration of one or more genes expressed by T cells, such as the CBLB gene and/or PTPN6, the FAS gene and/or BID, the CTLA4 gene and/or PDCDI and/or TRAC and/or TRBC.

Chimeric antigen receptor (CAR)19 T cells have been shown to have anti-leukemic effects in patients with malignancies. However, leukemia patients often have insufficient T cells to collect, so treatment must include modified T cells from donors. Accordingly, there is interest in establishing a donor T cell bank. 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) describe the modification of CAR19 T cells to eliminate the risk of graft-versus-host disease by disrupting T cell receptor expression and targeting CD52. Furthermore, CD52 cells were targeted so that they were refractory to alemtuzumab, thereby contributing to alemtuzumab's ability to prevent host-mediated rejection of human leukocyte antigen (HLA)-mismatched CAR19 T cells. The researchers used a third-generation self-inactivating lentiviral vector encoding 4g7 CAR19 (CD19 scFv-4-1BB-CD3ζ) linked to RQR8, then electroporated the cells with two pairs of TALEN mRNAs to multiplexly target the T cell receptor (TCR) constant alpha chain locus and the CD52 gene locus. Cells that still expressed TCR after ex vivo expansion were depleted by CliniMacs α/β TCR depletion, resulting in a T cell product (UCART19) with <1% TCR expression, 85% of which were CAR19 and 64% were negative for CD52. The CAR19-modified T cells were administered to treat relapsed acute lymphoblastic leukemia in patients. The concepts provided herein provide efficient ways to modify cells, for example, to remove and modulating CD52 or other targets, thus they can be used in combination with modification of the administration of T cells or other cells to patients for the treatment of malignancies.

In Watts, “Hematopoietic Stem Cell Expansion and Gene Therapy,” Cytotherapy 13(10):1164–1171. doi:10.3109/14653249.2011.620748 (2011), incorporated by reference herein, together with the documents it lists, as if they were set out in full, discusses hematopoietic stem cell (HSC) gene therapy, such as viral-mediated HSC gene therapy, as a promising treatment option for many disorders, including hematological conditions, types of immunodeficiency including HIV/AIDS, and other inherited disorders such as lysosomal storage diseases including SCID-X1, ADA-SCID, X-linked β-thalassemia CGD, Wiskott-Aldrich syndrome, Fanconi anemia, adrenoleukodystrophy (ALD), and metachromatic leukodystrophy (MLD).

U.S. Patent Application Publication Nos. 20110225664, 20110091441, 20100229252, 20090271881, and 20090222937 assigned to Cellectis relate to CREI variants wherein at least one of the two I-CreI monomers has at least two substitutions, one in each of the two functional subdomains of the LAGLIDADG core domain located at positions 26-40 and 44-77 of I-CreI, respectively, wherein said variant is capable of cleaving a target DNA sequence of the human interleukin-2 receptor gamma chain gene (IL2RG), also referred to as the cytokine receptor common gamma chain gene or gamma-C gene. The target sequences disclosed in U.S. Patent Application Publication Nos. 20110225664, 20110091441, 20100229252, 20090271881, and 20090222937 can be used for the nucleic acid targeting system of the present invention.

Severe combined immunodeficiency (SCID) results from defective T-lymphocyte maturation, in all cases associated with defective B-lymphocyte function (Cavazzana-Calvo et al., Annu. Rev. Med., 2005, 56, 585–602; Fischer et al., Immunol. Rev., 2005, 203, 98–109). The overall incidence is estimated at 1 in 75,000 births. Patients with untreated SCID are susceptible to multiple opportunistic infections and typically survive for less than 1 year. SCID can be treated by allogeneic transfer of hematopoietic stem cells from a related donor. The degree of histocompatibility with the donor can vary widely. In cases of adenosine deaminase (ADA) deficiency, a form of SCID, patients can be treated with an injection of recombinant adenosine deaminase enzyme.

Since the ADA gene has been shown to be mutated in patients with SCID (Giblett et al., 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 of 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 gamma-C protein (Noguchi, et al., Cell, 1993, 73, 147-157), a common component of at least five interleukin receptor complexes. These receptors activate several targets via the kinase JAK3 (Macchi et al., Nature, 1995, 377, 65-68), whose inactivation results in the same syndrome as gamma-C inactivation. (ii) Mutation in the ADA gene results in defective purine metabolism, causing death of lymphocyte precursors, which in turn leads to the apparent 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 genes 1 and 2 (RAG1 and RAG2) and Artemis, three genes involved in this process, result in the absence of mature T and B lymphocytes. (iv) Mutations in other genes such as CD45, involved in T cell-specific signaling, have also been reported, although they represent a minority of cases (Cavazzana-Calvo et al., Annu. 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 model for gene therapy approaches (Fischer et al., Immunol. Rev., 2005, 203, 98-109) for two main reasons. First, as with all blood disorders, ex vivo treatment may be considered. Hematopoietic stem cells (HSCs) can be isolated from bone marrow and retained in their pluripotency properties for several cell divisions. They can then be processed in vitro and then reinjected into the patient, where they repopulate the bone marrow. Second, because lymphocyte maturation is impaired in SCID patients, corrected cells have a selective advantage. Thus, a small number of corrected cells may restore immune function. This hypothesis has been supported several times by (i) partial restoration of immune functions associated with reversion of mutations in SCID patients (Hirschhorn 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. Sci. USA, 2000, 97, 274-278; Wada et al., Proc. Natl. Acad. Sci. USA, 2001, 98, 8697-8702; Nishikomori et al., Blood, 2004, 103, 4565-4572), (ii) correction of SCID-X1 deficiency forms in vitro in hematopoietic cells (Candotti et al., 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 (Soudais et al., Blood, 2000, 95, 3071-3077; Tsai et al., Blood, 2002, 100, 72-79), JAK-3 (Bunting et al., 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 in animal models and (iv) the result of clinical trials of gene therapy (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).

U.S. Patent Application Publication No. 20110182867 assigned to Children's Medical Center Corporation and the President and Board of Governors of Harvard University relates to methods of modulating fetal hemoglobin (HbF) expression and uses thereof in hematopoietic progenitor cells using inhibitors of BCL11A expression or activity, such as RNAi agents and antibodies. The targets disclosed in U.S. Patent Application Publication No. 20110182867, such as BCL11A, can be targeted using the CRISPR Cas9 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 18 November 2011: Vol. 334 no. 6058 pp. 993-996) for additional targets of BCL11A.

Having knowledge in the art and the ideas of the present invention, a person skilled in the art can correct HSC in relation to an inherited hematological disorder, for example, β-thalassemia, hemophilia or a genetic lysosomal storage disease.

Treatment of diseases of the brain, central nervous system and immune system

The present invention also provides for the delivery of a CRISPR-Cas system to the brain or neurons. For example, RNA interference (RNAi) provides therapeutic options for the treatment of this disorder 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), and therefore the inventor contemplates that it can be used with and/or adapted to a CRISPR-Cas system. The CRISPR-Cas system can be designed using an algorithm to reduce the possibility of off-target effects of antisense sequences. The CRISPR-Cas sequences can target a sequence in exon 52 of mouse, rhesus macaque, or human huntingtin and be expressed by a viral vector, such as an AAV-based one. Animals, including humans, can be administered by approximately three microinjections per hemisphere (six injections total): the first 1 mm rostral to the anterior commissure (12 μl) and two remaining injections (12 μl and 10 μl, respectively) 3 and 6 mm caudal to the first injection, with 1e12 vg/ml AAV at a rate of approximately 1 μl/minute, with the needle left in place for an additional 5 minutes to allow diffusion of the injected substance from the needle tip.

DiFiglia et al. (PNAS, October 23, 2007, vol. 104, no. 43, 17204-17209) observed that a single intrastriatal injection of siRNA targeting Htt could silence mutated Htt, attenuate neuronal pathology, and delay the development of the abnormal behavioral phenotype observed in a rapid-onset virus-derived transgenic mouse model of HD. DiFiglia injected mice intrastriatally with 2 μl of Cy3-labeled cc-siRNA-Htt or unconjugated siRNA-Htt at 10 μM. A similar dose of Htt-targeting CRISPR-Cas can be provided in the present invention for a human, for example, approximately 5-10 ml of 10 μM Htt-targeting CRISPR-Cas can be injected into the striatum.

In another example, Boudreau et al. (Molecular Therapy vol. 17 no. 6 June 2009) injected 5 μl of recombinant AAV serotype 2/1 vectors expressing htt-specific virus for RNAi (at 4 x 10 ¹² viral genomes/ml) into the striatum. A similar dose of Htt-targeting CRISPR-Cas can be envisaged in the present invention for humans, for example, about 10-20 ml of 4 x 10 ¹² viral genomes/ml, wherein Htt-targeting CRISPR-Cas can be injected into the striatum.

In another example, CRISPR-Cas targeting HTT can be administered continuously (see, e.g., Yu et al., Cell 150, 895-908, August 31, 2012). Yu et al. used osmotic pumps capable of delivering 0.25 ml/h (model 2004) to deliver 300 mg/day of ss-siRNA or phosphate-buffered saline (PBS) (Sigma Aldrich) for 28 days, and pumps capable of delivering 0.5 μl/h (model 2002) were used to deliver 75 mg/day of MOE ASO positive control for 14 days. Pumps (Durect Corporation) were filled with ss-siRNA or MOE diluted with sterile PBS and then incubated at 37 C for 24 or 48 (Model 2004) hours prior to implantation. Mice were anesthetized with 2.5% isoflurane and a midline incision was made at the base of the skull. Using stereotaxic probes, a cannula was implanted into the lateral right ventricle and secured with Loctite glue. A catheter attached to an Alzet osmotic minipump was attached to the cannula and the pump was placed subcutaneously between the scapulae. The incision was closed with sutures using 5.0 nylon. A similar dose of Htt-targeting CRISPR-Cas can be envisaged in the present invention for humans, for example, about 500 to 1000 g/day of Htt-targeting CRISPR-Cas can be administered.

In another example of continuous infusion, Stiles et al (Experimental Neurology 233 (2012) 463–471) implanted an intraparenchymal catheter with a titanium needle tip into the right putamen. The catheter was connected to a SynchroMed® II pump (Medtronic Neurological, Minneapolis, MN) implanted subcutaneously in the abdomen. After 7 days of phosphate-buffered saline infusion at 6 μL/day, the pumps were refilled with study drug and programmed for continuous delivery for 7 days. Approximately 2.3 to 11.52 mg/day of siRNA were infused at varying infusion rates from approximately 0.1 to 0.5 μL/min. A similar dosage of Htt-targeted CRISPR-Cas may be provided in the present invention for humans, such as about 20 to 200 mg/day of Htt-targeted CRISPR-Cas may be administered. In another example, the methods of U.S. Patent Publication No. 20130253040 (WO2013130824) assigned to Sangamo may also be adapted from TALES to the CRISPR-Cas system of the present invention for the treatment of Huntington's disease. WO2015089354 A1 by The Broad Institute et al., incorporated herein by reference, describes targets for Huntington's disease (HP). In Huntington's disease, potential target genes for the CRISPR complex include PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4, and TGM2.

Accordingly, one or more of PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4 and TGM2 may be selected as targets for Huntington's disease in some embodiments of the present invention.

Other trinucleotide repeat disorders. These may include any of the following. Category I includes Huntington's disease (HD) and the spinocerebellar ataxias; category II expansions are phenotypically diverse with heterogeneous expansions that are typically small in size but also occur in gene exons; and category III includes fragile X syndrome, myotonic dystrophy, two of the spinocerebellar ataxias, juvenile myoclonus epilepsy, and Friedreich's ataxia.

An additional aspect of the present invention relates to the use of the CRISPR-Cas system to correct defects in the EMP2A and EMP2B genes that have been found to be associated with Lafora disease. Lafora disease is an autosomal recessive condition characterized by progressive myoclonus epilepsy that may begin with epileptic seizures in adolescence. Some cases of the disease may be caused by mutations in genes that have already been identified. The disease causes seizures, muscle spasms, difficulty walking, dementia, and ultimately death. There is currently no therapy that has shown efficacy in the progression of the disease. Other genetic disorders associated with epilepsy can also be targeted using the CRISPR-Cas system, and the underlying genetic mechanisms are further described in Genetics of Epilepsy and Genetic Epilepsies, edited by Giuliano Avanzini, Jeffrey L. Noebels, Mariani Foundation Paediatric Neurology:20; 2009).

The methods of U.S. Patent Publication No. 20110158957 assigned to Sangamo BioSciences, Inc., related to the inactivation of T cell receptor (TCR) genes, can also be modified for use with the CRISPR-Cas system of the present invention. In another example, the methods of U.S. Patent Application Publication No. 20100311124 assigned to Sangamo BioSciences, Inc. and U.S. Patent Application Publication No. 20110225664 assigned to Cellectis, both of which relate to the inactivation of glutamine synthetase gene expression, can also be modified for use with the CRISPR-Cas system of the present invention.

Treatment of hearing related diseases

The present invention also provides for delivery of the CRISPR-Cas system to one ear or both ears.

Researchers are considering whether gene therapy could be used to support existing treatments for deafness, namely cochlear implants. Deafness is often caused by loss or damage of hair cells, which fail to transmit signals to auditory neurons. In such cases, cochlear implants can be used to provide a response to sound and transmit electrical signals to nerve cells. However, these neurons often degenerate and experience retraction of processes in the cochlea because the damaged hair cells release fewer growth factors.

US Patent Application 20120328580 describes injection of a pharmaceutical composition into the ear (e.g., by aural administration), such as into the lumens of the cochlea (e.g., the cochlear duct, scala vestibuli, and scala tympani), for example, using a syringe, such as a single dose syringe. For example, one or more compounds described herein can be administered by intratympanic injection (e.g., into the middle ear) and/or injections into the outer, middle, and/or inner ear. Such methods are routinely used in the art, for example, to administer steroids and antibiotics into the ears of humans. The injection can be performed, for example, through the round window of the ear or through the cochlear capsule. Other methods of administration into the inner ear are known in the art (see, for example, Salt and Plontke, Drug Discovery Today, 10:1299-1306, 2005).

In another method of administration, the pharmaceutical composition can be administered in situ using a catheter or pump. The catheter or pump can, for example, direct the pharmaceutical composition into the lumen of the cochlea, or the round window of the ear, and/or the lumen of the colon. An exemplary drug delivery apparatus and methods suitable for administering one or more compounds described herein to an ear, such as a human ear, are described by McKenna et al. (U.S. Patent Application Publication No. 2006/0030837) and Jacobsen et al. (U.S. Patent No. 7,206,639). In some embodiments, the catheter or pump can be positioned, for example, in an ear (e.g., an outer, middle, and/or inner ear) of a patient during surgery. In some embodiments, the catheter or pump can be positioned, for example, in an ear (e.g., an outer, middle, and/or inner ear) of a patient without the need for surgery.

Alternatively or in addition, one or more of the compounds described herein can be administered in combination with a mechanical device, such as a cochlear implant or a hearing aid, that is worn in the outer ear. An exemplary cochlear implant suitable for use in the present invention is described by Edge et al. (US Patent Application Publication No. 2007/0093878).

In some embodiments, the routes of administration described above may be combined in any order and may be administered simultaneously or alternately.

Alternatively or in addition, the present invention can be used in accordance with any of the methods approved by the Food and Drug Administration, such as those described in CDER Standards Handbook, Version 004 (available at fda.give/cder/dsm/DRG/drg00301.htm).

In general, the cell therapy methods described in U.S. Patent Application 20120328580 can be used to stimulate complete or partial differentiation of cells into or toward a particular mature inner ear cell type (e.g., hair cells) in vitro. The cells obtained from such methods can then be transplanted or implanted into a patient in need of such treatment. Cell culture methods necessary for practicing these methods, including methods for identifying and selecting suitable cell types, methods for stimulating complete or partial differentiation of selected cells, methods for identifying fully or partially differentiated cell types, and methods for implanting fully or partially differentiated cells, are described below.

Cells suitable for use in the present invention include, but are not limited to, cells capable of completely or partially differentiating into mature inner ear cells, such as hair cells (e.g., inner and/or outer hair cells), when contacted, such as in vitro, with one or more of the compounds described herein. Exemplary 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-derived stem cells, embryonic stem cells, mesenchymal stem cells, skin stem cells, iPS cells, and adipose-derived stem cells), progenitor cells (e.g., inner ear progenitor cells), support cells (e.g., Deiters cells, pillar cells, inner phalangeal cells, tectal cells, and Hensen cells), and/or germ cells. The use of stem cells to replace inner ear sensory cells is described by Li et al. (U.S. Patent Application Publication No. 2005/0287127) and Li et al. (U.S. Patent Serial No. 11/953797). The use of bone marrow-derived stem cells to replace inner ear sensory cells is described by Edge et al. in PCT/US2007/084654. iPS cells are 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 (e.g., 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. Protein detection techniques involve staining proteins (e.g., using cell extracts or whole cells) with antibodies to the relevant antigen. In this case, the relevant antigen is the protein product of tissue-specific gene expression. Although in principle the primary antibody (i.e. the antibody that binds the antigen) can be labeled, more common (and improves visualization) is the use of a second antibody directed against the first (e.g. an antibody to IgG). This second antibody is conjugated to fluorochromes, or to appropriate enzymes for colorimetric reactions, or to gold beads (for electron microscopy), or to the biotin-avidin system, so that the location of the primary antibody and hence the antigen can be determined.

The CRISPR-Cas molecules of the present invention can be delivered to the ear by directly applying the pharmaceutical composition to the outer ear using modified compositions of published U.S. Patent Application 20110142917. In some embodiments, the pharmaceutical composition is applied to the outer ear canal. Delivery to the ear may also be referred to as intra-aural or aural delivery.

In some embodiments, the RNA molecules of the present invention are delivered in liposomal or Lipofectin-based formulations and the like, and can be prepared using methods well known to those skilled in the art. Such methods are described, for example, in U.S. Patent Nos. 5,593,972, 5,589,466, and 5,580,859, incorporated herein by reference.

Delivery systems specifically designed to enhance the efficiency and improve the delivery of siRNA into 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 they can be used in the present invention. Recently, siRNA has been successfully used to inhibit gene expression in primates (see, e.g., Tolentino et al., Retina 24(4):660, which may also be applicable to the present invention).

Qi et al. disclose methods for efficiently delivering siRNA into the inner ear through an intact round window by transfection using a novel protein delivery technology that can be applied to the nucleic acid targeting system of the present invention (see, e.g., Qi et al., Gene Therapy (2013), 1-9). In particular, the use of TAT double-stranded RNA binding domains (TAT-DRBDs) that can transfect Cy3-labeled siRNA into inner ear cells, including inner and outer hair cells, crista ampullae, maculae ellipticae, and maculae sphericae, by penetrating through the intact round window was successful in delivering double-stranded siRNA in vivo for the treatment of various inner ear diseases and preserving auditory function. About 40 μl of 10 mM RNA can be provided as a dose for administration to the ear.

According to Rejali et al. (Hear Res. 2007 Jun;228(1-2):180-7), the performance of cochlear implants can be improved by properly preserving the spiral ganglion neurons that are the target of the implant's electrical stimulation, and brain-derived neurotrophic factor (BDNF) has previously been shown to enhance spiral ganglion survival in ears with experimentally induced deafness. Rejali et al. tested a modified cochlear implant electrode design coated with fibroblast cells transduced with a viral vector containing the BDNF gene insert. To achieve this type of ex vivo gene transfer, Rejali et al. transduced guinea pig fibroblasts with an adenovirus inserting a BDNF gene cassette and found that these cells secreted BDNF, then attached the BDNF-secreting cells to a cochlear implant electrode using an agarose gel and implanted the electrode into the scala tympani of the cochlea. Rejali et al. found that BDNF-expressing electrodes were able to preserve significantly more spiral ganglion neurons in the basal turns of the cochlea 48 days after implantation compared to control electrodes and demonstrated the feasibility of cochlear implant therapy in combination with ex vivo gene transfer to enhance the survival of spiral ganglion neurons. Such a system can be used to deliver the nucleic acid targeting system of the present invention to the ear.

Mukherjea et al. (Antioxidants & Redox Signaling, Volume 13, Number 5, 2010) document that knockdown of NOX3 using short interfering (si)RNA abrogated cisplatin ototoxicity as evidenced by protection of OHCs from injury and reduction in threshold shifts in brainstem auditory evoked potentials (ABRs). Rats were treated with different doses of siNOX3 (0.3, 0.6, and 0.9 μg) and NOX3 expression was assessed by real-time RT-PCR. The lowest dose of NOX3 siRNA used (0.3 μg) did not show any inhibition of NOX3 mRNA compared to transtympanic administration of scrambled siRNA or no cochlea treatment. However, administration of higher doses of siRNA for NOX3 (0.6 and 0.9 μg) reduced NOX3 expression compared to the control scrambled siRNA. Such a system can be used for transtympanic administration of the CRISPR-Cas system of the present invention at a dose of about 2 mg to about 4 mg of CRISPR-Cas for administration to a human.

Jung et al. (Molecular Therapy, vol. 21 no. 4, 834-841 Apr. 2013) demonstrate that Hes5 levels in the utricle were reduced after siRNA treatment and that the number of hair cells in these utricles was significantly greater than after control treatment. The data suggest 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 inhibiting specific gene expression. Jung et al. injected 8 μg of Hes5 siRNA in a 2 μl volume, prepared by adding sterile normal saline to lyophilized siRNA, into the vestibular epithelium of the ear. Such a system can be used to administer the nucleic acid targeting system of the present invention to the vestibular epithelium of the ear at a dose of from about 1 to about 30 mg CRISPR-Cas for administration to a human.

Treatment of eye diseases

The present invention also provides for delivery of the CRISPR-Cas9 system to one eye or both eyes.

In another aspect of the present invention, the CRISPR-Cas9 system can be used to correct eye defects that result from several genetic mutations, further described in Genetic Diseases of the Eye, Second Edition, edited by Elias I. Traboulsi, Oxford University Press, 2012.

Lentiviral vectors, particularly equine infectious anemia virus (EIAV), are particularly preferred for ocular administration.

In another embodiment, minimal lentiviral vectors for non-primate organisms based on equine infectious anemia virus (EIAV) are also provided, especially for gene therapy of eye diseases (see, for example, Balagaan, J Gene Med 2006; 8: 275-285, published online November 21, 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jgm.845). It is envisaged that the vectors have a cytomegalovirus (CMV) promoter driving expression of a target gene. Any of the intracameral, subretinal, intraocular, and intravitreal injections are also included (see, e.g., Balagaan, J Gene Med 2006; 8: 275–285, published online November 21, 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 bulged by gentle digital pressure and the fundus can be visualized using a contact lens system consisting of a drop of contact medium solution on the cornea covered with a slide cover slip. For subretinal injections, the tip of a 34-gauge 10-mm needle attached to a 5-μl Hamilton syringe can be advanced through the equatorial region of the superior sclera tangentially toward the posterior pole of the globe under direct visualization until the needle aperture is visible in the subretinal space. 2 μl of vector suspension can then be injected, causing a bullous superior retinal detachment, thus confirming subretinal injection of the vector. This approach creates a self-sealing scleral incision that allows the vector suspension to remain in the subretinal space until it is absorbed by the RPE, usually within 48 h after the procedure. This procedure can be repeated in the inferior hemisphere, causing an inferior retinal detachment. This technique results in exposure of approximately 70% of the neurosensory retina and the RPE to the vector suspension. For intravitreal injections, the needle tip can be advanced through the sclera 1 mm posterior to the corneoscleral limbus and 2 μl of the vector suspension can be injected into the vitreous cavity. For intracameral injections, the needle tip can be advanced through the paracentesis of the corneoscleral limbus towards the central cornea and 2 μl of the vector suspension can be injected. For intracameral injections, the needle tip can be advanced through the paracentesis of the corneoscleral limbus towards the central cornea and 2 μl of the vector suspension can be injected. These vectors can be injected at titers of 1.0-1.4 x 10 ¹⁰ or 1.0-1.4 x 10 ⁹ transducing units (TU)/ml.

Another embodiment also provides RetinoStat®, an equine infectious anemia virus-based lentiviral gene therapy vector expressing the angiostatic proteins endostatin and angiostatin, which is delivered by subretinal injection for the treatment of wet 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 the CRISPR-Cas9 system of the present invention. Each eye can be treated with any RetinoStat® at a dose of 1.1 x 10 ⁵ transducing units per eye (TU/eye), in a total volume of 100 μl.

In one embodiment, reference should be made to WO 2015/153780, which provides for the treatment or prevention of primary acute-angle glaucoma (POAG) by targeting the coding sequence of the MYOC gene. Some of the target mutations that lead to POAG include, but are not limited to, P370 (e.g., P370L); I477 (e.g., I477N or I477S); T377 (e.g., TE77R); Q368 (Q368stop) -- all in the MYOC gene. The target mutation may also include a mutagenesis hotspot between amino acid sequence positions 246-252 in the MYOC gene. In one embodiment, the target mutation is a mutagenesis hotspot between amino acid sequence positions, such as amino acids 368-380, amino acids 368-370 + 377-380, amino acids 364-380, or amino acids 347-380 in the MYOC gene. In one embodiment, the target mutation is a mutagenesis hotspot between amino acid sequence positions 423-437 (e.g., amino acids 423-426, amino acids 423-427, and amino acids 423-437) in the MYOC gene. In one embodiment, the target mutation is a mutagenesis hotspot between amino acid sequence positions 477-502 in the MYOC gene (see, e.g., WO 2015/153780).

In another embodiment, an adenoviral vector with a deletion of E1 and a partial deletion of E3 and E4 can be provided for delivery to the eye. Twenty-eight patients with advanced neovascular age-related macular degeneration (AMD) received a single intravitreal injection of an adenoviral vector with a deletion of E1 and a partial deletion of E3 and E4 expressing human pigment epithelial factor (AdPEDF.ll) (see, e.g., Campochiaro et al., Human Gene Therapy 17:167-176 (February 2006)). Doses ranging from ¹⁰⁶ to ^109.5 unit particles (PU) were studied and no AdPEDF.ll-related serious adverse events or dose-limiting toxicity were observed (see, e.g., Campochiaro et al., Human Gene Therapy 17:167-176 (February 2006)). Adenoviral vector-mediated gene transfer to the eye appears to be a viable approach for the treatment of ocular disorders and may be applicable to the CRISPR Cas9 system.

In another embodiment, the sd-rxRNA® system from RXi Pharmaceuticals can be used to deliver CRISPR Cas9 to the eye and/or adapted thereto. In this system, a single intravitreal administration of 3 μg of sd-rxRNA results in a sequence-specific reduction in PPIB mRNA levels for 14 days. The sd-rxRNA® system can be used in relation to the nucleic acid targeting system of the present invention, involving the administration to a human of a CRISPR dose of about 3-20 mg.

Millington-Ward et al. (Molecular Therapy, vol. 19 no. 4, 642-649 April 2011) describe adeno-associated virus (AAV) vectors for delivery of a rhodopsin suppressor that functions by RNA interference (RNAi) and a codon-engineered rhodopsin replacement gene that is resistant to suppression due to nucleotide changes at degenerate positions within the RNAi target site. Subretinal injection of either 6.0 x 10 ⁸ vp or 1.8 x 10 ¹⁰ vp AAV was performed into eyes according to Millington-Ward et al. The AAV vectors according to Millington-Ward et al. can be used in relation to the CRISPR Cas9 system of the present invention, providing for administration to a human of a dose of from about 2 x 10 ¹¹ to about 6 x 10 ¹³ vp.

Dalkara et al. (Sci Transl Med 5, 189ra76 (2013)) also turn to in vivo directed evolution to construct an AAV-based vector that delivers wild-type variants of defective genes throughout the retina after harmless injection into the aqueous humor of the eye. Dalkara describes a 7-mer peptide display library and an AAV library constructed by DNA shuffling the cap genes of AAV1, 2, 4, 5, 6, 8, and 9. rcAAV libraries and rAAV-based vectors expressing GFP under the control of the CAG or Rho promoter were packaged and titers of DNase-resistant genomes were generated using qPCR. Libraries were pooled and two rounds of evolution were performed, each consisting of diversification of the initial library followed by three rounds of in vivo selection. At each such step, rho-GFP-expressing P30 mice were injected intravitreally with 2 ml of the iodixanol-purified, phosphate-buffered saline (PBS)-dialyzed library at a genome titer of approximately 1 x 10 ¹² vg/ml. The AAV-based vectors of Dalkara et al. can be used with the nucleic acid targeting system of the present invention, providing for administration to a human of a dose of from about 1 x 10 ¹⁵ to about 1 x 10 ¹⁶ vg/ml.

In another embodiment, the rhodopsin gene can be targeted for the treatment of retinitis pigmentosa (RP), wherein the system of U.S. Patent Application Publication No. 20120204282 assigned to Sangamo BioSciences, Inc. can be modified in the image of the CRISPR Cas9 system of the present invention.

In another embodiment, the methods of U.S. Patent Application Publication No. 20130183282 assigned to Cellectis, directed to methods for cleaving a target sequence of the human rhodopsin gene, may also be modified for the nucleic acid targeting system of the present invention.

U.S. Patent Application Publication No. 20130202678 assigned to Academia Sinica relates to methods for treating forms of retinopathy and vision-threatening ophthalmologic disorders relating to the delivery of the Puf-A gene (expressed in retinal ganglion and pigment cells in ocular tissues and exhibiting unique anti-apoptotic activity) to the subretinal or intravitreal space of the eye. Specifically, desired targets are zgc:193933, prdm1a, spata2, tex10, rbb4, ddx3, zp2.2, Blimp-1, and HtrA2, all of which can be targeted using the nucleic acid targeting system of the present invention.

Wu (Cell Stem Cell, 13:659–62, 2013) developed a guide RNA that targets Cas9 to the location of a single base pair mutation that causes forms of cataracts in mice, where it induces DNA cleavage. Then, using another wild-type allele or oligonucleotides introduced into zygotes, repair mechanisms correct the sequence of the damaged allele and correct the genetic defect that causes cataracts in the mutant mouse.

U.S. Patent Application 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 leading cause of visual impairment in the elderly, but is also a hallmark of childhood diseases such as Stargardt disease, Sorsby ocular dystrophy, and fatal childhood neurodegenerative diseases, with onset occurring in infancy. Macular degeneration results in vision loss in the center of the visual field (the macula) due to damage to the retina. Currently available animal models do not reproduce the key hallmarks of the disease as seen in humans. Available animal models containing mutant genes encoding proteins associated with MD also produce highly variable phenotypes, moving toward understanding the human disease and developing therapeutics.

One aspect of U.S. Patent Application Publication No. 20120159653 relates to editing any chromosomal sequences that encode proteins associated with MD, which may be used with respect to the nucleic acid targeting system of the present invention. Proteins associated with MD are typically selected based on an experimentally established relationship of the MD-associated protein to the MD disorder. For example, the rate of production or circulating concentration of a protein associated with MD may be increased or decreased in a population with an MD disorder compared to a population without an MD disorder. Differences in protein levels may be assessed using proteomic techniques, including, but not limited to, Western blotting, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, proteins associated with MD can be identified by obtaining gene expression profiles for protein-coding genes using genomic analysis techniques, including but not limited to DNA microarray analysis, sequence-based analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).

By way of non-limiting example, proteins associated with MD include, but are not limited to, the following proteins: ATP-binding cassette subfamily A (ABC1) member 4 (ABCA4), ACHM1 - achromatopsia (rod monochromatism)-associated protein 1, ApoE - apolipoprotein E (ApoE), C1QTNF5 (CTRP5) - C1q/tumor necrosis factor-related protein 5 (C1QTNF5), C2 - complement component 2 (C2), complement component C3 (C3), CCL2 - chemokine ligand 2 (CCL2), CCR2 - chemokine receptor 2 (CCR2), CD36 - cluster of differentiation 36, CFB - complement factor B, CFH - complement factor H complement (CFH), CFHR1 - complement factor H-related protein 1, CFHR3 - complement factor H-related protein 3, CNGB3 - cyclic nucleotide-regulated ion channel beta-3, CP - ceruloplasmin (CP), CRP - C-reactive protein (CRP) CST3 - cystatin C or cystatin 3 (CST3), CTSD - cathepsin D (CTSD), CX3CR1 - receptor for chemokine 1 (with a C-X3-C motif), ELOVL4 - very long-chain fatty acid elongation protein 4, ERCC6 - excision repair cross-complementing protein correcting rodent excision repair deficiency, complementation group 6, FBLN5 - fibulin-5, FBLN5 - fibulin 5, FBLN6 - fibulin 6, FSCN2 - fascin (FSCN2), HMCN1 - hemicentrin 1, HMCN1 - hemicentrin 1, HTRA1 - serine peptidase HtrA 1 (HTRA1), HTRA1 - serine peptidase HtrA 1, IL-6 - interleukin 6, IL-8 - interleukin 8, LOC387715 - hypothetical protein, PLEKHA1 - pleckstrin homology domain-containing protein, family A member 1 (PLEKHA1), PROM1 - prominin 1 (PROM1 or CD133), PRPH2 - peripherin-2, RPGR - retinitis pigmentosa-associated GTPase regulator, SERPING1 - serine peptidase inhibitor, clade G member 1 (C1-inhibitor), TCOF1 - Treacle, TIMP3 - inhibitor of metalloproteinase 3 (TIMP3), TLR3 - Toll-like receptor 3.

The identity of the MD-associated protein whose chromosomal sequence is edited can and will vary. In a preferred embodiment, the MD-associated proteins whose chromosomal sequences are edited can be the ATP-binding cassette subfamily A (ABC1) member 4 (ABCA4) protein encoded by the ABCR gene, the apolipoprotein E (APOE) protein encoded by the APOE gene, the chemokine (C-C motif) ligand 2 (CCL2) protein encoded by the CCL2 gene, the chemokine (C-C motif) receptor 2 (CCR2) protein encoded by the CCR2 gene, the ceruloplasmin (CP) protein encoded by the CP gene, the cathepsin D (CTSD) protein encoded by the CTSD gene, or the metalloproteinase inhibitor 3 (TIMP3) protein encoded by the TIMP3 gene. In an exemplary embodiment, the genetically modified animal is a rat and the edited chromosomal sequences encoding a protein associated with MD may be the following: NM_000350 (ABCA4) for ATP-binding cassette subfamily A member 4 (ABC1), NM_138828 (APOE) for APOE apolipoprotein E, NM_031530 (CCL2) for CCL2 chemokine (C-C motif) ligand 2, NM_021866 (CCR2) for CCR2 chemokine (C-C motif) receptor 2, NM_012532 (CP) for CP ceruloplasmin, NM_134334 (CTSD) for CTSD cathepsin D, NM_012886 (TIMP3) for a TIMP3 inhibitor of 3 metalloproteinases. The animal or cell may contain 1, 2, 3, 4, 5, 6, 7 or more chromosomal sequences with a disrupted structure encoding a protein associated with MD, and zero, 1, 2, 3, 4, 5, 6, 7 or more chromosomally integrated sequences encoding a protein with a disrupted structure associated with MD.

The edited or integrated chromosomal sequence can be modified to encode an altered protein associated with MD. Several mutations in chromosomal sequences associated with MD have been associated with MD. Non-limiting examples of mutations in chromosomal sequences associated with MD include those mutations 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 1129 is replaced by 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 is replaced by arginine); in the CCR2 protein - V64I (i.e., 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 replaced by cysteine), S170C (i.e. serine at position 170 is replaced by cysteine), Y172C (i.e. tyrosine at position 172 is replaced by cysteine) and S181C (i.e. serine at position 181 is replaced by cysteine). Other relationships between gene variants of genes associated with MD and the disease are known from the prior art.

Treatment of cardiovascular and muscular diseases

The present invention also provides for delivery of the CRISPR-Cas system described herein, such as the Cas9 effector protein systems, to the heart. Preferred for the heart is a myocardial-tropic adeno-associated virus (AAVM), particularly AAVM41, which has been shown to preferentially transfer genes to the heart (see, e.g., Lin-Yanga et al., PNAS, March 10, 2009, vol. 106, no. 10). Administration may be systemic or local. A dose of about 1-10 x 10 ¹⁴ vector genomes is contemplated for systemic administration. See also, e.g., Eulalio et al. (2012) Nature 492: 376 and Somasuntharam et al. (2013) Biomaterials 34: 7790.

For example, U.S. Patent Application Publication No. 20110023139 describes the use of zinc finger nucleases to genetically modify cells, animals, and proteins associated with cardiovascular disease. Cardiovascular diseases typically include high blood pressure, heart attacks, heart failure, and stroke, as well as TIA. Any chromosomal sequence associated with cardiovascular disease, or a protein encoded by any chromosomal sequence associated with cardiovascular disease, can be used in the methods described herein. Proteins associated with cardiovascular disease are typically selected based on an experimentally established association of a protein associated with cardiovascular disease with the development of cardiovascular disease. For example, the rate of production or circulating concentration of a protein associated with cardiovascular disease may be increased or decreased in a population with cardiovascular disease compared to a population without cardiovascular disease. Differences in protein levels can be assessed using proteomic techniques, including but not limited to Western blotting, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, proteins associated with cardiovascular disease can be identified by generating gene expression profiles for the genes encoding the proteins using genomic analysis techniques, including but not limited to DNA microarray analysis, sequence-based analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).

As an example, the chromosomal sequence may include, but is not limited to, 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 (prostacyclin 12 (IP) receptor), KCNJ11 (inward rectifier potassium channel, subfamily J, member 11), INS (insulin), CRP (pentraxin-related C-reactive protein), PDGFRB (platelet-derived growth factor, beta polypeptide), CCNA2 (cyclin A2), PDGFB (platelet-derived growth factor beta polypeptide oncogene homolog (simian sarcoma virus (v-sis))), KCNJ5 (inward-rectifying potassium channel, subfamily J, member 5), KCNN3 (intermediate/low-conducting calcium-activated potassium channel, subfamily N, member 3), CAPN10 (calpain 10), PTGES (prostaglandin E synthase), ADRA2B (alpha-2B-adrenergic receptor), ABCG5 (ATP-binding cassette, subfamily G (WHITE), member 5), PRDX2 (peroxiredoxin 2), CAPN5 (calpain 5), PARP14 (poly (ADP-ribose) polymerase family, member 14), MEX3C (C mex-3 homolog C (C. elegans)), ACE angiotensin I-converting enzyme (peptidyl dipeptidase A) 1), TNF (tumor necrosis factor (TNF superfamily, member 2)), IL6 (interleukin 6 (interferon, beta 2)), STN (statin), SERPINE1 (serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1), ALB (albumin), ADIPOQ (adiponectin containing C1Q and collagen domain), APOB (apolipoprotein B (including Ag(x))), APOE (apolipoprotein E), LEP (leptin), MTHFR (5,10-methylenetetrahydrofolate reductase (NADPH)), APOA1 (apolipoprotein A-I), EDN1 (endothelin 1), NPPB (precursor natriuretic peptide B), NOS3 (synthase nitric oxide type 3 (endothelial cell)), PPARG (peroxisome proliferator-activated receptor gamma), PLAT (tissue plasminogen activator), PTGS2 (prostaglandin endoperoxide synthase type 2 (prostaglandin G/H synthase and cyclooxygenase)), CETP (plasma cholesteryl ester transport protein), AGTR1 (anti-otensin II receptor type 1), HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase), IGF1 (insulin-like growth factor 1 (somatomedin C)), SELE (selectin E), REN (renin), PPARA (peroxisome proliferator-activated receptor alpha), PON1 (paraoxonase 1), KNG1 (kininogen 1), CCL2 (chemokine ligand 2 (with C-C motif)), LPL (lipoprotein lipase), VWF (von Willebrand factor), F2 (coagulation factor II (thrombin)), ICAM1 (intercellular adhesion molecule 1), TGFB1 (transforming growth factor, beta 1), NPPA (precursor natriuretic peptide A), IL10 (interleukin 10), EPO (erythropoietin), SOD1 (superoxide dismutase 1, soluble), VCAM1 (vascular endothelial adhesion molecule type 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 type 2), MMP9 (matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa collagenase type IV)), SERPINC1 (serpin peptidase inhibitor, clade C (antithrombin), member 1), F8 (coagulation factor VIII, procoagulant component), HMOX1 (heme oxygenase (decyclic) type 1), APOC3 (apolipoprotein C-III), IL8 (interleukin 8), PROK1 (prokineticin 1), CBS (cystathionine beta synthase), NOS2 (sodium oxide synthase type 2, inducible), TLR4 (toll-like receptor type 4), SELP (selectin P (140 kDa granule membrane protein, CD62 antigen)), ABCA1 (ATP-binding cassette, subfamily A (ABC1), member 1), AGT (angiotensin (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 type 1 (neuronal)), NR3C1 (nuclear receptor, subfamily 3, group C, member 1 (glucocorticoid receptor)), FGB (fibrinogen beta chain), HGF (hepatocyte growth factor (hepatopoietin A; scattering factor)), IL1A (interleuin 1, alpha), RETN (resistin), AKT1 (murine thymoma virus oncogene homolog type 1 v-akt), LIPC (lipase, liver), HSPD1 (heat shock protein type 1 of 60 kDa (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 superfamily, member 11b), EDNRA (endothelin receptor type A), EGFR (epidermal growth factor receptor (v-erb-b oncogene homolog, avian)), MMP2 (matrix metallopeptidase 2 (gelatinase A, 72-kDa gelatinase, 72-kDa collagenase type IV)), PLG (plasminogen), NPY (neuropeptide Y), RHOD (ras homolog gene family, member D), MAPK8 (mitogen-activated protein kinase 8), MYC (v-myc myelocystomatosis virus oncogene homolog, avian)), FN1 (fibronectin 1), CMA1 (chymase 1, mast cell), PLAU (plasminogen activator, urokinase), GNB3 (guanine nucleotide-binding protein (G protein), beta polypeptide type 3), ADRB2 (adrenergic beta-2 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 1), PARP1 (poly (ADP-ribose) polymerase type 1), CD40LG (CD40 ligand), PON2 (paraoxonase 2), AGER (receptor for advanced glycation end products), IRS1 (insulin receptor substrate type 1), PTGS1 (prostaglandin endoperoxide synthase type 1 (prostaglandin G/H synthase and cyclooxygenase)), ECE1 (endothelin-converting enzyme type 1), F7 (coagulation factor VII (serum thrombin converting accelerator)), URN (interleukin 1 receptor antagonist), EPHX2 (epoxide hydrolase type 2, cytoplasmic), IGFBP1 (insulin-like growth factor binding protein type 1), MAPK10 (mitogen-activated protein kinase 10), FAS (Fas (TNF receptor superfamily, member 6)), ABCB1 (ATP-binding cassette, subfamily B (MDR/TAP), member 1), JUN (jun oncogene), IGFBP3 (insulin-like growth factor binding protein type 3), CD14 (CD14 molecule), PDE5A (phosphodiesterase 5A, cGMP-specific), AGTR2 (receptor angiotensin II, type 2), CD40 (CD40 molecule, member of the TNF receptor superfamily 5), LCAT (lecithin-cholesterol acyltransferase), CCR5 (chemokine receptor type 5 (with a C-C motif)), MMP1 (matrix metallopeptidase 1 (interstitial collagenase)), TIMP1 (TIMP inhibitor of metallopeptidase type 1), ADM (adrenomedullin), DYT10 (dystonia 10), STAT3 (signal transducer and activator of transcription type 3 (acute phase response factor)), MMP3 (matrix metallopeptidase 3 (stromelysin 1, progelatinase)), ELN (elastin), USF1 (transcription factor binding upstream of the transcription initiation site 1), CFH (complement factor H), HSPA4 (warm shock protein 4 of 70 kDa), MMP12 (matrix metallopeptidase 12 (macrophage elastase)), MME (membrane metalloendopeptidase), F2R (coagulation factor II (thrombin) receptor), SELL (selectin L), CTSB (cathepsin B), ANXA5 (annexin A5), ADRB1 (adrenergic beta-1 receptor), CYBA (cytochrome b-245, alpha peptide), FGA (fibrinogen alpha chain), GGT1 (gamma-glutamyl transferase 1), LIPG (lipase, endothelial), HIF1A (hypoxia-inducible factor 1, alpha subunit (helix-loop-helix basic structure transcription factor)), CXCR4 (chemokine receptor 4 (with C-X-C motif)), PROC (protein C (coagulation factor Va and VIIIa inactivator)), SCARB1 (phagocytic receptor, class B, member 1), CD79A (CD79a molecule, immunoglobulin-associated alpha), PLTP (phospholipid transfer protein), ADD1 (adducin 1 (alpha)), FGG (fibrinogen gamma chain), SAA1 (serum amyloid A1), KCNH2 (potassium voltage-gated channel, family H (eag-related), member 2), DPP4 (dipeptidyl peptidase 4), G6PD (glucose-6-phosphate dehydrogenase), NPR1 (natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A)), VTN (vitronectin), KIAA0101 (KIAA0101), FOS (homolog murine osteosarcoma virus oncogene FBJ), TLR2 (toll-like receptor 2), PPIG (peptidylproline isomerase G (cycloproline G)), IL1R1 (interleukin I receptor), 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-hexacysteine methyltransferase), RBP4 (retinol-binding protein type 4, plasma), APOA4 (apolipoprotein A-IV), CDKN2A (cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)), FGF2 (fibroblast growth factor 2 (basic)), EDNRB (endothelin receptor type B), ITGA2 (integrin, alpha 2 (CD49B, alpha 2 subunit of the VLA-2 receptor)), CABIN1 (calcineurin-binding protein 1), SHBG (sex hormone-binding globulin), HMGB1 (high-mobility group 1 protein), HSP90B2P (heat shock protein 90 kDa 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, caveolae protein, 22 kDa), ESR2 (estrogen receptor 2 (ER beta)), LTA (lymphotoxin alpha (TNF superfamily, member 1)), GDF15 (growth and differentiation factor 15), BDNF (brain-derived neurotrophic factor), CYP2D6 (cytochrome P450, family 2, subfamily D, polypeptide 6), NGF (nerve growth factor (beta polypeptide)), SP1 (transcription factor Sp1), TGIF1 (TGFB-inducible factor homeobox 1), SRC (sarcoma virus oncogene homolog v-src (Schmidt-Ruppin A-2) (avian)), EGF (epidermal growth factor (beta-urogastron)), PIK3CG (phosphoinositide 3-kinase, catalytic, gamma polypeptide), HLA-A (major histocompatibility complex, class I, A), KCNQ1 (potassium voltage-gated channel, KQT-like family, member 1), CNR1 (cannabinoid receptor 1 (brain)), FBN1 (fibrillin 1), CHKA (choline kinase alpha), BEST1 (bestrophin 1), APP (amyloid precursor protein beta (A4)), CTNNB1 (catenin (cadherin-associated protein), beta 1, 88 kDa), IL2 (interleukin 2), CD36 (CD36 molecule (thrombospondin receptor)), PRKAB1 (protein kinase, AMP-activated, beta 1 non-catalytic subunit), TPO (thyroid peroxidase), ALDH7A1 (aldehyde dehydrogenase family 7, member A1), CX3CR1 (chemokine receptor 1 (with C-X3-C motif)), TH (tyrosine hydroxylase), F9 (coagulation factor IX), GH1 (growth hormone 1), TF (transferrin), HFE (hemochromatosis), IL17A (interleukin 17A), PTEN (phosphatase tensin homolog), GSTM1 (glutathione S-transferase mu 1), DMD (dystrophin), GATA4 (GATA binding protein type 4), F13A1 (coagulation factor XIII, polypeptide A1), TTR (transthyretin), FABP4 (fatty acid binding protein type 4, adipocyte), PON3 (paraoxonase 3), APOC1 (apolipoprotein C-I), INSR (insulin receptor), TNFRSF1B (tumor necrosis factor receptor superfamily, member 1B), HTR2A (5-hydroxytryptamine (serotonin) receptor 2A), 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 (receptor kinase insertion domain-containing (receptor tyrosine kinase type III)), PLA2G2A (phospholipase A2, group IIA (platelets, synovial fluid)), B2M (beta-2-microglobulin), THBS1 (thrombospondin 1), GCG (glucagon), RHOA (ras homolog gene family, member A), ALDH2 (aldehyde dehydrogenase 2 family (mitochondrial)), TCF7L2 (transcription factor 7, factor 2-like (T-cell-specific, HMG-box)), BDKRB2 (bradykinin receptor B2), NFE2L2 (nuclear factor 2-like (erythroid 2)), NOTCH1 (Notch 1 homolog, translocation-associated (Drosophila)), UGT1A1 (UDP-glucuronyl transferase family 1, polypipeline A1), IFNA1 (interferon, alpha 1), PPARD (peroxisome proliferator-activated receptor delta), SIRT1 (sirtuin 1 (silent information regulation homolog 2) (S. cerevisiae)), GNRH1 (gonadotropin-releasing hormone 1 (luteinizing-releasing hormone)), PAPPA (pregnancy-associated plasma protein A, papalysin 1), ARR3 (arrestin 3, retinal (X-arrestin)), NPPC (precursor natriuretic peptide C), AHSP (alpha-hemoglobin-stabilizing protein), PTK2 (protein tyrosine kinase type 2 PTK2), IL13 (interleukin 13), MTOR (mechanism of action target of rapamycin (serine/threorine kinase)), ITGB2 (intergreen, beta 2 (subunit of complement component 3 receptor 3 and 4)), GSTT1 (glutathione-S-transferase theta 1), IL6ST (interleukin 6 signal transducer (gp130, oncostatin M receptor)), CPB2 (carboxypeptidase B2 (plasma)), CYP1A2 (cytochrome P450, family 1, subfamily A, polypeptide 2), HNF4A (hepatocyte nuclear factor 4, alpha), SLC6A4 (solute carrier family 6 (neurotransmitter transporter, serotonin), member 4), PLA2G6 (phospholipase A2, group VI (cytosolic, calcium-independent)), TNFSF11 (tumor growth factor superfamily (ligand), member 11), SLC8A1 (solute carrier family 8 (sodium-calcium antiporter), member 1), F2RL1 (coagulation factor II receptor-like 1 (thrombin)), AKR1A1 (aldoketo reductase family 1, member A1 (aldehyde reductase)), ALDH9A1 (aldehyde dehydrogenase family 9, member A1), BGLAP (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 (P2RY purinergic receptor G protein-coupled 12), RNLS (renalase, FAD-dependent amine oxidase), CREB1 (cAMP-responsive element-binding protein 1), POMC (pro-opiomelanocortin), RAC1 (ras-related botulinum toxin substrate 1) C3 (rho family, small GTP binding protein Rac1)), LMNA (lamin NC), CD59 (CD59 molecule, complement regulatory protein), SCN5A (sodium channel, voltage-gated, type V, alpha subunit), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), MIF (macrophage migration inhibitory factor (glycosylation inhibitory factor)), MMP13 (matrix metallopeptidase 13 (collagenase 3)), TIMP2 (TIMP inhibitor of metallopeptidase 2), CYP19A1 (cytochrome P450, family 19, subfamily A, polypeptide 1), CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2), PTPN22 (protein tyrosine phosphatase, non-receptor, 22 type (lymphoid)), MYH14 (myosin, heavy chain 14, non-muscle), MBL2 (mannose-binding lectin (protein C) 2, soluble (opsonin defect)), SELPLG (selectin P ligand), AOC3 (amine oxidase, copper-containing 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, MSK12)), CAST (calpastatin), CXCL12 (chemokine ligand 12 (with C-X-C motif) (stromal cell factor 1)), IGHE (heavy epsilon chain), KCNE1 (potassium voltage-gated channel, Isk-related family, member 1), TFRC (transferrin receptor (p90, CD71)), COL1A1 (collagen type 1, alpha 1), COL1A2 (collagen type I, alpha 2), IL2RB (interleukin 2 receptor, beta), PLA2G10 (pro-phospholipidase A2, group X), ANGPT2 (angiopoietin 2), PROCR (protein C receptor, endothelial (EPCR)), NOX4 (NADPH oxidase 4), HAMP (hepcidin antimicrobial peptide), PTPN11 (protein tyrosine phosphatase, non-receptor, type 11), SLC2A1 (solute carrier family 2 (facilitated diffusion glucose transporter), member 1), IL2RA (interleukin 2 receptor, alpha), CCL5 (chemokine ligand 5 (with C-C motif)), IRF1 (interferon regulatory factor 1), CFLAR (CASP8 and FADD-like regulator of apoptosis), CALCA (calcitonin-related polypeptide alpha), EIF4E (eukaryotic translation initiation factor 4E), GSTP1 (pi-1-glutathione S-transferase), JAK2 (Janus kinase 2), CYP3A5 (cytochrome P450, family 3, subfamily A, polypeptide 5), HSPG2 (heparin sulfate proteoglycan 2), CCL3 (chemokine ligand 3 (with C-C motif)), MYD88 (myeloid differentiation primary response gene (88)), VIP (vasoactive intestinal peptide), SOAT1 (sterol O-acyltransferase 1), ADRBK1 (adrenergic beta 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 hydrolase 1), EPRS (glutamyl propyl-tRNA synthetase), PPARGC1A (peroxisome proliferator-activated receptor gamma coactivator 1 alpha), F12 (coagulation factor XII (Hageman factor)), PECAM1 (platelet/endothelial cell adhesion molecule), CCL4 (chemokine (C-C motif) ligand 4), SERPINA3 (serpin peptidase inhibitor, clade A (alpha-1-antiproteinase, antitrypsin), member 3), CASR (calcium-sensing 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 (cardiotropin 1), SGCB (sarcoglycan, beta (dystrophin-associated glycoprotein of 43 kDa)), YME1L1 (YME1-like factor 1 (S. cerevisiae)), CAMP (cathelicidin antimicrobial peptide), ZC3H12A (containing zinc finger factor CCCH-type 12A), AKR1B1 (aldo-keto reductase family 1, 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, alpha, member 1), UGT1A (UDP-glucuronyl transferase family 1, polypeptide complex locus A), PRKCA (protein kinase C, alpha), COMT (catechol-beta-methyltransferase), S100B (S100 calcium-binding protein B), EGR1 (early response growth factor 1), PRL (prolactin), IL15 (interleukin 15), DRD4 (dopamine D4 receptor), CAMK2G (calcium/calmodulin-dependent protein kinase II gamma), SLC22A2 (solute carrier family 22 (organic cation transporter), member 2), CCL11 (chemokine ligand 11 (C-C motif)), PGF (placental growth factor B321), THPO (thrombopoietin), GP6 (glycoprotein VI (platelet)), TACR1 (tachykine receptor 1), NTS (neurotensin), HNF1A (HNF1 homeobox A), SST (somatostatin), KCND1 (potassium voltage-gated channel, 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-lipogenase), AHSG (alpha-2-HS-glycoprotein), BHMT (betaine-homocysteine methyltransferase), GJA4 (gap junction protein, alpha 4, 37 kDa), SLC25A4 (solute carrier family 25 (mitochondrial transporter; adenine nucleotide translocator), member 4), ACLY (ATP citrate lyase), ALOX5AP (arachidonate-5-lipoxygenase activating protein), NUMA1 (nuclear protein of the mitotic apparatus 1), CYP27B1 (cytochrome P450, family 27, subfamily B, polypeptide 1), CYSLTR2 (cysteinyl leukotriene receptor 2), SOD3 (superoxide dismutase 3, extracellular), LTC4S (leukotriene C4 synthase), UCN (urocortin), GHRL (ghrelin/obestatin prepropeptide), APOC2 (apolipoprotein C-II), CLEC4A (C-type lectin domain family 4, member A), KBTBD10 (kelch repeat and BTB domain-containing (POZ) 10), TNC (tenaskin C), TYMS (thymidylate synthetase), SHCl (SHC-transforming protein 1 (Src-homology domain-containing 2)), LRP1 (low-density lipoprotein receptor-related protein 1), SOCS3 (suppressor of cytokine signaling 3), ADH1B (alcohol dehydrogenase 1B (class I), beta polypeptide), KLK3 (kallikrein-related peptidase 3), HSD11B1 (hydroxysteroid (11-beta) dehydrogenase 1), VKORC1 (vitamin K epoxide reductase complex, subunit 1), SERPINB2 (serpin peptidase inhibitor, clade B (ovalbumin), member 2), TNS1 (tensin 1), RNF19A (ring zinc finger protein 19A), EPOR (erythropoietin receptor), ITGAM (integrin, alpha M (subunit of complement component 3 receptor 3)), PITX2 (paired homeodomain-like 2), MAPK7 (mitogen-activated protein kinase 7), FCGR3A (Fc fragment of IgG, low affinity 111a, receptor (CD16a)), LEPR (leptin receptor), ENG (endoglin), GPX1 (glutathione peroxidase 1), GOT2 (oxaloacetic transaminase glutamic acid type 2, mitochondrial (aspartate aminotransferase type 2)), HRH1 (histamine H1 receptor), NR112 (nuclear receptor family 1, group I, member 2), CRH (corticotropin-releasing hormone), HTR1A (5-hydroxytryptamine (serotonin) receptor 1A), VDAC1 (voltage-gated anion channel 1), HPSE (heparanase), SFTPD (surface-active protein D), TAP2 (ATP-binding cassette transporter 2, subfamily B (MDR/TAP)), RNF123 (ring 123 zinc finger protein), PTK2B (PTK2B protein tyrosine kinase 2 beta), NTRK2 (neurotrophic tyrosine kinase receptor, type 2), IL6R (interleukin 6 receptor), ACHE (acetylcholinesterase (blood group Yt)), GLP1R (glucagon-like peptide 1 receptor), GHR (growth hormone receptor), GSR (glutathione reductase), NQO1 (NAD(P)H dehydrogenase, quinone 1), NR5A1 (nuclear receptor family 5, group A, member 1), GJB2 (gap junction protein, beta 2, 26 kDa), SLC9A1 (solute carrier family 9 (sodium hydrogen antiporter), member 1), MAOA (monoamine oxidase A), PCSK9 (proprotein convertase subtilisin-kexin type 9), FCGR2A (Fc fragment of IgG, low affinity IIa, receptor (CD32)), SERPINF1 (serpin peptidase inhibitor, clade F (alpha-2-antiplasmin, pigment epithelial factor), member 1), EDN3 (endothelin 3), DHFR (dihydrofolate reductase), GAS6 (growth arrest-specific factor 6), SMPD1 (sphingomyelin phosphodiesterase 1, acidic lysosomal), UCP2 (unpaired protein 2 (mitochondrial, proton transporter)), TFAP2A (transport factor AP-2 alpha (activating enhancer binding protein 2 alpha)), C4BPA (complement component 4 binding protein alpha), SERPINF2 (serpin peptidase inhibitor, clade F (alpha-2-antilasmin, pigment epithelial factor), member 2), TYMP (thymidine phosphorylase), ALPP (alkaline phosphatase, placental (Regan isozyme)), CXCR2 (chemokine receptor 2 (with C-X-C motif)), SLC39A3 (solute carrier 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 (coagulation factor XI), ATP7A (ATPase, Cu++ transporter, alpha polypeptide), CR1 (complement component receptor 1 (3b/4b) (Knops blood groups)), GFAP (glial fibrillary alkaline protein), ROCK1 (Rho-associated double-stranded protein kinase 1), MECP2 (methyl CpG-binding protein 2 (Rett syndrome)), MYLK (myosin light chain), BCHE (butyrylcholinesterase), LIPE (lipase, hormone-sensitive), PRDX5 (peroxiredoxin 5), ADORA1 (adenosine A1 receptor), WRN (Werner syndrome, RecQ helicase-like), CXCR3 (chemokine receptor 3 (with C-X-C motif)), CD81 (CD81 molecule), SMAD7 (SMAD family, member 7), LAMC2 (laminin, gamma 2), MAP3K5 (mitogen-activated protein kinase kinase 5), CHGA (chromogranin A (parathyroid secretory protein 1)), IAPP (islet amyloid peptide), RHO (rhodopsin), ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), PTHLH (parathyroid hormone-like hormone), 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 (coagulation factor II (thrombin) receptor-like 3), CX3CL1 (chemokine (C-X3-C motif) ligand 1), BDKRB1 (bradykine receptor B1), ADAMTS13 (ADAM metallopeptidase with thrombospondin motif type 1, 13), ELANE (neutrophil-expressed elastase), ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase 2), CISH (cytokine-inducible SH2-containing protein), GAST (gastrin), MYOC (myocilin, trabecular meshwork-inducible glucocorticoid response), ATP1A2 (ATPase, Na+/K+ transport, alpha 2 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 factor 42 (GTP-binding protein, 25 kDa)), KRT18 (keratin 18), HSF1 (heat shock protein transcription factor 1), MYB (myeloblastosis virus oncogene homolog v-myb (avian)), PRKAA2 (protein kinase, AMP-activated, catalytic subunit alpha 2), ROCK2 (Rho-associated double-stranded protein kinase 2), TFPI (tissue factor pathway inhibitor (lipoprotein-associated inhibitor of coagulation)), PRKG1 (protein kinase, cGMP-dependent, type I), BMP2 (bone morphogenetic protein 2), CTNND1 (catenin (cadherin-associated protein), delta 1), CTH (cystathionase (cystathionine gamma-lyase)), CTSS (cathepsin S), VAV2 (guanine nucleotide exchange factor vav 2), NPY2R (neuropeptide Y receptor Y2), IGFBP2 (insulin-like growth factor binding protein 2, 36 kDa), CD28 (CD28 molecule), GSTA1 (glutathione S-transferase, alpha 1), PPIA (peptidylprolyl 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 family 1, group H, member 3), SCD (stearoyl-CoA desaturase (delta-9-desaturase)), GIP (gastric inhibitory peptide), 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 (hydroxysteroid (11-beta) dehydrogenase 2), CALCRL (calcitonin receptor-like), GALNT2 (UDP-N-acetyl-alpha-D-galactosamine:N-acetylgalactosaminyltransferase 2 polypeptide (GalNAc-T2)), ANGPTL4 (angiopoietin-like 4), KCNN4 (calcium-activated potassium channel, intermediate/low conduction, subfamily N, member 4), PIK3C2A (phosphoinositidine 3-kinase, class 2, alpha polypeptide), HBEGF (heparin-binding EGF-like growth factor), CYP7A1 (cytochrome P450, family 7, subfamily A, polypeptide 1), HLA-DRB5 (major histocompatibility complex, class II, DR beta 5), BNIP3 (19-molecular-weight protein 3), kDa, interacting with BCL2/adenovirus E1B), GCKR (glucokinase (hexokinase) regulator 4), S100A12 (S100 calcium-binding protein A12), PADI4 (peptidylarginine deiminase, type IV), HSPA14 (70 kDa heat shock protein 14), CXCR1 (chemokine receptor 1 (with a C-X-C motif)), H19 (H19, maternally expressed peptide imprinted (non-coding protein)), KRTAP19-3 (keratin-associated protein 19-3), IDDM2 (insulin-dependent diabetes mellitus type 2 factor), RAC2 (ras-related botulinum toxin C3 substrate 2 (rho family, small GTP binding protein Rac2)), RYR1 (ryanodine receptor 1 (muscle)), CLOCK (gene homolog (mouse)), NGFR (nerve growth factor receptor (TNFR superfamily, member 16)), DBH (dopamine beta-hydroxylase (dopamine beta-monooxygenase)), CHRNA4 (cholinergic receptor, nicotinic, alpha 4), CACNA1C (calcium channel, voltage-gated, type L, subunit alpha 1C), PRKAG2 (protein kinase, AMP-activated, gamma 2 noncatalytic subunit), CHAT (choline acetyltransferase), PTGDS (prostaglandin D2 synthase 21 kDa (brain)), NR1H2 (nuclear receptor family 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, NFKB activator), HSPA9 (70 kDa heat shock protein 9 (mortalin)), CYSLTR1 (cysteinyl leukotriene receptor 1), MAT1A (methionine adenosyltransferase I alpha), OPRL1 (opiate receptor-like 1), IMPA1 (insitol (myo)-1 (or 4)-monophosphatase 1), CLCN2 (chloride ion transporter channel 2), DLD (dihydrolipoamide dehydrogenase), PSMA6 (proteasome subunit (prosome, macropain), alpha type 6), PSMB8 (proteasome subunit (prosome, macropain), beta type 8 (large multifunctional peptidase 7)), CHI3L1 (chitinase 3-like factor 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, subfamily C (CFTR/MRP), member 6), RGS2 (regulator of G protein-mediated signaling 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, hip-shoulder muscular dystrophy 2B (autosomal recessive)), FDFT1 (farnesyl-diphosphate farnelystransferase 1), EDN2 (endothelin 2), CCR6 (chemokine receptor 6 (with a C-C motif)), GJB3 (gap junction protein, beta 3, 31 kDa), IL1RL1 (interleukin 1 receptor-like factor 1), ENTPD1 (ectonucleoside triphosphate diphosphohydrolase 1), BBS4 (Bardet-Biedl syndrome factor 4), CELSR2 (cadherin, seven-channel G-type receptor 2 EGF LAG (flamingo homolog, Drosophila)), F11R (F11 receptor), RAPGEF3 (Rap guanine nucleotide exchange factor (GEF) 3), HYAL1 (hyaluronoglycosaminidase 1), ZNF259 (zinc finger protein 259), ATOX1 (ATX1 antioxidant protein 1 homolog (yeast)), ATF6 (transcription activating factor 6), KHK (ketohexokinase (fructokinase)), SAT1 (spermidine/spermine N1-acetyltransferase 1), GGH (gamma-glutamyl hydrolase (conjugase, folylpolygamma-glutamyl hydrolase)), TIMP4 (TIMP inhibitor of metallopeptidase 4), SLC4A4 (solute carrier family 4, sodium bicarbonate cotransporter, member 4), PDE2A (phosphodiesterase 2A, cGMP-stimulated), PDE3B (phosphodiesterase 3B, cGMP-inhibited), FADS1 (fatty acid desaturase 1), FADS2 (fatty acid desaturase 2), TMSB4X (thymosin beta 4, X-linked), TXNIP (thioredoxin-interacting protein), LIMS1 (LIM-like and senescent cell antigen-like domains 1), RHOB (ras homolog gene family, member B), LY96 (lymphocyte antigen 96), FOXO1 (forkhead box O1), PNPLA2 (patatin-like phospholipidase domain-containing factor 2), TRH (thyrotropin-releasing hormone), GJC1 (gap junction protein gamma 1, 45 kDa), SLC17A5 (solute carrier family 17 (anion and sugars), member 5), FTO (fat mass and obesity-associated factor), GJD2 (gap junctional protein delta 2, 36 kDa), PSRC1 (proline-serine-rich double-stranded factor 1), CASP12 (caspase 12 (gene/pseudogene)), GPBAR1 (G protein-coupled bile acid receptor 1), PXK (PX domain-containing serine/threonine kinase), IL33 (interleukin 33), TRIB1 (tribbles homolog 1 (Drosophila)), PBX4 (pre-B-cell leukemia homeobox 4), NUPR1 (nuclear transcriptional regulator protein 1), 15-Sep (15 kDa selenoprotein), CILP2 (cartilage intermediate layer protein 2), TERC (RNA component of telomerase), GGT2 (gamma-glutamyl transferase 2), MT-CO1 (cytochrome c oxidase I, encoded by the mitochondrial genome) and UOX (urate oxidase, a pseudogene). Any of these sequences can be targeted by the CRISPR-Cas system, for example, to study mutation.

In a further embodiment, the chromosomal sequence may also be selected from the following: Pon1 (paraoxonase 1), LDLR (LDL receptor), ApoE (apolipoprotein E), Apo B-100 (apolipoprotein B-100), ApoA (apolipoprotein(a)), ApoA1 (apolipoprotein A1), CBS (cystathionine B synthase), glycoprotein IIb/IIb, MTHRF (5,10-methylenetetrahydrofolate reductase (NADPH) and combinations thereof. In one case, the chromosomal sequences and proteins encoded by the chromosomal sequences associated with cardiovascular disease may be selected from Cacna1C, Sod1, Pten, Ppar(alpha), Apo E, leptin and combinations thereof as the target(s) for the CRISPR-Cas system.

Treatment of liver and kidney diseases

The present invention also provides for the delivery of the CRISPR-Cas system described herein, such as the Cas9 effector protein systems, to the liver and/or kidneys. Delivery strategies for inducing cellular uptake of a therapeutic nucleic acid include the use of physical forces or vector systems, such as delivery using viruses, lipids, or complexes, or nanocarriers. From initial applications of little potential clinical relevance in the case of nucleic acid delivery into kidney cells via high-pressure hydrodynamic systemic injection, a wide range of viral and non-viral therapeutic carriers have already been used to target post-transcriptional events in various animal models of kidney disease in vivo (Csaba Révész 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 at: http://www.intechopen.com/books/gene-therapy-applications/delivery-methods-to-target-rnas-inthe-kidney). Kidney delivery methods may include those of Yuan et al. (Am J Physiol Renal Physiol 295: F605–F617, 2008) investigated whether in vivo delivery of small interfering RNA (siRNA) targeting the 12/15-lipoxygenase (12/15-LO) pathway of arachidonic acid metabolism could result in attenuated renal injury and diabetic nephropathy (DN) in a streptozotocin-injected mouse model of type 1 diabetes. To achieve greater in vivo availability and expression of siRNA in the kidney, Yuan et al. used cholesterol-conjugated double-stranded siRNA oligonucleotides to 12/15-LO. Approximately 400 μg of siRNA was administered to mice by subcutaneous injection. The method according to Yuang et al. can be applied to the CRISPR-Cas system of the present invention, which involves subcutaneous injection into a human of 1-2 g of CRISPR-Cas conjugated to cholesterol for delivery to the kidneys.

Molitoris et al. (J Am Soc Nephrol 20: 1754–1764, 2009) used proximal tubular cells (PTCs) as a site of oligonucleotide reabsorption in the kidney to investigate the efficacy of siRNA targeting p53, a key protein in the apoptotic pathway, in preventing kidney injury. Naked synthetic siRNA to p53, administered by intravenous injection 4 h after ischemic injury, provided maximal protection of both PTCs and kidney function. The data of Molitoris et al. indicate that intravenous administration is followed by rapid delivery of siRNA to proximal tubular cells. For dose-response analysis, rats were injected with siP53 doses of 0.33; 1, 3 or 5 mg/kg administered at the same four time points, resulting in total doses of 1.32, 4, 12 and 20 mg/kg, respectively. All siRNA doses tested resulted in a SCr lowering effect on day one, with higher doses being effective for approximately five days compared to PBS-treated ischemic control rats. Total doses of 12 and 20 mg/kg provided the best protective effect. The method of Molitoris et al. can be applied to the nucleic acid targeting system of the present invention, which provides for total doses of 12 and 20 mg/kg to be administered to humans for delivery to the kidney.

Thompson et al. (Nucleic Acid Therapeutics, Volume 22, Number 4, 2012) reported the toxicological and pharmacokinetic properties of the synthetic small interfering RNA I5NP following intravenous administration to rodents and non-human primates. I5NP is designed to act via the RNA interference (RNAi) pathway to transiently inhibit expression of the pro-apoptotic protein p53 and is designed to protect cells from acute ischemia/reperfusion-related injury, such as acute kidney injury that may occur following major cardiac surgery and delayed graft function that may occur following kidney transplantation. Doses of 800 mg/kg I5NP in rodents and 1000 mg/kg I5NP in non-human primates were required to produce adverse effects, which in monkeys were limited to direct effects on the blood, including asymptomatic complement activation and slightly prolonged clotting times. No additional adverse effects were observed in rats with the rat analogue of I5NP, indicating that these effects likely represent effects related to the class of synthetic RNA duplexes rather than toxicity due to the targeted pharmacologic activity of I5NP. Taken together, these data are consistent with a clinical study using intravenous I5NP to preserve renal function after acute ischemia/reperfusion injury. The no observed adverse effect level (NOAEL) in monkeys was 500 mg/kg. No effects on cardiovascular, respiratory, or neuronal parameters were observed in monkeys following intravenous administration at dose levels up to 25 mg/kg. Therefore, a similar dose could be envisaged for intravenous administration of CRISPR-Cas to the human kidney.

Shimizu et al. (J Am Soc Nephrol 21: 622–633, 2010) developed a system for targeted delivery of siRNA to glomeruli using poly(ethylene glycol) and poly(L-lysine)-based vehicles. The diameter of the siRNA/nanocarrier complex was approximately 10 to 20 nm, a size that would allow it to pass through the fenestrated endothelium to enter the mesangium. Following intraperitoneal injection of fluorescently labeled siRNA/nanocarrier complexes, Shimizu et al. detected siRNA in the circulation for a prolonged period. Repeated intraperitoneal administration of mitogen-activated protein kinase 1 (MAPK1) siRNA/nanocarrier complex suppressed MAPK1 mRNA and protein expression in glomeruli in a mouse model of glomerulonephritis. To study the accumulation of siRNA, Cy5-labeled siRNA complexed with PIC nanocarriers (0.5 ml, 5 nmol siRNA content), naked Cy5-labeled siRNA (0.5 ml, 5 nmol) or Cy5-labeled siRNA encapsulated in HVJ-E (0.5 ml, 5 nmol siRNA content) were administered to BALBc mice. The method of Shimizu et al. can be applied to the nucleic acid targeting system of the present invention, which provides a dose of about 10-20 μmol CRISPR-Cas complexed with nanocarriers per about 1-2 liters for intraperitoneal administration to humans and delivery to the kidneys.

Treatment of epithelial and lung diseases

The present invention also provides for delivery of a CRISPR-Cas system described herein, such as Cas9 systems, to one lung or both lungs.

Although AAV-2-based vectors were initially proposed for delivery of CFTR to the CF airway, other serotypes, such as AAV-1, AAV-5, AAV-6, and AAV-9, have demonstrated improved gene transfer efficiency in a number of lung epithelial models (see, e.g., Li et al., Molecular Therapy, vol. 17 no. 12, 2067-2077 Dec 2009). AAV-1 was shown to be ~100-fold more efficient than AAV-2 and AAV-5 in transducing human airway epithelial cells in vitro, although the transduction efficiency of murine tracheal airway epithelium in vivo using AAV-1 was equal to that of AAV-5. Other studies have demonstrated that AAV-5 was 50-fold more efficient than AAV-2 at delivering genes into human airway epithelium (HAE) in vitro and significantly more efficient in mouse lung airway epithelium in vivo. AAV-6 was also shown to be more efficient than AAV-2 in human airway epithelial cells in vitro and mouse airways in vivo. A more recently discovered isolate, AAV-9, was shown to exhibit greater gene transfer efficiency than AAV-5 in mouse nasal and alveolar epithelium in vivo, with gene expression detectable for over 9 months, suggesting that AAV can provide long-lasting gene expression in vivo, a necessary property for a CFTR gene delivery vector. Furthermore, it was demonstrated that AAV-9 could be repeatedly administered into mouse lungs without loss of CFTR expression and with minimal immune-related sequelae. CF and non-CF HAE cultures can be inoculated at the apical surface with 100 μl of AAV-based vectors for several hours (see, e.g., Li et al., Molecular Therapy, vol. 17 no. 12, 2067-2077 Dec 2009). The MOI can range from 1 x 10 ³ to 4 x 10 ⁵ vector genomes/cell, depending on the virus concentration and the experimental goals. The above-mentioned vectors are contemplated for delivery and/or administration according to the present invention.

Zamora et al. (Am J Respir Crit Care Med Vol 183. pp 531-538, 2011) presented an example of the use of RNAi-based therapeutics to treat human infectious diseases, as well as a randomized trial of an antiviral drug in lung transplant recipients infected with respiratory syncytial virus (RSV). Zamora et al. conducted a randomized, double-blind, placebo-controlled trial in LTX recipients with RSV respiratory tract infection. Patients were given the option of receiving standard of care anti-RSV treatment. ALN-RSV01 aerosol (0.6 mg/kg) or placebo was administered daily for 3 days. This study demonstrated that an RNAi-based therapeutic targeting RSV can be safely administered to LTX recipients with RSV infection. Three daily doses of ALN-RSV01 did not result in any exacerbation of airway symptoms or impairment of lung function and did not exhibit any systemic pro-inflammatory effects such as cytokine or CRP induction. Pharmacokinetic studies demonstrated only low-level transient systemic exposure following inhalation, consistent with preclinical animal studies demonstrating that ALN-RSV01 administered intravenously or by inhalation undergoes rapid clearance from the bloodstream via exonuclease-mediated cleavage and renal excretion. The method according to Zamora et al. can be used in relation to the nucleic acid targeting system of the present invention, and wherein CRISPR-Cas in the form of an aerosol, for example at a dose of 0.6 mg/kg, can be provided according to the present invention.

Schwank et al. (Cell Stem Cell, 13:653–58, 2013) used CRISPR-Cas9 to correct a defect associated with cystic fibrosis in human stem cells. The researchers targeted the ion channel gene cystic fibrosis transmembrane conductance receptor (CFTR). A deletion in CFTR results in misfolding of the protein 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 correct the defect using CRISPR along with a donor plasmid containing the repair sequence to be inserted. The researchers then grew the cells into small intestinal or gut “organoids” and demonstrated that they functioned normally. In this case, approximately half of the clonal organoids had the proper correction of their hereditary material.

Treatment of diseases of the muscular system

The present invention also provides for delivery of a CRISPR-Cas system described herein, such as Cas9 systems, to muscle(s).

Bortolanza et al. (Molecular Therapy vol. 19 no. 11, 2055-2064 Nov. 2011) demonstrated that systemic delivery of RNAi expression cassettes to FRG1 mice after the onset of facioscapulohumeral muscular dystrophy (FSHD) resulted in dose-dependent, long-term knockdown of FRG1 without toxicity symptoms. Bortolanza et al. found that a single intravenous injection of 5 × 10 ¹² vg (vector genomes) of rAAV6-sh1FRG1 rescued muscle histopathology and muscle function in FRG1 mice. In more detail, 200 μl containing 2 × 10 ¹² or 5 × 10 ¹² vg of vector in saline was injected into the tail vein using a Terumo syringe with a 25-gauge needle. The method according to Bortolanza et al. can be applied to AAV expressing CRISPR Cas and administered to a human by injection at a dose of approximately 2 × 10 ¹⁵ or 2 × 10 ¹⁶ vg of vector.

Dumonceaux et al. (Molecular Therapy vol. 18 no. 5, 881-887 May 2010) inhibited the myostatin pathway using RNA interference directed against the myostatin receptor AcvRIIb mRNA (sh-AcvRIIb). Quasi-dystrophin restoration was mediated by targeted U7 exon skipping (U7-DYS). Adeno-associated virus vectors carrying either the sh-AcvrIIb construct alone, the U7-DYS construct alone, or a combination of both constructs were injected into the tibialis anterior (TA) muscle of dystrophic mdx mice. Injections were performed using 10 ¹¹ AAV genomes. The method is according to Dumonceaux et al. can be used with respect to an AAV expressing CRISPR Cas and administered to a human by injection, for example, at a dose of from about 10 ¹⁴ to about 10 ¹⁵ vg of the vector.

Kinouchi et al. (Gene Therapy (2008) 15, 1126–1130) reported the efficacy of in vivo siRNA delivery to skeletal muscle of normal or diseased mice via the formation of nanoparticles from chemically unmodified atelocollagen-coated siRNA (ATCOL). ATCOL-mediated local application of siRNA targeting myostatin, a negative regulator of skeletal muscle growth, resulted in significant increases in muscle mass within weeks of administration when injected into mouse skeletal muscle or intravenously. These results indicate that ATCOL-mediated siRNA delivery is a powerful tool for further therapeutic application in diseases including muscle atrophy. Mst-siRNA (final concentration, 10 mM) was mixed with ATCOL (final concentration for local administration, 0.5%) (AteloGene, Kohken, Tokyo, Japan) according to the manufacturer's instructions. After anesthetizing mice (male C57BL/6, 20 weeks old) with Nembutal (25 mg/kg, i.p.), the Mst-siRNA/ATCOL complex was injected into the masseter and biceps femoris muscles. The method of Kinouchi et al. can be applied to CRISPR-Cas and administered to humans by injection, for example, at a dose of approximately 500 to 1000 mL of a 40 μM solution into the muscle. Hagstrom et al. (Molecular Therapy Vol. 10, No. 2, August 2004) described a virus-free intravascular technique that provides efficient and reproducible delivery of nucleic acids into muscle cells (muscle fibers) of mammalian limb muscles. The technique involves injection of naked plasmid DNA or siRNA into a distal limb vein temporarily isolated by a tourniquet or pneumatic cuff. Delivery of nucleic acid to muscle fibers is achieved by rapid injection of the nucleic acid in a volume sufficient to allow leakage of the nucleic acid solution into the muscle tissue. High levels of transgene expression in skeletal muscle were achieved in both small and large animals with minimal toxicity. Evidence of siRNA delivery to limb muscle was also obtained. For intravenous injection of plasmid DNA into a rhesus macaque, a three-way stopcock was attached to two syringe pumps (Model PHD 2000; Harvard Instruments), each holding one syringe. Five minutes after injection, papaverine was administered by injection of pDNA (15.5-25.7 mg in 40-100 ml of saline) at a rate of 1.7 or 2.0 ml/s. This can be scaled up for plasmid DNA expressing the CRISPR Cas of the present invention, with injection into humans of approximately 300 to 500 mg in 800-2000 ml of saline. For injection of adenoviral vector into a rat, 2 x 10 ⁹ infectious particles in 3 ml of normal saline (NSS) were injected. This can be scaled up for an adenoviral vector expressing the CRISPR Cas of the present invention, with an injection of approximately 1 x 10 ¹³ infectious particles in 10 liters of NSS into a human. For siRNA, 12.5 μg of siRNA was injected into the great saphenous vein in a rat, and 750 μg of siRNA was injected into the great saphenous vein in a primate. This can be scaled up for the CRISPR Cas of the present invention, for example, by injecting approximately 15 to approximately 50 mg into the great saphenous vein in a human.

See also, e.g., Duke University published application WO2013163628 A2, "Genetic Correction of Mutated Genes," which describes attempts to correct, e.g., a frameshift mutation that causes a premature stop codon and truncation of the gene product, which can be corrected by nuclease-mediated non-homologous end joining, such as that causing Duchenne muscular dystrophy ("DMD"), a recessive, lethal, X-linked disorder resulting in muscle degeneration associated with mutations in the dystrophin gene. Most of the dystrophin gene mutations that cause DMD are exon deletions that disrupt the reading frame and cause premature termination of translation of the dystrophin gene. Dystrophin is a cytoplasmic protein that provides structural stability to the cell membrane dystroglycan complex responsible for regulating muscle cell integrity and function. The dystrophin gene, or "DMD gene" as used interchangeably herein, spans 2.2 million base pairs at the Xp21 locus. The primary transcript is approximately 2,400 kb in size, with the mature mRNA being approximately 14 kb. 79 exons encode a protein of over 3,500 amino acids. Exon 51 is frequently adjacent to out-of-frame deletions in patients with DMD and has been targeted by oligonucleotide-based exon skipping in clinical trials. Recently, a clinical trial using exon 51 skipping with the compound eterlipsen reported a significant functional benefit over 48 weeks with a mean dystrophin-positive fiber count of 47% compared to baseline. Mutations in exon 51 are ideal for durable correction via NHEJ-based genome editing.

The methods of U.S. Patent Application Publication No. 20130145487 assigned to Cellectis, which relate to meganuclease variants for cleaving a target sequence of the human dystrophin gene (DMD), may also be modified for the nucleic acid targeting system of the present invention.

Treatment of skin diseases

The present invention also provides for delivery of the CRISPR-Cas system described herein, such as the Cas9 effector protein systems, to the skin.

Hickerson et al. (Molecular Therapy-Nucleic Acids (2013) 2, e129) address a powered microneedle array device for skin delivery designed for self-directed (sd) delivery of siRNA into human and mouse skin. A major challenge in translating siRNA-based skin therapeutics into clinical practice is the development of efficient delivery systems. Considerable effort has been devoted to developing a number of skin delivery techniques, with limited success. In a clinical trial in which skin was treated with siRNA, the acute pain associated with hypodermic needle injection prevented the enrollment of additional patients, highlighting the need for improved, more “patient-friendly” (i.e., causing little or no pain) delivery systems. Microneedles provide an efficient means of delivering large charged cargo molecules, including siRNA, across the primary barrier, the stratum corneum, and are generally considered to be less painful than conventional hypodermic needles. Powered "punch-type" microneedle devices, including the powered microneedle array (MMNA) device used by Hickerson et al., have been shown to be safe in nude mouse studies, causing little or no pain, as evidenced by (i) widespread use in cosmetics and (ii) limited testing in which virtually all volunteers rated the device as much less painful than influenza vaccination, suggesting that siRNA delivery using this device will be much less painful than that experienced in previous clinical studies using hypodermic needles. The MMNA device (commercially available as Triple-M or Tri-M from Bomtech Electronic Co, Seoul, South Korea) was adapted for siRNA delivery into mouse and human skin. The sd-siRNA solution (up to 300 μl of 0.1 mg/ml RNA) was injected into the chamber of a disposable injection cartridge with Tri-M needles (Bomtech) that were set at a depth of 0.1 mm. For human skin processing, de-identified skin (obtained immediately after surgical procedures) was manually stretched and pinned to a cork table 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 are according to 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, into the skin.

Leachman et al. (Molecular Therapy, vol. 18 no. 2, 442-446 Feb. 2010) report a phase Ib clinical trial targeting the rare skin disorder pachyonychia congenita (PC), an autosomal dominant syndrome that involves blocking plantar keratoderma, using the first short interfering RNA (siRNA) therapeutic for the skin. This siRNA, called TD101, specifically and efficiently targets mutant keratin 6a (K6a) N171K mRNA without affecting wild-type K6a mRNA.

Zheng et al. (PNAS, July 24, 2012, vol. 109, no. 30, 11975-11980) demonstrated that spherical nanoparticle nucleic acid conjugates (SNA-NCs), which are gold cores surrounded by a dense shell of tightly oriented, covalently immobilized siRNA, readily penetrate nearly 100% of keratinocytes in vitro, mouse skin, and human epidermis within hours of application. Zheng et al. demonstrated that a single application of 25 nM SNA-NCs to the epidermal growth factor receptor (EGFR) for 60 h demonstrated efficient gene knockdown in human skin. A similar dose could be envisaged for CRISPR-Cas immobilized in SNA-NCs for delivery to the skin.

General provisions of gene therapy

Examples of disease-associated genes and polynucleotides and specific disease-related information are available from the McKusick-Nathans Institute of Genetic Medicine at Johns Hopkins University (Baltimore, MD) and the National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD), both available on the World Wide Web.

Mutations in these genes and pathways can result in the production of inappropriate proteins or proteins in inappropriate amounts that affect function. Additional examples of genes, diseases, and proteins are hereby incorporated by reference from U.S. Provisional Patent Application No. 61/736,527, filed December 12, 2012. Such genes, proteins, and pathways can be a target polynucleotide for the CRISPR complex of the present invention.

Embodiments of the present invention also relate to methods and compositions related to gene knockout, gene amplification, and repair of specific mutations associated with DNA repeat instability and neurological disorders (Robert D. Wells, Tetsuo Ashizawa, Genetic Instabilities and Neurological Diseases, Second Edition, Academic Press, Oct 13, 2011 - Medical). Certain 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. McIvor EI, Polak U, Napierala M. RNA Biol. 2010 Sep-Oct;7(5):551-8). The effector protein systems of the present invention can be adapted to correct such genomic instability defects.

Certain additional aspects of the present invention relate to the correction of defects associated with a wide range of inherited disorders, which are further described on the National Institutes of Health website under the topic heading "Genetic Disorders" (website at health.nih.gov/topic/GeneticDisorders). Inherited brain disorders may include, but are not limited to, adrenoleukodystrophy, agenesis of the corpus callosum, Aicardi syndrome, Alpers syndrome, Alzheimer's disease, Barth syndrome, Batten disease, CADASIL, cerebellar degeneration, Fabry disease, Gerstmann-Straussler-Scheinker syndrome, Huntington's disease and other triplet repeat-related disorders, Leigh disease, Lesch-Nyhan syndrome, Menkes disease, NINDS types of mitochondrial myopathy, and colpocephaly. These disorders are further described on the National Institutes of Health website under the topic "Genetic Brain Disorders."

Illustrative applications of the CRISPR Cas system

The present invention relates to a non-naturally occurring or engineered composition, or one or more polynucleotides encoding components of said composition, or a vector or delivery systems comprising one or more polynucleotides encoding components of said composition, for use in modifying a target cell in vivo, ex vivo or in vitro, and they can be made using a method that alters the cell such that after modification, the progeny or cell line of the cell modified by CRISPR retains the altered phenotype. The modified cells and progeny can be part of a multicellular organism, such as a plant or an animal, using an ex vivo or in vivo CRISPR system against desired cell types. The CRISPR invention can be a therapeutic method of treatment. The therapeutic method of treatment can include gene or genome editing or gene therapy.

Modification of a target using the CRISPR-Cas system or complex

In one aspect, the present invention relates to methods for modifying a target polynucleotide in a eukaryotic cell, which may occur in vivo, ex vivo or in vitro. In some embodiments, the method comprises selecting a cell or a population of cells from a human or non-human animal and modifying the cell or cells. The culturing may be performed at any stage ex vivo. The cell or cells may even be reintroduced into a non-human animal or into a plant. With respect to the cells reintroduced, it is particularly preferred that these cells are stem cells.

In some embodiments, the method includes providing binding of a CRISPR complex to a target polynucleotide to effect cleavage of said target polynucleotide, thereby providing modification of the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence that hybridizes or is capable of hybridizing with a target sequence in said target polynucleotide, wherein said guide sequence is associated with a mate tracr sequence that, in turn, hybridizes with the tracr sequence.

In one aspect of the present invention, there is provided a method for modifying the expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises causing a CRISPR complex to bind to the polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence that hybridizes or is capable of hybridizing to a target sequence in said polynucleotide, wherein said guide sequence is linked to a tracr mate sequence that in turn hybridizes to the tracr sequence. Similar factors and conditions apply to the methods for modifying a target polynucleotide set forth above. Indeed, these sampling, culturing, and reintroduction options are encompassed by aspects of the present invention.

Indeed, in any aspect of the present invention, the CRISPR complex may comprise a CRISPR enzyme complexed with a guide sequence that hybridizes or is capable of hybridizing to a target sequence, wherein said guide sequence may be associated with a mate tracr sequence that, in turn, may hybridize to the tracr sequence.

Similar factors and conditions apply to the methods of modifying a target polynucleotide described above. Thus, any of the non-naturally occurring CRISPR enzymes described herein contain at least one modification, and thus the enzyme is characterized by certain improved properties. In particular, any of the enzymes is capable of forming a CRISPR complex with a guide RNA. When such a complex is formed, the guide RNA is capable of binding to a target polynucleotide sequence, and the enzyme is capable of modifying the target locus. In addition, the enzyme in the CRISPR complex is characterized by a reduced ability to modify one or more non-target loci compared to the unmodified enzyme.

In addition, the modified CRISPR enzymes described herein include enzymes wherein in a CRISPR complex the enzyme is characterized by an increased ability to modify one or more target loci compared to an unmodified enzyme. Such functionality may be provided alone or provided in combination with the above-described functionality of reduced ability to modify one or more off-target loci. Any such enzymes may be provided with one of the additional modifications of the CRISPR enzyme as described herein, such as in combination with the activity provided by one or more associated heterologous functional domains, any additional mutations to reduce nuclease activity, etc.

In preferred embodiments of the present invention, a modified CRISPR enzyme is provided with a reduced ability to modify one or more off-target loci compared to an unmodified enzyme and an increased ability to modify one or more target loci compared to an unmodified enzyme. In combination with additional modifications of the enzyme, significantly increased specificity can be achieved. For example, a combination of such preferred embodiments with one or more additional mutations is provided, wherein the one or more additional mutations are in one or more catalytically active domains. Such additional catalytic mutations can confer nickase functionality, as described in detail elsewhere herein. In such enzymes, increased specificity can be achieved by increased specificity in terms of enzymatic activity.

Modifications to reduce off-target effects and/or enhance on-target effects as described above can be performed on amino acid residues located in the positively charged region/groove located between the RuvC-III and HNH domains. It is expected that any of the functional effects described above can be achieved by modifying amino acids in the aforementioned groove, but also by modifying amino acids near or outside the groove.

Additional functional properties that can be engineered into modified CRISPR enzymes as described herein may include the following. 1. Modified CRISPR enzymes that disrupt DNA-protein interactions without disrupting the tertiary or secondary structure of the proteins. This includes residues that contact any part of the RNA:DNA duplex. 2. Modified CRISPR enzymes that weaken interactions between proteins that hold Cas9 in a conformation required for nuclease cleavage in response to DNA binding (on-target or off-target). For example, a modification that slightly inhibits but still allows the nuclease conformation of the HNH domain (located at the cleavable phosphate). 3. Modified CRISPR enzymes that strengthen interactions between proteins that hold Cas9 in a conformation that inhibits activity in response to DNA binding (on-target or off-target). For example: a modification that stabilizes the HNH domain in a conformation beyond the scissile phosphate. Any such additional functional enhancement may be provided in combination with any other modification of the CRISPR enzyme, as described in detail elsewhere in this document.

Any of the improved functional properties described herein may be performed with respect to any CRISPR enzyme, such as a Cas9 enzyme. The Cas9 enzymes described herein are derived from Cas9 enzymes from S. pyogenes and S. aureus. However, it is contemplated that any of the functional properties described herein may be engineered into Cas9 enzymes from other orthologs, including chimeric enzymes containing fragments from multiple orthologs.

Nucleic acids, amino acids and proteins, regulatory sequences, vectors and others

In the present invention, nucleic acids are used to bind target DNA sequences. This is advantageous because nucleic acids are much easier and cheaper to produce than proteins, and the specificity can vary with fragment length if homology is desired. For example, complex 3-D determination of the positions of multiple domains is not required. The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably. They denote a polymeric form of nucleotides of any length, both deoxyribonucleotides and ribonucleotides or their analogs. Polynucleotides can have any spatial structure and can perform any function, known or unknown. Non-limiting examples of polynucleotides are: coding or non-coding regions of a gene or gene fragment, loci(s) determined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNAs, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. The term also encompasses nucleic acid-like structures with synthetic backbones, see, for example, Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO 96/39154; 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 be introduced before or after assembly of the polymer. The nucleotide sequence may be interrupted by components other than nucleotides. The polynucleotide may be further modified after polymerization, such as by coupling with a labeling component. As used herein, the term "wild type" is a term of art understood by one skilled in the art and means the typical form of an organism, strain, gene or characteristic that occurs in nature, as distinguished from mutant or variant forms. "Wild type" represents the backbone. As used herein, the term "variant" is to be understood to mean the expression of properties that are characterized by a pattern that differs from that found in nature. The expressions "non-naturally occurring" and "engineered" are used interchangeably and indicate human intervention. The expressions, when applied 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 and found in nature. "Complementarity" means the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence, either by conventional Watson-Crick pairing or other non-conventional types. Percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 would be 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfect complementarity" means that all contiguous residues in the nucleic acid sequence will hydrogen bond with the same number of contiguous residues in the second nucleic acid sequence. As used herein, the term "substantially complementary" means a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% within a region of 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" in relation to hybridization means conditions under which a nucleic acid with complementarity to a target sequence preferentially hybridizes to the target sequence and substantially does not hybridize to non-targeted sequences. Stringent conditions are generally 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 described in detail 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 If a polynucleotide sequence is envisaged, complementary or partially complementary sequences are also envisaged. These sequences are preferably capable of hybridizing to the reference sequence under conditions of high stringency. Typically, to maximize the hybridization rate, hybridization conditions of relatively low stringency are selected: a temperature of approximately 20-25°C below the melting point temperature ( _Tm ). _Tm is the temperature at which 50% of a specific target sequence hybridizes to a perfectly complementary probe in solution at a given ionic strength and pH. Typically, if at least about 85% nucleotide complementarity of the hybridized sequences is desired, very stringent wash conditions are selected with a temperature of approximately 5-15°C lower than _Tm . If at least about 70% nucleotide complementarity of the hybridized sequences is desired, moderately stringent wash conditions are selected with a temperature of approximately 15-30°C lower than _Tm . Highly permissive (very low stringency) wash conditions may have a lowest temperature of 50°C below _Tm , which allows a high level of mismatches between the hybridized sequences. Those skilled in the art will appreciate that other physical and chemical parameters during the hybridization and wash steps may also be varied to influence the resulting detectable hybridization signal based on the particular level of homology between the target and probe sequences. Preferred high stringency conditions include incubation in 50% formamide, 5x SSC, and 1% SDS at 42°C, or incubation in 5x SSC and 1% SDS at 65°C with a wash of 0.2x 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 may occur via Watson-Crick base pairing, Hoogsteen bonding, or any other sequence-specific method. The complex may comprise two strands forming a duplex structure, three or more strands forming a multistrand complex, a single self-hybridizing strand, or any combination thereof. The hybridization reaction may be a step in a larger process, such as the initial step of PCR or the cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing to a given sequence is called a "complementary sequence" for the sequence. As used herein, the term "genomic locus" or "locus" (plural loci) is a specific position of a gene or DNA sequence on a chromosome. "Gene" refers to fragments of DNA or RNA that encode a polypeptide chain or RNA that plays a functional role in an organism, and therefore it is the molecular unit of heredity in living organisms. For the purpose of the present invention, genes may be considered to contain regions that regulate the formation of a gene product, whether the regulatory sequences are adjacent to coding and/or transcribed sequences or not. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences, such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and locus regulatory regions. As used herein, the term "expression of a genomic locus" or "gene expression" refers to the process by which the information of a gene is used in the synthesis of a functional gene product. Gene expression products are often proteins, but for non-protein-coding 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 necessary for survival. As used herein, "expression" of a gene or nucleic acid encompasses not only the expression of genes in cells, but also the transcription and translation of nucleic acid(s) in cloning systems and in any other context. As used herein, the term "expression" also refers to the process by which a polynucleotide is transcribed from a DNA template (such as to form an mRNA or other RNA transcript) and/or the process by which transcribed mRNA is further translated to form peptides, polypeptides, or proteins. The transcripts and encoded polypeptides may be referred to collectively as the "gene product." If the polynucleotide is derived from genomic DNA, expression may involve splicing of mRNA 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, it may contain modified amino acids, and its structure may be interrupted by non-amino acid components. The terms also encompass a polymer of amino acids that has been modified; for example, by disulfide bonding, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation such as coupling to a labeling component. As used herein, the term "amino acid" includes natural and/or non-natural or synthetic amino acids, including glycine and both D- and L-optical isomers, and 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 in aspects of the present invention, sequence identity refers to sequence homology. Homology comparisons can be made by eye or, more commonly, using readily available sequence comparison programs. These commercially available computer programs can calculate the percentage (%) of homology between two or more sequences, and can also calculate the sequence identity between two or more amino acid sequences or nucleic acid sequences.

In aspects of the present invention, the term "guide RNA" refers to a polynucleotide sequence comprising one or more putative or identified tracr sequences or putative or identified crRNA sequences or guide sequences. In particular embodiments, the "guide RNA" comprises a putative or identified crRNA sequence or guide sequence. In further embodiments, the guide RNA does not comprise a putative or identified tracr sequence.

As used herein, the term "wild type" is a term of art understood by one skilled in the art and refers to the typical form of an organism, strain, gene, or characteristic that occurs in nature, as distinguished from mutant or variant forms. "Wild type" represents the base.

As used in this document, the term "variant" should be understood to mean the manifestation of qualities that are characterized by a pattern that differs from that found in nature.

The expression "non-naturally occurring" or "engineered" are used interchangeably and indicate human intervention. The expressions, when they relate 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 occurs in nature. In all aspects and embodiments, whether or not they include these expressions, it is clear that they may preferably be optional and thus are preferably included or not preferably not included. Furthermore, the expressions "non-naturally occurring" and "engineered" may be used interchangeably and thus may be used alone or in combination, and one or the other may replace the reference to both together. In particular, "engineered" is preferred over "non-naturally occurring" or "non-naturally occurring and/or engineered".

Sequence homology values can be obtained using any of a number of computer programs known in the art, such as BLAST or FASTA, etc. A suitable computer program for performing such alignments is the GCG Wisconsin Bestfit software package (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST software package (see Ausubel et al., 1999 ibid - Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410), and the GENEWORKS software package as comparison tools. Offline and online searching is available in both BLAST and FASTA (see Ausubel et al., 1999 ibid, pages 7-58 - 7-60). However, the GCG Bestfit program is preferred. Percentage (%) sequence homology can be calculated for contiguous sequences, i.e. one sequence is aligned with another sequence and each amino acid or nucleotide in one sequence is directly compared to the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called a "gapless" alignment. Typically, such gapless alignments are only performed for a relatively small number of residues. Although this method is very simple and consistent, it does not take into account the fact that, for example, in a pair of otherwise identical sequences, a single insertion or deletion may cause the following amino acid residues to be ignored in the alignment, thus potentially resulting in a significant decrease in % homology when the global alignment is performed. Therefore, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology or identity score. This is achieved by inserting "gaps" into the sequence alignment in an attempt to maximize local homology or identity. However, these more sophisticated methods assign "gap penalties" to each gap that occurs in the alignment, so that for the same number of identical amino acids, a sequence alignment with the fewest possible gaps, reflecting the higher degree of relatedness between the two sequences being compared, may result in a higher score than an alignment with a larger number of gaps. Typically, "affine gap penalty values for related sequences" are used, which assign a relatively high value for the existence of a gap and a lower penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. Of course, high gap penalties may result in optimized alignments with fewer gaps. Most alignment programs allow gap penalties to be varied. However, it is preferable to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit software package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each residue in its continuation. Calculating the maximum % homology therefore requires initially obtaining an optimal alignment that takes the gap penalties into account. A suitable computer program for performing such an alignment is the GCG Wisconsin Bestfit software package (Devereux et al., 1984 Nuc. Acids Research 12 p387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 Short Protocols in Molecular Biology, ^4th Ed. - Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol. 403-410), and the GENEWORKS package as comparison tools. Both BLAST and FASTA offer offline and online searching (see Ausubel et al., 1999, Short Protocols in Molecular Biology, pages 7-58 - 7-60). However, for some tasks, the GCG Bestfit program may be preferable. A new tool called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol Lett. 1999 174(2): 247-50; FEMS Microbiol Lett. 1999 177(1): 187-8 and the National Center for Biotechnology Information web site at the National Institutes of Health web site). Although the final % homology can be measured in units of identity, the alignment method itself is typically not based on an all-or-none comparison of pairs. Instead, a substitution matrix with a similarity scale is typically used, assigning scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix most commonly used is the BLOSUM62 matrix, the default matrix for the BLAST suite of programs. The GCG Wisconsin programs generally use either publicly available defaults or custom character comparison tables if provided (see the user manual for further details). For some purposes it is preferable to use the publicly available defaults for the GCG package or, in the case of other software, a default matrix such as BLOSUM62. Alternatively, percent homology values can be calculated using the multiple alignment function in DNASIS™ (Hitachi Software) using an algorithm similar to CLUSTAL (Higgins DG & Sharp PM (1988) Gene 73(1), 237-244). Once the software has provided an optimal alignment, % homology, preferably % sequence identity, can be calculated. The software generally does this as part of the sequence comparison and provides a numerical result. Sequences may also have deletions, insertions or substitutions of amino acid residues that result in a silent change and result in a functionally equivalent substance. Intentional amino acid substitutions can be made based on similarities in amino acid properties (e.g. polarity, charge, solubility, hydrophobicity, hydrophilicity and/or amphipathic nature of 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 alone. However, it is also more useful to include mutation data. The amino acid groups obtained in this way are likely to be conservative for structural reasons. These groups can be described in the form of a Venn diagram (Livingstone CD and Barton GJ (1993) "Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation" Comput. Appl. Biosci. 9: 745-756) (Taylor WR (1986) "The classification of amino acid conservation" J. Theor. Biol. 119; 205-218). Conservative substitutions can be made, for example, according to the table below, which describes the generally accepted 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 Small A G S

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 included are tissues, cells, and progeny thereof, of a biological organism, whether obtained in vivo or cultured in vitro.

The terms "therapeutic agent," "agent producing a therapeutic effect," or "agent for treating" are used interchangeably and mean a molecule or compound that produces some beneficial effect when administered to a subject. Beneficial effects include the ability to make diagnostic determinations; alleviate a disease, symptom, disorder, or condition; reduce or prevent the onset of a disease, symptom, disorder, or condition; and generally counteract a disease, symptom, disorder, or condition.

As used herein, "treating" or "treating" or "alleviating" or "alleviating" are used interchangeably. These terms refer to an approach to obtaining beneficial or desired results, including but not limited to a therapeutic effect and/or a prophylactic effect. A therapeutic effect is understood to mean any therapeutically significant improvement or effect with respect to one or more diseases, conditions or symptoms being treated. For a prophylactic effect, the compositions may be administered to a subject at risk of developing a particular disease, condition or symptom or to a subject who reports one or more physiological symptoms of the disease, even though the disease, condition or symptom may not have manifested yet.

The term "effective amount" or "therapeutically effective amount" means an amount of an agent that is sufficient to provide favorable or desired results. A therapeutically effective amount may vary depending on one or more of: the subject and disease state being treated, the weight and age of the subject, the severity of the disease state, the route of administration, and the like, as one of skill in the art can readily determine. The term also applies to a dose that can be imaged for determination by any one of the imaging methods described herein. The specific dose may vary depending on one or more of: the particular agent selected, the dosing regimen followed, whether it is administered in combination with other agents, the timing of administration, the tissue being imaged, and the physical delivery system in which it is contained.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA technology, all of which are within the skill of 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)); METHODS IN ENZYMOLOGY series (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)).

Some aspects of the present invention relate to vector systems comprising one or more vectors, or vectors per se. Vectors can be designed to express CRISPR transcripts (e.g., 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 further discussed in Goeddel, GENE expression TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, a recombinant expression vector can be transcribed and translated in vitro, such as with the aid of T7 promoter and T7 polymerase regulatory sequences.

Embodiments of the present invention encompass sequences (both polynucleotide and polypeptide) that may comprise a homologous substitution (as used herein, both substitution and replacement mean the exchange of an existing amino acid residue or nucleotide for an alternative residue or nucleotide), which may occur, i.e., in the case of amino acids, a like-for-like substitution, such as basic for basic, acidic for acidic, polar for polar, etc. Non-homologous substitution may also occur, i.e., a residue from one class for a residue from another, or, alternatively, involving the inclusion of amino acids other than naturally occurring, such as ornithine (hereinafter referred to as Z), ornithinediaminobutyric acid (hereinafter referred to as B), norleucineornithine (hereinafter referred to as O), pyridylalanine, thienylalanine, naphthylalanine, and phenylglycine. Variant amino acid sequences may contain suitable spacer groups that can be inserted between any two amino acid residues of the sequence, including alkyl groups, for example, methyl, ethyl or propyl groups, in addition to amino acid spacers such as glycine or β-alanine residues. Another form of variation, which involves the presence of one or more amino acid residues in peptoid form, may be well understood by those skilled in the art. For the avoidance of doubt, "peptoid form" is used to refer to variant amino acid residues wherein the substituent group for the α-carbon is located on the nitrogen atom of the residue rather than on the α-carbon. Methods for producing 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.

Homology modeling. Corresponding residues in other Cas9 orthologs 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)—the protein–protein interaction (PPI) computational method for predicting interactions mediated by domain–motif boundaries. PrePPI (predicted PPI), a framework based on the PPI prediction method, combines structural evidence with non-structural evidence using the concept of Bayesian statistics. The method involves taking a pair of proteins and applying a structural alignment to identify structural elements that match either their experimentally determined structures or homology models. Structural alignment is further used to identify both nearby and distant structural neighbors through global and local geometric relationships. Whenever two neighboring structural elements form a complex described in the Protein Data Bank, it defines a matrix for modeling the interaction between the two proteins of interest. Models of the complex are created by superimposing the element structures onto their corresponding structural neighbor in the matrix. This approach is further described in Dey et al., 2013 (Prot Sci; 22: 359-66).

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 accomplished using natural or recombinant DNA polymerases, such as TaqGold™, T7 DNA polymerase, the Klenow fragment of E. coli DNA polymerase, and reverse transcriptase. The preferred amplification method is PCR.

In certain aspects, the present invention encompasses vectors. As used herein, a "vector" is a vehicle that enables or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another segment of DNA can be inserted to thereby effect replication of the inserted segment. Typically, a vector is capable of replication if associated with appropriate control elements. In general, the term "vector" refers to a nucleic acid molecule capable of transferring 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, no free ends (e.g., circular); nucleic acid molecules that contain DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular loop of double-stranded DNA into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, where virally derived DNA or RNA sequences are present in the vector for packaging into the virus (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses (AAV)). Viral vectors also include polynucleotides carried by the virus for transfection into a host cell. Certain vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and mammalian episomal vectors). Other vectors (e.g., mammalian vectors other than episomal vectors) integrate into the host cell genome upon introduction into the host cell and are thus replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are functionally linked. Such vectors are referred to herein as "expression vectors". Common expression vectors suitable for recombinant DNA techniques are often in the form of plasmids.

Recombinant expression vectors may comprise a nucleic acid of the present invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors comprise one or more regulatory elements, which may be selected taking into account the host cells that are intended to be used for expression, which are operably linked to the nucleic acid sequence that is intended to be expressed. In the context of a recombinant expression vector, the term "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) such that expression of the nucleotide sequence is allowed (e.g. in an in vitro transcription/translation system or in a host cell upon introduction of the vector into the host cell). With respect to recombination and cloning methods, reference should be made to U.S. Patent Application No. 10/815,730, published September 2, 2004 as US 2004-0171156 A1, the contents of which are incorporated herein by reference in their entirety.

Aspects of the present invention relate to bicistronic vectors for chimeric RNA and Cas9. Bicistronic expression vectors for chimeric RNA and Cas9 are preferred. In general, and particularly in this embodiment, Cas9 is preferably driven by the CBh promoter. The chimeric RNA may preferably be driven by a Pol III promoter, such as the U6 promoter. Optimally, a combination of both. The chimeric guide RNA typically consists of a 20 bp guide sequence (Ns), which may be linked to a tracr sequence (extending from the first "U" in the lower strand to the end of the transcript). The tracr sequence may be truncated at various indicated positions. The guide and tracr sequences are separated by a tracr pair sequence, which may be GUUUUAGAGCUA. This may be followed by the shown GAAA loop sequence. Both of these are preferred examples. The present inventors demonstrated Cas9-mediated insertions/deletions at the human EMX1 and PVALB loci using SURVEYOR nuclease assays. ChiRNAs are indicated by their "+n" designation, and crRNA refers to a hybrid RNA in which the guide and tracr sequences are expressed as separate transcripts. Throughout this application, chimeric RNA may also be referred to as single guide or synthetic guide RNA (sgRNA).

In some embodiments, a loop is provided in the guide RNA. This may be a stem loop or a tetra-loop. The loop is preferably GAAA, but is not limited to this sequence, or indeed only 4 bp in length. Indeed, preferred loop-forming sequences for use in hairpin structures are four nucleotides in length, and most preferably have the sequence GAAA. However, longer or shorter loop sequences, as well as alternative sequences, may be used. The sequences preferably include a nucleotide triplet (e.g., AAA) and an additional nucleotide (e.g., C or G). Examples of loop-forming sequences include CAAA and AAAG. In practicing any of the methods disclosed herein, a suitable vector can be introduced into a cell or embryo by one or more methods known in the art, including but not limited to microinjection, electroporation, sonoporation, ballistic transfection, calcium phosphate-mediated transfection, cationic lipid particle transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impalefection, optical transfection, proprietary agent-stimulated nucleic acid uptake, and liposome, immunoliposome, virosome, or artificial virion delivery. In some methods, the vector is introduced into the embryo by microinjection. The vector or vectors can be microinjected into the nucleus or cytoplasm of the embryo. In some methods, the vector or vectors can be introduced into the cell by nucleofection.

The term "regulatory element" is intended to encompass promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., 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 direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct expression preferentially in a target tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g., liver, pancreas), or specific cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a time-dependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, which may or may not also be tissue-specific or cell type-specific. In some embodiments, the vector comprises one or more pol III promoters (e.g., 1, 2, 3, 4, 5 or more pol III promoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5 or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5 or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, the U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase gene promoter, the β-actin gene promoter, the glycerophosphate kinase (PGK) gene promoter, and the EF1α promoter. Also encompassed by the term "regulatory element" are enhancer elements such as the WPRE; CMV; the R-U5' segment of the LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), pp. 466-472, 1988); the SV40 enhancer; and the intronic sequence between exons 2 and 3 of the rabbit β-globin gene (Proc. Natl. Acad. Sci. USA., Vol. 78(3), pp. 1527-31, 1981). Those skilled in the art will appreciate that the configuration of the expression vector may depend on factors such as the choice of host cell to be transformed, the desired expression level, etc. The vector can be introduced into host cells to thereby produce transcripts, proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids described herein (e.g., clustered regularly interspaced short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.). With respect to regulatory sequences, reference is made to U.S. Patent Application No. 10/491,026, 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 U.S. Patent Application No. 12/511,940, the contents of which are incorporated herein by reference in their entireties.

Vectors can be designed to express CRISPR transcripts (e.g., 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 further discussed 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 regulatory sequences of the T7 promoter and T7 polymerase.

Vectors can be introduced and propagated in a prokaryote or a prokaryotic cell. In some embodiments, the prokaryote is used to amplify copies of a vector that is to be introduced into a eukaryotic cell, or as an intermediate vector in the preparation of a vector that is to be introduced into a eukaryotic cell (e.g., by amplifying a plasmid as part of a viral vector packaging system). In some embodiments, the prokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism. Expression of proteins in prokaryotes is most often accomplished in Escherichia coli using vectors containing constitutive or inducible promoters that direct the expression of either fusion proteins or proteins other than fusion proteins. Fusion vectors add a number of amino acids to the protein encoded therein, such as to the amino terminus of a recombinant protein. Such fusion vectors may serve one or more purposes, such as: (i) to enhance expression of the recombinant protein; (ii) to enhance the solubility of the recombinant protein; and (iii) to aid in purification of the recombinant protein by functioning as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced into fusion expression vectors at the junction of the fusion fragment and the recombinant protein to facilitate separation of the recombinant protein from the fusion fragment after purification of the fusion protein. Such enzymes and their cognate recognition sequences include factor Xa, thrombin, and enterokinase. Illustrative fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA), and pRIT5 (Pharmacia, Piscataway, NJ), in which glutathione S-transferase (GST), maltose-binding protein E, or protein A, respectively, are fused to the target recombinant protein. Examples of suitable inducible non-fusion expression vectors for E. coli include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89). In some embodiments, the vector is a yeast expression vector. Examples of vectors for expression in the yeast Saccharomyces cerivisae include pYepSec1 (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). In some embodiments, the vector directs protein expression in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc group (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL group (Lucklow and Summers, 1989. Virology 170: 31-39).

In some embodiments, the vector is capable of directing the 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 the expression vector are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma virus, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells, see, for example, Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In some embodiments, recombinant mammalian expression vectors are capable of directing expression of a nucleic acid preferentially in a particular cell type (e.g., 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 gene promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid tissue-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), particularly the 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), neuron-specific promoters (e.g., the neurofilament gene promoter; Byrne and Ruddle, 1989). Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreatic cell-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary cell-specific promoters (e.g., the whey promoter; U.S. Patent No. 4,873,316 and European Patent Application Publication No. 264,166). Developmental stage-regulated promoters are also included, e.g., the mouse hox gene promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the alpha-fetoprotein gene promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546). With respect to these prokaryotic and eukaryotic vectors, reference should be made to U.S. Patent No. 6,750,059, the contents of which are incorporated herein by reference in their entirety. Other embodiments of the present invention may relate to viral vectors, which are mentioned in U.S. Patent Application Ser. No. 13/092,085, the contents of which are incorporated herein by reference in their entirety. Tissue-specific regulatory elements are known in the art and, in this regard, reference should be made to U.S. Patent No. 7,776,321, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the regulatory element is operably linked to one or more elements of the CRISPR system so as to direct the expression of one or more elements of the CRISPR system. In general, CRISPR (clustered regularly interspaced short palindromic repeats), also known as SPIDR (spacer-interrupted direct repeats), constitute a family of DNA loci that are typically specific to a particular bacterial species. The CRISPR locus includes a particular class of interspersed short sequence repeats (SSRs) that were discovered 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 alternating SSRs 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]; and Mojica et al., Mol. Microbiol., 17:85–93 [1995]). CRISPR loci are typically distinguished from other SSRs by the structure of repeats, which have been termed regularly interspaced short 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 elements that occur in groups that are regularly separated by unique intervening sequences of nearly constant length (Mojica et al., [2000], supra). Although the repeat sequences are highly conserved between strains, a number of interspersed repeats and spacer region sequences typically differ between strains (van Embden et al., J. Bacteriol., 182:2393–2401 [2000]). CRISPR loci have been identified in over 40 prokaryotes (see, e.g., Jansen et al., Mol. Microbiol., 43:1565–1575 [2002]; and Mojica et al., [2005]), including but not limited to Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema and Thermotoga.

In general, "nucleic acid targeting system" as used herein refers collectively to transcripts and other elements involved in the expression or control of the activity of CRISPR-associated ("Cas") nucleic acid targeting genes (also referred to herein as effector protein), including sequences encoding a nucleic acid targeting Cas (effector) protein and a guide RNA (comprising a crRNA sequence and a trans-activating RNA (tracrRNA) sequence of the CRISPR/Cas system), or other sequences and transcripts from a nucleic acid targeting CRISPR locus. In some embodiments, one or more elements of the nucleic acid targeting system are derived from a type V/type VI nucleic acid targeting CRISPR system. In some embodiments, one or more elements of the nucleic acid targeting system are derived from a particular organism that contains an endogenous nucleic acid targeting CRISPR system. In general, the nucleic acid targeting system is characterized by elements that facilitate the formation of a nucleic acid targeting complex at the site of a target sequence. In the context of forming a nucleic acid targeting complex, a "target sequence" refers to a sequence to which a guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide RNA facilitates the formation of a DNA or RNA targeting complex. Perfect complementarity is not required, so long as there is sufficient complementarity to allow hybridization and facilitate the formation of a nucleic acid targeting complex. The target sequence may comprise RNA polynucleotides. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, the target sequence may be in an organelle of a eukaryotic cell, such as a mitochondrion or a chloroplast. A sequence or template that can be used for recombination at a target locus containing target sequences is called an "editing template" or "editing RNA" or "editing sequence". In aspects of the present invention, exogenous template DNA may be called an editing template. In one aspect of the present invention, the recombination is homologous recombination.

Typically, in the context of an endogenous nucleic acid targeting system, formation of a nucleic acid targeting complex (comprising a guide RNA that hybridizes to a target sequence and forms a complex with one or more nucleic acid targeting effector proteins) results in cleavage of one or both strands of the RNA at (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs) or near the target sequence. In some embodiments, one or more vectors that direct expression of one or more elements of the nucleic acid targeting system are introduced into a host cell such that expression of the elements of the nucleic acid targeting system directs formation of the nucleic acid targeting complex at one or more target sites. For example, both the nucleic acid targeting effector protein and the guide RNA can be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined in 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 elements of the nucleic acid targeting system that are combined in a single vector can be positioned in any suitable orientation, such as one element positioned 5' ("upstream") relative to a second element or 3' ("downstream") relative to a second element. The coding sequence of one element can be positioned on the same or opposite strand relative to the coding sequence of the second element and oriented in the same or opposite direction. In some embodiments, a single promoter drives the expression of a transcript encoding a nucleic acid targeting effector protein and a guide RNA embedded in one or more intronic sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the nucleic acid targeting effector protein and the guide RNA are operably linked to and expressed from the same promoter.

In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to hybridize to the target sequence and direct sequence-specific binding of a nucleic acid targeting 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 about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. An optimal alignment can be determined using any suitable algorithm for the sequences to be aligned, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transform (e.g., Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, the length of 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 some embodiments, the length of the guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 or less nucleotides. The ability of the guide sequence to direct sequence-specific binding of the nucleic acid targeting complex to the target sequence can be assessed using any suitable assay. For example, components of a nucleic acid targeting system sufficient to form a nucleic acid targeting complex, including a guide sequence to be tested, can be delivered to a host cell with an appropriate target sequence, such as by transfection with vectors encoding CRISPR nucleic acid targeting sequence components, followed by assessment of preferential cleavage within or near the target sequence, such as by the Surveyor nuclease 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, components of a nucleic acid targeting complex, including a guide sequence to be tested, and a control guide sequence different from the test guide sequence, and comparing binding or the extent of cleavage at or near the target sequence in reactions with the test and control guide sequences. Other assays are possible and can be performed by those skilled in the art.

The guide sequence may be selected to specifically target any target sequence. In some embodiments, the target sequence is a sequence within a transcript or mRNA.

In some embodiments, the target sequence is a sequence within the genome of the cell.

In some embodiments, the guide sequence is selected to reduce the proportion of secondary structure in the guide sequence. The secondary structure can be determined using any suitable polynucleotide folding algorithm. Some programs are based on the calculation of the minimum Gibbs free energy. An example of one such algorithm is mFold, which is described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example of a folding algorithm is the online web server RNAfold, developed at the Institute for Theoretical Chemistry at the University of Vienna, which uses a centroid-based structure prediction algorithm (see, e.g., A. R. Gruber et al., 2008, Cell 106(1): 23-24 and P. A. Carr and G. M. Church, 2009, Nature Biotechnology 27(12): 1151-62). Additional algorithms can be found in U.S. Patent Application Serial No. 61/836,080), incorporated herein by reference.

In some embodiments, a recombination template is also provided. The recombination template may be a component of another vector as described herein, may be contained in a separate vector, or may be provided as a separate polynucleotide. In some embodiments, the recombination template is designed to serve as a template for homologous recombination, such as within or near a target sequence, nicked or cleaved by an enzyme with a nucleic acid targeting effector protein as part of a nucleic acid targeting complex. The template polynucleotide may be any suitable length, such as about or greater than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000 nucleotides or more. In some embodiments, the template polynucleotide is complementary to a portion of a polynucleotide comprising a target sequence. When optimally aligned, the template polynucleotide may overlap with one or more nucleotides of the target sequences (e.g., with about or more than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more nucleotides). In some embodiments, when a template sequence and a polynucleotide comprising a target sequence are optimally aligned, the closest nucleotide of the template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000 or more nucleotides of the target sequence.

In some embodiments, the nucleic acid targeting effector protein is part of a fusion protein comprising one or more domains of a heterologous protein (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains in addition to the nucleic acid targeting effector protein). In some embodiments, the effector protein/CRISPR enzyme is part of a fusion protein comprising one or more domains of a heterologous protein (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains in addition to the CRISPR enzyme). The fusion protein comprising the CRISPR enzyme may comprise any additional protein sequence and, optionally, a linker sequence between any two domains. Examples of protein domains that can be fused to a CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains with one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcript release 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 acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). The CRISPR enzyme may be fused to a gene sequence encoding a protein or protein fragment that binds to DNA molecules or binds to other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA-binding domain (DBD) fusions, GAL4 DNA-binding domain fusions, and herpes simplex virus (HSV) BP16 fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US20110059502, incorporated herein by reference. In some embodiments, a labeled CRISPR enzyme is used to identify the location of a target sequence.

In some embodiments, the CRISPR enzyme may form a component of an inducible system. The inducible nature of the system will allow for spatiotemporal control of gene editing or gene expression using a form of 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.), or light-inducible systems (phytochrome, LOV, or cryptochrome domains). In one embodiment, the CRISPR enzyme may be part of a light-inducible transcriptional effector (LITE) to drive changes in transcriptional activity in a sequence-specific manner. Components of a light-inducible system may include a CRISPR enzyme, a light-sensitive cytochrome heterodimer (e.g., from Arabidopsis thaliana), and a transcriptional activation/repression domain. Additional examples of inducible DNA binding proteins and methods of using them are provided in US 61/736,465 and US 61/721,283 and WO 2014/018423 and US8889418, US8895308, US20140186919, US20140242700, US20140273234, US20140335620, WO2014093635, which are incorporated herein by reference in their entireties.

In some aspects, the present invention provides methods comprising delivering to a host cell one or more polynucleotides, such as, for example, or one or more vectors as described herein, one or more transcripts thereof, and/or one or more proteins transcribed therefrom. In some aspects, the present invention further provides cells produced by such methods and organisms (such as animals, plants, or fungi) comprising or derived from such cells. In some embodiments, a nucleic acid targeting effector protein in combination with (and optionally complexed with) a guide RNA is delivered to the 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 in culture or in a host organism. Non-viral vector delivery systems include plasmid DNA, RNA (e.g., the vector transcript 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 genomes in episomal form or integrated genomes after delivery into the cell. For reviews of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & 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, 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 Böhm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).

Methods for non-viral delivery of nucleic acids include lipofection, nucleofection, microinjection, ballistic transfection, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced DNA uptake. Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient lipofection with polynucleotide receptor recognition include those of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).

The preparation of lipid:nucleic acid complexes, including targeting liposomes such as immunolipid complexes, is well known in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., 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); U.S. Patent Nos. 4,186,183, 4,217,344, 4235871, 4261975, 4485054, 4501728, 4774085, 4837028 and 4946787).

The use of RNA or DNA virus-based systems for nucleic acid delivery takes advantage of carefully designed methods for ensuring targeting of the virus to specific cells in the body and translocation of the viral payload sequences into the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to manipulate cells in vitro and the modified cells can optionally be administered to patients (ex vivo). Traditional virus-based gene transfer systems can include retroviral, lentiviral, adenoviral, adeno-associated virus, and herpes simplex virus vectors. Integration into the host genome is possible using retroviral, lentiviral, and adeno-associated virus gene transfer methods, often resulting in long-term expression of the integrated transgene. In addition, high transduction efficiencies have been observed in many different cell types and target tissues.

Retroviral tropism can be altered by incorporating foreign envelope proteins to broaden the possible target cell population. Lentiviral vectors are retroviral vectors that are capable of transducing or infecting non-dividing cells and typically yield high viral titers. The choice of retroviral gene transfer system will therefore depend on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats with the packaging capacity of up to 6-10 kb of foreign sequence. Minimal cis-acting LTRs are sufficient for vector replication and packaging, which are then used to integrate the therapeutic gene into the target cell to produce permanent transgene expression. Commonly used retroviral vectors include those 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 applications where transient expression is preferred, adenovirus-based systems can be used. Adenovirus vectors can exhibit very high transduction efficiency in many cell types and do not require cell division. High titers and expression levels have been obtained using such vectors. Such a vector can be produced in large quantities in a relatively simple system. Adeno-associated virus ("AAV") vectors can also be used to transduce target nucleic acids into cells, such as for the in vitro production of nucleic acids and peptides and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Patent No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). The creation of recombinant AAV vectors has been described in a number of publications, including U.S. 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 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822–3828 (1989).

Delivery in general

The present invention also encompasses at least one component of a CRISPR complex, such as RNA, delivered via at least one nanoparticle complex. In some aspects, the present invention relates to methods comprising delivering to a host cell one or more polynucleotides, such as, for example, or one or more vectors as described herein, one or more transcripts thereof, and/or one or more proteins transcribed therefrom. In some aspects, the present invention further provides cells produced by such methods and animals comprising or derived from such cells. In some embodiments, a CRISPR enzyme in combination with (and optionally complexed with) a guide sequence 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 CRISPR system into cells in culture or in a host organism. Non-viral vector delivery systems include plasmid DNA, RNA (e.g., the vector transcript 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 genomes in episomal form or integrated genomes after delivery into the cell. For reviews of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & 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, 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 Böhm (eds) (1995) and Yu et al., Gene Therapy 1:13-26 (1994).

Methods for non-viral delivery of nucleic acids include lipofection, microinjection, ballistic transfection, virosome-mediated delivery via liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced DNA uptake. Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient lipofection with polynucleotide receptor recognition include those of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration).

The preparation of lipid:nucleic acid complexes, including targeting liposomes such as immunolipid complexes, is well known in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., 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); U.S. Patent Nos. 4,186,183, 4,217,344, 4235871, 4261975, 4485054, 4501728, 4774085, 4837028 and 4946787).

RNA and DNA virus-based systems for nucleic acid delivery employ carefully designed methods to ensure targeting of the virus to specific cells in the body and translocation of the viral payload sequences into the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to manipulate cells in vitro and the modified cells can optionally be administered to patients (ex vivo). Traditional virus-based gene transfer systems can include retroviral, lentiviral, adenoviral, adeno-associated virus, and herpes simplex virus vectors. Integration into the host genome is possible using retroviral, lentiviral, and adeno-associated virus gene transfer methods, often resulting in long-term expression of the inserted transgene. In addition, high transduction efficiencies have been observed in many different cell types and target tissues.

Retroviral tropism can be altered by incorporating foreign envelope proteins to broaden the possible target cell population. Lentiviral vectors are retroviral vectors that are capable of transducing or infecting non-dividing cells and typically yield high viral titers. The choice of retroviral gene transfer system will therefore depend on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats with the packaging capacity of up to 6-10 kb of foreign sequence. Minimal cis-acting LTRs are sufficient for vector replication and packaging, which are then used to integrate the therapeutic gene into the target cell to produce permanent transgene expression. Commonly used retroviral vectors include those 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 another embodiment, pseudotyped retroviral vector particles based on the envelope of the vesiculovirus Cocal (see, e.g., U.S. Patent Application Publication No. 20120164118 assigned to the Fred Hutchinson Cancer Research Center) are provided. Cocal virus is a member of the genus Vesiculovirus and is the causative agent of vesicular stomatitis in mammals. Cocal virus was originally isolated from ticks in Trinidad (Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964)), and infections have been identified in Trinidad, Brazil, and Argentina in insects, cattle, and horses. Many vesiculoviruses that infect mammals have been isolated from naturally infected arthropods, suggesting that they are transmissible vectors. Antibodies to vesiculoviruses are common in people living in rural areas where the viruses are endemic and laboratory-associated; human infections typically result in influenza-like symptoms. The cocal virus envelope glycoprotein shares 71.5% amino acid identity with Indiana VSV-G, and phylogenetic comparison of vesiculovirus envelope genes has shown that cocal virus is serologically distinct from, but most closely related to, Indiana VSV-G strains of vesiculoviruses. 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). Pseudotyped retroviral vector particles based on the Cocal vesiculovirus envelope may include, for example, lentiviral, alpharetroviral, betaretroviral, gammaretroviral, deltaretroviral and epsilonretroviral vector particles, which may comprise retroviral Gag, Pol and/or one or more accessory proteins and the Cocal vesiculovirus envelope protein. In certain aspects of these embodiments, the Gag, Pol and accessory proteins are lentiviral and/or gammaretroviral. The present invention provides an AAV that comprises or consists essentially of an exogenous nucleic acid molecule encoding a CRISPR system, such as a plurality of cassettes comprising or consisting essentially of a first cassette comprising or consisting essentially of a promoter, a nucleic acid molecule encoding a CRISPR-associated (Cas) protein (putative nuclease or helicase proteins), such as Cas9, and a terminator, and two or more, preferably up to the packaging limit of the vector, such as a total of (including the first cassette) five cassettes comprising or consisting essentially of a promoter, a nucleic acid molecule encoding a guide RNA (gRNA), and a terminator (e.g., each cassette is schematically represented as a promoter-gRNA1-terminator, a promoter-gRNA2-terminator ... a promoter-gRNA(N)-terminator (where N is the number that can be inserted, being at the upper limit of the packaging limit of the vector)), or two or more separate rAAVs, each comprising one or more cassettes of the CRISPR system, such as a first rAAV comprising a first cassette comprising or consisting essentially of a promoter, a nucleic acid molecule encoding a Cas, such as Cas (Cas9), and a terminator, and a second rAAV comprising multiple, four cassettes, cassettes comprising or consisting essentially of a promoter, a nucleic acid molecule encoding a guide RNA (gRNA), and a terminator (e.g., each cassette is schematically represented as a promoter-gRNA1-terminator, a promoter-gRNA2-terminator ... a promoter-gRNA(N)-terminator (where N is the number that can be inserted, being at the upper limit of the vector packaging limit)). Since rAAV is a DNA virus, nucleic acid molecules in the discussion herein with respect to AAV or rAAV are predominantly DNA. In some embodiments, the promoter is predominantly the human synapsin I (hSyn) promoter. Additional methods for delivering nucleic acids into cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference.

In some embodiments, the host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, the cell is transfected while it is in vivo in the subject. In some embodiments, the cell that is transfected is derived from the subject. In some embodiments, the cell is derived from cells derived from the subject, such as cell lines. A variety of cell lines are known in the art for use in tissue culture. Examples of 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, Bcl-1, BC-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 epithelial cells BS-C-1, mouse embryonic fibroblasts BALB/3T3, 3T3 Swiss, 3T3-L1, human fetal fibroblasts 132-d5; 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 cells 293, BxPC3, 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, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, 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 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic variants thereof. Cell lines are available from a variety of sources known to those skilled in the art (see, e.g., American Type Culture Collection (ATCC) (Manassas, VA)). In some embodiments, a cell transfected with one or more vectors described herein is used to generate a new cell line comprising one or more vector-derived sequences. In some embodiments, a cell transiently transfected with components of the CRISPR system described herein (such as by transient transfection with one or more vectors or transfection using RNA) and modified with the activity of the CRISPR complex is used to generate a new cell line comprising cells with the modification but without any other exogenous sequence. In some embodiments, cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells, are used in the evaluation of one or more test compounds.

In some embodiments, one or more vectors described herein are used to produce a non-human transgenic animal or transgenic plant. In some embodiments, the transgenic animal is a mammal, such as a mouse, rat, or rabbit. Methods for producing transgenic animals and plants are known in the art and typically begin with a cell transfection method such as described herein. In another embodiment, a needle array fluid delivery device (see, e.g., U.S. Patent Application Publication No. 20110230839 assigned to the Fred Hutchinson Cancer Research Center) may be provided for delivering CRISPR-Cas to solid tissue. The device of U.S. Patent Application Publication No. 20110230839 for delivering fluid to solid tissue may comprise a plurality of needles arranged in an array; a plurality of reservoirs, each of which is in fluid communication with a respective one of the plurality of needles; and a plurality of actuators operatively connected to the respective containers of the plurality of containers and configured to regulate the fluid pressure in the container. In certain embodiments, each of the plurality of actuators may comprise one of the plurality of pistons, wherein the first end portion of each of the plurality of pistons is located in the respective one container of the plurality of containers, and in certain further embodiments, the pistons of the plurality of pistons are operatively connected together at the respective second end portions with the ability to be pressed simultaneously. In certain other further embodiments, a control element for the pistons may be provided, configured to press all of the plurality of pistons at a selectively changing speed. In other embodiments, each of the plurality of actuators may comprise one of the plurality of fluid flow lines having first and second end portions, wherein the first end portion of each of the plurality of fluid flow lines is connected to the respective one container of the plurality of containers. In other embodiments, the device may comprise a fluid pressure source, and wherein each of the plurality of actuators provides a fluid coupling between the fluid pressure source and the respective one container of the plurality of containers. In further embodiments, the fluid pressure source may include at least one of the following: a compressor, a vacuum accumulator, a peristaltic pump, a main cylinder, a microfluidic pump, and a valve. In another embodiment, each of the plurality of needles may comprise a plurality of holes distributed along its length.

Delivery to the kidneys

Delivery routes to the kidney are summarized below.

Delivery method Carrier Target RNA Disease Model Functional analyses Author Hydrodynamic/lipid In Vivo TransIT Gene Delivery System, DOTAP p85α Acute kidney injury Ischemia-reperfusion Absorption, biodistribution Larson et al., Surgery, (Aug 2007), Vol. 142, No. 2, pp. (262-269) Hydrodynamic/lipid Lipofectamine 2000 Fas Acute kidney injury Ischemia-reperfusion Blood urea nitrogen, Fas immunohistochemistry, apoptosis, histological evaluation Hamar et al., Proc Natl Acad Sci, (Oct 2004), Vol. 101, No. 41, pp. (14883-14888) Hydrodynamic Not applicable Elements of the apoptotic cascade Acute kidney injury Ischemia-reperfusion Not applicable Zheng et al., Am J Pathol, (Oct 2008), Vol. 173, No. 4, pp. (973-980) Hydrodynamic Not applicable Nuclear factor kappa B (NFkB) Acute kidney injury Ischemia-reperfusion Not applicable Feng et al., Transplantation, (May 2009), Vol. 87, No. 9, pp. (1283-1289) Hydrodynamic/viral Lipofectamine 2000 Anti-apoptosis transcription factor (AATF) Acute kidney injury Ischemia-reperfusion Apoptosis, oxidative stress, caspase activation, membrane lipid peroxidation Xie & Guo, Am Soc Nephrol, (Dec 2006), Vol. 17, No. 12, pp. (3336-3346) Hydrodynamic pBAsi mU6 Neo/TransIT-EE Hydrodynamic Delivery System Gremlin Diabetic nephropathy Streptozotosin-induced diabetes Proteinuria, serum creatinine, glomerular and tubular diameter, collagen type IV/BMP7 expression Q. Zhang et al., PloS ONE, (Jul 2010), Vol. 5, No. 7, e11709, pp. (1-13) Viral/
lipid pSUPER/Lipofectamine Vector TGF-β receptor type II Interstitial fibrosis of the kidney Unilateral urethral obstruction α-SMA expression, collagen content Kushibikia et al., J Controlled Release, (Jul 2005), Vol. 105, No. 3, pp. (318-331) Viral Adeno-associated virus-2 Mineralocorticoid receptor Kidney damage caused by hypertension Cold-induced hypertension Blood pressure, serum albumin, serum urea nitrogen, serum creatinine, kidney weight, urine sodium Wang et al., Gene Therapy, (Jul 2006), Vol. 13, No. 14, pp. (1097-1103) Hydrodynamic/
viral Vector pU6 Luciferase Not applicable Not applicable Absorption Kobayashi et al., Journal of Pharmacology and Experimental Therapeutics, (Feb 2004), Vol. 308, No. 2, pp. (688-693) Lipid Lipoproteins, albumin apoB1, apoM Not applicable Not applicable Absorption, binding affinity to lipoproteins and albumin Wolfrum et al., Nature Biotechnology, (Sep 2007), Vol. 25, No. 10, pp. (1149-1157) Lipid Lipofectamine 2000 p53 Acute kidney injury Acute ischemia-induced cisplatin injury Histological assessment, apoptosis Molitoris et al., J Am Soc Nephrol, (Aug 2009), Vol. 20, No. 8,
pp. (1754-1764) Lipid DOTAP/DOPE, DOTAP/DOPE/DOPE-PEG2000 COX-2 Adenocarcinoma of the mammary gland MDA-MB-231 mammary tumor xenograft-bearing mouse Cell viability, absorption 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, urine creatinine, histology, collagen types I and IV, 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 Mitochondrial membrane protein 44 (TIM44) Diabetic nephropathy Streptozotocin-induced diabetes Cell proliferation and apoptosis, histological examination, ROS, mitochondrial import of Mn-SOD and glutathione peroxidase, cell membrane polarization Y. Zhang et al., J Am Soc Nephrol, (Apr 2006), Vol. 17, No. 4, pp. (1090-1101) Hydrodynamic/
lipid Proteoliposome RLI76 Renal carcinoma Caki-2 renal tumor xenograft bearing mouse Absorption Singhal et al., Cancer Res, (May 2009), Vol. 69, No. 10, pp. (4244-4251) Polymeric PEGylated PEI Luciferase pGL3 Not applicable Not applicable Absorption, biodistribution, erythrocyte aggregation Malek et al., Toxicology and Applied Pharmacology, (Apr 2009), Vol. 236, No. 1, pp. (97-108) Polymeric PEGylated poly-L-lysine MAPK1 Lupus glomerulonephritis Glomerulonephritis Proteinuria, glomerulosclerosis, TGF-β, fibronectin, plasminogen activator inhibitor 1 Shimizu et al., J Am Soc Nephrology, (Apr 2010), Vol. 21, No. 4, pp. (622-633) Polymer/nanoparticle based Hyaluronic acid/quantum dot/PEI VEGF Kidney cancer/melanoma B16F1 melanoma-bearing mouse Biodistribution, cytotoxicity, tumor volume, endocytosis Jiang et al., Molecular Pharmaceutics, (May-Jun 2009), Vol. 6, No. 3, pp. (727-737) Polymer/nanoparticle based PEGylated polycaprolactone nanofiber GAPDH Not applicable Not applicable Cell viability, absorption Cao et al., J Controlled Release, (Jun 2010), Vol. 144, No. 2, pp. (203-212) Aptamer Spiegelmer
mNOX-E36 CC-chemokine ligand 2 Glomerulosclerosis Unilaterally nephrectomized mouse Urine albumin, urine creatinine, histopathology, glomerular filtration rate, macrophage count, serum Ccl2, Mac- 2+, Ki-67+ Ninichuk et al., Am J Pathol, (Mar 2008), Vol. 172, No. 3, pp. (628-637) Aptamer Aptamer NOX-F37 Vasopressin (AVP) Congestive heart failure Not applicable D-AVP binding affinity, inhibition of AVP signaling, urine osmolality and sodium concentration Purschke et al., Proc Natl Acad Sci, (Mar 2006), Vol. 103, No. 13, pp. (5173-5178)

Delivery to the brain

Options for delivery to the brain include encapsulating the CRISPR enzyme and guide RNA in the form of DNA or RNA in liposomes and conjugating to "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 brain of non-human primates. The same approach could be used to deliver 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) describe the possibility of delivering short interfering RNAs (siRNAs) to cells in culture and in vivo using a combined receptor-specific monoclonal antibody (mAb) and avidin-biotin technology. The authors also report that since the association between the targeting mAb and siRNA is stable with avidin-biotin technology, RNAi effects are observed in vivo in distant sites such as the brain following intravenous administration of the targeting siRNA.

Zhang et al. (Mol Ther. 2003 Jan;7(1):11-8) describe how expression plasmids encoding reporters such as luciferase were encapsulated within an "artificial virus" comprising a pegylated 85 nm immunoliposome targeted to the rhesus macaque brain in vivo using a monoclonal antibody (MAb) to the human insulin receptor (HIR). The HIR MAb allows the liposome carrying the exogenous gene to undergo transcytosis across the blood-brain barrier and endocytosis across the plasma membrane of neurons following intravenous injection. The level of luciferase gene expression in the rhesus macaque brain was 50-fold higher than in the rat. Widespread expression of the beta-galactosidase gene in neurons of the primate brain was demonstrated using both histochemistry and confocal microscopy. The authors indicate that this approach allows for reversible expression of the transgene in adult animals within 24 hours. Accordingly, immunoliposomes are preferred. They can be used in combination with antibodies to target specific tissues or cell surface proteins.

HSC delivery and editing in hematopoietic stem cells; certain conditions

The term "hematopoietic stem cell" or "HSC" includes, in a broad sense, those cells that are considered to be HSCs, such as blood cells that give rise to all other blood cells and are derived from the mesoderm; located in the red bone marrow contained in the interior of most bones. HSCs of the present invention include cells having the phenotype of hematopoietic stem cells, identified by their small size, the absence of linear (lin) markers and markers that belong to a cluster of differentiation series, such as: CD34, CD38, CD90, CD133, CD105, CD45, and c-kit, a stem cell factor receptor. Hematopoietic stem cells are negative for markers that are used to detect differentiation commitment and are thus called Lin-; and during their purification by FACS of a number of up to 14 markers of mature blood cell lineages, such as CD13 and CD33 for myeloid, CD71 for erythroid, CD19 for B cells, CD61 for megakaryocytic cells, etc., such as for humans; and B220 (mouse CD45) for B cells, Mac-1 (CD11b/CD18) for monocytes, Gr-1 for granulocytes, Ter119 for erythroid cells, 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+, Thy1/CD90+, CD38lo/-, C-kit/CD117+, and lin-. HSC are identified by the markers. Thus, in embodiments described herein, HSC can be CD34+ cells. HSC 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 the art to be HSC, are within the scope of the present invention, as well as CD133+ cells, which are similarly considered to be HSC in the art.

A CRISPR-Cas system (e.g., Cas9) can be designed to target a gene locus or loci in an HSC. A protein (e.g., Cas9) that is advantageously codon-optimized for a eukaryotic cell and in particular a mammalian cell, such as a human cell, such as an HSC, and an sgRNA that targets a locus or loci in an HSC, such as the EMX1 gene, can be provided. They can be delivered via particles. The particles can be formed by a Cas protein (e.g., Cas9) and an added sgRNA. A mixture of sgRNA and a Cas protein (e.g., Cas9) can be mixed, for example, with a mixture comprising or consisting essentially of or consisting of a surfactant, a phospholipid, a biodegradable polymer, a lipoprotein, and an alcohol, whereby particles comprising sgRNA and a Cas protein (e.g., Cas9) can be formed. The present invention encompasses particles formed in this manner and particles obtained by such a method, as well as their uses.

In a more general sense, the particles can be formed using an effective method. First of all, a Cas protein (e.g., Cas9) and an sgRNA targeting an EMX1 gene or a LacZ control gene can be mixed together at a suitable molar ratio, such as 3:1-1:3, or 2:1-1:2, or 1:1, at a suitable temperature, such as 15-30C, such as 20-25C, such as room temperature, for a suitable period of time, such as 15-45, such as 30 minutes, preferably in a sterile nuclease-free buffer, such as 1X PBS. Individually, the particle components, such as or including a surfactant, such as a cationic lipid, such as 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); a phospholipid, such as dimyristoylphosphatidylcholine (DMPC); a biodegradable polymer such as an ethylene glycol polymer or PEG and a lipoprotein such as a low density lipoprotein, for example cholesterol, can be dissolved in an alcohol, preferably a C1-6 alkyl alcohol such as methanol, ethanol, isopropanol, for example 100% ethanol. The two solutions can be mixed together to form particles comprising Cas (e.g. Cas9)-sgRNA complexes. In certain embodiments, the particle can comprise an HDR template. This can be a particle co-administered with a particle comprising sgRNA+Cas protein (e.g. Cas9), i.e. in addition to contacting the HSC with the particle comprising sgRNA+Cas protein (e.g. Cas9), the HSC is contacted with the particle comprising the HDR template; or the HSC is contacted with a particle comprising all of sgRNA, Cas (e.g. Cas9) and the HDR template. The HDR template can be introduced via a separate vector, in which case the particle enters the HSC cell and a separate vector also enters the cell where the HSC genome is modified with sgRNA+Cas (e.g. Cas9) and the HDR template is also present, in which case the genomic locus is modified via HDR; for example, this can lead to the correction of a mutation.

Following particle formation, HSC in 96-well plates can be transfected with 15 μg of Cas protein (e.g., Cas9) per well. Three days after transfection, HSC can be harvested and the number of insertions and deletions (indels) in the EMX1 locus can be determined.

This illustrates how HSCs can be modified using CRISPR-Cas (e.g., Cas9) targeting a genomic locus or loci of interest within the HSC. The HSCs to be modified may be in vivo, such as within an organism, such as a human or a non-human eukaryotic organism, such as an animal, such as a fish, such as a zebrafish, a mammal, such as a primate, such as a great ape, a chimpanzee, a macaque, a rodent, such as a mouse, rabbit, rat, cat, or dog, a livestock (cow/bull, ram/sheep, goat, or pig), or a wild or domesticated bird, such as a chicken. The HSCs to be modified may be in vitro, i.e., outside of such an organism. Also, the modified HSCs can be used ex vivo, i.e., one or more such HSCs of such an organism can be obtained or isolated from the organism, optionally the HSCs can be expanded, the HSCs are modified using a composition comprising a CRISPR-Cas (e.g., Cas9) that targets a gene locus or loci in the HSCs, such as by contacting the HSCs with a composition, such as where the composition comprises a CRISPR enzyme and one or more sgRNAs that target a gene locus or loci in the HSCs, such as a particle obtained or obtainable by mixing sgRNA and a Cas protein (e.g., Cas9) with a mixture comprising or consisting essentially of or consisting of a surfactant, a phospholipid, a biodegradable polymer, a lipoprotein and an alcohol (wherein the one or more sgRNAs target a gene locus or loci in the HSCs), optionally expanding the obtained modified HSCs and introducing the obtained modified HSCs into the organism. In some examples, the isolated or obtained HSCs can be from a first organism, such as an organism of the same species as the second organism, and the second organism can be an organism into which the obtained modified HSCs are introduced, for example, the first organism can be a donor (for example, a relative, such as a parent or sibling) for the second organism. The modified HSCs can have genetic mutations for treating, alleviating or reducing the symptoms of a disease or condition of an individual or subject or patient. The modified HSCs, for example, in the case where the first organism is a donor for the second organism, can have genetic modifications so that the HSCs have one or more proteins, such as surface markers or proteins, that are more similar than those of the second organism. The modified HSCs can have genetic modifications to mimic a disease or condition of an individual or subject or patient and can be reintroduced into a non-human organism to form an animal model. Expanding HSC is within the skill of the art based on the present invention 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.

As indicated, to enhance the activity of sgRNA, the sgRNA can be pre-complexed with a Cas protein (e.g., Cas9) before the entire complex is assembled into the particle. The formulations can be prepared with different molar ratios of various components known to facilitate the delivery of nucleic acids into cells (e.g., 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), polyethyleneglycol (PEG), and cholesterol). For example, the molar 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 invention accordingly encompasses mixing of sgRNA, Cas protein (e.g. Cas9) and components that form a particle; as well as particles resulting from such addition.

In a preferred embodiment, particles containing a Cas (e.g., Cas9)-sgRNA complex can be formed by mixing a Cas protein (e.g., Cas9) and one or more sgRNAs together, preferably at a molar ratio of enzyme:guide RNA of 1:1. Separately, various components known to facilitate nucleic acid delivery (e.g., DOTAP, DMPC, PEG, and cholesterol) are dissolved, preferably in ethanol. The two solutions can be mixed together to form particles containing the Cas (e.g., Cas9)-sgRNA complexes. After formation of the particles, the Cas (e.g., Cas9)-sgRNA complexes can be transfected into cells (e.g., HSCs). Barcoding can be used. The particles, Cas-9, and/or sgRNA can be barcoded.

The present invention in one embodiment provides a method of producing a particle comprising a complex of an sgRNA and a Cas protein (e.g., Cas9), comprising mixing a mixture of an sgRNA and a Cas protein (e.g., Cas9) with a mixture comprising, consisting essentially of, or consisting of a surfactant, a phospholipid, a biodegradable polymer, a lipoprotein, and an alcohol. The embodiment encompasses a particle comprising a complex of an sgRNA and a Cas protein (e.g., Cas9) produced by the method. The present invention in an embodiment encompasses use of the particle in a method of modifying a genomic locus of interest, or an organism, or a non-human organism by manipulating a target sequence in the genomic locus of interest, comprising contacting a cell comprising the genomic locus of interest with the particle, wherein the sgRNA targets the genomic locus of interest; or a method for modifying a genomic locus of interest, or an organism, or a non-human organism by manipulating a target sequence in the genomic locus of interest, comprising contacting a cell containing the genomic locus of interest with a particle wherein the sgRNA targets the genomic locus of interest. In these embodiments, the genomic locus of interest is preferably a genomic locus in an HSC.

Factors to Consider for Therapeutic Applications A factor in genome editing-based therapeutics is the choice of a sequence-specific nuclease, such as a Cas9 nuclease variant. Each nuclease variant may have its own unique set of strengths and weaknesses, many of which must be balanced in the context of a treatment to maximize therapeutic benefit. To date, two nuclease-based editing approaches for therapeutic intent have shown significant promise: gene disruption and gene correction. Gene disruption involves the stimulation of NHEJ to create targeted insertions/deletions in genetic elements, often resulting in loss-of-function mutations that are beneficial to patients. In contrast, gene correction utilizes HDR to directly regress disease-causing mutations, restoring function while maintaining physiological regulation of the corrected element. HDR can also be used to insert a therapeutic transgene into a defined “safe” locus in the genome to restore missing gene function. For a specific editing therapy to be effective, a high enough level of modification must be achieved in the target cell populations to reverse disease symptoms. This "threshold" of therapeutic modification is determined by determining the fitness of the edited cells after treatment and the amount of gene product required to reverse symptoms. With respect to fitness, editing allows for three possible outcomes for the treated cells compared to their unedited counterparts: increased, neutral, or decreased fitness. In the case of increased fitness, such as in the treatment of SCID-X1, the modified hematopoietic progenitor cells expand selectively compared to their unedited counterparts. SCID-X1 is a disease caused by mutations in the IL2RG gene, the function of which is required for the proper development of the lymphocyte lineage of hematopoiesis [Leonard, W. J., et al. Immunological reviews 138, 61-86 (1994); Kaushansky, K. & Williams, W. J. Williams hematology, (McGraw-Hill Medical, New York, 2010)]. In clinical trials with patients receiving viral gene therapy for SCID-X1, and in a rare example of spontaneous correction of the SCID-X1 mutation, the corrected hematopoietic progenitor cells, compared with their disease-affected counterparts, could overcome this developmental block and expanded, contributing to the therapy [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, H.B., et al. Lancet 364, 2181-2187 (2004)]. In this case, where edited cells have a selection advantage, even small numbers of edited cells can be expanded through expansion, providing therapeutic benefit to the patient. In contrast, editing for other hematopoietic diseases, such as chronic granulomatous disease (CGD), will not induce fitness changes for edited hematopoietic progenitor cells, raising the threshold for therapeutic modification. CGD is caused by mutations in genes encoding phagocytic oxidase proteins, which are normally used by neutrophils to generate pathogen-killing reactive oxygen species [Mukherjee, S. & Thrasher, A.J. Gene 525, 174–181 (2013)]. Because dysfunction of these genes does not affect the fitness or development of hematopoietic progenitor cells, but only the ability of mature hematopoietic hematopoiesis to fight infections, there is likely to be no preferential expansion of edited cells in this disease. Indeed, no selection advantage was observed for CGD-corrected cells in gene therapy trials, leading to difficulties in long-term cell engraftment [Malech, H.L., et al. Proceedings of the National Academy of Sciences of the United States of America 94, 12133–12138 (1997); Kang, H.J., et al. Molecular therapy: the journal of the American Society of Gene Therapy 19, 2092–2101 (2011)]. As such, significantly higher levels of editing will be required to treat diseases such as CGD, where the editing confers predominantly neutral fitness, compared to diseases where the editing confers increased fitness on the target cells. If the editing introduces a fitness disadvantage, as would be the case if tumor suppressor gene function were restored in cancer cells, the disease-damaged counterparts would outcompete the modified cells, resulting in a treatment benefit that is lower relative to the rate of editing. This latter class of diseases would be particularly challenging to treat with genome-editing therapies.

In addition to cell suitability, the amount of gene product required to treat a disease also influences the minimum level of therapeutic genome editing that must be achieved to reverse symptoms. Hemophilia B is one of the diseases in which a small change in gene product levels can lead to a large change in clinical outcome. The disease is caused by mutations in the gene encoding factor IX, a protein normally secreted by 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 amount of factor IX activity. Whereas severe disease is associated with activity less than 1% of normal, milder forms of the disease are associated with greater than 1% of factor IX activity [Kaushansky, K. & Williams, W.J. Williams hematology, (McGraw-Hill Medical, New York, 2010); Lofqvist, T., et al. Journal of internal medicine 241, 395-400 (1997)]. This suggests that editing therapies that can restore factor IX expression in liver cells to even a small percentage may have a major impact on clinical outcomes. A study using ZFN to correct a mouse model of hemophilia B shortly after birth demonstrated that 3-7% correction was sufficient to reverse disease symptoms, providing preclinical support for this hypothesis [Li, H., et al. Nature 475, 217-221 (2011)].

Disorders in which small changes in gene product levels can impact clinical outcomes and diseases where there is a fitness advantage of edited cells represent excellent targets for genome editing-based therapies because the threshold for therapeutic modification is low enough to provide a high probability of success given current technologies. Targeting these diseases has resulted in successes in editing-based therapies to date in preclinical and phase I clinical trials. Improvements in DSB repair manipulation and nuclease delivery are needed to extend these promising results to diseases with a neutral fitness advantage of edited cells or where larger amounts of gene product are needed for treatment. The table below shows some examples of applications of genome editing in therapeutic models and the references in the table below and the documents listed in those references are incorporated by reference herein as if set forth in full.

Type of disease The nuclease platform used Therapeutic strategy Links Hemophilia B ZFN HDR-mediated insertion of the correct gene sequence ^{Li, H., et al. Nature 475, 217-221 (2011)} SCID ZFN HDR-mediated insertion of the correct gene sequence ^{Genovese, P., et al. Nature 510, 235-240 (2014)} Hereditary tyrosinemia CRISPR HDR-mediated correction of mutation in the liver ^{Yin, H., et al. Nature biotechnology 32, 551-553 (2014)}

Treating each of such conditions from the preceding table using a CRISPR-Cas system (e.g., Cas9) for targeting by HDR-mediated correction of a mutation or HDR-mediated insertion of an appropriate gene sequence, preferably via a delivery system as described herein, e.g., a particle delivery system, is within the skill of the art based on the present invention and the knowledge in the art. Thus, an embodiment encompasses contacting an HSC carrying a mutation leading to hemophilia B, SCID (e.g., SCID-X1, ADA-SCID) or congenital tyrosinemia with an sgRNA and a Cas protein (e.g., Cas9) targeting a genomic locus of interest associated with hemophilia B, SCID (e.g., SCID-X1, ADA-SCID) or congenital tyrosinemia (e.g., as described in Li, Genovese or Yin). The particle may also contain a suitable HDR template to correct the mutation; or the HSC may be contacted with a second particle or vector that contains or delivers a HDR template. In this regard, it is mentioned that hemophilia B is an X-linked recessive disorder caused by loss-of-function mutations in the gene encoding factor IX, an important component of the blood coagulation cascade. Restoration of factor IX activity to approximately 1% of its levels in severely affected individuals may transform the disease into a significantly milder form, since prophylactic infusion of recombinant factor IX in such patients from an early age to achieve such levels significantly alleviates the severity of clinical complications. A person skilled in the art, using the knowledge in the art and the teachings of the present invention, can correct HSCs for hemophilia B using a CRISPR-Cas system (e.g., Cas9) that targets and corrects a mutation (an X-linked recessive disorder caused by loss-of-function mutations in the gene encoding factor IX) (e.g., using a suitable template for HDR that delivers a coding sequence for factor IX); in particular, an sgRNA can target a mutation that leads to hemophilia B, and HDR can provide encoding that leads to proper expression of factor IX. An sgRNA that targets a mutation and a particle containing a Cas protein (e.g., Cas9) contacts HSCs bearing the mutation. The particle can also contain a suitable template for HDR to correct the mutation in order to properly express factor IX; or the HSC may be contacted with a second particle or vector that contains or delivers the HDR matrix. The cells so contacted may be administered and optionally processed and expanded; see Cartier, described herein.

Cartier's publication "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 by reference together with the documents that it would list if they were set forth in full, provides evidence that allogeneic hematopoietic stem cell transplantation (HSCT) has been used to deliver a normal lysosomal enzyme to the brain of a patient with Hurler disease and describes HSC gene therapy for the treatment of ALD. From two patients, peripheral CD34+ cells were collected after granulocyte colony-stimulating factor (G-CSF) activation and transduced with a lentiviral vector (MND)-ALD with the murine myeloproliferative sarcoma virus enhancer, the negative control region deleted, and the dl587rev primer binding site replaced. Patients' CD34+ cells were transduced with the MND-ALD vector for 16 h in the presence of low concentrations of cytokines. Transduced CD34+ cells were frozen after transduction to perform a battery of safety tests on 5% of the cells, which included, inter alia, three assays for the presence of replication-competent lentiviruses (RCL). Transduction efficiencies of CD34+ cells ranged from 35% to 50%, with a mean number of integrated lentivirus copies of 0.65-0.70. After thawing the transduced CD34+ cells, patients were re-infused with more than 4.106 transduced CD34+ cells/kg, followed by complete disruption of the myelin sheaths using busulfan and cyclophosphamide. The patient's HSCs were disrupted, allowing engraftment of the genetically corrected HSCs. Hematological recovery occurred on days 13-15 for two patients. Near-complete immunological recovery occurred at 12 months for the first patient and at 9 months for the second patient. In contrast to the use of lentivirus, one skilled in the art, using knowledge in the art and the teachings of the present invention, can correct HSCs for ALD using the CRISPR-Cas (Cas9) system, which targets and corrects the mutation (e.g., with an appropriate template for HDR); in particular, the sgRNA can target mutations in ABCD1, a gene located on the X chromosome that encodes ALD, a peroxisomal membrane transport protein, and HDR can provide encoding that results in proper expression of the protein. The sgRNA that targets the mutation and the particle containing the Cas protein (Cas9) is contacted with HSCs, such as CD34+ cells, carrying the mutation, as described in Cartier. The particle can also contain a suitable HDR template for correcting the mutation to express the peroxisomal membrane transport protein; or the HSC can be contacted with a second particle or vector that contains or delivers the HDR template. The cells thus contacted can optionally be treated as described in Cartier. The cells thus contacted can be administered as described in Cartier.

Reference should be made to WO 2015/148860, in the teachings set forth therein, the present invention contemplates the methods and materials of these documents used in combination with the teachings set forth herein. In one aspect, gene therapy for diseases associated with the blood system, methods and compositions for the treatment of beta-thalassemia can be adapted to the CRISPR-Cas system of the present invention (see, for example, WO 2015/148860). In one embodiment, WO 2015/148860 provides for the treatment or prevention of beta-thalassemia, or its symptoms, for example, by altering the B-cell CLL/lymphoma 11A (BCL11A) gene. The BCL11A gene is also known as B-cell CLL/lymphoma gene 11A, BCL11A-L, BCL11A-S, BCL11AXL, CTIP 1, HBFQTL5, and ZNF. BCL11A encodes a zinc finger protein that is involved in regulating globin gene expression. When the BCL11A gene is altered (e.g., one or both alleles of the BCL11A gene), gamma globin levels may increase. Gamma globin can replace beta globin in the hemoglobin complex and efficiently deliver oxygen to tissues, thereby normalizing the disease phenotypes of beta thalassemia.

WO 2015/148863 should be mentioned, and in the teachings set forth in this document, the present invention contemplates the methods and materials of these documents, which can be adapted to the CRISPR-Cas system of the present invention. In an aspect of treating or preventing sickle cell anemia, which is an inherited hematological blood disorder, WO 2015/148863 provides for altering the BCL11A gene. By altering the BCL11A gene (for example, one or both alleles of the BCL11A gene), gamma globin levels can be increased. Gamma globin can replace beta globin in the hemoglobin complex and effectively deliver oxygen to tissues, thereby normalizing the phenotypes of sickle cell anemia.

In one aspect of the present invention, methods and compositions that involve editing a target nucleic acid sequence or modulating the expression of a target nucleic acid sequence and their uses in connection with cancer immunotherapy are understood by adapting the CRISPR-Cas system of the present invention. Reference is made to the use of gene therapy in WO 2015/161276, which provides methods and compositions that can be used to disrupt the proliferation, survival and/or function of T cells by altering one or more genes expressed by T cells, such as one or more of the FAS, BID, CTLA4, PDCD1, CBLB, PTPN6, TRAC and/or TRBC genes. In related aspects, T cell proliferation may be impaired by alteration of one or more genes expressed by T cells, such as the CBLB gene and/or PTPN6, the FAS gene and/or BID, the CTLA4 gene and/or PDCDI and/or TRAC and/or TRBC.

Chimeric antigen receptor (CAR)19 T cells have been shown to have anti-leukemic effects in patients with malignancies. However, leukemia patients often have insufficient T cells to collect, so treatment must include modified T cells from donors. Accordingly, there is interest in establishing a donor T cell bank. 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) describe the modification of CAR19 T cells to eliminate the risk of graft-versus-host disease by disrupting T cell receptor expression and targeting CD52. Furthermore, CD52 cells were targeted so that they were refractory to alemtuzumab, thereby contributing to alemtuzumab's ability to prevent host-mediated rejection of human leukocyte antigen (HLA)-mismatched CAR19 T cells. The researchers used a third-generation self-inactivating lentiviral vector encoding 4g7 CAR19 (CD19 scFv-4-1BB-CD3ζ) linked to RQR8, then electroporated the cells with two pairs of TALEN mRNAs to multiplexly target the T cell receptor (TCR) constant alpha chain locus and the CD52 gene locus. Cells that still expressed TCR after ex vivo expansion were depleted by CliniMacs α/β TCR depletion, resulting in a T cell product (UCART19) with <1% TCR expression, 85% of which were CAR19 and 64% were negative for CD52. The CAR19-modified T cells were administered to treat relapsed acute lymphoblastic leukemia in patients. The ideas presented herein provide efficient methods for producing modified hematopoietic stem cells and their progeny, including but not limited to cells of the myeloid and lymphoid blood lineage, including T cells, B cells, monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells and megakaryocytes or platelets, and natural killer cells and their precursors and progeny. Such cells can be modified by knocking out, knocking in or otherwise modulating targets, such as by removing or modulating CD52 as described herein and other targets such as but not limited to CXCR4 and PD-1. Such compositions, cells and the method of the present invention can be used to modulate immune responses and to treat but not limited to malignancies, viral infections and immune disorders, in combination with the administration of T cells or other cells to patients.

Reference should be made to WO 2015/148670 and in the teachings set forth herein, the present invention pertains to the methods and materials of this document used in combination with the teachings set forth herein. In one aspect of gene therapy, methods and compositions for editing a target sequence associated with or in association with the human immunodeficiency virus (HIV) and acquired immunodeficiency syndrome (AIDS) are pertained. In a related aspect, the present invention described herein pertains to 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. The CCR5 gene is also known as CKR5, CCR-5, CD195, CKR-5, CCCKR5, CMKBR5, IDDM22 and CC-CKR-5. In a further aspect, the present invention described herein involves use for preventing or reducing infection with HIV and/or preventing or reducing the ability of HIV to enter host cells, such as in subjects who are already infected. Exemplary host cells for HIV include, but are not limited to, CD4 cells, T cells, gut-associated lymphoid tissue (GALT), macrophages, dendritic cells, myeloid progenitor cells, and microglia. Entry of the virus into host cells requires interaction of the viral glycoproteins gp41 and gp120 with the CD4 receptor and a coreceptor, such as CCR5. If the coreceptor, such as CCR5, is not present on the surface of the host cells, the virus cannot bind to and enter the host cells. Thus, disease progression is impeded. By knocking out or knocking down CCR5 in host cells, for example by introducing a protective mutation (such as the CCR5 delta 32 mutation), the HIV virus is prevented from entering the host cells.

X-linked chronic granulomatous disease (CGD) is an inherited immune disorder due to absent or decreased activity of phagocytic NADPH oxidase. By using the CRISPR-Cas9 system, which targets and corrects the mutation (absent or decreased activity of phagocytic NADPH oxidase) (e.g., using a suitable template for HDR that delivers the coding sequence for phagocytic NADPH oxidase); in particular, sgRNA can target the mutation that leads to CGD (defective phagocytic NADPH oxidase), and HDR can provide coding for the proper expression of phagocytic NADPH oxidase. sgRNA that targets the mutation and a particle containing the Cas protein (Cas9) contacts HSCs carrying the mutation. The particle may also contain a suitable HDR matrix to correct the mutation for proper expression of phagocytic NADPH oxidase; or the HSC may be contacted with a second particle or vector that contains or delivers the HDR matrix. The cells so contacted may be administered and optionally processed and expanded; see Cartier.

Fanconi Anemia Mutations in at least 15 genes (FANCA, FANCB, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ/BACH1/BRIP1, FANCL/PHF9/POG, FANCM, FANCN/PALB2, FANCO/Rad51C, and FANCP/SLX4/BTBD12) can cause Fanconi anemia. The proteins produced by the expression of these genes are involved in a cellular process known as the FA pathway. The FA pathway is turned on (activated) when the process of making new copies of DNA, called “DNA replication,” is blocked by DNA damage. The FA pathway directs certain proteins to the site of damage that trigger DNA repair so DNA replication can continue. The FA pathway specifically responds to a certain type of DNA damage known as interstrand crosslinks (ICLs). ICLs occur when DNA building blocks (nucleotides) on opposite strands of DNA abnormally join or link together, stopping DNA replication. ICLs can be caused by the buildup of toxic substances produced in the body or by treatment with certain anticancer drugs. Eight proteins associate with the Fanconi anemia group to form a complex known as the FA core complex. The FA core complex activates two proteins called FANCD2 and FANCI. Activation of these two proteins results in DNA repair proteins being recruited to the ICL region so that the crosslink can be removed and DNA replication can continue with the help of the FA core complex. More specifically, the FA core complex, a nuclear multiprotein complex consisting of FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, and FANCM, functions as an E3 ubiquitin ligase and mediates the activation of the ID complex, which is a heterodimer consisting of FANCD2 and FANCI. Once monoubiquitinated, it interacts with classical tumor suppressors downstream of the FA pathway, including FANCD1/BRCA2, FANCN/PALB2, FANCJ/BRIP1, and FANCO/Rad51C, and thus participates in DNA repair through homologous recombination (HR). Eighty to 90 percent of FA cases are due to mutations in one of three genes, FANCA, FANCC, and FANCG. These genes carry the information for the production of components of the FA core complex. Mutations in such genes associated with the FA core complex will result in loss of functionality of the complex and disruption of the entire FA pathway. As a result, DNA damage is not effectively repaired, and ICL accumulates over time. Geiselhart's "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 FA and an animal experiment involving intrafemoral administration of a lentivirus encoding the FANCC gene, which resulted in correction of HSCs in vivo. By means of a CRISPR-Cas (Cas9) system that targets one or more mutations associated with FA, for example, a CRISPR-Cas (Cas9) system having an sgRNA and an HDR template(s) that respectively target one or more of the FANCA, FANCC or FANCG mutations that lead to FA and provide corrected expression of one or more of FANCA, FANCC or FANCG; for example, the sgRNA can target a mutation, for example, FANCC, and the HDR can provide encoding for proper expression of FANCC. The sgRNA that targets the mutation(s) (for example, one or more involved in FA, such as the mutation(s) in a particle containing any one or more of FANCA, FANCC or FANCG) and the Cas (Cas9) protein are contacted with HSCs bearing the mutation(s). The particle may also contain suitable HDR matrix(es) for correcting a mutation to properly express one or more of the proteins involved in FA, such as one or more of FANCA, FANCC, or FANCG; or the HSC may be contacted with a second particle or vector that contains or delivers the HDR matrix. The cells so contacted may be administered and optionally processed and expanded; see Cartier.

A particle in the description of this document (e.g. comprising an sgRNA and a Cas (Cas9), optionally an HDR template(s), or an HDR template(s); e.g. in the case of hemophilia B, SCID, SCID-X1, ADA-SCID, hereditary tyrosinemia, β-thalassemia, X-linked CGD, Wiskott-Aldrich syndrome, Fanconi anemia, adrenoleukodystrophy (ALD), metachromatic leukodystrophy (MLD), HIV/AIDS, an immunodeficiency, a hematological condition, or a genetic lysosomal storage disease) is preferably obtained or obtainable by mixing a mixture of an sgRNA and a Cas (Cas9) protein (optionally comprising an HDR template(s), or such mixture only comprising an HDR template(s) in the case where the individual particles (with respect to the matrix(es) are desirable) with a mixture comprising, or consisting essentially of, or consisting of a surfactant, a phospholipid, a biodegradable polymer, a lipoprotein, and an alcohol (wherein one or more sgRNAs target a gene locus or loci in an HSC).

Indeed, the present invention is particularly suitable for the treatment of hematopoietic inherited disorders by genome editing and immunodeficiencies, such as inherited immunodeficiencies, especially using the particle-based technology described herein. Hereditary immunodeficiencies are diseases where the genome editing procedures of the present invention can be successful. The reasons are that hematopoietic cells, a subset of which are immune cells, are therapeutically accessible. They can be removed from the body and transplanted autologously or allogeneically. In addition, certain inherited immunodeficiencies, such as severe combined immunodeficiency (SCID), result in a defect in the proliferation of immune cells. Correction of inherited disorders causing SCID by rare spontaneous "reverse" mutations suggests that correction of even a single lymphocyte precursor may be sufficient to restore immune function in patients .../../../Users/t_kowalski/AppData/Local/Microsoft/Windows/Temporary Internet Files/Content.Outlook/GA8VY8LK/Treating SCID for Ellen.docx - _ENREF_1, 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 edited cells ensures that even low levels of editing result in therapeutic effect. This effect according to the present invention can be observed in SCID, Wiskott-Aldrich syndrome and other conditions mentioned herein, including inherited hematopoietic disorders such as alpha- and beta-thalassemia, where hemoglobin deficiencies negatively affect the fitness of red blood cell precursors.

The DSB repair activity of NHEJ and HDR varies considerably depending on the cell type and cell state. NHEJ is not tightly regulated by the cell cycle and is effective in all cell types, ensuring the presence of high levels of gene disruption in accessible target cell populations. In contrast, HDR acts primarily during the S/G2 phase and is thus restricted to cells that are actively dividing, limiting the application of therapies that require precise genomic modifications prior to mitotic cells [Ciccia, A. & Elledge, S.J. Molecular cell 40, 179–204 (2010); Chapman, J.R., et al. Molecular cell 47, 497–510 (2012)].

The efficiency of HDR correction may be controlled by the epigenetic state or sequence of the targeted locus or the configuration of the specific repair template used (single-stranded versus double-stranded, long versus short homology arms) [Hacein-Bey-Abina, S., et al. The New England journal of medicine 346, 1185–1193 (2002); Gaspar, H.B., et al. Lancet 364, 2181–2187 (2004); Beumer, K.J., et al. G3 (2013)]. The relative activity of NHEJ and HDR machinery in target cells may also influence the efficiency of gene correction, as these pathways may compete for DSB repair [Beumer, K.J., et al. HDR also introduces a delivery problem not seen with NHEJ strategies, as it requires simultaneous delivery of nucleases and repair templates. In practice, these limitations have so far resulted in low levels of HDR in therapeutically relevant cell types. The transition to clinical application has thus largely focused on NHEJ strategies for disease treatment, although preclinical proof-of-concept studies have just been described for mouse models of hemophilia B and congenital tyrosinemia [Li, H., et al. Nature 475, 217–221 (2011); Yin, H., et al. Nature biotechnology 32, 551–553 (2014)].

Any of the above genome editing applications may involve combinations of proteins, small RNA molecules, and/or repair templates, making delivery of these multiple parts significantly more challenging than delivery of small molecule drugs. Two main strategies have been developed for delivering genome editing tools: ex vivo and in vivo. In ex vivo treatments, diseased cells are removed from the body, edited, and then transplanted back into the patient. Ex vivo editing has the advantage of allowing for proper targeting of the cell population and precise dosing of therapeutic molecules. The latter consideration may be particularly important when off-target modifications are of particular interest, as tailoring the amount of nuclease may result in lower levels of such mutations (Hsu et al., 2013). Another advantage of ex vivo approaches is the typically high editing yields that can be achieved due to the development of efficient delivery systems for proteins and nucleic acids into cells in culture for research and gene therapy applications.

There may be two major drawbacks to ex vivo approaches that limit their application to a small number of diseases. For example, the target cells must be able to survive manipulation outside the body. For many tissues, such as the brain, culturing cells outside the body is a major challenge because the cells either fail to survive or lose the properties necessary for their function in vivo. Thus, from the perspective of the present invention and the knowledge in the art, ex vivo therapy of tissues with adult stem cell populations that are amenable to ex vivo culturing and manipulation, such as the hematopoietic system, using the CRISPR-Cas (Cas9) system is feasible. [Bunn, H.F. & Aster, J. Pathophysiology of blood disorders, (McGraw-Hill, New York, 2011)]

In vivo genome editing involves direct delivery of editing systems into their native tissues. In vivo editing allows for the treatment of diseases in which the affected cell population is not amenable to ex vivo manipulation. Furthermore, in situ delivery of nucleases to cells creates the potential for treatment of multiple tissues and cell types. These properties likely make in vivo treatments applicable to a broader range of diseases than ex vivo therapies.

To date, in vivo editing has been achieved largely through the use of viral vectors with defined, tissue-specific tropism. Such vectors are currently limited in capacity and tropism, limiting this type of therapy to organ systems where transduction with clinically useful vectors is effective, such as the liver, muscle, and eye [Kotterman, M.A. & Schaffer, D.V. Nature reviews. Genetics 15, 445–451 (2014); Nguyen, T.H. & Ferry, N. Gene therapy 11 Suppl 1, S76–84 (2004); Boye, S.E., et al. Molecular therapy : the journal of the American Society of Gene Therapy 21, 509–519 (2013)].

A major potential barrier to in vivo delivery is the immune response that may be generated in response to the large quantities of virus required for treatment, but this phenomenon is not unique to genome editing and has been observed in other viral-based gene therapies [Bessis, N., et al. Gene therapy 11 Suppl 1, S10-17 (2004)]. It is also likely that the peptides from the editing nucleases themselves are presented on MHC class I molecules to stimulate an immune response, although there is little evidence that this occurs at the preclinical level. Another major challenge with this type of therapy is controlling the distribution and hence dosage of the genome editing nucleases in vivo, resulting in off-target mutation profiles that can be difficult to predict. However, in view of the present invention and the knowledge in the art, including the use of virus- and particle-based therapies used in the treatment of cancer, in vivo modification of HSC, for example by delivery of a particle or virus, is within the skill of the art.

Ex vivo editing-based therapies Long-term clinical experience with purification, culture, and transplantation of hematopoietic cells has identified diseases affecting the blood system, such as SCID, Fanconi anemia, Wiskott-Aldrich syndrome, and sickle cell anemia, as priority areas for ex vivo editing-based therapies. Another reason for focusing on hematopoietic cells is that, thanks to previous efforts to develop gene therapies for blood disorders, delivery systems with relatively high efficiencies already exist. Given these advantages, this type of therapy may be applicable to diseases where the edited cells have a fitness advantage, such that a small number of engrafted, edited cells can proliferate and provide treatment for the disease. One such disease is HIV, in which infection results in a fitness deficiency of CD4+ T cells.

Ex vivo editing therapies have recently been expanded to include gene correction strategies. Barriers to ex vivo HDR have been overcome, as demonstrated by the recent work of Genovese et al., who achieved correction of the mutated IL2RG gene in hematopoietic stem cells (HSCs) derived from a patient suffering from SCID-X1 [Genovese, P., et al. Nature 510, 235-240 (2014)]. Genovese et al. performed gene correction in HSCs using a multimodal strategy. First, HSCs were transduced with an integration-deficient lentivirus containing an HDR template encoding a therapeutic cDNA for IL2RG. Following transduction, cells were electroporated with mRNA encoding ZFNs that target a mutagenesis hotspot in IL2RG to promote HDR-based gene correction. To enhance HDR rates, culture conditions were optimized using small molecules that promote HSC division. Using optimized culture conditions, nucleases, and HDR templates, gene-corrected HSCs from a SCID-X1 patient were obtained in culture at therapeutically relevant levels. HSCs from unaffected individuals that underwent the same gene correction procedure could maintain long-term hematopoiesis in mice, which is the gold standard for HSC function. HSCs are capable of giving rise to all hematopoietic cell types and can be autologously transplanted, making them an extremely important cell population for all inherited hematopoietic disorders [Weissman, I.L. & Shizuru, J.A. Blood 112, 3543-3553 (2008)]. In principle, gene-corrected HSCs could be used to treat a wide range of genetic blood disorders, making this study an important discovery for therapeutic genome editing.

In vivo editing-based therapy. In vivo editing can be advantageously applied based on the present invention and the knowledge in the art. For organ systems to which delivery is effective, there are already a number of impressive preclinical therapeutic successes. The first example of successful in vivo editing-based therapy was demonstrated 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 loss-of-function mutations in the gene encoding factor IX, an important component of the blood coagulation cascade. Restoring factor IX activity to approximately 1% of its levels in severely affected individuals can transform the disease into a significantly milder form, since prophylactic infusion of recombinant factor IX in such patients from an early age to achieve such levels significantly alleviates the severity of clinical complications [Lofqvist, T., et al. Journal of internal medicine 241, 395-400 (1997)]. Thus, extremely low levels of HDR-mediated gene correction are required to alter clinical outcomes in patients. Furthermore, factor IX is synthesized and secreted by the liver, an organ that can be efficiently transduced by viral vectors encoding editing systems.

Using hepatotropic adeno-associated virus (AAV) serotypes encoding ZFN and an HDR proofreading template, up to 7% correction of the mutated, humanized factor IX gene in mouse liver was achieved [Li, H., et al. Nature 475, 217–221 (2011)]. This resulted in improved coagulation kinetics, a measure of the function of the blood coagulation cascade, demonstrating for the first time that in vivo editing-based therapy is not only possible but also effective. As described in this paper, one skilled in the art is guided by the teachings of this paper and knowledge in the art, such as Li in the case of treating hemophilia B with a particle containing an HDR template and the CRISPR-Cas (Cas9) system that targets a mutation in an X-linked recessive disorder to reverse a loss-of-function mutation.

Building on this study, other groups have recently applied in vivo liver genome editing using CRISPR-Cas to successfully treat a mouse model of congenital tyrosinemia and to generate mutations that confer protection against cardiovascular disease. These two separate applications demonstrate the generality of this approach for disorders that involve liver dysfunction [Yin, H., et al. Nature biotechnology 32, 551–553 (2014); Ding, Q., et al. Circulation research 115, 488–492 (2014)]. Applications of in vivo editing to other organ systems are needed to confirm the broad applicability of this strategy. Efforts to optimize both viral and non-viral vectors are currently underway to expand the spectrum of disorders that can be treated with this therapy [Kotterman, M.A. & Schaffer, D.V. Nature reviews. Genetics 15, 445-451 (2014); Yin, H., et al. Nature reviews. Genetics 15, 541-555 (2014)]. As described in this document, one skilled in the art is guided by the teachings of this document and knowledge in the art, such as Yin in the case of treating hereditary tyrosinemia with a particle containing an HDR template and a CRISPR-Cas (Cas9) system that targets a mutation.

Targeted deletion, therapeutic uses. Targeted deletion of genes may be preferred. Thus, genes involved in an immunodeficiency, hematological condition or genetic lysosomal storage disease are preferred, for example, hemophilia B, SCID, SCID-X1, ADA-SCID, hereditary tyrosinemia, β-thalassemia, X-linked CGD, Wiskott-Aldrich syndrome, Fanconi anemia, adrenoleukodystrophy (ALD), metachromocytic leukodystrophy (MLD), HIV/AIDS, other metabolic disorders, genes encoding misfolded proteins involved in diseases, genes leading to loss of function involved in the disease, mutations that can be targeted to HSCs using any of the delivery systems described herein, with a particle system being preferred.

In the present invention, the immunogenicity of a CRISPR enzyme in particular can be reduced following the approach first described by Tangri et al. for erythropoietin and subsequently developed. Accordingly, directed evolution or rational engineering can be used to reduce the immunogenicity of a CRISPR enzyme (e.g., Cas9) in a host species (human or other species).

Genome editing. The CRISPR/Cas (Cas9) systems of the present invention can be used to correct genetic mutations, which has previously been attempted with limited success using TALENs and ZFNs, as well as lentiviruses, including as described herein; see also WO2013163628.

Types of adoptive cell therapy

The present invention also provides for the use of the CRISPR-Cas system described herein, such as the Cas9 effector protein systems, to modify cells for adoptive therapies. Aspects of the present invention include the adoptive transfer of immune system cells, such as T cells, specific for certain 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 cancer, Science Vol. 348 no. 6230 pp. 62-68; 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 altering the specificity of the T cell receptor (TCR), such as by introducing new TCR α- and β-chains with specificity for certain peptides (see U.S. Patent No. 8,697,854; PCT Patent Application Publications: WO2003020763, WO2004033685, WO2004044004, WO2005114215, WO2006000830, WO2008038002, WO2008039818, WO2004074322, WO2005113595, WO2006125962, WO2013166321, WO2013039889, WO2014018863, WO2014083173; US Patent No. 8088379).

As an alternative or in addition to TCR modifications, chimeric antigen receptors (CARs) can be used to generate immunoresponsive cells, such as T cells, specific for certain targets, such as cancer cells, with a wide variety of receptor chimeric constructs having been described (see U.S. Patent Nos. 5,843,728; 5,851,828; 5,912,170; 6,004,811; 6,284,240; 6,392,013; 6,410,014; 6,753,162; 8,211,422 and PCT publication WO9215322). Alternative CAR constructs can be characterized as belonging to subsequent generations. First generation CARs typically consist of a single-chain variable fragment of an antibody specific for an antigen, e.g., comprising a V _L linked to a V _H of a particular antibody, linked via a flexible linker, e.g., via the CD8α hinge domain and the CD8α transmembrane domain to the transmembrane and intracellular signaling domains of CD3ζ or FcRγ (scFv-CD3ζ or scFv-FcRγ; see U.S. Patent No. 7,741,465; U.S. Patent No. 5,912,172; U.S. Patent No. 5,906,936). Second-generation CARs encompass the intracellular domains of one or more costimulatory molecules such as CD28, OX40 (CD134), or 4-1BB (CD137) in the endodomain (e.g., scFv-CD28/OX40/4-1BB-CD3ζ; see U.S. Patent Nos. 8,911,993; 8,916,381; 8,975,071; 9,101,584; 9,102,760; 9,102,761). Third-generation CARs include a combination of costimulatory endodomains such as the signaling domains of the CD3ζ chain, CD97, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, or CD28 (e.g., scFv-CD28-4-1BB-CD3ζ or scFv-CD28-OX40-CD3ζ; see U.S. Patent No. 8,906,682; U.S. Patent No. 8,399,645; U.S. Patent No. 5,686,281; PCT Publication No. WO2014134165; PCT Publication No. WO2012079000). Alternatively, costimulation can be regulated by CAR expression in antigen-specific T cells selected for activation and expansion by engagement of their native αβTCR, e.g., by antigen on professional antigen-presenting cells via concomitant costimulation. In addition, additional engineered receptors can be provided on immunoreactive cells, e.g., to improve targeting of T cell attack and/or minimize side effects.

Alternative techniques can be used to transform the target immunoresponsive cells, such as protoplast fusion, lipofection, transfection, or electroporation. A wide variety of vectors can be used, such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, plasmids, or transposons such as the Sleeping Beauty transposon (see U.S. Patent Nos. 6,489,458; 7,148,203; 7,160,682; 7,985,739; 8,227,432), and can be used to administer CARs, such as by generating 2nd generation antigen-specific CARs that signal through CD3ζ and CD28 or CD137. Viral vectors can include, for example, HIV, SV40, EBV, HSV, or BPV-based vectors.

The cells to be targeted for transformation may include, for example, T cells, natural killer (NK) cells, 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 differentiate. T cells expressing the desired CAR can, for example, be selected by co-cultivation with γ-irradiated activating and dividing cells (AaPC) that co-express a cancer antigen and costimulatory molecules. The engineered CAR T cells can be expanded, for example, by co-cultivation on AaPC in the presence of solubilized factors such as IL-2 and IL-21. Such expansion can, for example, be carried out with the aim of obtaining CAR+ memory T cells (which can, for example, be analyzed using non-enzymatic digital array and/or multi-panel flow cytometry). In this regard, CAR T cells can be obtained that are characterized by specific cytotoxic activity towards antigen-bearing tumors (optionally in combination with the formation of desired chemokines, such as interferon-γ). CAR T cells of this type can, for example, be used in animal models, for example for the treatment of tumor xenografts.

Approaches such as those described above can be adapted to provide methods for treating and/or improving the survival of a subject having a disease, such as a neoplasm, for example, by administering an effective amount of immunoresponsive cells comprising an antigen recognition receptor that binds to a particular antigen, wherein the binding activates the immunoresponsive cell, thereby treating or preventing the disease (such as a neoplasm, a pathogenic infection, an autoimmune disease, or a reaction to an allogeneic transplant). Dosing in CAR T cell-based therapies can, for example, involve administering 10 ⁶ to 10 ⁹ cells/kg, with or without a course of anti-lymphoma therapy, such as cyclophosphamide.

In one embodiment, the treatment can be administered to patients undergoing immunosuppressive treatment. The cells or population of cells can be rendered resistant to at least one immunosuppressive agent by inactivating a gene encoding a receptor for such an immunosuppressive agent. Without being limited by theory, the immunosuppressive treatment should facilitate the selection and expansion of immunoreactive cells or T cells in accordance with the present invention in the patient.

The administration of the cells or cell population according to the present invention can be performed by any convenient method, including by aerosol inhalation, injection, absorption, transfusion, implantation or transplantation. The cells or cell population can be administered to the patient subcutaneously, intradermally, intratumorally, intranodularly, intramedullary, intramuscularly, by intravenous or intralymphatic injection or intraperitoneally. In one embodiment, the cellular compositions of the present invention are preferably administered by intravenous injection.

The administration of the cells or cell population may comprise the administration of 10 ⁴ - 10 ⁹ cells per kg body weight, preferably 10 ⁵ to 10 ⁶ cells/kg body weight, including integer cell numbers within these ranges. Dosing in CAR T cell-based therapies may, for example, comprise the administration of 10 ⁶ to 10 ⁹ cells/kg, with or without a course of anti-lymphoma therapy, such as cyclophosphamide. The cells or cell population may be administered in one or more doses. In another embodiment, an effective amount of cells is administered in 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 discretion of the treating physician and depends on the clinical condition of the patient. The cells or cell population may be obtained from any source, such as a blood bank or a donor. Given that individual needs vary, it is within the skill of the art to determine optimal ranges of effective amounts of a particular cell type for a particular disease or condition. An effective amount is an amount that provides a therapeutic or prophylactic benefit. The dose administered may depend on the age, health, and weight of the recipient, the type of concomitant treatment, if any, the frequency of treatment, and the nature of the desired effect.

In another embodiment, an effective amount of cells or a composition containing such cells is administered parenterally. The administration may be intravenous administration. The administration may be performed directly by injection into the tumor.

To prevent potential adverse reactions, engineered immunoresponsive cells can be equipped with a safety device in the form of a transgene that renders the cells susceptible to the action of a specific signal. For example, the herpes simplex virus thymidine kinase (TK) gene can be used in this regard, for example by introducing it 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, introduction of a nucleoside prodrug such as ganciclovir or acyclovir induces cell death. Alternative safety device designs include inducible caspase 9, for example activated by introduction of a small molecule dimer that joins two non-functional icasp9 molecules to form the active enzyme. A wide variety of alternative approaches have been described for controlling cell proliferation (see U.S. Patent Application Publication No. 20130071414; PCT Patent Application Publication WO2011146862; PCT Patent Application Publication WO2014011987; PCT Patent Application Publication WO2013040371; Zhou et al. BLOOD, 2014, 123/25:3895-3905; Di Stasi et al., The New England Journal of Medicine 2011; 365:1673-1683; Sadelain M, The New England Journal of Medicine 2011; 365:1735-173; Ramos et al., Stem Cells 28(6):1107-15 (2010)).

In further improving adoptive therapies, genome editing can be used to adapt immunoresponsive cells to alternative embodiments, such as to produce edited CAR T cells (see Poirot et al., 2015, Multiplex genome edited T-cell manufacturing platform for "off-the-shelf" adoptive T-cell immunotherapies, Cancer Res 75 (18): 3853). The cells can be edited using the CRISPR system and method of using it described herein. The CRISPR systems can be delivered to the immune cell using any method described herein. In preferred embodiments, the cells are edited ex vivo and transferred to a subject in need thereof. Immunoresponsive cells, CAR T cells, or any cells for adoptive cell transfer can be edited. Editing may be performed to eliminate potential alloreactive T cell receptors (TCRs), disrupt the target of a chemotherapeutic agent, block an immune checkpoint, activate a T cell, and/or enhance the differentiation and/or proliferation of functionally exhausted or dysfunctional CD8+ T cells (see PCT Patent Publications WO2013176915, WO2014059173, WO2014172606, WO2014184744, and WO2014191128). Editing may result in gene inactivation.

By gene inactivation is meant that the gene of interest is not expressed as a functional protein. In a specific embodiment, the CRISPR system specifically catalyzes cleavage in one target gene, thereby inactivating said target gene. The resulting nucleic acid strand breaks are typically repaired by various mechanisms of homologous recombination or non-homologous end joining (NHEJ). However, NHEJ is an imperfect repair process that often results in changes in the DNA sequence at the cleavage site. Non-homologous end joining (NHEJ) repair often results in small insertions or deletions (insertions/deletions) and can be used to obtain specific gene knockouts. Cells in which the cleavage-induced mutagenesis phenomenon has occurred can be identified and/or selected using well-known methods in the art.

T cell receptors (TCRs) are cell surface receptors that are involved in the activation of T cells in response to antigen presentation. The TCR typically consists of two chains, α and β, that assemble to form a heterodimer and associate with CD3 transducing subunits to form the T cell receptor complex present on the cell surface. The α and β targets of the TCR each consist of an immunoglobulin-like N-terminal variable (V) and constant (C) region, a hydrophobic transmembrane domain, and a short cytoplasmic region. As with immunoglobulin molecules, the variable region of the α and β targets is generated by V(D)J recombination, creating a wide variety of antigen specificities within the T cell population. However, unlike immunoglobulins, which recognize intact antigen, T cells are activated by processed peptide fragments in combination with an MHC molecule, introducing an additional region for T cell antigen recognition known as the MHC restriction site. Recognition of MHC mismatches between donor and recipient via the T cell receptor leads to T cell proliferation and potential development of graft-versus-host disease (GVHD). Inactivation of TCRα or TCRβ can result in the elimination of TCR from the T cell surface, preventing alloantigen recognition and thus GVHD. However, disruption of the TCR typically results in the elimination of the CD3 signaling component and alters the pathways for further T cell expansion.

Allogeneic cells are rapidly rejected by the host immune system. Allogeneic leukocytes present in non-irradiated blood products have been shown to survive for no more than 5-6 days (Boni, Muranski et al. 2008 Blood 1;112(12):4746-54). Thus, to prevent rejection of allogeneic cells, the host immune system typically needs to be suppressed to some extent. However, in the case of adoptive cell transfer, the use of immunosuppressive drugs also has a detrimental effect on the therapeutically administered T cells. Thus, for an adoptive immunotherapy approach to be effective in these settings, the administered cells must be resistant to immunosuppressive treatment. Thus, in a specific embodiment, the present invention further provides the step of modifying the T cells to render them resistant to an immunosuppressive agent, preferably by inactivating at least one gene encoding an immunosuppressive agent. An immunosuppressant is an agent that suppresses immune function through one of several mechanisms of action. An immunosuppressant may be, but is not limited to, a calcineurin inhibitor, a target of rapamycin, an interleukin 2 receptor alpha chain blocker, an inosine monophosphate dehydrogenase inhibitor, a dihydrofolic acid reductase inhibitor, a corticosteroid, or an immunosuppressive antimetabolite. The present invention provides for rendering T cells resistant to immunosuppressants for immunotherapy by inactivating a target of an immunosuppressant in T cells. As non-limiting examples, targets of an immunosuppressant may be a receptor for an immunosuppressant such as CD52, a glucocorticoid receptor (GR), a member of the FKBP gene family, and a member of the cyclophilin gene family.

Immune checkpoints are inhibitory pathways that slow or stop immune responses and prevent excessive tissue destruction due to uncontrolled immune cell activity. In certain embodiments, the target immune checkpoint is programmed death gene 1 (PD-1 or CD279) (PDCD1). In other embodiments, the immune checkpoint being targeted is cytotoxic T lymphocyte-associated antigen (CTLA-4). In further embodiments, the target immune checkpoint is another member of the CD28 and CTLA4 Ig superfamily, such as BTLA, LAG3, ICOS, PDL1, or KIR. In further embodiments, the target immune checkpoint is a member of the TNFR superfamily, such as CD40, OX40, CD137, GITR, CD27, or TIM-3.

Additional immune checkpoints include Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) (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 therefore not amenable to antibody-mediated therapies, but its role in activation and proliferation makes it an attractive target for genetic manipulation in adoptive transfer strategies such as chimeric antigen receptor (CAR) T cells. Immune checkpoints may also include the T-cell immunoreceptor with Ig and ITIM domains (TIGIT/Vstm3/WUCAM/VSIG9) and VISTA (Le Mercier I, et al., (2015) Beyond CTLA-4 and PD-1, the generation Z of negative checkpoint regulators. Front. Immunol. 6:418).

WO2014172606 relates to the use of MT1 and/or MT1 inhibitors to enhance the proliferation and/or activity of exhausted CD8+ T cells and to reduce CD8+ T cell exhaustion (e.g., a decrease in the functional properties of exhausted or unresponsive CD8+ immune cells). In certain embodiments, metallothioneins are targeted by gene editing in adoptively transferred T cells.

In certain embodiments, gene editing targets may be at least one target locus involved in the expression of an immune checkpoint protein. Such targets may include, but are not limited to, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, ICOS (CD278), PDL1, KIR, LAG3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244 (2B4), TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, VISTA, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, MT1, MT2, CD40, OX40, CD137, GITR, CD27, SHP-1, or TIM-3. In preferred embodiments, a gene locus involved in the expression of the PD-1 or CTLA-4 genes is targeted. In other preferred embodiments, combinations of genes are targeted, such as, but not limited to, PD-1 and TIGIT.

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 TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ.

Regardless of whether the modification of T cells is pre- or post-modification, T cells can be activated and expanded, typically using methods described in, for example, U.S. Patents 6,352,694; 6,534,055; 6,905,680; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and 7,572,631. T cells can be expanded in vitro or in vivo.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA technology, all of which are within the skill of the art. See MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989) (Sambrook, Fritsch and Maniatis); MOLECULAR CLONING: A LABORATORY MANUAL, 4th edition (2012) (Green and Sambrook); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (1987) (F. M. Ausubel, et al. eds.); METHODS IN ENZYMOLOGY series (Academic Press, Inc.); PCR 2: A PRACTICAL APPROACH (1995) (M.J. MacPherson, B.D. Hames and G.R. Taylor eds.); ANTIBODIES, A LABORATORY MANUAL (1988) (Harlow and Lane, eds.); ANTIBODIES A LABORATORY MANUAL, 2nd edition (2013) (E.A. Greenfield ed.); and ANIMAL CELL CULTURE (1987) (R.I. Freshney, ed.).

The practice of the present invention involves, unless otherwise indicated, standard techniques for producing genetically modified mice. See Marten H. Hofker and Jan van Deursen, TRANSGENIC MOUSE METHODS AND PROTOCOLS, 2nd edition (2011).

ALS

U.S. Patent Application Publication No. 20110023144 describes the use of zinc finger nucleases to genetically modify cells, animals, and proteins associated with the disease amyotrophic lateral sclerosis (ALS). ALS is characterized by gradual, progressive degeneration of certain nerve cells in the cerebral cortex, brainstem, and spinal cord that are involved in voluntary movement.

With respect to a disorder associated with motor neurons, proteins associated with these disorders are a diverse group of proteins that influence susceptibility to developing a disorder associated with motor neurons, the presence of a disorder associated with motor neurons, the severity of a disorder associated with motor neurons, or any combination thereof. The present disclosure contemplates editing any chromosomal sequences that encode proteins associated with an ALS-related disease, a specific disorder associated with motor neurons. Proteins associated with ALS are typically selected based on an experimentally established relationship of ALS-related proteins with an ALS-type disorder. For example, the rate of formation or the concentration in the bloodstream of a protein associated with ALS may be increased or decreased in a population with ALS compared to a population without ALS. Differences in protein levels can be assessed using proteomic 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 gene expression profiling of protein-coding genes using genomic analysis techniques, including but not limited to DNA microarray analysis, sequence-based analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).

By way of non-limiting example, proteins associated with ALS include, but are not limited to, the following proteins: SOD1 - soluble superoxide dismutase 1, ALS3 - amyotrophic lateral sclerosis-associated protein 3, SETX - senataxin, ALS5 - amyotrophic lateral sclerosis-associated protein 5, FUS - FUS RNA-binding protein (fused in sarcoma), ALS7 - amyotrophic lateral sclerosis-associated protein 7, ALS2 - amyotrophic lateral sclerosis-associated protein 2, DPP6 - dipeptidyl peptidase 6, NEFH - neurofilament heavy polypeptide, PTGS1 - prostaglandin endoperoxide synthase 1, SLC1A2 - solute carrier family 1 (glial cell high-affinity glutamate transporter), member 2, TNFRSF10B - tumor necrosis factor receptor superfamily, member 10b, PRPH - peripherin, HSP90AA1 - 90 kDa heat shock protein alpha (cytosolic), class A member 1, GRIA2 - glutamate receptor, ionotropic, AMPA 2, IFNG - interferon, gamma, S100B - S100, calcium-binding protein B, FGF2 - fibroblast growth factor 2, AOX1 - aldehyde oxidase 1, CS - citrate synthase, TARDBP - TAR DNA-binding protein, TXN - thioredoxin, RAPH1 - Ras-associated protein (RaIGDS/AF-6), and pleckstrin-homology domain kinase 5 1, MAP3K5 - mitogen-activated protein kinase, NBEAL1 - neurobichin-like protein 1, GPX1 - glutathione peroxidase 1, ICA1L - islet cell autoantigen 1.69 kDa-like, RAC1 - ras-related protein, botulinum C3 toxin substrate 1-like, MAPT - microtubule-associated protein tau, ITPR2 - inositol-1,4,5-triphosphate receptor type 2, ALS2CR4 - amyotrophic lateral sclerosis (juvenile)-associated protein 2 candidate region on chromosome 4, GLS - glutaminase, ALS2CR8 - amyotrophic lateral sclerosis (juvenile)-associated protein 2 candidate region on chromosome 8, CNTFR - ciliary neurotrophic factor receptor, ALS2CR11 - chromosome 11 candidate region for 2, associated with amyotrophic lateral sclerosis (juvenile), FOLH1 - folate hydrolase 1, FAM117B - protein family with sequence similarity to protein 117, member B, P4HB - prolyl 4-hydroxylase, polypeptide beta, CNTF - ciliary neurotrophic factor, SQSTM1 - sequestosome 1, STRADB - STE20-related kinase, beta-adaptor, NAIP - NLR family apoptosis-associated inhibitory protein, YWHAQ - tyrosinase/tryptophan-5-monooxygenase activating protein, polypeptide theta, SLC33A1 - solute carrier (acetyl-CoA) transporter family 33, member 1, TRAK2 - transport protein, kinesin-binding 2, Fig. 4, SAC1 lipid phosphatase domain-containing homolog, NIF3L1 - NIF3 NGG1-interacting factor 3-like 1, INA - internexin, neuronal intermediate filament protein, alpha, PARD3B - par-3 (partitioning defective 3) protein, homolog B, COX8A - cytochrome c oxidase, subunit VIIIA, CDK15 - cyclin-dependent kinase, HECW1 HECT - C2 and WW 15 domain-containing protein, E3 - ubiquitin protein ligase 1, NOS1 - nitric oxide synthase 1, MET - met proto-oncogene, SOD2 - mitochondrial superoxide dismutase 2, HSPB1 - 27 kDa heat shock protein 1, NEFL - neurofilament light polypeptide, CTSB - cathepsin B, ANG - angiogenin, ribonuclease ANG - angiogenin, ribonuclease, RNase family 5, HSPA8 - 70 kDa heat shock protein 8, VAPB VAMP (vesicle-associated membrane protein)-associated proteins B and C, ESR1 - estrogen receptor 1, SNCA - synuclein, alpha, HGF - hepatocyte growth factor, CAT - catalase, ACTB - actin, beta, NEFM - medium-sized neurofilament polypeptide, TH - tyrosine hydroxylase, BCL2 - B-cell protein 2, CLL/lymphoma-associated protein 2, FAS - Fas (TNF receptor superfamily, member 6), CASP3 - caspase 3, apoptosis-associated cysteine peptidase, CLU - clusterin, SMN1 - motor neuron survival-associated protein, G6PD - glucose-6-phosphate dehydrogenase 1, BAX BCL2-associated protein X, HSF1 - heat shock protein transcription factor 1, RNF19A - ring domain protein 19A, JUN - jun oncogene, ALS2CR12 - amyotrophic lateral sclerosis (juvenile)-associated protein 2 candidate region on chromosome 12, HSPA5 - 70 kDa heat shock protein 5, MAPK14 - mitogen-activated protein kinase 14, IL10 - interleukin 10, APEX1 - APEX nuclease (multifunctional DNA repair enzyme), TXNRD1 - thioredoxin reductase 1, NOS2 - inducible nitric oxide synthase 2, TIMP1 - TIMP - inhibitor of metallopeptidase 1, CASP9 - caspase 9, apoptosis-associated cysteine peptidase, XIAP - X-linked inhibitor of apoptosis, GLG1 - Golgi glycoprotein 1, EPO - erythropoietin, VEGFA - vascular endothelial growth factor A, ELN - elastin, GDNF - glial cell-derived neurotrophic factor, NFE2L2 - nuclear factor (erythroid)-like protein 2, SLC6A3 - solute carrier family 6 member 3 (neurotransmitter transporter, dopamine transporter), HSPA4 - 70 kDa heat shock protein 4, APOE - apolipoprotein E, PSMB8 - proteasome (prosome, macropain) subunit, type beta, 8, DCTN1 - dynactin 1, TIMP3 - TIMP - metallopeptidase inhibitor 3, KIFAP3 - kinesin-associated protein 3, SLC1A1 - solute carrier family 1 member 1 (high-affinity neuronal/epithelial cell glutamate transporter, Xag system), SMN2 - motor neuron survival centromeric protein 2, CCNC - cyclin C, MPP4 - palmitoylated membrane protein 4, STUB1 - STIP1-homologous and U-box-containing protein 1, ALS2 - amyloid beta (A4) precursor protein, PRDX6 - peroxiredoxin 6, SYP - synaptophysin, CABIN1 - calcineurin-binding protein 1, CASP1 - caspase 1, apoptosis-associated cysteine peptidase, GART - phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase, CDK5 - cyclin-dependent kinase 5, ATXN3 - ataxin 3, RTN4 - reticulon 4, C1QB complement component 1, subcomponent q, chain B, VEGFC - nerve growth factor receptor, HTT - huntingtin, PARK7 - Parkinson's disease-associated protein 7, XDH - xanthine dehydrogenase, GFAP - glial fibrillary acidic protein, MAP2 - microtubule-associated protein 2, CYCS - cytochrome c, somatic cells, FCGR3B - Fc fragment of IgG receptor IIIb with low affinity, CCS - copper-containing chaperone of superoxide dismutase, UBL5 - ubiquitin-like protein 5, MMP9 - matrix metallopeptidase 9, SLC18A3 - member 3 of solute carrier family 18 (vesicular, acetylcholine), TRPM7 - cation channel of transient receptor potential, subfamily M, member 7, HSPB2 - 27 kDa heat shock protein 2, AKT1 - murine thymoma virus v-akt oncogene homolog 1, DERL1- member 1 of protein family with Der1-like domain, CCL2 - chemokine ligand 2 (C--C motif), NGRN - neugrin, associated with axon growth, GSR - glutathione reductase, TPPP3 - member 3 of proteins that promote tubulin polymerization, APAF1 - factor 1, activating apoptotic peptidase, BTBD10 - protein 10, containing BTB domain (POZ), GLUD1 - glutamate dehydrogenase 1, CXCR4 - chemokine receptor 4 (C--X--C motif), SLC1A3 - solute carrier family 1 member 3 (high-affinity glutamate transporter of glial cells), FLT1 - fms-related tyrosine kinase 1, PON1 - paraoxonase 1, AR - androgen receptor, LIF - leukemia-associated inhibitory factor, ERBB3 - v-erb-b2 oncogene homolog 3 of erythroblastic leukemia virus, LGALS1 - lectin, galactoside-binding, soluble, protein 1, CD44 - CD44 molecule, TP53 - tumor protein p53, TLR3 - toll-like receptor 3, GRIA1 - glutamate receptor, ionotropic, AMPA 1, GAPDH - glyceraldehyde-3-phosphate dehydrogenase, GRIK1 - glutamate receptor, ionotropic, kainate protein 1, DES - desmin, CHAT - choline acetyltransferase, FLT4 - fms-related tyrosine kinase 4, CHMP2B - chromatin modifying protein 2B, BAG1 - BCL2-associated athanogen, MT3 - metallothionein 3, CHRNA4 - cholinergic receptor, nicotinic, alpha 4, GSS - glutathione synthetase, BAK1 - BCL2 antagonist/killer 1, KDR - receptor tyrosine kinase insertion domain (receptor tyrosine kinase type III), GSTP1 - glutathione-S-transferase pi 1, OGG1 - 8-oxoguanine DNA glycosylase, IL6 - interleukin 6 (interferon, beta 2).

The animal or cell may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chromosomal sequences with a disrupted structure encoding a protein associated with ALS, and zero, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chromosomally integrated sequences encoding a protein with a disrupted structure associated with ALS. Preferred proteins associated with ALS include SOD1 (superoxide dismutase 1), ALS2 (amyotrophic lateral sclerosis-associated protein 2), FUS (FUS RNA-binding protein), TARDBP (TAR-DNA binding protein), VAGFA (vascular endothelial growth factor A), VAGFB (vascular endothelial growth factor B) and VAGFC (vascular endothelial growth factor C), and any combination thereof.

Autism

U.S. Patent Application Publication No. 20110023145 describes the use of zinc finger nucleases to genetically modify cells, animals, and proteins associated with autism spectrum disorders (ASD). Autism spectrum disorders (ASD) are a group of disorders characterized by qualitative impairments in social interaction and communication, as well as restricted, repetitive, and stereotyped patterns of behavior, interests, and activities. The three disorders, autism, Asperger syndrome (AS), and pervasive developmental disorder not otherwise specified (PDD-NOS), refer to the same disorder with varying degrees of severity associated with intellectual functioning and medical conditions. ASD is predominantly a disorder that is determined by heritable factors, with a heritability of approximately 90%.

U.S. Patent Application Publication No. 20110023145 contemplates editing of any chromosomal sequences that encode proteins associated with ASD, which may be applied to the CRISPR-Cas system of the present invention. Proteins associated with ASD are typically selected based on an experimentally established association of the protein associated with ASD with the occurrence or symptom of ASD. For example, the rate of production or circulating concentration of a protein associated with ASD may be increased or decreased in a population with ASD compared to a population without ASD. Differences in protein levels may be assessed using proteomic techniques, including, but not limited to, Western blotting, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, proteins associated with ASD can be identified by obtaining gene expression profiles for protein-coding genes using genomic analysis techniques, including but not limited to DNA microarray analysis, sequence-based analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).

Non-limiting examples of disease states or disorders that may be associated with ASD-associated proteins include autism, Asperger 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 proteins: ATP10C - aminophospholipid transfer ATPase (ATP10C), MET - MET receptor tyrosine kinase, 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 - contactin-associated protein-like protein 2 (CNTNAP2), SEMA5A - neuroligin-3, DHCR7 - 7-dehydrocholesterol reductase (DHCR7), NLGN4X - neuroligin-4 X-linked, NLGN4Y - neuroligin-4 Y-linked, DOC2A - alpha-protein containing double C2-like domain, DPP6 - dipeptidylaminopeptidase-like protein 6, NLGN5 - neuroligin-5, EN2 - homeobox-encoded protein 2 (EN2), NRCAM - neuronal cell adhesion molecule (NRCAM), MDGA2, associated with mental retardation linked to fragile X chromosome (MDGA2), NRXN1 - neurexin-1, FMR2 (AFF2) - member 2 of the AF4/FMR2 family, OR4M2 - olfactory bulb receptor 4M2, FOXP2 - protein encoded by forkhead box P2 (FOXP2), OR4N4 - olfactory bulb receptor 4N4, FXR1 - fragile X-linked mental retardation autosomal homolog 1 (FXR1), OXTR - oxytocin receptor (OXTR), FXR2 - fragile X-linked mental retardation autosomal homolog 2 (FXR2), PAH - phenylalanine hydroxylase (PAH), GABRA1 - gamma-aminobutyric acid receptor alpha-1 subunit (GABRA1), PTEN - phosphatase and tensin homolog (PTEN), GABRA5 - GABAA (gamma-aminobutyric acid) receptor alpha-5 subunit (GABRA5), PTPRZ1 - protein tyrosine phosphatase-zeta receptor type (PTPRZ1), GABRB1 - gamma-aminobutyric acid receptor beta-1 subunit (GABRB1), RELN - reelin, GABRB3 - GABAA (gamma-aminobutyric acid) receptor beta-3 subunit (GABRB3), RPL10 - 60S ribosomal protein L10, GABRG1 - gamma-aminobutyric acid receptor gamma-1 subunit (GABRG1), SEMA5A - semaphorin-5A (SEMA5A), HIRIP3 - HIRA-interacting protein 3, SEZ6L2 - seizure-associated protein 6 homolog-like protein 2 (mouse), HOXA1 - Hox homeobox A1 (HOXA1)-encoded protein, SHANK3 - SH3 and multiple ankyrin repeat domain-containing protein 3 (SHANK3), IL6 - interleukin-6, SHBZRAP1 - SH3 and multiple repeat domain-containing protein 3 ankyrin (SHBZRAP1), LAMB1 - laminin, beta-1 subunit (LAMB1), SLC6A4 - serotonin transporter (SERT), MAPK3 - mitogen-activated protein kinase 3, TAS2R1 - taste receptor type 2, member 1 (TAS2R1), MAZ - Myc-associated zinc finger protein, TSC1 - tuberous sclerosis-associated protein 1, MDGA2 - glycosylphosphatidylinositol-related protein 2, anchor form 2, containing MAM domain (MDGA2), TSC2 - tuberous sclerosis-associated protein 2, MECP2 - methyl-CpG-binding protein 2 (MECP2), UBE3A - ubiquitin protein ligase E3A (UBE3A), MECP2 - methyl-CpG-binding protein 2 (MECP2), WNT2 - MMTV integration site of the Wingless type, representative of the 2nd family (WNT2).

The identity of the ASD-associated protein whose chromosomal sequence is edited can and will vary. In preferred embodiments, the ASD-associated proteins whose chromosomal sequence editing is performed may be benzodiazepine receptor-associated protein 1 (BZRAP1), encoded by the BZRAP1 gene, AF4/FMR2 family member 2 (AFF2), encoded by the AFF2 gene (also referred to as MFR2), fragile X-linked mental retardation-associated autosomal homolog 1 (FXR1), encoded by the FXR1 gene, or fragile X-linked mental retardation-associated autosomal homolog 2 (FXR2), encoded by the FXR2 gene, glycosylphosphatidylinositol-related protein containing MAM domain anchor form 2 (MDGA2), encoded by the MDGA2 gene, methyl-CpG binding protein 2 (MECP2), encoded by the MECP2 gene, metabotropic glutamate receptor 5 (MGLUR5), encoded by the MGLUR5-1 gene (also called GRM5), neurexin 1 protein, encoded by the NRXN1 gene, or semaphorin-5A protein (SEMA5A), encoded by the SEMA5A gene. In an exemplary embodiment, the genetically modified animal is a rat and the edited chromosomal sequences encoding a protein associated with ASD are listed below: BZRAP1 - benzodiazepine receptor (peripheral) associated protein 1 (BZRAP1) - XM_002727789, XM_213427, XM_002724533, XM_001081125, AFF2 (FMR2) - AF4/FMR2 family member 2 (AFF2) - XM_219832, XM_001054673, FXR1 - fragile X-linked mental retardation associated autosomal homolog 1 (FXR1) - NM_001012179, FXR2 - autosomal homolog 2, associated with fragile X-linked mental retardation (FXR2) - NM_001100647, MDGA2 - glycosylphosphatidylinositol-related protein containing MAM domain, anchor form 2 (MDGA2) - NM_199269, MECP2 - methyl-CpG-binding protein 2 (MECP2) - NM_022673, MGLUR5 - metabotropic glutamate receptor 5 (GRM5) (MGLUR5) - NM_017012, NRXN1 - neurexin-1 - NM_021767, SEMA5A - semaphorin-5A (SEMA5A) - NM_001107659.

Trinucleotide repeat expansion (TRE) disorders

U.S. Patent Application Publication No. 20110016540 describes the use of zinc finger nucleases to genetically modify cells, animals, and proteins associated with trinucleotide repeat expansion disorders. Trinucleotide repeat expansion disorders are complex, progressive disorders that affect neurodevelopmental biology and often impair cognitive function as well as sensorimotor function.

Trinucleotide repeat expansion-associated proteins are a diverse group of proteins associated with susceptibility to developing a trinucleotide repeat expansion disorder, the presence of a trinucleotide repeat expansion disorder, the severity of a trinucleotide repeat expansion disorder, or any combination thereof. Trinucleotide repeat expansion disorders are divided into two categories determined by the type of repeat. The most common repeat is the CAG triplet, which, when present in the coding region of a gene, codes for the amino acid glutamine (Q). These disorders are thus termed polyglutamine (poly-Q) repeat expansion disorders and include the following disorders: Huntington disease (HD); spinal muscular atrophy (SBMA); forms of spinocerebellar ataxia (SCA types 1, 2, 3, 6, 7, and 17) and dentato-rubro-pallido-Lewis atrophy (DRPLA). The remaining trinucleotide repeat expansion disorders in which the CAG triplet is not involved or the CAG triplet is not in the coding region of the gene are thus termed non-polyglutamine repeat expansion disorders. Non-polyglutamine repeat expansion disorders include fragile X syndrome (FRAXA); fragile X-linked mental retardation syndrome (FRAXE); Friedreich's ataxia (FRDA); myotonic dystrophy (DM), and forms of spinocerebellar ataxia (SCA types 8 and 12).

Proteins associated with trinucleotide repeat expansion disorders are typically selected based on an experimentally established association between the trinucleotide repeat expansion disorder-associated protein and the trinucleotide repeat expansion disorder. For example, the rate of production or circulating concentration of a protein associated with a trinucleotide repeat expansion disorder may be increased or decreased in a population with a trinucleotide repeat expansion disorder compared to a population without a trinucleotide repeat expansion disorder. Differences in protein levels can be assessed using proteomic techniques, including but not limited to Western blotting, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, proteins associated with trinucleotide repeat expansion disorders can be identified by generating gene expression profiles for protein-coding genes using genomic analysis techniques, including but not limited to DNA microarray analysis, sequence-based analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).

Non-limiting examples of proteins associated with trinucleotide repeat expansion disorders include AR (androgen receptor), FMR1 (Fragile X-linked mental retardation-associated protein 1), HTT (huntingtin), DMPK (myotonic dystrophy-associated protein kinase), FXN (frataxin), ATXN2 (ataxin 2), ATN1 (utrophin 1), FEN1 (flap structure-specific endonuclease 1), TNRC6A (trinucleotide repeat-containing gene 6A protein), PABPN1 (nuclear poly(A)-binding protein 1), JPH3 (junctophilin 3), MED15 (mediator complex subunit 15), ATXN1 (ataxin 1), ATXN3 (ataxin 3), TBP (TATA box binding protein), CACNA1A (alpha-1A subunit of P/Q voltage-dependent calcium channel), ATXN80S (protein synthesized from the opposite strand of ATXN8 (non-coding protein)), PPP2R2B (beta isoform of protein phosphatase 2 regulatory subunit B), ATXN7 (ataxin 7), TNRC6B (protein encoded by gene 6B containing trinucleotide repeats), TNRC6C (protein encoded by gene 6C containing trinucleotide repeats), CELF3 (CUGBP, member 3 of Elav-like proteins family), MAB21L1 (mab-21-like protein 1 (C. elegans)), MSH2 (mutS homolog 2, associated with nonpolyposis colorectal cancer type 1 (E. coli)), TMEM185A (transmembrane protein 185A), SIX5 (protein encoded by homeobox 5 SIX), CNPY3 (homolog of Canopy 3 (zebrafish)), FRAXE (folate deficiency rare fragile site-associated protein, fra(X)(q28)E), GNB2 (guanine nucleotide binding protein (G protein) beta polypeptide 2), RPL14 (ribosomal protein L14), ATXN8 (ataxin 8), INSR (insulin receptor), TTR (transthyretin), EP400 (E1A-binding protein p400), GIGYF2 (GYF 2 protein interacting with GRB10), OGG1 (8-oxoguanine DNA glycosylase), STC1 (stanniocalcin 1), CNDP1 (carnosine dipeptidase 1 (M20 family metallopeptidase)), C10orf2 (chromosome 10 open reading frame 2 protein), MAML3 (mastermind-like protein 3 (Drosophila)), DKC1 (dyskeratosis congenita-associated protein 1, dyskerin), PAXIP1 (PAX-interacting protein 1 (with transcription activation domain)), CaSK (calcium/calmodulin-dependent serine protein kinase (MAGUK family)), MAPT (microtubule-associated protein tau), SP1 (Sp1 transcription factor), POLG (polymerase gamma (DNA-directed)), AFF2 (AF4/FMR2 family member 2), THBS1 (thrombospondin 1), TP53 (p53 tumor protein), ESR1 (estrogen receptor 1), CGGBP1 (CGG triplet repeat-binding protein 1), ABT1 (basal transcription activator 1), KLK3 (kallikrein-related peptidase 3), PRNP (prion protein), JUN (jun oncogene), KCNN3 (medium/small-conductance calcium-activated potassium channel, member 3 of the N subfamily), BAX (BCL2-associated protein X), FRAXA (folate deficiency rare fragile site-associated protein, fra(X)(q27.3) A (macro-orchidism, mental retardation)), KBTBD10 (Kelch repeat and BTB domain-containing protein 10 (POZ)), MBNL1 (muscleblind-like protein (Drosophila)), RAD51 (RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae)), NCOA3 (nuclear receptor coactivator 3), ERDA1 (repeat domain expansion protein, CAG/CTG 1), TSC1 (tuberous sclerosis-associated protein 1), COMP (cartilage oligomeric matrix protein), GCLC (catalytic subunit of glutamate cysteine ligase), RRAD (Ras-related protein associated with diabetes mellitus), MSH3 (mutS homolog 3 (E. coli)), DRD2 (dopamine D2 receptor), CD44 (CD44 molecule (Indian blood group system)), CTCF (CCCTC-binding factor (protein with "zinc (with fingers")), CCND1 (cyclin D1), CLSPN (claspin homolog (Xenopus laevis)), MEF2A (myocyte enhancer factor 2A), PTPRU (protein tyrosine phosphatase receptor type U), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TRIM22 (triple motif-containing protein 22), WT1 (Wilms tumor protein 1), AHR (aryl hydrocarbon receptor), GPX1 (glutathione peroxidase 1), TPMT (thiopurine-S-methyltransferase), NDP (Norrie disease (pseudoglioma)-associated protein), ARX (aristaless-related homeobox protein), MUS81 (MUS81 endonuclease homolog (S. cerevisiae)), TYR (tyrosinase (oculocutaneous albinism IA)), EGR1 (early growth response protein 1), UNG (uracil DNA glycosylase), NUMBL (numb homolog-like protein (Drosophila)), FABP2 (fatty acid binding protein 2 in the intestine), EN2 (engrailed homeobox 2-derived protein), CRYGC (gamma-C-crystallin), SRP14 (signal recognition particle homologous protein 14 kDa Alu RNA-binding protein), CRYGB (gamma-B-crystallin), PDCD1 (programmed cell death protein 1), HOXA1 (homeobox A1-derived protein), ATXN2L (ataxin-2-like protein), PMS2 (PMS2, postmeiotic segregation enhancement counteracting protein 2 (S. cerevisiae)), GLA (alpha-galactosidase), CBL (a protein encoded by a sequence transforming with the ecotropic retrovirus Cas-Br-M (mouse)), FTH1 (ferritin heavy subunit polypeptide 1), IL12RB2 (interleukin 12 receptor beta-2 subunit), OTX2 (a protein encoded by orthodenticle homeobox 2), HOXA5 (a protein encoded by homeobox A5), POLG2 (an accessory gamma-2 subunit of the polymerase (DNA-directed)), DLX2 (a protein encoded by distal-less 2), SIRPA (signal regulatory protein alpha), OTX1 (a protein encoded by orthodenticle homeobox 1), AHRR (aryl hydrocarbon receptor repressor), MANF (mesencephalic neurotrophic factor-derived astrocytes), TMEM158 (transmembrane protein 158 (gene/pseudogene)) and ENSG00000078687.

Preferred proteins associated with trinucleotide repeat expansion disorders include HTT (huntingtin), AR (androgen receptor), FXN (frataxin), Atxn3 (ataxin), Atxn1 (ataxin), Atxn2 (ataxin), Atxn7 (ataxin), Atxn10 (ataxin), DMPK (myotonic dystrophy-associated protein kinase), Atn1 (utrophin 1), CBP (creb-binding protein), VLDLR (very low density lipoprotein receptor), and any combination thereof.

Alzheimer's disease

U.S. Patent Application Publication No. 20110023153 describes the use of zinc finger nucleases to genetically modify cells, animals, and proteins associated with Alzheimer's disease. Once modified, the cells and animals can be further assayed using known methods to study the effects of targeted mutations on the development and/or progression of AD using measures commonly used in AD research, such as, but not limited to, learning and memory, anxiety, depression, addiction, and sensorimotor function, as well as assays that measure behavioral, functional, pathological, metabolic, and biochemical characteristics.

The present disclosure contemplates editing any chromosomal sequences that encode proteins associated with AD. Proteins associated with AD are typically selected based on experimentally confirmed association of the AD-associated protein with AD. For example, the rate of production or circulating concentration of an AD-associated protein may be increased or decreased in a population with AD compared to a population without AD. Differences in protein levels can be assessed using proteomic techniques, including but not limited to Western blotting, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, proteins associated with AD can be identified by generating gene expression profiles for genes encoding the proteins using genomic analysis techniques, including but not limited to DNA microarray analysis, sequence-based analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).

Examples of Alzheimer's disease-associated proteins may include, for example, the very low density lipoprotein receptor protein (VLDLR) encoded by the VLDLR gene, the ubiquitin-like modifier activating enzyme 1 (UBA1) encoded by the UBA1 gene, or the NEDD8-activating enzyme E1 catalytic subunit protein (UBE1C) encoded by the UBA3 gene.

By way of non-limiting example, proteins associated with AD include, but are not limited to, the proteins listed below: chromosomally encoded protein ALAS2, delta-aminolevulinate synthase 2 (ALAS2), ABCA1 - ATP-binding cassette transporter (ABCA1), ACE - angiotensin I-converting enzyme (ACE), APOE - apolipoprotein E precursor (APOE), APP - amyloid precursor protein (APP), AQP1 - aquaporin 1 protein (AQP1), BIN1 - Myc-box-dependent interacting protein 1 or adaptor protein integrator 1 (BIN1), BDNF - brain-derived neurotrophic factor (BDNF), BTNL8 - butyrophilin-like protein 8 (BTNL8), C1ORF49 - chromosome 1 open reading frame 49 protein, CDH4 - cadherin-4, CHRNB2 - neuronal acetylcholine receptor, subunit beta-2, CKLFSF2 - CKLF-like protein 2 containing MARVEL transmembrane domain (CKLFSF2), CLEC4E - C-type lectin domain, family 4, member e (CLEC4E), CLU - clusterin protein (also known as apolipoprotein J), CR1 - erythrocyte complement receptor 1 (CR1, also known as CD35, the C3b/C4b receptor, and the immune adhesion receptor), CR1L - erythrocyte complement receptor 1 (CR1L), CSF3R - granulocyte colony-stimulating factor receptor 3 (CSF3R), CST3 - cystatin C or cystatin 3, CYP2C - cytochrome P450 2C, DAPK1 - death-associated protein kinase 1 (DAPK1), ESR1 - estrogen receptor 1, FCAR - Fc fragment of the receptor for IgA (FCAR, also known as CD89), FCGR3B - Fc fragment of the receptor IIIb for IgG, low affinity (FCGR3B or CD16b), FFA2 - free fatty acid receptor 2 (FFA2), FGA - fibrinogen (factor I), GAB2 - GRB2-associated binding protein 2 (GAB2), GAB2 - GRB2-associated binding protein 2 (GAB2), GALP - galanin-like peptide, GAPDHS - glyceraldehyde-3-phosphate dehydrogenase of spermatogenic cells (GAPDHS), GMPB - GMBP, HP - haptoglobin (HP), HTR7 - 5-hydroxytryptamine (serotonin) receptor 7 (adenyl cyclase-coupled), IDE - insulin degrading enzyme IF127 IF127, IFI6 - interferon alpha-inducible protein 6 (IFI6), IFIT2 - interferon-inducible protein with tetratricopeptide repeats 2 (IFIT2), IL1RN - interleukin-1 receptor antagonist (IL-1RA), IL8RA - interleukin 8 receptor, alpha (IL8RA or CD181), IL8RB - interleukin 8 receptor, beta (IL8RB), JAG1 - Jagged 1 protein (JAG1), KCNJ15 - potassium influx channel, subfamily J, member 15 (KCNJ15), LRP6 - low-density lipoprotein receptor-related protein 6 (LRP6), MAPT - microtubule-associated protein tau (MAPT), MARK4 - MAP/microtubule affinity-regulated kinase 4 (MARK4), MPHOSPH1 - M-phase phosphoprotein 1, MTHFR - 5,10-methylenetetrahydrofolate reductase, MX2 - interferon-inducible GTP-binding protein Mx2, NBN - nibrin, also known as NBN, NCSTN - nicastrin, NIACR2 - niacin receptor 2 (NIACR2, also known as GPR109B), NMNAT3 - nicotinamide nucleotide adenylyltransferase 3, NTM - neurotrimin (or HNT), ORM1 - orosomucoid 1 (ORM1) or alpha-1-acid glycoprotein 1, P2RY13 - P2Y purinergic receptor 13 (P2RY13), PBEF1 - nicotinamide phosphoribosyltransferase (NAmPRTase or Nampt), also known as pre-B-cell colony-stimulating factor 1 (PBEF1) or visfatin, PCK1 - phosphoenolpyruvate carboxykinase, PICALM - phosphatidylinositol-binding protein involved in the formation of clathrin complexes (PICALM), PLAU - urokinase-type plasminogen activator (PLAU), PLXNC1 - plexin C1 (PLXNC1), PRNP - prion protein, PSEN1 - presenilin 1 protein (PSEN1), PSEN2 - presenilin 2 protein (PSEN2), PTPRA - protein tyrosine receptor phosphatase type A (PTPRA), RALGPS2 - Ral GEF with PH domain and SH3-binding motif 2 (RALGPS2), RGSL2 - G protein-coupled signaling regulator 2-like protein (RGSL2), SELENBP1 - selenium-binding protein 1 (SELNBP1), SLC25A37 - mitoferrin-1, SORL1 - sortilin-related receptor L (DLR class) repeat-containing protein A (SORL1), TF - transferrin, TFAM - mitochondrial transcription factor A, TNF - tumor necrosis factor, TNFRSF10C - tumor necrosis factor receptor superfamily member 10C (TNFRSF10C), TNFSF10 - tumor necrosis factor receptor superfamily (TRAIL) member 10a (TNFSF10), UBA1 - ubiquitin-like modifier-activating enzyme 1 (UBA1), UBA3 - NEDD8-activating enzyme E1 catalytic subunit protein (UBE1C), UBB - ubiquitin B protein (UBB), UBQLN1 - ubiquilin-1, UCHL1 - ubiquitin carboxy-terminal esterase protein L1 (UCHL1), UCHL3 - ubiquitin carboxy-terminal hydrolase L3 isoenzyme protein (UCHL3), VLDLR - very low density lipoprotein receptor protein (VLDLR).

In exemplary embodiments, the AD-associated proteins whose chromosomal sequence editing is performed can be the very low density lipoprotein receptor (VLDLR) protein encoded by the VLDLR gene, the ubiquitin-like modifier activating enzyme 1 (UBA1) enzyme encoded by the UBA1 gene, the NEDD8 activating enzyme E1 catalytic subunit protein (UBE1C) protein encoded by the UBA3 gene, the aquaporin 1 (AQP1) protein encoded by the AQP1 gene, the ubiquitin carboxy-terminal esterase L1 protein (UCHL1) protein encoded by the UCHL1 gene, the ubiquitin carboxy-terminal hydrolase isoenzyme L3 protein (UCHL3) encoded by the UCHL3 gene, the ubiquitin B (UBB) protein encoded by the UBB gene, the microtubule-associated tau protein (MAPT) encoded by the MAPT gene, protein tyrosine phosphatase receptor type A (PTPRA), encoded by the PTPRA gene, phosphatidylinositol-binding protein involved in clathrin complex formation (PICALM), encoded by the PICALM gene, clusterin protein (also known as apolipoprotein J), encoded by the CLU gene, presenilin 1 protein, encoded by the PSEN1 gene, presenilin 2 protein, encoded by the PSEN2 gene, sortilin-related receptor L (DLR class), protein containing repeats A (SORL1), encoded by the SORL1 gene, amyloid precursor protein (APP), encoded by the APP gene, apolipoprotein E precursor (APOE), encoded by the APOE gene, or brain-derived neurotrophic factor (BDNF), encoded by the BDNF gene. In an exemplary embodiment, the genetically modified animal is a rat and the edited chromosomal sequences encoding a protein associated with AD are as follows: APP - amyloid precursor protein (APP) - NM_019288, AQP1 - aquaporin 1 protein (AQP1) - NM_012778, BDNF - brain-derived neurotrophic factor - NM_012513, CLU - clusterin protein (also known as apolipoprotein J) - NM_053021, MAPT - microtubule-associated protein tau (MAPT) - NM_017212, PICALM - phosphatidylinositol-binding protein involved in the formation of clathrin complexes (PICALM) - NM_053554, PSEN1 - presenilin protein 1 (PSEN1) - NM_019163, PSEN2 - presenilin 2 protein (PSEN2) - NM_031087, PTPRA - protein tyrosine phosphatase receptor type A (PTPRA) - NM_012763, SORL1 - sortilin-related receptor L (DLR class), protein containing repeats A (SORL1) - NM_053519, XM_001065506, XM_217115, UBA1 - ubiquitin-like modifier activating enzyme 1 (UBA1) - NM_001014080, UBA3 - protein that is the catalytic subunit of NEDD8-activating enzyme E1 (UBE1C) - NM_057205, UBB - ubiquitin B protein (UBB) - NM_138895, UCHL1 - ubiquitin carboxy-terminal esterase protein L1 (UCHL1) - NM_017237, UCHL3 - ubiquitin carboxy-terminal hydrolase protein L3 isoenzyme (UCHL3) - NM_001110165, VLDLR - very low density lipoprotein receptor protein (VLDLR) - NM_013155.

The animal or cell may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more chromosomal sequences with a disrupted structure encoding a protein associated with AD, and zero, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more chromosomally integrated sequences encoding a protein associated with AD.

The edited or integrated chromosomal sequence can be modified to encode an altered protein associated with AD. A number of mutations in chromosomal sequences associated with AD have been associated with AD. For example, the missense mutation V7171 (i.e., valine at position 717 is replaced by isoleucine) in APP results in a familial form of AD. Several mutations in the presenilin-1 protein, such as H163R (i.e., histidine at position 163 is replaced by arginine), A246E (i.e., alanine at position 246 is replaced by glutamate), L286V (i.e., leucine at position 286 is replaced by valine), and C410Y (i.e., cysteine at position 410 is replaced by tyrosine), result in a familial form of Alzheimer's disease type 3. Mutations in the presenilin-2 protein, such as N141I (i.e., asparagine at position 141 is replaced by isoleucine), M239V (i.e., methionine at position 239 is replaced by valine), and D439A (i.e., aspartate at position 439 is replaced by alanine) lead to familial Alzheimer's disease type 4. Other associations of gene variants in genes associated with AD and disease are known in the art. See, for example, Waring et al. (2008) Arch. Neurol. 65:329-334, the disclosure of which is incorporated herein by reference in its entirety.

Examples of disease-associated genes

Examples of disease-associated genes and polynucleotides are shown in Tables A and B. Examples of biochemical signaling pathway-associated genes and polynucleotides are shown in Table C.

Table A DISEASE/DISORBORS GENE(GENES) Neoplasia PTEN; ATM; ATR; EGFR; ERBB2; ERBB3; ERBB4; Notch1; Notch2; Notch3; Notch4; AKT; AKT2; AKT3; HIF; HIF1a; HIF3a; Met; HRG; Bcl2; PPAR alpha; PPAR gamma; WT1 (Wilms tumor); members of the FGF receptor family (5 representatives: 1, 2, 3, 4, 5); CDKN2a; APC; RB (retinoblastoma); MEN1; VHL; BRCA1; BRCA2; AR (androgen receptor); TSG101; IGF; IGF receptor; Igf1 (4 option); Igf2 (3 variants); Igf receptor 1; Igf receptor 2; Bax; Bcl2; caspase family (9 members: 1, 2, 3, 4, 6, 7, 8, 9, 12); Kras; Apc Age-related degeneration Abcr; Ccl2; Cc2; cp (ceruloplasmin); Timp3; cathepsin D; yellow spot Vldlr;Ccr2 Schizophrenic Neuregulin 1 (Nrg1); Erb4 (receptor for neuregulin); Complexin 1 (Cplx1); Tph1, tryptophan hydroxylase; Tph2, tryptophan hydroxylase 2; neurexin 1; GSK3; GSK3a; GSK3b violations 5-HTT (Slc6a4); COMT; DRD (Drd1a); SLC6A3; DAOA; DTNBP1; Dao (Dao1) Associated with trinucleotide repeats HTT (Huntington's disease); SBMA/SMAX1/AR (Huntington's disease) violations Kennedy); FXN/X25 (Friedreich's ataxia); ATX3 (Machado's disease- Joseph); ATXN1 and ATXN2 (forms of spinocerebellar ataxia); DMPK (myotonic dystrophy); utrophin-1 and Atn1 (DRPLA disease); CBP (Creb-BP - general instability); VLDLR (Alzheimer's); Atxn7; Atxn10 Fragile X syndrome FMR2;FXR1;FXR2;mGLUR5 Associated with secretase activity APH-1 (alpha and beta); presenilin (Psen1); nicastrine violations (Ncstn); PEN-2 Other Nos1; Parp1; Nat1; Nat2 Prion-related disorders Prp ALS SOD1; ALS2; STEX; FUS; TARDBP; VEGF (VEGF-a; VEGF-b; VEGF-c) Addiction to drugs Prkce (alcohol); Drd2; Drd4; ABAT (alcohol); GRIA2; Grm5; Grin1; Htr1b; Grin2a; Drd3; Pdyn; Gria1 (alcohol) Autism Mecp2; BZRAP1; MDGA2; Sema5A; neurexin 1; fragile X (FMR2 (AFF2); FXR1; FXR2; Mglur5) Alzheimer's disease E1; CHIP; UCH; UBB; Tau; LRP; PICALM; clusterin; PS1; SORL1; CR1; Vldlr; Uba1; Uba3; CHIP28(Aqp1, aquaporin 1); Uchl1; Uchl3; APP Inflammation IL-10; IL-1 (IL-1a; IL-1b); IL-13; IL-17 (IL-17a (CTLA8); IL- 17b; IL-17c; IL-17d; IL-17f); II-23; Cx3cr1; ptpn22; TNFa; NOD2/CARD15 for IBD; IL-6; IL-12 (IL-12a; IL-12b); CTLA4;Cx3cl1 Parkinson's disease x-synuclein; DJ-1; LRRK2; parkin; PINK1

Table B Diseases and disorders associated with the circulatory system and blood 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, RING11, 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 (F11); factor XII deficiency (F12, HAF); factor XIIIA deficiency (F13A1, F13A); factor XIIIB deficiency (F13B); Fanconi syndrome (FANCA, FACA, FA1, FA, FAA, FAAP95, FAAP90, FLJ34064, FANCB, FANCC, FACC, BRCA2, FANCD1, FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BRIP1, BACH1, FANCJ, PHF9, FANCL, FANCM, KIAA1596); hemophagocytic lymphohistiocytosis disorders (PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3, HLH3, FHL3); hemophilia A (F8, F8C, HEMA); hemophilia B (F9, HEMB), hemorrhagic disorders (PI, ATT, F5); leukocyte-related deficiencies and disorders (ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4); sickle cell anemia (HBB); thalassemia (HBA2, HBB, HBD, LCRB, HBA1). Cellular dysregulation-related diseases and disorders and cancer diseases and disorders B-cell non-Hodgkin lymphoma (BCL7A, BCL7); leukemia (TAL1, 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, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT, LPP, NPM1, NUP214, D9S46E, CAN, CAIN, RUNX1, CBFA2, AML1, WHSC1L1, NSD3, FLT3, AF1Q, NPM1, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF10, CALM, CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF, WSS, NFNS, PTPN11, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA, GATA1, GF1, ERYF1, NFE1, ABL1, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN, CAIN). Inflammation and immune system related diseases and disorders AIDS (KIR3DL1, NKAT3, NKB1, AMB11, KIR3DS1, IFNG, CXCL12, SDF1); autoimmune lymphoproliferative syndrome (TNFRSF6, APT1, FAS, CD95, ALPS1A); combined immunodeficiency (IL2RG, SCIDX1, SCIDX, IMD4); HIV-1 (CCL5, SCYA5, D17S136E, TCP228), HIV susceptibility or HIV infection (IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5)); types of immunodeficiency (CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSF5, CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI); inflammation (IL-10, IL-1 (IL-1a, IL-1b), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-17f), II-23, Cx3cr1, ptpn22, TNFa, NOD2/CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cl1); severe combined immunodeficiency (SCID) types (JAK3, JAKL, DCLRE1C, ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDX1, SCIDX, IMD4). Metabolic, hepatic, renal and protein metabolism diseases and disorders Amyloid neuropathy (TTR, PALB); amyloidosis (APOA1, APP, AAA, CVAP, AD1, GSN, FGA, LYZ, TTR, PALB); cirrhosis (KRT18, 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); liver adenoma, 142330 (TCF1, HNF1A, MODY3), liver failure, early-onset and with neurologic impairment (SCOD1, SCO1), hepatic lipase deficiency (LIPC), hepatoblastoma, cancer and carcinoma forms (CTNNB1, PDGFRL, PDGRL, PRLTS, AXIN1, AXIN, CTNNB1, TP53, P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5); medullary cystic nephropathy disease (UMOD, HNFJ, FJHN, MCKD2, ADMCKD2); phenylketonuria (PAH, PKU1, QDPR, DHPR, PTS); polycystic kidney and liver disease (FCYT, PKHD1, ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63). Muscle/Bone Diseases and Disorders Becker myopathy (DMD, BMD, MYF6), Duchenne myopathy (DMD, BMD); Emery-Dreifuss muscular dystrophy (LMNA, LMN1, EMD2, FPLD, CMD1A, HGPS, LGMD1B, LMNA, LMN1, EMD2, FPLD, CMD1A); facioscapulohumeral myopathy (FSHMD1A, FSHD1A); muscular dystrophy (FKRP, MDC1C, LGMD2I, LAMA2, LAMM, LARGE, KIAA0609, 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, TRIM32, HT2A, LGMD2H, FKRP, MDC1C, LGMD2I, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1); osteoporosis (LRP5, BMND1, LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTM1, GL, TCIRG1, TIRC7, OC116, OPTB1); muscle atrophy (VAPB, VAPC, ALS8, SMN1, SMA1, SMA2, SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2, CATF1, SMARD1). Neurological and neuronal diseases and disorders ALS (SOD1, ALS2, STEX, FUS, TARDBP, VEGF (VEGF-a, VEGF-b, VEGF-c); Alzheimer's disease (APP, AAA, CVAP, AD1, APOE, AD2, PSEN2, AD4, STM2, APBB2, FE65L1, NOS3, PLAU, URK, ACE, DCP1, ACE1, MPO, PACIP1, PAXIP1L, PTIP, A2M, BLMH, BMH, PSEN1, AD3); autism (Mecp2, BZRAP1, MDGA2, Sema5A, neurexin 1, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2); fragile X syndrome (FMR2, FXR1, FXR2, mGLUR5); Huntington's disease and Huntington-like disorders (HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17); Parkinson's disease (NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJ1, PARK7, LRRK2, PARK8, PINK1, PARK6, UCHL1, PARK5, SNCA, NACP, PARK1, 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 (Nrg1), Erb4 (neuregulin receptor), complexin 1 (Cplx1), Tph1, tryptophan hydroxylase, Tph2, tryptophan hydroxylase 2, neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), COMT, DRD (Drd1a), SLC6A3, DAOA, DTNBP1, Dao (Dao1)); secretase activity-related disorders (APH-1 (alpha and beta), presenilin (Psen1), nicastrin, (Ncstn), PEN-2, Nos1, Parp1, Nat1, Nat2); trinucleotide repeat-related disorders (HTT (Huntington's disease), SBMA/SMAX1/AR (Kennedy disease), FXN/X25 (ataxia Friedreich's disease), ATX3 (Machado-Joseph disease), ATXN1 and ATXN2 (forms of spinocerebellar ataxia), DMPK (myotonic dystrophy), utrophin-1 and Atn1 (DRPLA-type disease), CBP (Creb-BP - general instability), VLDLR (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, MP19, CRYGD, CRYG4, BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM, MIP, AQP0, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA, CRYA1, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1); corneal opacity and dystrophy (APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3, CDG2, TACSTD2, TROP2, M1S1, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD); 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 amaurosis (CRB1, RP12, CRX, CORD2, CRD, RPGRIP1, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4, GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3); macular dystrophy (ELOVL4, ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, VMD2).

Table C CELLULAR FUNCTION GENES Signal transduction involving PI3K/AKT PRKCE; ITGAM; ITGA5; IRAK1; PRKAA2; EIF2AK2; PTEN; EIF4E; 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; ITGB1; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; CHUK; PDPK1; PPP2R5C; CTNNB1; MAP2K1; NFKB1; PAK3; ITGB3; CCND1; GSK3A; FRAP1; SFN; ITGA2; TTK; CSNK1A1; BRAF; GSK3B; AKT3; FOXO1; SGK; HSP90AA1;RPS6KB1 Signal transduction involving ERK/MAPK PRKCE; ITGAM; ITGA5; HSPB1; IRAK1; PRKAA2; EIF2AK2; RAC1; RAP1A; TLN1; EIF4E; ELK1; GRK6; MAPK1; RAC2; PLK1; AKT2; PIK3CA; CDK8; CREB1; PRKCI; PTK2; FOS; RPS6KA4; PIK3CB; PPP2R1A; PIK3C3; MAPK8; MAPK3; ITGA1; ETS1; KRAS; MYCN; EIF4EBP1; PPARG; PRKCD; PRKAA1; MAPK9; SRC; CDK2; PPP2CA; PIM1; PIK3C2A; ITGB7; YWHAZ; PPP1CC; KSR1; PXN; RAF1; FYN; DYRK1A; ITGB1; MAP2K2; PAK4; PIK3R1; STAT3; PPP2R5C; MAP2K1; PAK3; ITGB3; ESR1; ITGA2; MYC; TTK; CSNK1A1; CRKL; BRAF; ATF4; PRKCA; SRF; STAT1; SGK Signal transmission RAC1; TAF4B; EP300; SMAD2; TRAF6; PCAF; ELK1; with the participation of the glucocorticoid receptor MAPK1; SMAD3; AKT2; IKBKB; NCOR2; UBE2I; PIK3CA; CREB1; FOS; HSPA5; NFKB2; BCL2; MAP3K14; STAT5B; PIK3CB; PIK3C3; MAPK8; BCL2L1; MAPK3; TSC22D3; MAPK10; NRIP1; KRAS; MAPK13; RELA; STAT5A; MAPK9; NOS2A; PBX1; NR3C1; PIK3C2A; CDKN1C; TRAF2; SERPINE1; NCOA3; MAPK14; TNF; RAF1; IKBKG; MAP3K7; CREBBP; CDKN1A; MAP2K2; JAK1; IL8; NCOA2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; TGFBR1; ESR1; SMAD4; CEBPB; JUN; AR; AKT3; CCL2; MMP1; STAT1; IL6; HSP90AA1 Signal transduction for axonal guidance PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; ADAM12; IGF1; RAC1; RAP1A; EIF4E; PRKCZ; NRP1; NTRK2; ARHGEF7; SMO; ROCK2; MAPK1; PGF; RAC2; PTPN11; GNAS; AKT2; PIK3CA; ERBB2; PRKCI; PTK2; CFL1; GNAQ; PIK3CB; CXCL12; PIK3C3; WNT11; PRKD1; GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PIK3C2A; ITGB7; GLI2; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; ADAM17; AKT1; PIK3R1; GLI1; WNT5A; ADAM10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; CRKL; RND1; GSK3B; AKT3;PRKCA Ephrin receptor-mediated signaling PRKCE; ITGAM; ROCK1; ITGA5; CXCR4; IRAK1; PRKAA2; EIF2AK2; RAC1; RAP1A; GRK6; ROCK2; MAPK1; PGF; RAC2; PTPN11; GNAS; PLK1; AKT2; DOK1; CDK8; CREB1; PTK2; CFL1; GNAQ; MAP3K14; CXCL12; MAPK8; GNB2L1; ABL1; MAPK3; ITGA1; KRAS; RHOA; PRKCD; PRKAA1; MAPK9; SRC; CDK2; PIM1; ITGB7; PXN; RAF1; FYN; DYRK1A; ITGB1; MAP2K2; PAK4; AKT1; JAK2; STAT3; ADAM10; MAP2K1; PAK3; ITGB3; CDC42; VEGFA; ITGA2; EPHA8; TTK; CSNK1A1; CRKL; BRAF; PTPN13; ATF4; AKT3;SGK Signal transmission ACTN4; PRKCE; ITGAM; ROCK1; ITGA5; IRAK1; with the participation of the actin cytoskeleton PRKAA2; EIF2AK2; RAC1; INS; ARHGEF7; GRK6; ROCK2; MAPK1; 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; ITGB1; MAP2K2; PAK4; PIP5K1A; PIK3R1; MAP2K1; PAK3; ITGB3; CDC42; APC; ITGA2; TTK; CSNK1A1; CRKL; BRAF;VAV3;SGK Signal transmission PRKCE; IGF1; EP300; RCOR1; PRKCZ; HDAC4; TGM2; for Huntington's disease MAPK1; 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; AKT3; PRKCA; CLTC; SGK; HDAC6; CASP3 Signal transduction during apoptosis PRKCE; ROCK1; BID; IRAK1; PRKAA2; EIF2AK2; BAK1; BIRC4; GRK6; MAPK1; CAPNS1; PLK1; AKT2; IKBKB; CAPN2; CDK8; FAS; NFKB2; BCL2; MAP3K14; MAPK8; BCL2L1; CAPN1; MAPK3; CASP8; KRAS; RELA; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; TP53; TNF; RAF1; IKBKG; RELB; CASP9; DYRK1A; MAP2K2; CHUK; APAF1; MAP2K1; NFKB1; PAK3; LMNA; CASP2; BIRC2; TTK; CSNK1A1; BRAF; BAX; PRKCA; SGK; CASP3; BIRC3; PARP1 B-cell receptor signaling RAC1; PTEN; LYN; ELK1; MAPK1; RAC2; PTPN11; AKT2; IKBKB; PIK3CA; CREB1; SYK; NFKB2; CAMK2A; MAP3K14; PIK3CB; PIK3C3; MAPK8; BCL2L1; ABL1; MAPK3; ETS1; KRAS; MAPK13; RELA; PTPN6; MAPK9; EGR1; PIK3C2A; BTK; MAPK14; RAF1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1; PIK3R1; CHUK; MAP2K1; NFKB1; CDC42; GSK3A; FRAP1; BCL6; BCL10; JUN; GSK3B; ATF4; AKT3; VAV3; RPS6KB1 Signal transmission ACTN4; CD44; PRKCE; ITGAM; ROCK1; CXCR4; CYBA; with leukocyte diapedesis RAC1; RAP1A; PRKCZ; ROCK2; RAC2; PTPN11; MMP14; PIK3CA; PRKCI; PTK2; PIK3CB; CXCL12; PIK3C3; MAPK8; PRKD1; ABL1; MAPK10; C.Y.B.B.; MAPK13; RHOA; PRKCD; MAPK9; SRC; PIK3C2A; BTK; MAPK14; NOX1; PXN; VIL2; VASP; ITGB1; MAP2K2; CTNND1; PIK3R1; CTNNB1; CLDN1; CDC42; F11R; ITK; CRKL; VAV3; CTTN; PRKCA; MMP1; MMP9 Integrin-mediated signal transduction ACTN4; ITGAM; ROCK1; ITGA5; RAC1; PTEN; RAP1A; TLN1; ARHGEF7; MAPK1; RAC2; CAPNS1; AKT2; CAPN2; PIK3CA; PTK2; PIK3CB; PIK3C3; MAPK8; CAV1; CAPN1; ABL1; MAPK3; ITGA1; KRAS; RHOA; SRC; PIK3C2A; ITGB7; PPP1CC; ILK; PXN; VASP; RAF1; FYN; ITGB1; MAP2K2; PAK4; AKT1; PIK3R1; TNK2; MAP2K1; PAK3; ITGB3; CDC42; RND3; ITGA2; CRKL; BRAF; GSK3B; AKT3 Signal transmission IRAK1; SOD2; MYD88; TRAF6; ELK1; MAPK1; PTPN11; in acute phase response AKT2; IKBKB; PIK3CA; FOS; NFKB2; MAP3K14; PIK3CB; MAPK8; RIPK1; MAPK3; IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; FTL; NR3C1; TRAF2; SERPINE1; MAPK14; TNF; RAF1; PDK1; IKBKG; RELB; MAP3K7; MAP2K2; AKT1; JAK2; PIK3R1; CHUK; STAT3; MAP2K1; NFKB1; FRAP1; CEBPB; JUN; AKT3; IL1R1; IL6 Signal transduction involving PTEN ITGAM; ITGA5; RAC1; PTEN; PRKCZ; BCL2L11; MAPK1; RAC2; AKT2; EGFR; IKBKB; CBL; PIK3CA; CDKN1B; PTK2; NFKB2; BCL2; PIK3CB; BCL2L1; MAPK3; ITGA1; KRAS; ITGB7; ILK; PDGFRB; INSR; RAF1; IKBKG; CASP9; CDKN1A; ITGB1; MAP2K2; AKT1; PIK3R1; CHUK; PDGFRA; PDPK1; MAP2K1; NFKB1; ITGB3; CDC42; CCND1; GSK3A; ITGA2; GSK3B; AKT3; FOXO1; CASP3; RPS6KB1 p53-mediated signal transduction PTEN; EP300; BBC3; PCAF; FASN; BRCA1; GADD45A; BIRC5; AKT2; PIK3CA; CHEK1; TP53INP1; BCL2; PIK3CB; PIK3C3; MAPK8; THBS1; ATR; BCL2L1; E2F1; PMAIP1; CHEK2; TNFRSF10B; TP73; RB1; HDAC9; CDK2; PIK3C2A; MAPK14; TP53; LRDD; CDKN1A; HIPK2; AKT1; PIK3R1; RRM2B; APAF1; CTNNB1; SIRT1; CCND1; PRKDC; ATM; SFN; CDKN2A; JUN; SNAI2; GSK3B; BAX; AKT3 Signal transmission HSPB1; EP300; FASN; TGM2; RXRA; MAPK1; NQO1; with the participation of the aryl-hydrocarbon receptor NCOR2; SP1; ARNT; CDKN1B; FOS; CHEK1; SMARCA4; NFKB2; MAPK8; ALDH1A1; ATR; E2F1; MAPK3; NRIP1; CHEK2; RELA; TP73; GSTP1; RB1; SRC; CDK2; AHR; NFE2L2; NCOA3; TP53; TNF; CDKN1A; NCOA2; APAF1; NFKB1; CCND1; ATM; ESR1; CDKN2A; MYC; JUN; ESR2; BAX; IL6; CYP1B1; HSP90AA1 Signal transmission PRKCE; EP300; PRKCZ; RXRA; MAPK1; NQO1; in xenobiotic metabolism NCOR2; PIK3CA; ARNT; PRKCI; NFKB2; CAMK2A; PIK3CB; PPP2R1A; PIK3C3; MAPK8; PRKD1; ALDH1A1; MAPK3; NRIP1; KRAS; MAPK13; PRKCD; GSTP1; MAPK9; NOS2A; ABCB1; AHR; PPP2CA; FTL; NFE2L2; PIK3C2A; PPARGC1A; MAPK14; TNF; RAF1; CREBBP; MAP2K2; PIK3R1; PPP2R5C; MAP2K1; NFKB1; KEAP1; PRKCA; EIF2AK3; IL6; CYP1B1; HSP90AA1 Signal transmission involving SAPK/JNK PRKCE; IRAK1; PRKAA2; EIF2AK2; RAC1; ELK1; GRK6; MAPK1; GADD45A; RAC2; PLK1; AKT2; PIK3CA; FADD; CDK8; PIK3CB; PIK3C3; MAPK8; RIPK1; GNB2L1; IRS1; MAPK3; MAPK10; DAXX; KRAS; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; TRAF2; TP53; LCK; MAP3K7; DYRK1A; MAP2K2; PIK3R1; MAP2K1; PAK3; CDC42; JUN; TTK; CSNK1A1; CRKL; BRAF; SGK Signal transmission involving PPAr/RXR 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; MAPK14; INSR; RAF1; IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; JAK2; CHUK; MAP2K1; NFKB1; TGFBR1; SMAD4; JUN; IL1R1; PRKCA; IL6; HSP90AA1; ADIPOQ Signal transduction involving NF-KB IRAK1; EIF2AK2; EP300; INS; MYD88; PRKCZ; TRAF6; 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; NFKB1; TLR2; BCL10; GSK3B; AKT3; TNFAIP3; IL1R1 Neuregulin-mediated signal transduction ERBB4; PRKCE; ITGAM; ITGA5; PTEN; PRKCZ; ELK1; MAPK1; PTPN11; AKT2; EGFR; ERBB2; PRKCI; CDKN1B; STAT5B; PRKD1; MAPK3; ITGA1; KRAS; PRKCD; STAT5A; SRC; ITGB7; RAF1; ITGB1; MAP2K2; ADAM17; AKT1; PIK3R1; PDPK1; MAP2K1; ITGB3; EREG; FRAP1; PSEN1; ITGA2; MYC; NRG1; CRKL; AKT3; PRKCA; HSP90AA1; RPS6KB1 Signal transmission CD44; EP300; LRP6; DVL3; CSNK1E; GJA1; SMO; with the participation of Wnt and beta-catenin AKT2; PIN1; CDH1; BTRC; GNAQ; MARK2; PPP2R1A; WNT11; SRC; DKK1; PPP2CA; SOX6; SFRP2; ILK; LEF1; SOX9; TP53; MAP3K7; CREBBP; TCF7L2; AKT1; PPP2R5C; WNT5A; LRP5; CTNNB1; TGFBR1; CCND1; GSK3A; DVL1; APC; CDKN2A; MYC; CSNK1A1; GSK3B; AKT3; SOX2 Insulin receptor mediated signal transduction PTEN; INS; EIF4E; PTPN1; PRKCZ; MAPK1; TSC1; PTPN11; AKT2; CBL; PIK3CA; PRKCI; PIK3CB; PIK3C3; MAPK8; IRS1; MAPK3; TSC2; KRAS; EIF4EBP1; SLC2A4; PIK3C2A; PPP1CC; INSR; RAF1; FYN; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; PDPK1; MAP2K1; GSK3A; FRAP1; CRKL; GSK3B; AKT3; FOXO1; SGK; RPS6KB1 Signaling involving IL-6 HSPB1; TRAF6; MAPKAPK2; ELK1; MAPK1; PTPN11; IKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK3; MAPK10; IL6ST; KRAS; MAPK13; IL6R; RELA; SOCS1; MAPK9; ABCB1; TRAF2; MAPK14; TNF; RAF1; IKBKG; RELB; MAP3K7; MAP2K2; IL8; JAK2; CHUK; STAT3; MAP2K1; NFKB1; CEBPB; JUN; IL1R1; SRF; IL6 Hepatic cholestasis PRKCE; IRAK1; INS; MYD88; PRKCZ; TRAF6; PPARA; RXRA; IKBKB; PRKCI; NFKB2; MAP3K14; MAPK8; PRKD1; MAPK10; RELA; PRKCD; MAPK9; ABCB1; TRAF2; TLR4; TNF; INSR; IKBKG; RELB; MAP3K7; IL8; CHUK; NR1H2; TJP2; NFKB1; ESR1; SREBF1; FGFR4; JUN;IL1R1;PRKCA;IL6 Signal transduction involving IGF-1 IGF1; PRKCZ; ELK1; MAPK1; PTPN11; NEDD4; AKT2; PIK3CA; PRKCI; PTK2; FOS; PIK3CB; PIK3C3; MAPK8; IGF1R; IRS1; MAPK3; IGFBP7; KRAS; PIK3C2A; YWHAZ; PXN; RAF1; CASP9; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; IGFBP2; SFN; JUN; CYR61; AKT3; FOXO1; SRF; CTGF; RPS6KB1 NRF2-mediated response to PRKCE; EP300; SOD2; PRKCZ; MAPK1; SQSTM1; oxidative stress NQO1; PIK3CA; PRKCI; FOS; PIK3CB; PIK3C3; MAPK8; PRKD1; MAPK3; KRAS; PRKCD; GSTP1; MAPK9; FTL; NFE2L2; PIK3C2A; MAPK14; RAF1; MAP3K7; CREBBP; MAP2K2; AKT1; PIK3R1; MAP2K1; PPIB; JUN; KEAP1; GSK3B; ATF4; PRKCA; EIF2AK3; HSP90AA1 Liver fibrosis/activation EDN1; IGF1; KDR; FLT1; SMAD2; FGFR1; MET; PGF; hepatic stellate cells SMAD3; EGFR; FAS; CSF1; NFKB2; BCL2; MYH9; IGF1R; IL6R; RELA; TLR4; PDGFRB; TNF; RELB; IL8; PDGFRA; NFKB1; TGFBR1; SMAD4; VEGFA; BAX; IL1R1; CCL2; HGF; MMP1; STAT1; IL6; CTGF; MMP9 Signal transduction involving PPAR EP300; INS; TRAF6; PPARA; RXRA; MAPK1; IKBKB; NCOR2; FOS; NFKB2; MAP3K14; STAT5B; MAPK3; NRIP1; KRAS; PPARG; RELA; STAT5A; TRAF2; PPARGC1A; PDGFRB; TNF; INSR; RAF1; IKBKG; RELB; MAP3K7; CREBBP; MAP2K2; CHUK; PDGFRA; MAP2K1; NFKB1; JUN; IL1R1; HSP90AA1 Signal Transduction Involving Fc-epsilon-RI PRKCE; RAC1; PRKCZ; LYN; MAPK1; RAC2; PTPN11; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3; MAPK8; PRKD1; MAPK3; MAPK10; KRAS; MAPK13; PRKCD; MAPK9; PIK3C2A; BTK; MAPK14; TNF; RAF1; FYN; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; AKT3; VAV3;PRKCA Receptor-mediated signal transduction, PRKCE; RAP1A; RGS16; MAPK1; GNAS; AKT2; IKBKB; G protein coupled PIK3CA; CREB1; GNAQ; NFKB2; CAMK2A; PIK3CB; PIK3C3; MAPK3; KRAS; RELA; SRC; PIK3C2A; RAF1; IKBKG; RELB; FYN; MAP2K2; AKT1; PIK3R1; CHUK; PDPK1; STAT3; MAP2K1; NFKB1; BRAF; ATF4; AKT3; PRKCA Metabolism PRKCE; IRAK1; PRKAA2; EIF2AK2; PTEN; GRK6; inositol phosphate MAPK1; PLK1; AKT2; PIK3CA; CDK8; PIK3CB; PIK3C3; MAPK8; MAPK3; PRKCD; PRKAA1; MAPK9; CDK2; PIM1; PIK3C2A; DYRK1A; MAP2K2; PIP5K1A; PIK3R1; MAP2K1; PAK3; ATM; TTK; CSNK1A1; BRAF; SGK Signal transduction involving PDGF EIF2AK2; ELK1; ABL2; MAPK1; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; CAV1; ABL1; MAPK3; KRAS; SRC; PIK3C2A; PDGFRB; RAF1; MAP2K2; JAK1; JAK2; PIK3R1; PDGFRA; STAT3; SPHK1; MAP2K1; MYC; JUN; CRKL; PRKCA; SRF; STAT1; SPHK2 VEGF-mediated signaling ACTN4; ROCK1; KDR; FLT1; ROCK2; MAPK1; PGF; AKT2; PIK3CA; ARNT; PTK2; BCL2; PIK3CB; PIK3C3; BCL2L1; MAPK3; KRAS; HIF1A; NOS3; PIK3C2A; PXN; RAF1; MAP2K2; ELAVL1; AKT1; PIK3R1; MAP2K1; SFN; VEGFA; AKT3; FOXO1; PRKCA Natural killer cell signaling PRKCE; RAC1; PRKCZ; MAPK1; RAC2; PTPN11; KIR2DL3; AKT2; PIK3CA; SYK; PRKCI; PIK3CB; PIK3C3; PRKD1; MAPK3; KRAS; PRKCD; PTPN6; PIK3C2A; LCK; RAF1; FYN; MAP2K2; PAK4; AKT1; PIK3R1; MAP2K1; PAK3; AKT3; VAV3; PRKCA Regulation at the cell cycle checkpoint: HDAC4; SMAD3; SUV39H1; HDAC5; CDKN1B; BTRC; G1/S ATR; ABL1; E2F1; HDAC2; HDAC7A; RB1; HDAC11; HDAC9; CDK2; E2F2; HDAC3; TP53; CDKN1A; CCND1; E2F4; ATM; RBL2; SMAD4; CDKN2A; MYC; NRG1; GSK3B; RBL1; HDAC6 T cell receptor signaling RAC1; ELK1; MAPK1; IKBKB; CBL; PIK3CA; FOS; NFKB2; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; RELA; PIK3C2A; BTK; LCK; RAF1; IKBKG; RELB; FYN; MAP2K2; PIK3R1; CHUK; MAP2K1; NFKB1; ITK; BCL10; JUN;VAV3 Death receptor signaling CRADD; HSPB1; BID; BIRC4; TBK1; IKBKB; FADD; FAS; NFKB2; BCL2; MAP3K14; MAPK8; RIPK1; CASP8; DAXX; TNFRSF10B; RELA; TRAF2; TNF; IKBKG; RELB; CASP9; CHUK; APAF1; NFKB1; CASP2; BIRC2; CASP3; BIRC3 Signal transduction involving FGF RAC1; FGFR1; MET; MAPKAPK2; MAPK1; PTPN11; AKT2; PIK3CA; CREB1; PIK3CB; PIK3C3; MAPK8; MAPK3; MAPK13; PTPN6; PIK3C2A; MAPK14; RAF1; AKT1; PIK3R1; STAT3; MAP2K1; FGFR4; CRKL; ATF4; AKT3; PRKCA; HGF Signal transduction involving GM-CSF LYN; ELK1; MAPK1; PTPN11; AKT2; PIK3CA; CAMK2A; STAT5B; PIK3CB; PIK3C3; GNB2L1; BCL2L1; MAPK3; ETS1; KRAS; RUNX1; PIM1; PIK3C2A; RAF1; MAP2K2; AKT1; JAK2; PIK3R1; STAT3; MAP2K1; CCND1; AKT3; STAT1 Signal transmission BID; IGF1; RAC1; BIRC4; PGF; CAPNS1; CAPN2; in amyotrophic lateral
sclerosis PIK3CA; BCL2; PIK3CB; PIK3C3; BCL2L1; CAPN1; PIK3C2A; TP53; CASP9; PIK3R1; RAB5A; CASP1; APAF1; VEGFA; BIRC2; BAX; AKT3; CASP3; BIRC3 JAK/Stat-mediated signal transduction PTPN1; MAPK1; PTPN11; AKT2; PIK3CA; STAT5B; PIK3CB; PIK3C3; MAPK3; KRAS; SOCS1; STAT5A; PTPN6; PIK3C2A; RAF1; CDKN1A; MAP2K2; JAK1; AKT1; JAK2; PIK3R1; STAT3; MAP2K1; FRAP1; AKT3; STAT1 Metabolism PRKCE; IRAK1; PRKAA2; EIF2AK2; GRK6; MAPK1; nicotinol and nicotinamide PLK1; AKT2; CDK8; MAPK8; MAPK3; PRKCD; PRKAA1; PBEF1; MAPK9; CDK2; PIM1; DYRK1A; MAP2K2; MAP2K1; PAK3; NT5E; TTK; CSNK1A1; BRAF; SGK Chemokine-mediated signal transduction CXCR4; ROCK2; MAPK1; PTK2; FOS; CFL1; GNAQ; CAMK2A; CXCL12; MAPK8; MAPK3; KRAS; MAPK13; RHOA; CCR3; SRC; PPP1CC; MAPK14; NOX1; RAF1; MAP2K2; MAP2K1; JUN; CCL2; PRKCA Signaling involving IL-2 ELK1; MAPK1; PTPN11; AKT2; PIK3CA; SYK; FOS; STAT5B; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; SOCS1; STAT5A; PIK3C2A; LCK; RAF1; MAP2K2; JAK1; AKT1; PIK3R1; MAP2K1; JUN; AKT3 Long-term synaptic PRKCE; IGF1; PRKCZ; PRDX6; LYN; MAPK1; GNAS; suppression PRKCI; GNAQ; PPP2R1A; IGF1R; PRKD1; MAPK3; KRAS; GRN; PRKCD; NOS3; NOS2A; PPP2CA; YWHAZ; RAF1; MAP2K2; PPP2R5C; MAP2K1; PRKCA Signal transmission TAF4B; EP300; CARM1; PCAF; MAPK1; NCOR2; with the participation of the estrogen receptor SMARCA4; MAPK3; NRIP1; KRAS; SRC; NR3C1; HDAC3; PPARGC1A; RBM9; NCOA3; RAF1; CREBBP; MAP2K2; NCOA2; MAP2K1; PRKDC; ESR1; ESR2 Path TRAF6; SMURF1; BIRC4; BRCA1; UCHL1; NEDD4; protein ubiquitination CBL; UBE2I; BTRC; HSPA5; USP7; USP10; FBXW7; USP9X; STUB1; USP22; B2M; BIRC2; PARK2; USP8; USP1; VHL; HSP90AA1; BIRC3 Signaling involving IL-10 TRAF6; CCR1; ELK1; IKBKB; SP1; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; MAPK14; TNF; IKBKG; RELB; MAP3K7; JAK1; CHUK; STAT3; NFKB1; JUN;IL1R1;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-beta signaling EP300; SMAD2; SMURF1; MAPK1; SMAD3; SMAD1; FOS; MAPK8; MAPK3; KRAS; MAPK9; RUNX2; SERPINE1; RAF1; MAP3K7; CREBBP; MAP2K2; MAP2K1; TGFBR1; SMAD4; JUN; SMAD5 Toll-like receptor signaling IRAK1; EIF2AK2; MYD88; TRAF6; PPARA; ELK1; IKBKB; FOS; NFKB2; MAP3K14; MAPK8; MAPK13; RELA; TLR4; MAPK14; IKBKG; RELB; MAP3K7; CHUK; NFKB1; TLR2; JUN Signal transduction involving p38 MAPK HSPB1; IRAK1; TRAF6; MAPKAPK2; ELK1; FADD; FAS; CREB1; DDIT3; RPS6KA4; DAXX; MAPK13; TRAF2; MAPK14; TNF; MAP3K7; TGFBR1; MYC; ATF4; IL1R1; SRF;STAT1 Neurotrophin/TRK signaling NTRK2; MAPK1; PTPN11; PIK3CA; CREB1; FOS; PIK3CB; PIK3C3; MAPK8; MAPK3; KRAS; PIK3C2A; RAF1; MAP2K2; AKT1; PIK3R1; PDPK1; MAP2K1; CDC42;JUN;ATF4 FXR/RXR activation INS; PPARA; FASN; RXRA; AKT2; SDC1; MAPK8; APOB; MAPK10; PPARG; MTTP; MAPK9; PPARGC1A; TNF; CREBBP; AKT1; SREBF1; FGFR4; AKT3; FOXO1 Long-term synaptic PRKCE; RAP1A; EP300; PRKCZ; MAPK1; CREB1; potentiation PRKCI; GNAQ; CAMK2A; PRKD1; MAPK3; KRAS; PRKCD; PPP1CC; RAF1; CREBBP; MAP2K2; MAP2K1; ATF4;PRKCA Calcium-mediated signal transduction RAP1A; EP300; HDAC4; MAPK1; HDAC5; CREB1; CAMK2A; MYH9; MAPK3; HDAC2; HDAC7A; HDAC11; HDAC9; HDAC3; CREBBP; CALR; CAMKK2; ATF4; HDAC6 Signal transduction involving EGF ELK1; MAPK1; EGFR; PIK3CA; FOS; PIK3CB; PIK3C3; MAPK8; MAPK3; PIK3C2A; RAF1; JAK1; PIK3R1; STAT3; MAP2K1; JUN; PRKCA; SRF; STAT1 Signal transmission during hypoxia in EDN1; PTEN; EP300; NQO1; UBE2I; CREB1; ARNT; cardiovascular system HIF1A; SLC2A4; NOS3; TP53; LDHA; AKT1; ATM; VEGFA; JUN; ATF4; VHL; HSP90AA1 LPS/IL-1 mediated inhibition IRAK1; MYD88; TRAF6; PPARA; RXRA; ABCA1; RXR functions MAPK8; ALDH1A1; 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; LDLR; NR1H2; NFKB1; SREBF1; IL1R1; CCL2; IL6; MMP9 Amyloid processing PRKCE; CSNK1E; MAPK1; CAPNS1; AKT2; CAPN2; CAPN1; MAPK3; MAPK13; MAPT; MAPK14; AKT1; PSEN1; CSNK1A1; GSK3B; AKT3; APP Signaling involving IL-4 AKT2; PIK3CA; PIK3CB; PIK3C3; IRS1; KRAS; SOCS1; PTPN6; NR3C1; PIK3C2A; JAK1; AKT1; JAK2; PIK3R1; FRAP1; AKT3; RPS6KB1 Regulation of DNA damage EP300; PCAF; BRCA1; GADD45A; PLK1; BTRC; at the cell cycle checkpoint: CHEK1; ATR; CHEK2; YWHAZ; TP53; CDKN1A; G2/M PRKDC;ATM;SFN;CDKN2A Signal transduction involving nitric oxide in KDR; FLT1; PGF; AKT2; PIK3CA; PIK3CB; PIK3C3; cardiovascular system CAV1; PRKCD; NOS3; PIK3C2A; AKT1; PIK3R1; VEGFA; AKT3; HSP90AA1 Purine metabolism NME2; SMARCA4; MYH9; RRM2; ADAR; EIF2AK4; PKM2; ENTPD1; RAD51; RRM2B; TJP2; RAD51C; NT5E; POLD1; NME1 cAMP-mediated signaling RAP1A; MAPK1; GNAS; CREB1; CAMK2A; MAPK3; SRC; RAF1; MAP2K2; STAT3; MAP2K1; BRAF; ATF4 Mitochondrial dysfunction SOD2; MAPK8; CASP8; MAPK10; MAPK9; CASP9; PARK7; PSEN1; PARK2; APP; CASP3 Signal transduction involving Notch HES1; JAG1; NUMB; NOTCH4; ADAM17; NOTCH2; PSEN1; NOTCH3; NOTCH1; DLL4 The Path to Stress Associated with HSPA5; MAPK8; XBP1; TRAF2; ATF6; CASP9; ATF4; endoplasmic reticulum EIF2AK3;CASP3 Pyrimidine metabolism NME2; AICDA; RRM2; EIF2AK4; ENTPD1; RRM2B; NT5E; POLD1; NME1 Signaling in Parkinson's Disease UCHL1; MAPK8; MAPK13; MAPK14; CASP9; PARK7; PARK2;CASP3 Signal transmission GNAS; GNAQ; PPP2R1A; GNB2L1; PPP2CA; PPP1CC; in cardiac muscle cells and with the participation of beta-adrenergic receptors PPP2R5C Glycolysis/glyconeogenesis HK2; GCK; GPI; ALDH1A1; PKM2; LDHA; HK1 Interferon-mediated signal transduction IRF1; SOCS1; JAK1; JAK2; IFITM1; STAT1; IFIT3 Signal transmission involving Sonic Hedgehog ARRB2; SMO; GLI2; DYRK1A; GLI1; GSK3B; DYRK1B Metabolism PLD1; GRN; GPAM; YWHAZ; SPHK1; SPHK2 glycerophospholipids Destruction of phospholipids PRDX6; PLD1; GRN; YWHAZ; SPHK1; SPHK2 Tryptophan metabolism SIAH2; PRMT5; NEDD4; ALDH1A1; CYP1B1; SIAH1 Lysine Destruction SUV39H1; EHMT2; NSD1; SETD7; PPP2R5C Path ERCC5; ERCC4; XPA; XPC; ERCC1 in nucleotide excision repair Metabolism UCHL1; HK2; GCK; GPI; HK1 starch and sucrose Metabolism of amino sugars NQO1; HK2; GCK; HK1 Metabolism PRDX6; GRN; YWHAZ; CYP1B1 arachidonic acid Signal transmission involving the circadian rhythm CSNK1E;CREB1;ATF4;NR1D1 Coagulating system BDKRB1;F2R;SERPINE1;F3 Signal transmission PPP2R1A; PPP2CA; PPP1CC; PPP2R5C with the participation of the dopamine receptor Metabolism of glutathione IDH2;GSTP1;ANPEP;IDH1 Glycerolipid metabolism ALDH1A1;GPAM;SPHK1;SPHK2 Metabolism of linoleic acid PRDX6; GRN; YWHAZ; CYP1B1 Metabolism of methionine DNMT1; DNMT3B; AHCY; DNMT3A Metabolism of pyruvate GLO1;ALDH1A1;PKM2;LDHA Metabolism ALDH1A1;NOS3;NOS2A ascorbate and aldarate Eicosanoid-mediated signal transduction PRDX6;GRN;YWHAZ Metabolism HK2; GCK; HK1 fructose and mannose Metabolism of galactose HK2; GCK; HK1 Biosynthesis of stilbene, coumarin and PRDX6; PRDX1; TYR lignin Path CALR; B2M during antigen presentation Biosynthesis of steroids NQO1;DHCR7 Metabolism of butanoate ALDH1A1;NLGN1 Citrate cycle IDH2; IDH1 Fatty acid metabolism ALDH1A1;CYP1B1 Metabolism PRDX6;CHKA glycerophospholipids Histidine metabolism PRMT5;ALDH1A1 Inositol metabolism ERO1L;APEX1 Metabolism of xenobiotics GSTP1;CYP1B1 Methane Metabolism with the participation of cytochrome p450 PRDX6;PRDX1 Phenylalanine metabolism PRDX6;PRDX1 Metabolism of propanoate ALDH1A1;LDHA Metabolism PRMT5;AHCY selenoamino acids Sphingolipid metabolism SPHK1; SPHK2 Metabolism PRMT5 aminophosphonate Metabolism PRMT5 androgen and estrogen Metabolism ALDH1A1 ascorbate and aldarate Biosynthesis of bile acids ALDH1A1 Cysteine metabolism LDHA Biosynthesis of fatty acids FASN involving the glutamate receptor GNB2L1 Signal transmission NRF2-mediated response to PRDX1 oxidative stress Pentose phosphate GPI path Mutual transformation UCHL1 Mutual transformation Retinol metabolism ALDH1A1 Riboflavin metabolism TYR Tyrosine metabolism PRMT5, TYR Biosynthesis of ubiquinone PRMT5 Destruction of valine, leucine and ALDH1A1 isoleucine Metabolism of glycine, serine and CHKA threonine Lysine Destruction ALDH1A1 Pain/taste TRPM5; TRPA1 Pain TRPM7; TRPC5; TRPC6; TRPC1; Cnr1; cnr2; grk2; Trpa1; Pomc; Cgrp; Crf; Pka; era; Nr2b; TRPM5; Prkaca; Prkacb; Prkar1a; Prkar2a Mitochondrial function AIF; CytC; SMAC (Diablo); Aifm-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; burn-related proteins; Otx-2; Gbx2; FGF-8; Reelin; Dab1; unc-86 (Pou4f1 or Brn3a); Numb; Reln

List of illustrative target genes, target loci, target polynucleotides

As an example, the chromosomal sequence may include, but is not limited to, 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 (prostacyclin 12 (IP) receptor), KCNJ11 (inward rectifier potassium channel, subfamily J, member 11), INS (insulin), CRP (pentraxin-related C-reactive protein), PDGFRB (platelet-derived growth factor, beta polypeptide), CCNA2 (cyclin A2), PDGFB (platelet-derived growth factor beta polypeptide oncogene homolog (simian sarcoma virus (v-sis))), KCNJ5 (inward-rectifying potassium channel, subfamily J, member 5), KCNN3 (intermediate/low-conducting calcium-activated potassium channel, subfamily N, member 3), CAPN10 (calpain 10), PTGES (prostaglandin E synthase), ADRA2B (alpha-2B-adrenergic receptor), ABCG5 (ATP-binding cassette, subfamily G (WHITE), member 5), PRDX2 (peroxiredoxin 2), CAPN5 (calpain 5), PARP14 (poly (ADP-ribose) polymerase family, member 14), MEX3C (C mex-3 homolog C (C. elegans)), ACE angiotensin I-converting enzyme (peptidyl dipeptidase A) 1), TNF (tumor necrosis factor (TNF superfamily, member 2)), IL6 (interleukin 6 (interferon, beta 2)), STN (statin), SERPINE1 (serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1), ALB (albumin), ADIPOQ (adiponectin containing C1Q and collagen domain), APOB (apolipoprotein B (including Ag(x))), APOE (apolipoprotein E), LEP (leptin), MTHFR (5,10-methylenetetrahydrofolate reductase (NADPH)), APOA1 (apolipoprotein A-I), EDN1 (endothelin 1), NPPB (precursor natriuretic peptide B), NOS3 (synthase nitric oxide type 3 (endothelial cell)), PPARG (peroxisome proliferator-activated receptor gamma), PLAT (tissue plasminogen activator), PTGS2 (prostaglandin endoperoxide synthase type 2 (prostaglandin G/H synthase and cyclooxygenase)), CETP (plasma cholesteryl ester transport protein), AGTR1 (anti-otensin II receptor type 1), HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase), IGF1 (insulin-like growth factor 1 (somatomedin C)), SELE (selectin E), REN (renin), PPARA (peroxisome proliferator-activated receptor alpha), PON1 (paraoxonase 1), KNG1 (kininogen 1), CCL2 (chemokine ligand 2 (with C-C motif)), LPL (lipoprotein lipase), VWF (von Willebrand factor), F2 (coagulation factor II (thrombin)), ICAM1 (intercellular adhesion molecule 1), TGFB1 (transforming growth factor, beta 1), NPPA (precursor natriuretic peptide A), IL10 (interleukin 10), EPO (erythropoietin), SOD1 (superoxide dismutase 1, soluble), VCAM1 (vascular endothelial adhesion molecule type 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 type 2), MMP9 (matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa collagenase type IV)), SERPINC1 (serpin peptidase inhibitor, clade C (antithrombin), member 1), F8 (coagulation factor VIII, procoagulant component), HMOX1 (heme oxygenase (decyclic) type 1), APOC3 (apolipoprotein C-III), IL8 (interleukin 8), PROK1 (prokineticin 1), CBS (cystathionine beta synthase), NOS2 (sodium oxide synthase type 2, inducible), TLR4 (toll-like receptor type 4), SELP (selectin P (140 kDa granule membrane protein, CD62 antigen)), ABCA1 (ATP-binding cassette, subfamily A (ABC1), member 1), AGT (angiotensin (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 type 1 (neuronal)), NR3C1 (nuclear receptor, subfamily 3, group C, member 1 (glucocorticoid receptor)), FGB (fibrinogen beta chain), HGF (hepatocyte growth factor (hepatopoietin A; scattering factor)), IL1A (interleuin 1, alpha), RETN (resistin), AKT1 (murine thymoma virus oncogene homolog type 1 v-akt), LIPC (lipase, liver), HSPD1 (heat shock protein type 1 of 60 kDa (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 superfamily, member 11b), EDNRA (endothelin receptor type A), EGFR (epidermal growth factor receptor (v-erb-b oncogene homolog, avian)), MMP2 (matrix metallopeptidase 2 (gelatinase A, 72-kDa gelatinase, 72-kDa collagenase type IV)), PLG (plasminogen), NPY (neuropeptide Y), RHOD (ras homolog gene family, member D), MAPK8 (mitogen-activated protein kinase 8), MYC (v-myc myelocystomatosis virus oncogene homolog, avian)), FN1 (fibronectin 1), CMA1 (chymase 1, mast cell), PLAU (plasminogen activator, urokinase), GNB3 (guanine nucleotide-binding protein (G protein), beta polypeptide type 3), ADRB2 (adrenergic beta-2 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 1), PARP1 (poly (ADP-ribose) polymerase type 1), CD40LG (CD40 ligand), PON2 (paraoxonase 2), AGER (receptor for advanced glycation end products), IRS1 (insulin receptor substrate type 1), PTGS1 (prostaglandin endoperoxide synthase type 1 (prostaglandin G/H synthase and cyclooxygenase)), ECE1 (endothelin-converting enzyme type 1), F7 (coagulation factor VII (serum thrombin converting accelerator)), URN (interleukin 1 receptor antagonist), EPHX2 (epoxide hydrolase type 2, cytoplasmic), IGFBP1 (insulin-like growth factor binding protein type 1), MAPK10 (mitogen-activated protein kinase 10), FAS (Fas (TNF receptor superfamily, member 6)), ABCB1 (ATP-binding cassette, subfamily B (MDR/TAP), member 1), JUN (jun oncogene), IGFBP3 (insulin-like growth factor binding protein type 3), CD14 (CD14 molecule), PDE5A (phosphodiesterase 5A, cGMP-specific), AGTR2 (receptor angiotensin II, type 2), CD40 (CD40 molecule, member of the TNF receptor superfamily 5), LCAT (lecithin-cholesterol acyltransferase), CCR5 (chemokine receptor type 5 (with a C-C motif)), MMP1 (matrix metallopeptidase 1 (interstitial collagenase)), TIMP1 (TIMP inhibitor of metallopeptidase type 1), ADM (adrenomedullin), DYT10 (dystonia 10), STAT3 (signal transducer and activator of transcription type 3 (acute phase response factor)), MMP3 (matrix metallopeptidase 3 (stromelysin 1, progelatinase)), ELN (elastin), USF1 (transcription factor binding upstream of the transcription initiation site 1), CFH (complement factor H), HSPA4 (warm shock protein 4 of 70 kDa), MMP12 (matrix metallopeptidase 12 (macrophage elastase)), MME (membrane metalloendopeptidase), F2R (coagulation factor II (thrombin) receptor), SELL (selectin L), CTSB (cathepsin B), ANXA5 (annexin A5), ADRB1 (adrenergic beta-1 receptor), CYBA (cytochrome b-245, alpha peptide), FGA (fibrinogen alpha chain), GGT1 (gamma-glutamyl transferase 1), LIPG (lipase, endothelial), HIF1A (hypoxia-inducible factor 1, alpha subunit (helix-loop-helix basic structure transcription factor)), CXCR4 (chemokine receptor 4 (with C-X-C motif)), PROC (protein C (coagulation factor Va and VIIIa inactivator)), SCARB1 (phagocytic receptor, class B, member 1), CD79A (CD79a molecule, immunoglobulin-associated alpha), PLTP (phospholipid transfer protein), ADD1 (adducin 1 (alpha)), FGG (fibrinogen gamma chain), SAA1 (serum amyloid A1), KCNH2 (potassium voltage-gated channel, family H (eag-related), member 2), DPP4 (dipeptidyl peptidase 4), G6PD (glucose-6-phosphate dehydrogenase), NPR1 (natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A)), VTN (vitronectin), KIAA0101 (KIAA0101), FOS (homolog murine osteosarcoma virus oncogene FBJ), TLR2 (toll-like receptor 2), PPIG (peptidylproline isomerase G (cycloproline G)), IL1R1 (interleukin I receptor), 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-hexacysteine methyltransferase), RBP4 (retinol-binding protein type 4, plasma), APOA4 (apolipoprotein A-IV), CDKN2A (cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)), FGF2 (fibroblast growth factor 2 (basic)), EDNRB (endothelin receptor type B), ITGA2 (integrin, alpha 2 (CD49B, alpha 2 subunit of the VLA-2 receptor)), CABIN1 (calcineurin-binding protein 1), SHBG (sex hormone-binding globulin), HMGB1 (high-mobility group 1 protein), HSP90B2P (heat shock protein 90 kDa 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, caveolae protein, 22 kDa), ESR2 (estrogen receptor 2 (ER beta)), LTA (lymphotoxin alpha (TNF superfamily, member 1)), GDF15 (growth and differentiation factor 15), BDNF (brain-derived neurotrophic factor), CYP2D6 (cytochrome P450, family 2, subfamily D, polypeptide 6), NGF (nerve growth factor (beta polypeptide)), SP1 (transcription factor Sp1), TGIF1 (TGFB-inducible factor homeobox 1), SRC (sarcoma virus oncogene homolog v-src (Schmidt-Ruppin A-2) (avian)), EGF (epidermal growth factor (beta-urogastron)), PIK3CG (phosphoinositide 3-kinase, catalytic, gamma polypeptide), HLA-A (major histocompatibility complex, class I, A), KCNQ1 (potassium voltage-gated channel, KQT-like family, member 1), CNR1 (cannabinoid receptor 1 (brain)), FBN1 (fibrillin 1), CHKA (choline kinase alpha), BEST1 (bestrophin 1), APP (amyloid precursor protein beta (A4)), CTNNB1 (catenin (cadherin-associated protein), beta 1, 88 kDa), IL2 (interleukin 2), CD36 (CD36 molecule (thrombospondin receptor)), PRKAB1 (protein kinase, AMP-activated, beta 1 non-catalytic subunit), TPO (thyroid peroxidase), ALDH7A1 (aldehyde dehydrogenase family 7, member A1), CX3CR1 (chemokine receptor 1 (with C-X3-C motif)), TH (tyrosine hydroxylase), F9 (coagulation factor IX), GH1 (growth hormone 1), TF (transferrin), HFE (hemochromatosis), IL17A (interleukin 17A), PTEN (phosphatase tensin homolog), GSTM1 (glutathione S-transferase mu 1), DMD (dystrophin), GATA4 (GATA binding protein type 4), F13A1 (coagulation factor XIII, polypeptide A1), TTR (transthyretin), FABP4 (fatty acid binding protein type 4, adipocyte), PON3 (paraoxonase 3), APOC1 (apolipoprotein C-I), INSR (insulin receptor), TNFRSF1B (tumor necrosis factor receptor superfamily, member 1B), HTR2A (5-hydroxytryptamine (serotonin) receptor 2A), 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 (receptor kinase insertion domain-containing (receptor tyrosine kinase type III)), PLA2G2A (phospholipase A2, group IIA (platelets, synovial fluid)), B2M (beta-2-microglobulin), THBS1 (thrombospondin 1), GCG (glucagon), RHOA (ras homolog gene family, member A), ALDH2 (aldehyde dehydrogenase 2 family (mitochondrial)), TCF7L2 (transcription factor 7, factor 2-like (T-cell-specific, HMG-box)), BDKRB2 (bradykinin receptor B2), NFE2L2 (nuclear factor 2-like (erythroid 2)), NOTCH1 (Notch 1 homolog, translocation-associated (Drosophila)), UGT1A1 (UDP-glucuronyl transferase family 1, polypipeline A1), IFNA1 (interferon, alpha 1), PPARD (peroxisome proliferator-activated receptor delta), SIRT1 (sirtuin 1 (silent information regulation homolog 2) (S. cerevisiae)), GNRH1 (gonadotropin-releasing hormone 1 (luteinizing-releasing hormone)), PAPPA (pregnancy-associated plasma protein A, papalysin 1), ARR3 (arrestin 3, retinal (X-arrestin)), NPPC (precursor natriuretic peptide C), AHSP (alpha-hemoglobin-stabilizing protein), PTK2 (protein tyrosine kinase type 2 PTK2), IL13 (interleukin 13), MTOR (mechanism of action target of rapamycin (serine/threorine kinase)), ITGB2 (intergreen, beta 2 (subunit of complement component 3 receptor 3 and 4)), GSTT1 (glutathione-S-transferase theta 1), IL6ST (interleukin 6 signal transducer (gp130, oncostatin M receptor)), CPB2 (carboxypeptidase B2 (plasma)), CYP1A2 (cytochrome P450, family 1, subfamily A, polypeptide 2), HNF4A (hepatocyte nuclear factor 4, alpha), SLC6A4 (solute carrier family 6 (neurotransmitter transporter, serotonin), member 4), PLA2G6 (phospholipase A2, group VI (cytosolic, calcium-independent)), TNFSF11 (tumor growth factor superfamily (ligand), member 11), SLC8A1 (solute carrier family 8 (sodium-calcium antiporter), member 1), F2RL1 (coagulation factor II receptor-like 1 (thrombin)), AKR1A1 (aldoketo reductase family 1, member A1 (aldehyde reductase)), ALDH9A1 (aldehyde dehydrogenase family 9, member A1), BGLAP (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 (P2RY purinergic receptor G protein-coupled 12), RNLS (renalase, FAD-dependent amine oxidase), CREB1 (cAMP-responsive element-binding protein 1), POMC (pro-opiomelanocortin), RAC1 (ras-related botulinum toxin substrate 1) C3 (rho family, small GTP binding protein Rac1)), LMNA (lamin NC), CD59 (CD59 molecule, complement regulatory protein), SCN5A (sodium channel, voltage-gated, type V, alpha subunit), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), MIF (macrophage migration inhibitory factor (glycosylation inhibitory factor)), MMP13 (matrix metallopeptidase 13 (collagenase 3)), TIMP2 (TIMP inhibitor of metallopeptidase 2), CYP19A1 (cytochrome P450, family 19, subfamily A, polypeptide 1), CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2), PTPN22 (protein tyrosine phosphatase, non-receptor, 22 type (lymphoid)), MYH14 (myosin, heavy chain 14, non-muscle), MBL2 (mannose-binding lectin (protein C) 2, soluble (opsonin defect)), SELPLG (selectin P ligand), AOC3 (amine oxidase, copper-containing 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, MSK12)), CAST (calpastatin), CXCL12 (chemokine ligand 12 (with C-X-C motif) (stromal cell factor 1)), IGHE (heavy epsilon chain), KCNE1 (potassium voltage-gated channel, Isk-related family, member 1), TFRC (transferrin receptor (p90, CD71)), COL1A1 (collagen type 1, alpha 1), COL1A2 (collagen type I, alpha 2), IL2RB (interleukin 2 receptor, beta), PLA2G10 (pro-phospholipidase A2, group X), ANGPT2 (angiopoietin 2), PROCR (protein C receptor, endothelial (EPCR)), NOX4 (NADPH oxidase 4), HAMP (hepcidin antimicrobial peptide), PTPN11 (protein tyrosine phosphatase, non-receptor, type 11), SLC2A1 (solute carrier family 2 (facilitated diffusion glucose transporter), member 1), IL2RA (interleukin 2 receptor, alpha), CCL5 (chemokine ligand 5 (with C-C motif)), IRF1 (interferon regulatory factor 1), CFLAR (CASP8 and FADD-like regulator of apoptosis), CALCA (calcitonin-related polypeptide alpha), EIF4E (eukaryotic translation initiation factor 4E), GSTP1 (pi-1-glutathione S-transferase), JAK2 (Janus kinase 2), CYP3A5 (cytochrome P450, family 3, subfamily A, polypeptide 5), HSPG2 (heparin sulfate proteoglycan 2), CCL3 (chemokine ligand 3 (with C-C motif)), MYD88 (myeloid differentiation primary response gene (88)), VIP (vasoactive intestinal peptide), SOAT1 (sterol O-acyltransferase 1), ADRBK1 (adrenergic beta 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 hydrolase 1), EPRS (glutamyl propyl-tRNA synthetase), PPARGC1A (peroxisome proliferator-activated receptor gamma coactivator 1 alpha), F12 (coagulation factor XII (Hageman factor)), PECAM1 (platelet/endothelial cell adhesion molecule), CCL4 (chemokine (C-C motif) ligand 4), SERPINA3 (serpin peptidase inhibitor, clade A (alpha-1-antiproteinase, antitrypsin), member 3), CASR (calcium-sensing 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 (cardiotropin 1), SGCB (sarcoglycan, beta (dystrophin-associated glycoprotein of 43 kDa)), YME1L1 (YME1-like factor 1 (S. cerevisiae)), CAMP (cathelicidin antimicrobial peptide), ZC3H12A (containing zinc finger factor CCCH-type 12A), AKR1B1 (aldo-keto reductase family 1, 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, alpha, member 1), UGT1A (UDP-glucuronyl transferase family 1, polypeptide complex locus A), PRKCA (protein kinase C, alpha), COMT (catechol-beta-methyltransferase), S100B (S100 calcium-binding protein B), EGR1 (early response growth factor 1), PRL (prolactin), IL15 (interleukin 15), DRD4 (dopamine D4 receptor), CAMK2G (calcium/calmodulin-dependent protein kinase II gamma), SLC22A2 (solute carrier family 22 (organic cation transporter), member 2), CCL11 (chemokine ligand 11 (C-C motif)), PGF (placental growth factor B321), THPO (thrombopoietin), GP6 (glycoprotein VI (platelet)), TACR1 (tachykine receptor 1), NTS (neurotensin), HNF1A (HNF1 homeobox A), SST (somatostatin), KCND1 (potassium voltage-gated channel, 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-lipogenase), AHSG (alpha-2-HS-glycoprotein), BHMT (betaine-homocysteine methyltransferase), GJA4 (gap junction protein, alpha 4, 37 kDa), SLC25A4 (solute carrier family 25 (mitochondrial transporter; adenine nucleotide translocator), member 4), ACLY (ATP citrate lyase), ALOX5AP (arachidonate-5-lipoxygenase activating protein), NUMA1 (nuclear protein of the mitotic apparatus 1), CYP27B1 (cytochrome P450, family 27, subfamily B, polypeptide 1), CYSLTR2 (cysteinyl leukotriene receptor 2), SOD3 (superoxide dismutase 3, extracellular), LTC4S (leukotriene C4 synthase), UCN (urocortin), GHRL (ghrelin/obestatin prepropeptide), APOC2 (apolipoprotein C-II), CLEC4A (C-type lectin domain family 4, member A), KBTBD10 (kelch repeat and BTB domain-containing (POZ) 10), TNC (tenaskin C), TYMS (thymidylate synthetase), SHCl (SHC-transforming protein 1 (Src-homology domain-containing 2)), LRP1 (low-density lipoprotein receptor-related protein 1), SOCS3 (suppressor of cytokine signaling 3), ADH1B (alcohol dehydrogenase 1B (class I), beta polypeptide), KLK3 (kallikrein-related peptidase 3), HSD11B1 (hydroxysteroid (11-beta) dehydrogenase 1), VKORC1 (vitamin K epoxide reductase complex, subunit 1), SERPINB2 (serpin peptidase inhibitor, clade B (ovalbumin), member 2), TNS1 (tensin 1), RNF19A (ring zinc finger protein 19A), EPOR (erythropoietin receptor), ITGAM (integrin, alpha M (subunit of complement component 3 receptor 3)), PITX2 (paired homeodomain-like 2), MAPK7 (mitogen-activated protein kinase 7), FCGR3A (Fc fragment of IgG, low affinity 111a, receptor (CD16a)), LEPR (leptin receptor), ENG (endoglin), GPX1 (glutathione peroxidase 1), GOT2 (oxaloacetic transaminase glutamic acid type 2, mitochondrial (aspartate aminotransferase type 2)), HRH1 (histamine H1 receptor), NR112 (nuclear receptor family 1, group I, member 2), CRH (corticotropin-releasing hormone), HTR1A (5-hydroxytryptamine (serotonin) receptor 1A), VDAC1 (voltage-gated anion channel 1), HPSE (heparanase), SFTPD (surface-active protein D), TAP2 (ATP-binding cassette transporter 2, subfamily B (MDR/TAP)), RNF123 (ring 123 zinc finger protein), PTK2B (PTK2B protein tyrosine kinase 2 beta), NTRK2 (neurotrophic tyrosine kinase receptor, type 2), IL6R (interleukin 6 receptor), ACHE (acetylcholinesterase (blood group Yt)), GLP1R (glucagon-like peptide 1 receptor), GHR (growth hormone receptor), GSR (glutathione reductase), NQO1 (NAD(P)H dehydrogenase, quinone 1), NR5A1 (nuclear receptor family 5, group A, member 1), GJB2 (gap junction protein, beta 2, 26 kDa), SLC9A1 (solute carrier family 9 (sodium hydrogen antiporter), member 1), MAOA (monoamine oxidase A), PCSK9 (proprotein convertase subtilisin-kexin type 9), FCGR2A (Fc fragment of IgG, low affinity IIa, receptor (CD32)), SERPINF1 (serpin peptidase inhibitor, clade F (alpha-2-antiplasmin, pigment epithelial factor), member 1), EDN3 (endothelin 3), DHFR (dihydrofolate reductase), GAS6 (growth arrest-specific factor 6), SMPD1 (sphingomyelin phosphodiesterase 1, acidic lysosomal), UCP2 (unpaired protein 2 (mitochondrial, proton transporter)), TFAP2A (transport factor AP-2 alpha (activating enhancer binding protein 2 alpha)), C4BPA (complement component 4 binding protein alpha), SERPINF2 (serpin peptidase inhibitor, clade F (alpha-2-antilasmin, pigment epithelial factor), member 2), TYMP (thymidine phosphorylase), ALPP (alkaline phosphatase, placental (Regan isozyme)), CXCR2 (chemokine receptor 2 (with C-X-C motif)), SLC39A3 (solute carrier 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 (coagulation factor XI), ATP7A (ATPase, Cu++ transporter, alpha polypeptide), CR1 (complement component receptor 1 (3b/4b) (Knops blood groups)), GFAP (glial fibrillary alkaline protein), ROCK1 (Rho-associated double-stranded protein kinase 1), MECP2 (methyl CpG-binding protein 2 (Rett syndrome)), MYLK (myosin light chain), BCHE (butyrylcholinesterase), LIPE (lipase, hormone-sensitive), PRDX5 (peroxiredoxin 5), ADORA1 (adenosine A1 receptor), WRN (Werner syndrome, RecQ helicase-like), CXCR3 (chemokine receptor 3 (with C-X-C motif)), CD81 (CD81 molecule), SMAD7 (SMAD family, member 7), LAMC2 (laminin, gamma 2), MAP3K5 (mitogen-activated protein kinase kinase 5), CHGA (chromogranin A (parathyroid secretory protein 1)), IAPP (islet amyloid peptide), RHO (rhodopsin), ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), PTHLH (parathyroid hormone-like hormone), 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 (coagulation factor II (thrombin) receptor-like 3), CX3CL1 (chemokine (C-X3-C motif) ligand 1), BDKRB1 (bradykine receptor B1), ADAMTS13 (ADAM metallopeptidase with thrombospondin motif type 1, 13), ELANE (neutrophil-expressed elastase), ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase 2), CISH (cytokine-inducible SH2-containing protein), GAST (gastrin), MYOC (myocilin, trabecular meshwork-inducible glucocorticoid response), ATP1A2 (ATPase, Na+/K+ transport, alpha 2 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 factor 42 (GTP-binding protein, 25 kDa)), KRT18 (keratin 18), HSF1 (heat shock protein transcription factor 1), MYB (myeloblastosis virus oncogene homolog v-myb (avian)), PRKAA2 (protein kinase, AMP-activated, catalytic subunit alpha 2), ROCK2 (Rho-associated double-stranded protein kinase 2), TFPI (tissue factor pathway inhibitor (lipoprotein-associated inhibitor of coagulation)), PRKG1 (protein kinase, cGMP-dependent, type I), BMP2 (bone morphogenetic protein 2), CTNND1 (catenin (cadherin-associated protein), delta 1), CTH (cystathionase (cystathionine gamma-lyase)), CTSS (cathepsin S), VAV2 (guanine nucleotide exchange factor vav 2), NPY2R (neuropeptide Y receptor Y2), IGFBP2 (insulin-like growth factor binding protein 2, 36 kDa), CD28 (CD28 molecule), GSTA1 (glutathione S-transferase, alpha 1), PPIA (peptidylprolyl 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 family 1, group H, member 3), SCD (stearoyl-CoA desaturase (delta-9-desaturase)), GIP (gastric inhibitory peptide), 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 (hydroxysteroid (11-beta) dehydrogenase 2), CALCRL (calcitonin receptor-like), GALNT2 (UDP-N-acetyl-alpha-D-galactosamine:N-acetylgalactosaminyltransferase 2 polypeptide (GalNAc-T2)), ANGPTL4 (angiopoietin-like 4), KCNN4 (calcium-activated potassium channel, intermediate/low conduction, subfamily N, member 4), PIK3C2A (phosphoinositidine 3-kinase, class 2, alpha polypeptide), HBEGF (heparin-binding EGF-like growth factor), CYP7A1 (cytochrome P450, family 7, subfamily A, polypeptide 1), HLA-DRB5 (major histocompatibility complex, class II, DR beta 5), BNIP3 (19-molecular-weight protein 3), kDa, interacting with BCL2/adenovirus E1B), GCKR (glucokinase (hexokinase) regulator 4), S100A12 (S100 calcium-binding protein A12), PADI4 (peptidylarginine deiminase, type IV), HSPA14 (70 kDa heat shock protein 14), CXCR1 (chemokine receptor 1 (with a C-X-C motif)), H19 (H19, maternally expressed peptide imprinted (non-coding protein)), KRTAP19-3 (keratin-associated protein 19-3), IDDM2 (insulin-dependent diabetes mellitus type 2 factor), RAC2 (ras-related botulinum toxin C3 substrate 2 (rho family, small GTP binding protein Rac2)), RYR1 (ryanodine receptor 1 (muscle)), CLOCK (gene homolog (mouse)), NGFR (nerve growth factor receptor (TNFR superfamily, member 16)), DBH (dopamine beta-hydroxylase (dopamine beta-monooxygenase)), CHRNA4 (cholinergic receptor, nicotinic, alpha 4), CACNA1C (calcium channel, voltage-gated, type L, subunit alpha 1C), PRKAG2 (protein kinase, AMP-activated, gamma 2 noncatalytic subunit), CHAT (choline acetyltransferase), PTGDS (prostaglandin D2 synthase 21 kDa (brain)), NR1H2 (nuclear receptor family 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, NFKB activator), HSPA9 (70 kDa heat shock protein 9 (mortalin)), CYSLTR1 (cysteinyl leukotriene receptor 1), MAT1A (methionine adenosyltransferase I alpha), OPRL1 (opiate receptor-like 1), IMPA1 (insitol (myo)-1 (or 4)-monophosphatase 1), CLCN2 (chloride ion transporter channel 2), DLD (dihydrolipoamide dehydrogenase), PSMA6 (proteasome subunit (prosome, macropain), alpha type 6), PSMB8 (proteasome subunit (prosome, macropain), beta type 8 (large multifunctional peptidase 7)), CHI3L1 (chitinase 3-like factor 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, subfamily C (CFTR/MRP), member 6), RGS2 (regulator of G protein-mediated signaling 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, hip-shoulder muscular dystrophy 2B (autosomal recessive)), FDFT1 (farnesyl-diphosphate farnelystransferase 1), EDN2 (endothelin 2), CCR6 (chemokine receptor 6 (with a C-C motif)), GJB3 (gap junction protein, beta 3, 31 kDa), IL1RL1 (interleukin 1 receptor-like factor 1), ENTPD1 (ectonucleoside triphosphate diphosphohydrolase 1), BBS4 (Bardet-Biedl syndrome factor 4), CELSR2 (cadherin, seven-channel G-type receptor 2 EGF LAG (flamingo homolog, Drosophila)), F11R (F11 receptor), RAPGEF3 (Rap guanine nucleotide exchange factor (GEF) 3), HYAL1 (hyaluronoglycosaminidase 1), ZNF259 (zinc finger protein 259), ATOX1 (ATX1 antioxidant protein 1 homolog (yeast)), ATF6 (transcription activating factor 6), KHK (ketohexokinase (fructokinase)), SAT1 (spermidine/spermine N1-acetyltransferase 1), GGH (gamma-glutamyl hydrolase (conjugase, folylpolygamma-glutamyl hydrolase)), TIMP4 (TIMP inhibitor of metallopeptidase 4), SLC4A4 (solute carrier family 4, sodium bicarbonate cotransporter, member 4), PDE2A (phosphodiesterase 2A, cGMP-stimulated), PDE3B (phosphodiesterase 3B, cGMP-inhibited), FADS1 (fatty acid desaturase 1), FADS2 (fatty acid desaturase 2), TMSB4X (thymosin beta 4, X-linked), TXNIP (thioredoxin-interacting protein), LIMS1 (LIM-like and senescent cell antigen-like domains 1), RHOB (ras homolog gene family, member B), LY96 (lymphocyte antigen 96), FOXO1 (forkhead box O1), PNPLA2 (patatin-like phospholipidase domain-containing factor 2), TRH (thyrotropin-releasing hormone), GJC1 (gap junction protein gamma 1, 45 kDa), SLC17A5 (solute carrier family 17 (anion and sugars), member 5), FTO (fat mass and obesity-associated factor), GJD2 (gap junctional protein delta 2, 36 kDa), PSRC1 (proline-serine-rich double-stranded factor 1), CASP12 (caspase 12 (gene/pseudogene)), GPBAR1 (G protein-coupled bile acid receptor 1), PXK (PX domain-containing serine/threonine kinase), IL33 (interleukin 33), TRIB1 (tribbles homolog 1 (Drosophila)), PBX4 (pre-B-cell leukemia homeobox 4), NUPR1 (nuclear transcriptional regulator protein 1), 15-Sep (15 kDa selenoprotein), CILP2 (cartilage intermediate layer protein 2), TERC (RNA component of telomerase), GGT2 (gamma-glutamyl transferase 2), MT-CO1 (cytochrome c oxidase I, encoded by the mitochondrial genome) and UOX (urate oxidase, a pseudogene). Any of these sequences can be targeted by the CRISPR-Cas system, for example, to study mutation.

In a further embodiment, the chromosomal sequence may also be selected from the following: Pon1 (paraoxonase 1), LDLR (LDL receptor), ApoE (apolipoprotein E), Apo B-100 (apolipoprotein B-100), ApoA (apolipoprotein(a)), ApoA1 (apolipoprotein A1), CBS (cystathionine B synthase), glycoprotein IIb/IIb, MTHRF (5,10-methylenetetrahydrofolate reductase (NADPH) and combinations thereof. In one case, the chromosomal sequences and proteins encoded by the chromosomal sequences associated with cardiovascular disease may be selected from Cacna1C, Sod1, Pten, Ppar(alpha), Apo E, leptin and combinations thereof as the target(s) for the CRISPR-Cas system.

Disorders associated with secretase activity

U.S. Patent Application Publication No. 20110023146 describes the use of zinc finger nucleases to genetically modify cells, animals, and proteins associated with a disorder related to secretase activity. Secretases are required for the processing of precursor proteins into their biologically active forms. Defects in various components of the secretase pathways are associated with many disorders, particularly those characterized by amyloidogenesis or amyloid plaques, such as Alzheimer's disease (AD).

With respect to a disorder associated with secretase activity, proteins associated with these disorders are a diverse group of proteins that influence susceptibility to multiple disorders, the presence of a disorder, the severity of a disorder, or any combination thereof. The present disclosure contemplates editing any chromosomal sequences that encode proteins associated with a disorder associated with secretase activity. Proteins associated with a disorder associated with secretase activity are typically selected based on an experimentally established association of secretase-related proteins with the development of a disorder associated with secretase activity. For example, the rate of formation or the circulating concentration of a protein associated with a disorder associated with secretase activity may be increased or decreased in a population with a disorder associated with secretase activity compared to a population without a disorder associated with secretase activity. Differences in protein levels can be assessed using proteomic techniques including, but not limited to, Western blotting, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, the protein associated with a secretase activity disorder can be identified by generating gene expression profiles of protein-coding genes using genomic analysis techniques including, but not limited to, DNA microarray analysis, sequence-based analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).

By way of non-limiting example, proteins associated with secretase activity disorder include PSENEN (presenilin enhancer homolog 2 (C. elegans)), CTSB (cathepsin B), PSEN1 (presenilin 1), APP (amyloid beta (A4) protein precursor), APH1B (anterior laryngeal defect 1 homolog B (C. elegans)), PSEN2 (presenilin 2 (Alzheimer's disease type 4)), BACE1 (beta-site APP-cleaving enzyme 1), ITM2B (integral membrane protein 2B), CTSD (cathepsin D), NOTCH1 (Notch translocation-associated homolog 1 (Drosophila)), TNF (tumor necrosis factor (TNF family member 2)), INS (insulin), DYT10 (dysfunction factor 10 dystonia), ADAM17 (ADAM domain 17 metallopeptidase), APOE (apolipoprotein E), ACE (angiotensin I converting enzyme (peptidyl dipeptidase A) 1), STN (statin), TP53 (tumor protein p53), IL6 (interleukin 6 (interferon, beta 2)), NGFR (nerve growth factor receptor (TNFR family, member 16)), IL1B (interleukin 1, beta), ACHE (acetylcholinesterase (blood group Yt)), CTNNB1 (catenin (cadherin-associated protein), beta 1, 88 kDa), IGF1 (insulin-like growth factor 1 (somatomedin C)), IFNG (interferon, gamma), NRG1 (neuregulin 1), CASP3 (apoptosis-associated caspase 3 cysteine peptidase), MAPK1 (mitogen-activated protein kinase 1), CDH1 (cadherin 1, type 1, E-cadherin (epithelial)), APBB1 (amyloid precursor-binding protein beta (A4), family B, member 1 (Fe65)), HMGCR (3-hydroxy-3-methylglutaryl-coenzyme A reductase), CREB1 (cAMP-responsive element binding protein 1), PTGS2 (prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)), HES1 (hairy and enhancer of split 1 protein, (Drosophila)), CAT (catalase), TGFB1 (transforming growth factor, beta 1), ENO2 (enolase 2 (gamma, neuronal)), ERBB4 (ERBB4 oncogene homolog 4 virus erythroblastic leukemia v-erb-a (avian)), TRAPPC10 (particle protein migration complex 10), MAOB (monoamine oxidase B), NGF (nerve growth factor (beta-polypeptide)), 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 (basic)), IL3 (interleukin 3 (colony stimulating factor, multiple)), LRP1 (low-density lipoprotein receptor-related protein 1), NOTCH4 (Notch homolog 4 (Drosophila)), MAPK8 (mitogen-activated protein kinase 8), PREP (prolyl endopeptidase), NOTCH3 (Notch 3 homolog 3 (Drosophila)), PRNP (prion protein), CTSG (catapsin G), EGF (epidermal growth factor (beta-urogastron)), REN (renin), CD44 (CD44 molecule (Indian blood group)), SELP (selectin P (granular membrane protein with a mass of 140 kDa, CD62 antigen)), GHR (growth hormone receptor), ADCYAP1 (adentyl cyclase activating polypeptide 1 1 (pituitary)), INSR (insulin receptor), GFAP (glial fibrillary acidic protein), MMP3 (matrix metallopeptidase 3 (stromelysin 1, progelatinase)), MAPK10 (mitogen-activated protein kinase 10), SP1 (transcription factor Sp1), MYC (avian myelocytomatosis virus oncogene homolog v-myc), CTSE (cathepsin E), PPARA (peroxisome proliferator-activated receptor alpha), JUN (jun oncogene), TIMP1 (TIMP inhibitor of metallopeptidase 1), IL5 (interleukin 5 (colony stimulating factor, eosinophil)), IL1A (interleukin 1, alpha), MMP9 (matrix metallopeptidase 9 (gelatinase B, 92-kDa gelatinase, 92-kDa collagenase type IV)), HTR4 (5-hydroxytryptamine (serotonin) receptor type 4), HSPG2 (heparan sulfate proteoglycan 2), KRAS (Kirsten rat sarcoma virus oncogene homolog v-Ki-ras2), CYCS (cytochrome c, somatic), SMG1 (SMG1 homolog, phosphatidylinositol 3-kinase-related kinase (C. elegans)), IL1R1 (interleukin 1 receptor, type I), PROK1 (prokineticin 1), MAPK3 (mitogen-activated protein kinase 3), NTRK1 (neurotrophic tyrosine kinase receptor, type 1), IL13 (interleukin 13), MME (membrane metalloendopeptidase), TKT (transketolase), CXCR2 (chemokine receptor 2 (with a C-X-C motif)), IGF1R (insulin-like growth factor receptor 1), RARA (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 (ataxin 1), PAWR (PRKC, apoptotic, WT1, regulator), NOTCH2 (Notch homolog 2 (Drosophila)), 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 kinase 14), PRG2 (proteoglycan 2, bone marrow (activator of natural killer cells, major basic eosinophil granule protein)), PRKCA (protein kinase C, alpha), L1 CAM (cell adhesion molecule L1), CD40 (CD40 molecule, TNF receptor superfamily member 5), NR1I2 (nuclear receptor family 1, group I, member 2), JAG2 (jagged 2), CTNND1 (catenin (cadherin-associated protein), delta 1), CDH2 (cadherin 2, type 1, N-cadherin (neuronal)), CMA1 (chymase 1, mast cell), SORT1 (sortilin 1), DLK1 (delta-like 1 homolog (Drosophila)), THEM4 (thioesterase family member 4), JUP (intercellular contact plakoglobin), CD46 (CD46 molecule, complement regulatory protein), CCL11 (chemokine ligand 11 (with C-C motif)), CAV3 (caveolin 3), RNASE3 (ribonuclease, RNase, family A, 3 (eosinophil cationic protein)), HSPA8 (heat shock protein 8, with a mass of 70 kDa), CASP9 (caspase 9, apoptosis-associated cysteine peptidase), CYP3A4 (cytochrome P450, family 3, subfamily A, polypeptide 4), CCR3 (chemokine receptor 3 (with C-C motif)), TFAP2A (transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha)), SCP2 (sterol transfer protein 2), CDK4 (cyclin-dependent kinase 4), HIF1A (hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)), TCF7L2 (T-cell-specific transcription factor 7-like factor 2, HMG-box), IL1R2 (interleukin 1 receptor, type II), B3GALTL (beta 1,3-galactosyltransferase-like factors), MDM2 (Mdm2 homolog of p53-binding protein (mouse)), RELA (reticuloendotheliosis virus oncogene A homolog v-rel (avian)), CASP7 (caspase 7, apoptosis-associated cysteine peptidase), IDE (insulin-degrading enzyme), FABP4 (fatty acid-binding protein 4, adipocyte), CASK (calcium/calmodulin-dependent protein kinase (MAGUK family)), ADCYAP1R1 (adenylate cyclase activating receptor polypeptide 1 (pituitary), type I), ATF4 (activating transcription factor 4 (tax-responsive enhancer element B67)), PDGFA (platelet-derived growth factor alpha polypeptide), C21 or f33 (chromosome 21 open reading frame 33), SCG5 (secretogranin V (protein 7B2)), RNF123 (ring 123 zinc finger protein), NFKB1 (nuclear factor of B-cell kappa light chain polypeptide gene enhancer type 1), ERBB2 (erythroblastic leukemia virus oncogene 2 homolog v-erb-b2, oncogene homolog of neuro-/glioblastoma origin (avian)), CAV1 (caveolin 1, caveolae protein, 22 kDa), MMP7 (matrix metallopeptidase 7 (matrilysin, uterine)), TGFA (transforming growth factor, alpha), RXRA (retinoid X receptor, alpha), STX1A (syntaxin 1A (brain)), PSMC4 (proteasome subunit 26S (prosome, macropain) ATPase, 4), P2RY2 (pyrenergic receptor P2Y, G protein-coupled receptor, 2), TNFRSF21 (tumor necrosis factor receptor family, member 21), DLG1 (discs, large homolog 1 (Drosophila)), NUMBL (numb-like homolog (Drosophila)), SPN (sialophorin), PLSCR1 (phospholipid scramblease 1), UBQLN2 (ubiquitin 2), UBQLN1 (ubiquitin 1), PCSK7 (proprotein convertase subtilisin/kexin type 7), SPON1 (spondin 1, extracellular matrix protein), SILV (silver homolog (mouse)), QPCT (glutaminyl peptide cyclotransferase), HESS (hairy and enhancer of split 5 protein (Drosophila)), GCC1 (GRIP-containing double-stranded domain 1), and combinations thereof.

A genetically modified animal or cell may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chromosomal sequences with a disrupted structure encoding a protein associated with a disorder associated with secretase activity, and zero, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chromosomally integrated sequences encoding a protein with a disrupted structure associated with a disorder associated with secretase activity.

Targeting the liver and liver cells; hemophilia

Targeting of liver cells is envisaged. This can be done in vitro or in vivo. Hepatocytes are preferred. Delivery of the CRISPR protein can be done via viral vectors, especially AAV-based vectors (and in particular AAV2/6). These can be administered via intravenous injection.

The preferred liver target, whether in vitro or in vivo, is the albumin gene. It represents a so-called "safety zone" because albumin is expressed at very high levels, and therefore some reduction in albumin production after successful gene editing is acceptable. It is also preferred because the high expression levels seen with the albumin promoter/enhancer ensure that useful levels of correct or transgene production (from the inserted donor template) are achieved even when only a small proportion of hepatocytes are edited.

Intron 1 of albumin was shown by Wechsler et al. (presented at 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 posted December 6, 2015) to be a suitable target site. Their study used zinc fingers to cut DNA at the target site, and suitable guide sequences can be generated to direct cleavage at the same site using the CRISPR protein.

The use of targets in highly expressed genes (genes with highly active enhancers/promoters), such as albumin, can also allow the use of a promoter-free donor template, as described by Wechsler et al., and this is also widely applicable beyond liver targeting. Other examples of highly expressed genes are known.

Blood disorders involving the liver, in particular hemophilia, namely hemophilia B

Successful gene editing of hepatocytes has been achieved in mice (both in vitro and in vivo) and in non-human primates (in vivo), demonstrating that treatment of blood disorders through gene editing/genome engineering in hepatocytes is feasible. In particular, expression of the human F9 gene (hF9) in hepatocytes has been demonstrated in non-human primates, suggesting the possibility of treating hemophilia B in humans.

Wechsler et al. presented at the 57th Annual Meeting and Exposition of the American Society of Hematology (abstract posted December 6, 2015, and available online at https://ash.confex.com/ash/2015/webprogram/Paper86495.html) that they successfully expressed human F9 (hF9) from hepatocytes from non-human primates via in vivo gene editing. This was achieved using 1) two zinc finger nucleases (ZFNs) targeting intron 1 of the albumin locus and 2) a human F9 donor template construct. ZFN and donor template were encoded on separate hepatotropic adeno-associated virus serotype 2/6 (AAV2/6)-based vectors and administered intravenously, resulting in targeted insertion of a corrected copy of the hF9 gene into the albumin locus of a subset of liver hepatocytes.

The albumin locus was chosen as a "safety net" because production of this most abundant plasma protein exceeds 10 g/day and modest reductions in such levels are well tolerated. Genome-edited hepatocytes produced hFIX (hF9) in therapeutic amounts, in contrast to albumin driven by a highly active enhancer/promoter. Targeted integration of the hF9 transgene into the albumin locus and splicing of this gene into the albumin transcript were demonstrated.

Mouse studies: C57BL/6 mice were treated with backbone (n=20) or AAV2/6-based vectors (n=25) encoding mouse surrogate reagents at 1.0 ^x1013 vector genomes (vg)/kg via tail vein injection. Plasma hFIX ELISA analysis of treated mice showed peak levels of 50-1053 ng/mL that were maintained over the 6-month study. Analysis of FIX activity from mouse plasma confirmed bioactivity commensurate with expression levels.

Studies in non-human primates (NHPs): A single intravenous co-infusion of AAV2/6-based vectors encoding NHP albumin-targeting ZFNs and human F9 donor matrix at ^1.2x1013 vg/kg (n=5/group) resulted in levels >50 ng/mL (>1% of normal) in this large animal model. Administration of higher doses of AAV2/6 (up to and including ^1.5x1014 vg/kg) resulted in hFIX levels up to and including 1000 ng/mL (or 20% of normal) in several animals and up to 2000 ng/mL (or 50% of normal) in one animal over the course of the study (3 months).

The treatment was well tolerated in mice and NHPs, with no significant toxicological findings associated with AAV2/6 ZFN+ donor treatment in either species at therapeutic doses. Sangamo (CA, USA) has since applied to and received FDA approval to conduct the world’s first human clinical trial to apply in vivo genome editing. This is in addition to EMEA approval of Glybera, a gene therapy-based treatment for lipoprotein lipase deficiency.

Accordingly, it is preferred in some embodiments that any or all of the following are used:

AAV-based vectors (in particular AAV2/6), preferably administered by intravenous injection;

albumin as a target for gene editing/transgene/matrix insertion - especially in intron 1 of albumin;

donor matrix of human F9; and/or

promoter-free donor matrix.

Hemophilia B

Accordingly, in some embodiments, it is preferred that the present invention is used to treat hemophilia B. For example, it is preferred that a template is provided, and that it is a human F9 gene. It is believed that the hF9 template contains a wt or "error-free" version of hF9, so that the treatment is effective.

In an alternative embodiment, the hemophilia B version of F9 may be delivered to generate a model organism, cell, or cell line (e.g., a mouse or non-human primate model organism, cell, or cell line), a model organism, cell, or cell line having or carrying the hemophilia B phenotype, i.e., an inability to produce wt F9.

Hemophilia A

In some embodiments, the F9 (factor IX) gene may be replaced with the F8 (factor VIII) gene described herein, resulting in a treatment for hemophilia A (by producing an error-free F8 gene) and/or the creation of a model organism, cell, or cell line with hemophilia A (by producing an error-containing F8 gene, a version that results in hemophilia A).

Hemophilia C

In some embodiments, the F9 (factor IX) gene may be replaced with the F11 (factor XI) gene described herein, resulting in a treatment for hemophilia C (by producing an error-free F11 gene) and/or the creation of a model organism, cell, or cell line for hemophilia C (by producing an error-containing F11 gene, a version that results in hemophilia C).

Other conditions

Cystic fibrosis (CF)

In some embodiments, treatment, prevention and diagnosis of cystic fibrosis are provided. The target is preferably the SCNN1A or CFTR gene. This is described in WO2015157070, the disclosure of which is incorporated herein by reference.

Cancer and CAR-T

In some embodiments, treatment, prevention and diagnosis of cystic fibrosis are provided. 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 lymphoma, chronic lymphocytic leukemia (CLL), B-cell acute lymphocytic 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 cancer, lung cancer, esophageal cancer, hepatocellular carcinoma, pancreatic cancer, astrocytoma, mesothelioma, head and neck cancer, and medulloblastoma. This can be done using an engineered chimeric antigen receptor (CAR) T cell. This is described in WO2015161276, the disclosure of which is incorporated herein by reference.

Herpes simplex virus types 1 and 2

In some embodiments, treatment, prevention and diagnosis of HSV-1 (herpes simplex virus 1) are provided. The target is preferably the UL19, UL30, UL48 or UL50 gene in HSV-1. This is described in WO2015153789, the disclosure of which is incorporated herein by reference.

In other embodiments, treatment, prevention and diagnosis of HSV-2 (herpes simplex virus 2) are provided. The target is preferably the UL19, UL30, UL48 or UL50 gene in HSV-2. This is described in WO2015153791, the disclosure of which is incorporated herein by reference.

The present invention can be further illustrated and expanded based on the aspect of development and use of CFISPR-Cas9 as set forth in the following articles, hereby incorporated by reference herein and, in particular, it relates to delivery of the CRISPR protein complex and uses of the RNA-guided endonuclease in cells and organisms:

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 RJ, Scott DA, Church GM, Zhang F. Nature. Aug 22;500(7463):472-6. doi: 10.1038/Nature12466. Epub 2013 Aug 23 (2013);

Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity. Ran, F.A., Hsu, P.D., Lin, C.Y., Gootenberg, J.S., Konermann, S., Trevino, A.E., Scott, D.A., 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, F.A., Konermann, S., Agarwala, V., Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, T.J., Marraffini, L.A., Bao, G., & Zhang, F. Nat Biotechnol doi:10.1038/nbt.2647 (2013);

Genome engineering using the CRISPR-Cas9 system. Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott, D.A., Zhang, F. Nature Protocols Nov;8(11):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). [Online publication ahead of print];

Crystal structure of cas9 in complex with guide RNA and target DNA. Nishimasu, H., Ran, F.A., Hsu, P.D., Konermann, S., Shehata, S.I., Dohmae, N., Ishitani, R., Zhang, F., Nureki, O. 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 Biotechnol. Apr 20. doi: 10.1038/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 O, Eisenhaure TM, Jovanovic M, Graham DB, Jhunjhunwala S, Heidenreich M, Xavier RJ, Langer 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 3 September 2014) Nat Biotechnol. 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 Biotechnol. 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 Biotechnol. 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 PA. 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 O, 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/srep10833 (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/nature15521. 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 Cas9 nucleases with improved specificity, Slaymaker et al., Science 2016 Jan 1 351(6268): 84–88 doi: 10.1126/science.aad5227. Online publication 1 December 2015. [Online publication precedes print publication],

each of which is incorporated herein by reference, may be considered in the practice of the present invention and is described briefly below.

Cong et al. constructed type II CRISPR-Cas systems based on both Streptococcus thermophilus Cas9 and Streptococcus pyogenes Cas9 for use in eukaryotic cells and demonstrated that Cas9 nucleases can be guided by short RNAs to induce precise DNA cleavage in human and mouse cells. Their study further demonstrated that Cas9 engineered into a single-strand break-inducing enzyme can be used to facilitate homologous recombination repair in eukaryotic cells with minimal mutagenic activity. Furthermore, their study demonstrated that multiple guide sequences can be encoded in a single CRISPR array to enable simultaneous editing at multiple sites of endogenous genomic loci in the mammalian genome, demonstrating the facile programmability and broad applicability of RNA-guided nuclease technology. This ability to use RNA to program sequence-specific DNA cleavage in cells has defined a new class of genome engineering tools. These studies further demonstrate that other CRISPR loci are likely to be transplanted into mammalian cells and may also mediate mammalian genome cleavage. Importantly, further improvements to certain aspects of the CRISPR-Cas system are possible to enhance its efficiency and versatility.

Jiang et al. used the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 complexed with dual RNAs to introduce precise mutations into the genomes of Streptococcus pneumoniae and Escherichia coli. The approach relied on cleavage at the target site of the genome, guided by the dual RNA:Cas9 system, to kill unmutated cells and eliminated the need for selectable markers or negative selection systems. The study reported reprogramming the specificity of the dual RNA:Cas9 system by altering the CRISPR short RNA (crRNA) sequence to introduce single- or multi-nucleotide changes, driven by editing templates. The study showed that the simultaneous use of two crRNAs enabled multiplexed mutagenesis. Furthermore, when the approach was used in combination with recombinational engineering, in S. pneumoniae, nearly 100% of the cells recovered using the described approach contained the desired mutation, and in E. coli, 65% of the recovered cells contained the mutation.

Wang et al. (2013) used the CRISPR/Cas system to generate mice carrying mutations in multiple genes in a single step, which were traditionally generated in several steps, using serial recombination in embryonic stem cells and/or continuous intercrossing of mice with a single mutation. The CRISPR/Cas system will greatly accelerate the study of functionally redundant genes and epistatic gene interactions in vivo.

Konermann et al. (2013) explored the need in the field for flexible and robust technologies to enable optical and chemical modulation of the Cas9 CRISPR enzyme based on DNA-binding domains and transcription activator-like effectors.

Ran et al. (2013-A) described an approach in which a mutant Cas9 nickase was used in combination with paired guide RNAs to introduce targeted double-strand breaks. This addresses the issue that the Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a guide sequence that may tolerate some mismatches with the target DNA and thus promote unwanted off-target mutagenesis. Since individual single-strand breaks in the genome are subject to high-fidelity repair, simultaneous introduction of single-strand breaks by appropriately offset guide RNAs is necessary for double-strand break formation and increases the number of specifically recognized bases for target cleavage. The authors demonstrated that the use of pairwise introduction of single-strand breaks can reduce off-target activity in cell lines by 50- to 1500-fold and facilitate gene knockout in mouse zygotes without reducing the efficiency of on-target cleavage. This flexible strategy enables a wide variety of genome editing applications requiring high specificity.

Hsu et al. (2013) characterized the targeting specificity of SpCas9 in human cells to inform target site selection and avoid off-target effects. The study assessed >700 guide RNA variants and SpCas9-induced insertion/deletion mutation rates at >100 predicted off-target loci across the genome in 293T and 293FT cells. The authors showed that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner with sensitivity to the number, position, and distribution of mismatches. The authors further showed that SpCas9-mediated cleavage is not affected by DNA methylation and that the dosage of SpCas9 and sgRNA can be tailored to minimize off-target modifications. Furthermore, to facilitate applications in mammalian genome engineering, the authors reported the development of a web-based software tool to manage target sequence selection and confirmation, as well as off-target event analyses.

Ran et al. (2013-B) described a toolkit for Cas9-mediated genome editing via non-homologous end joining (NHEJ) or homologous recombination repair (HDR) in mammalian cells, and generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, the authors additionally described a strategy for introducing double-strand breaks using a mutant Cas9 nickase with paired guide RNAs. The protocol presented by the authors provides an experimentally derived guide to selecting target sites, assessing cleavage efficiency, and analyzing off-target activity. Studies have shown that from target engineering, gene modifications can be achieved in as little as 1-2 weeks, and modified clonal cell lines can be generated within 2-3 weeks.

Shalem et al. describe a new way to probe gene function at a genome-wide scale. Their studies showed that delivery of a genome-wide CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique targeting sequences enabled screening by both positive and negative selection in human cells. First, the authors demonstrated the use of the GeCKO library to identify genes essential for cell survival in cancer and pluripotent stem cells. Next, in a melanoma model, the authors screened for genes whose loss of function is involved in resistance to vemurafenib, a therapeutic that inhibits mutant protein kinase BRAF. Their studies showed that the top-ranking candidates included previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. The authors observed a high level of concordance between independent guide RNAs targeting the same gene and a high hit confirmation rate, thus demonstrating the promise of Cas9-based genome-scale screening.

Nishimasu et al. reported the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at a resolution of 2.5 Å. The structure revealed a bilobed architecture formed by the target recognition lobe and the nuclease lobe, which mediate the placement of the sgRNA:DNA heteroduplex in the positively charged groove at their interface. While the recognition lobe is essential for sgRNA and DNA binding, the nuclease lobe contains the HNH and RuvC nuclease domains, which are positioned appropriately to cleave the complementary and non-complementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxy-terminal domain responsible for interaction with the protospacer adjacent motif (PAM). These high-resolution structural analyses and accompanying functional analyses have revealed the molecular mechanism of RNA-guided Cas9 targeting of DNA, thus paving the way for the rational design of new general-purpose genome editing technologies.

Wu et al. performed genome-wide binding site mapping for catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes delivered with single guide RNAs (sgRNAs) into mouse embryonic stem cells (mESCs). The authors showed that each of the four sgRNAs tested targets dCas9 to tens to thousands of sites in the genome, often characterized by the presence of a 5-nucleotide seed region in the sgRNA and a protospacer adjacent motif (PAM) NGG. Chromatin inaccessibility reduces dCas9 binding to other sites with sequences complementary to the seed; thus, 70% of off-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 a mutation above background levels at only one site. The authors proposed a two-state model of Cas9 binding and cleavage in which a sequence complementary to a seed triggers binding, but cleavage requires multiple pairing with the target DNA.

Platt et al. generated a Cre-dependent Cas9 knockin mouse and demonstrated genome editing in vivo as well as ex vivo by delivering adeno-associated virus (AAV), lentivirus, or particle-based guide RNA to neurons, immune cells, and endothelial cells.

The publication by Hsu et al. (2014) is a review article that describes the overall history of CRISPR-Cas9 from yogurt to genome editing, including genetic screening of cells.

The publication by Wang et al. (2014) reported a pooled loss-of-function genetic screening approach applicable to both positive and negative selection using a whole-genome lentiviral single guide RNA (sgRNA) library.

Doench et al. generated a pool of sgRNAs covering all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantified their ability to form null alleles of their target gene using antibody staining and flow cytometry. The authors showed that PAM optimization increased activity and also provided an online tool for sgRNA design.

Swiech et al. show that AAV-mediated SpCas9 genome editing can enable reverse genetic studies of gene function in the brain.

Konermann et al. (2015) describe the ability to attach multiple effector domains, such as transcriptional activator, functional and epigenomic regulators, at specific positions on the leader sequence, such as the stem or tetra-loop, with and without linkers.

Zetsche et al. show that the Cas9 enzyme can be cleaved into two and thus the assembly of Cas9 for activation can be controlled.

The publication by Chen et al. advances the multiplex screen by demonstrating that a genome-wide in vivo CRISPR-Cas9 screen in mice identified genes that regulate lung metastasis.

The paper by Ran et al. (2015) addresses SaCas9 and its ability to edit genomes, demonstrating the impossibility of extrapolating from biochemical assays. The paper by Shalem et al. (2015) describes ways in which catalytically inactive Cas9 (dCas9) fusions are used to synthetically repress (CRISPRi) or activate (CRISPRa) expression, showing advances in the use of Cas9 for genome-wide screening, including ordered and pooled screens, knockout approaches that inactivate genomic loci, and strategies that modulate transcriptional activity.

The publication by Shalem et al. (2015) describes ways in which catalytically inactive Cas9 (dCas9) fusions are used to synthetically repress (CRISPRi) or activate (CRISPRa) expression, showing advances in the use of Cas9 for genome-wide screens, including ordered and pooled screens, knockout approaches that inactivate genomic loci, and strategies that modulate transcriptional activity.

Xu et al. (2015) evaluated DNA sequence characteristics that contribute to the efficiency of single guide RNA (sgRNA) in CRISPR-based screening. The authors examined the efficiency of CRISPR/Cas9 knockout and the nucleotide preference at the cleavage site. The authors also found that the sequence preference for CRISPRi/a was significantly different from that for CRISPR/Cas9 knockout.

Parnas et al. (2015) introduced whole-genome CRISPR-Cas9 pooled libraries into dendritic cells (DCs) to identify genes that control tumor necrosis factor (Tnf) induction by bacterial lipopolysaccharide (LPS). Known regulators of Tlr4 signaling and previously unknown candidates were identified and classified into three functional modules with distinct effects on classical LPS responses.

Ramanan et al (2015) demonstrated cleavage of viral episomal DNA (cccDNA) in infected cells. The HBV genome exists in the nuclei of infected hepatocytes as 3.2-kb double-stranded episomal DNA molecules called covalently linked circular DNA (cccDNA), which is a major component in the HBV life cycle and whose replication is not inhibited by existing therapies. The authors showed that sgRNA specifically targeting highly conserved regions of HBV consistently suppressed viral replication and cleaved cccDNA.

Nishimasu et al. (2015) reported crystal structures of SaCas9 in complex with single RNA (sgRNA) and its double-stranded DNA targets containing 5′-TTGAAT-3′ PAM and 5′-TTGGGT-3′ PAM. Structural comparison of SaCas9 with SpCas9 indicated both structural conservation and variability, explaining their different PAM specificities and orthologous sgRNA recognition.

Canver et al. (2015) demonstrated a CRISPR-Cas9-based functional study of non-coding genomic elements. The authors designed pooled CRISPR-Cas9 guide RNA libraries to perform in situ saturation mutagenesis of human and mouse BCL11A enhancers, revealing critical features of the enhancers.

Zetsche et al. (2015) described the characterization of Cpf1, a class 2 CRISPR nuclease from Francisella novicida U112, with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease that lacks tracrRNA, utilizes a protospacer-adjacent motif with high T content, and cleaves DNA via a staggered double-strand DNA break.

Shmakov et al. (2015) described three different class 2 CRISPR-Cas systems. The enzymes of two CRISPR systems (C2c1 and C2c3) contain RuvC-like endonuclease domains distinct from Cpf1. Unlike Cpf1, C2c1 depends on both crRNA and tracrRNA to cleave DNA. The third enzyme (C2c2) contains two putative HEPN RNase domains and is tracrRNA-independent.

Slaymaker et al. (2016) described the use of structure-guided protein engineering to improve the specificity of Streptococcus pyogenes Cas9 (SpCas9). The authors developed "enhanced specificity" SpCas9 (eSpCas9) variants that retained robust on-target cleavage with reduced off-target effects.

Also, the publication "Dimeric CRISPR RNA-guided FokI 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) refers to dimeric RNA-guided FokI nucleases that recognize extended sequences and can edit endogenous genes with high efficiency in human cells.

With respect to the general information on CRISPR-Cas systems, all components thereof and methods of delivering such components, including methods, materials, delivery vehicles, vectors, particles, AAVs and their production and use taking into account the amounts and compositions are applicable in the practice of the present invention, reference is hereby made to: U.S. Patent Nos. 8,697,359, 8,771,945, 8,795,965, 8,865,406, 8,871,445, 8,889,356, 8,889,418, 8,895,308, 8,906,616, 8,932,814, 8,945,839, 8,993,233 and 8,999,641; US Patent Application Publications US 2014-0310830 (US Patent Application Serial Number 14/105,031), US 2014-0287938 A1 (US Patent Application Serial Number 14/213,991), US 2014-0273234 A1 (US Patent Application Serial Number 14/293,674), US2014-0273232 A1 (US Patent Application Serial Number 14/290,575), US 2014-0273231 (US Patent Application Serial Number 14/259,420), US 2014-0256046 A1 (US Patent Application Serial Number 14/226274), US 2014-0248702 A1 (US Patent Application Serial Number 14/258458), US 2014-0242700 A1 (US Patent Application Serial Number 14/222930), US 2014-0242699 A1 (US Patent Application Serial Number 14/183512), US 2014-0242664 A1 (US Patent Application Serial Number 14/104990), US 2014-0234972 A1 (US Patent Application Serial Number 14/183471), US 2014-0227787 A1 (US Patent Application Serial Number 14/256,912), US 2014-0189896 A1 (US Patent Application Serial Number 14/105,035), US 2014-0186958 (US Patent Application Serial Number 14/105,017), US 2014-0186919 A1 (US Patent Application Serial Number 14/104,977), US 2014-0186843 A1 (US Patent Application Serial Number 14/104,900), US 2014-0179770 A1 (US Patent Application Serial Number 14/104,837), and US 2014-0179006 A1 (US Patent Application Serial Number 14/183,486), US 2014-0170753 (US patent application serial number 14/183,429); US 2015-0184139 (US patent application serial number 14/324,960); 14/054,414 European patent applications EP 2 771 468 (EP13818570.7), EP 2 764 103 (EP13824232.6) and EP 2 784 162 (EP14170383.5); 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 (PCT/US2013/074825), 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), WO 2014/204725 (PCT/US2014/041803), WO 2014/204726 (PCT/US2014/041804), WO 2014/204727 (PCT/US2014/041806), WO 2014/204728 (PCT/US2014/041808), WO 2014/204729 (PCT/US2014/041809), WO 2015/089351 (PCT/US2014/069897), WO 2015/089354 (PCT/US2014/069902), WO 2015/089364 (PCT/US2014/069925), WO 2015/089427 (PCT/US2014/070068), WO 2015/089462 (PCT/US2014/070127), WO 2015/089419 (PCT/US2014/070057), WO 2015/089465 (PCT/US2014/070135), WO 2015/089486 (PCT/US2014/070175), PCT/US2015/051691, PCT/US2015/051830. Reference is also made to U.S. Provisional Patent Applications 61/758,468; 61/802,174; 61/806,375; 61/814,263; 61/819,803; and 61/828,130, filed January 30, 2013; March 15, 2013; March 28, 2013; April 20, 2013; May 6, 2013; and May 28, 2013, respectively. Reference is also made to U.S. Provisional Patent Application No. 61/836,123, filed June 17, 2013. Reference is further made to U.S. Provisional Patent Applications 61/835,931, 61/835,936, 61/835,973, 61/836,080, 61/836,101, and 61/836,127, each filed June 17, 2013. Further reference is made to U.S. Provisional Patent Applications 61/862,468 and 61/862,355, filed August 5, 2013; 61/871,301, filed August 28, 2013; 61/960,777, filed September 25, 2013, and 61/961,980, filed October 28, 2013. Reference is further made to: PCT/US2014/62558, filed October 28, 2014, and U.S. Provisional Patent Applications Serial Nos. 61/915,148, 61/915,150, 61/915,153, 61/915,203, 61/915,251, 61/915,301, 61/915,267, 61/915,260, and 61/915,397, each filed December 12, 2013; 61/757,972 and 61/768,959, each filed January 29, 2013 and February 25, 2013; 62/010888 and 62/010879, each filed June 11, 2014; 62/010329, 62/010439, and 62/010441, each filed June 10, 2014; 61/939228 and 61/939242, each filed February 12, 2014; 61/980012, filed April 15, 2014; 62/038358, filed August 17, 2014; 62/055,484, 62/055,460, and 62/055,487, each filed September 25, 2014; and 62/069,243, filed October 27, 2014. Reference is made to a PCT application designating, among others, the United States of America, Application No. PCT/US14/41806, filed June 10, 2014. Reference is made to U.S. Provisional Patent Application No. 61/930,214, filed January 22, 2014. Reference is made to a PCT application designating, among others, the United States of America, Application No. PCT/US14/41806, filed June 10, 2014.

Also referenced is U.S. Patent Application Serial No. 62/180,709, filed June 17, 2015, PROTECTED GUIDE RNAS (PGRNAS); U.S. Patent Application Serial No. 62/091,455, filed December 12, 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. Patent Application Serial No. 62/096,708, filed December 24, 2014, PROTECTED GUIDE RNAS (PGRNAS); U.S. Patent Applications 62/091,462, filed December 12, 2014, 62/096,324, filed December 23, 2014, 62/180,681, filed June 17, 2015, and 62/237,496, filed October 5, 2015, DEAD GUIDES FOR CRISPR TRANSCRIPTION FACTORS; U.S. Patent Applications 62/091,456, filed December 12, 2014, and 62/180,692, filed June 17, 2015, ESCORTED AND FUNCTIONALIZED GUIDES FOR CRISPR-CAS SYSTEMS; U.S. Patent Application Serial No. 62/091,461, filed December 12, 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR GENOME EDITING AS TO HEMATOPOETIC STEM CELLS (HSC); U.S. Patent Application Serial No. 62/094,903, filed December 19, 2014, UNBIASED IDENTIFICATION OF DOUBLE-STRAND BREAKS AND GENOMIC REARRANGEMENT BY GENOME-WISE INSERT CAPTURE SEQUENCING; U.S. Patent Application Serial No. 62/096,761, filed December 24, 2014, ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED ENZYME AND GUIDE SCAFFOLDS FOR SEQUENCE MANIPULATION; U.S. Patent Application Nos. 62/098,059, filed December 30, 2014, 62/181,641, filed June 18, 2015, and 62/181,667, filed June 18, 2015, RNA-TARGETING SYSTEM; U.S. Patent Application Nos. 62/096,656, filed December 24, 2014, and 62/181,151, filed June 17, 2015, CRISPR HAVING OR ASSOCIATED WITH DESTABILIZATION DOMAINS; U.S. Patent Application No. 62/096,697, filed December 24, 2014, CRISPR HAVING OR ASSOCIATED WITH AAV; U.S. Patent Application 62/098,158, filed December 30, 2014, ENGINEERED CRISPR COMPLEX INSERTIONAL TARGETING SYSTEMS; U.S. Patent Application 62/151,052, filed April 22, 2015, CELLULAR TARGETING FOR EXTRACELLULAR EXOSOMAL REPORTING; U.S. Patent Application 62/054,490, filed September 24, 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING PARTICLE DELIVERY COMPONENTS; U.S. Patent Application Ser. No. 61/939,154, filed February 12, 2014, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. Patent Application Ser. No. 62/055,484, filed September 25, 2014, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. Patent Application Ser. No. 62/087,537, filed December 4, 2014, SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. Patent Application 62/054,651, filed September 24, 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OF MULTIPLE CANCER MUTATIONS IN VIVO; U.S. Patent Application 62/067,886, filed October 23, 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR MODELING COMPETITION OF MULTIPLE CANCER MUTATIONS IN VIVO; U.S. Patent Applications 62/054,675, filed September 24, 2014, and 62/181,002, filed June 17, 2015, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS IN NEURONAL CELLS/TISSUES; U.S. Patent Application 62/054,528, filed September 24, 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS IN IMMUNE DISEASES OR DISORDERS; U.S. Patent Application 62/055,454, filed September 25, 2014, DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TARGETING DISORDERS AND DISEASES USING CELL PENETRATION PEPTIDES (CPP); U.S. Patent Application 62/055,460, filed September 25, 2014, MULTIFUNCTIONAL-CRISPR COMPLEXES AND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; U.S. Patent Application Nos. 62/087,475, filed December 4, 2014, and 62/181,690, filed June 18, 2015, FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. Patent Application No. 62/055,487, filed September 25, 2014, FUNCTIONAL SCREENING WITH OPTIMIZED FUNCTIONAL CRISPR-CAS SYSTEMS; U.S. Patent Applications 62/087,546, filed December 4, 2014, and 62/181,687, filed June 18, 2015, MULTIFUNCTIONAL CRISPR COMPLEXES AND/OR OPTIMIZED ENZYME LINKED FUNCTIONAL-CRISPR COMPLEXES; and U.S. Patent Application 62/098,285, filed December 30, 2014, CRISPR MEDIATED IN VIVO MODELING AND GENETIC SCREENING OF TUMOR GROWTH AND METASTASIS.

Reference is made to U.S. Patent Applications 62/181,659, filed June 18, 2015, and 62/207,318, filed 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 U.S. Patent Applications 62/181,663, filed June 18, 2015, and 62/245,264, filed October 22, 2015, NOVEL CRISPR ENZYMES AND SYSTEMS, U.S. Patent Applications 62/181,675, filed June 18, 2015, 62/285,349, filed October 22, 2015, 62/296,522, filed February 17, 2016, and 62/320,231, filed April 8, 2016, NOVEL CRISPR ENZYMES AND SYSTEMS, U.S. Patent Application 62/232,067, filed September 24, 2015, U.S. Patent Application 14/975,085, filed December 18, 2015, European Patent Application No. 16150428.7, U.S. Patent Application 62/205,733, filed August 16, 2015, U.S. Patent Application 62/201542, filed August 5, 2015, U.S. Patent Application 62/193,507, filed July 16, 2015, and U.S. Patent Application 62/181,739, filed June 18, 2015, each entitled NOVEL CRISPR ENZYMES AND SYSTEMS, and U.S. Patent Application 62/245,270, filed October 22, 2015, NOVEL CRISPR ENZYMES AND SYSTEMS. Also referenced is U.S. Patent Application No. 61/939,256, filed February 12, 2014, and WO 2015/089473 (PCT/US2014/070152), filed December 12, 2014, each titled ENGINEERING OF SYSTEMS, METHODS AND OPTIMIZED GUIDE COMPOSITIONS WITH NEW ARCHITECTURES FOR SEQUENCE MANIPULATION. Also referenced are PCT/US2015/045504, filed August 15, 2015, U.S. Patent Application 62/180,699, filed June 17, 2015, and U.S. Patent Application 62/038,358, filed August 17, 2014, each of which is titled GENOME EDITING USING CAS9 NICKASES.

Each of these patents, patent publications, and applications, and all documents cited therein or during the pendency thereof (the "application cited documents"), and all documents cited or referred to in the documents cited in the application, together with any instructions, descriptions, product specifications, and flow charts for any products mentioned therein or in any document referred to therein and incorporated by reference herein, are hereby incorporated by reference herein and may be used in the practice of the present invention. All documents (e.g., these patents, patent publications, and applications, and documents cited in the applications) are hereby incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

Also cited is PCT application PCT/US14/70057, Attorney Docket No. 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 to one or more or all of U.S. Provisional Patent Applications 62/054,490, filed September 24, 2014; 62/010,441, filed June 10, 2014; and 61/915,118, 61/915,215, and 61/915,148, each filed December 12, 2013) ("the Particle Delivery PCT"), incorporated herein by reference, regarding a method for producing an sgRNA-and-Cas9 protein complex comprising a particle, comprising adding a mixture comprising sgRNA and Cas9 protein (and optionally a matrix for HDR) to a mixture comprising, consisting essentially of, or consisting of a surfactant, a phospholipid, a biodegradable polymer, a lipoprotein, and an alcohol; and the particles from such method. For example, wherein the Cas9 protein and sgRNA are mixed together at a suitable molar ratio, such as 3:1-1:3, or 2:1-1:2, or 1:1, at a suitable temperature, such as 15-30°C, such as 20-25°C, such as room temperature, for a suitable period of time, such as 15-45, such as 30 minutes, preferably in a sterile nuclease-free buffer, such as 1X PBS. Individually, particle components such as or including a surfactant, such as a cationic lipid, such as 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); a phospholipid, such as dimyristoylphosphatidylcholine (DMPC); a biodegradable polymer such as ethylene glycol polymer or PEG and a lipoprotein such as low density lipoprotein, for example cholesterol, were dissolved in an alcohol, preferably a C _1-6 alkyl alcohol such as methanol, ethanol, isopropanol, for example 100% ethanol. The two solutions were mixed together to form particles containing Cas9-sgRNA complexes. Accordingly, it is possible to provide a pre-complex of sgRNA with Cas9 protein before assembling the entire complex in the particle. The formulations can be prepared with different molar ratios of various components known to facilitate the delivery of nucleic acid to cells (e.g. 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), polyethylene glycol (PEG) and cholesterol). For example, the molar ratios of DOTAP: DMPC: PEG: cholesterol can be as follows: 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. This application accordingly encompasses mixing of sgRNA, Cas9 protein and components that form a particle; as well as particles obtained as a result of such addition. Aspects of the present invention can encompass particles; for example, particles using a method similar to that in "Particle Delivery PCT", for example by adding a mixture containing sgRNA and/or Cas9, as in the present invention, and components that form a particle, for example as in "Particle Delivery PCT", to form a particle and particles as a result of such mixing (or, of course, other particles containing sgRNA and/or Cas9, as in the present invention).

The present invention will now be further described by means of the following non-limiting examples.

Examples

The following examples serve to illustrate the present invention as described herein. It should also be understood that the scientific basis described below, which motivated the studies, should not be limited by the subject matter of the invention claimed in any way or impose any mechanistic or other requirement.

Scientific justification

Without wishing to be bound by any theory described herein, the inventors of the present invention have devised a strategy for modifying Streptococcus pyogenes Cas9 (SpCas9) to produce modified variants of the SpCas9 enzyme that have target specificity. The same rationale can be applied to any Cas9 ortholog. This increased specificity can be achieved in variants that have reduced activity at non-targeted loci while retaining adequate activity at the intended target locus. Activity in the assays described below, related to nuclease activity, is demonstrated by DNA cleavage as measured by the formation of INSERTIONS/DELETIONS. It is believed that such activity is related to the ability of the CRISPR complex (in other words, the complex between the Cas9 enzyme and the guide RNA) to bind to the appropriate site on DNA. Thus, the reduced activity at the off-target site might be expected to result from reduced binding of the CRISPR complex at that site. Modified Cas9 enzymes that exhibit reduced activity at off-target loci compared to unmodified (e.g., wild-type) enzymes may thus have poorer binding to off-target sites. The publication by Nishimasu et al. (Cell, 2014, 156(5), pp935-49) describes a crystal structure at 2.5 Å resolution of a variant SpCas9 enzyme in complex with a 98-nucleotide single guide RNA (sgRNA) and a 23-nucleotide target DNA region. Based on these structural data, we identified a positively charged region located between the RuvC-III and HNH domains. The authors reasoned that the groove may accommodate the non-targetable strand following disruption of normal Watson-Crick base pairing upon binding of the Cas9 enzyme to the target DNA region. Positively charged residues in this region of Cas9 may function to stabilize the interaction between the enzyme and DNA by interacting with the negatively charged phosphodiester backbone of the non-targetable DNA strand. The present inventors hypothesize that by substituting positively charged residues in Cas9, interactions with the non-targetable strand may be disrupted. Sufficient disruption of this interaction may retain adequate activity toward target sites but reduce enzyme activity toward non-targetable sites (which would typically be expected to have weaker interactions with the guide sequence due to one or more mismatches compared to the target sequence). The present inventors have surprisingly found that modification of Cas9 can indeed reduce off-target activity. See also Figure 1, and its description herein.

In addition to the discovery that substitution of positively charged residues disrupts DNA backbone interactions and results in reduced enzyme activity at non-targeted sites while maintaining adequate activity at target sites, Kleinstiver BP et al. describe a triple-substitution (R661A/Q695A/Q926A) and a quadruple-substitution (N497A/R661A/Q695A/Q926A) variant of SpCas9 containing mutations in the REC1 domain. The substitutions involve residues that are designed to disrupt hydrogen bonds in the phosphate backbone of DNA, and the mutants are reported to have high on-target activity and minimal off-target activity. (See, Kleinstiver BP et al., High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 2016 Jan 28;529(7587):490-5. doi: 10.1038/nature16526. Epub January 6, 2016

In addition, the inventors hypothesize that modification of amino acid residues of the CRISPR enzyme can be performed that can have the effect of increasing the stability of the interaction between the enzyme and the non-targeted strand after disruption of the normal Watson-Crick base pairing upon binding of the Cas9 enzyme to the corresponding region of DNA. For example, substitution of a positively charged amino acid for an amino acid residue that is not positively charged in the unmodified enzyme can have the effect of stabilizing the interaction between the enzyme and the non-targeted strand by increasing the net positive charge of the enzyme. Thus, it is assumed that a higher net positive charge in the corresponding regions of the enzyme provides a stronger interaction with the negatively charged phosphodiester backbone of the non-targeted strand of DNA. Amino acids that are not positively charged in the unmodified enzyme can be, for example, amino acids that are neutrally charged, negatively charged, hydrophobic, etc. Such amino acids can be replaced by a positively charged amino acid to achieve the desired effect. The above-described functional effects are based on complex and interrelated electrostatic and thermodynamic positions. Thus, it is assumed that the above-described functional effects can be combined. Thus, the CRISPR enzyme can be modified in such a way that the activity of the enzyme with respect to the target is increased, and also the activity with respect to one or more non-target elements is reduced. For example, it is assumed that modifications can be made that promote increased target activity, while modifications can be made that reduce non-target activity. In this way, synergistic effects can be obtained.

It is proposed that any of the functional effects described above can be achieved by modifying amino acids in the above-mentioned groove, but also by modifying amino acids near the groove or outside it.

Example 1. Materials and methods

Production of SpCas9 mutants

Although it should be obvious from the full context of the present invention, the abbreviation "SpCas9" refers to Streptococcus pyogenes Cas9 and the abbreviation "SaCas9" refers to Staphyloccocus aureus Cas9. Modified variants of SpCas9 and SaCas9, for example modified alanine variants, have been created by PCR-based mutagenesis using known techniques. Other techniques may include obtaining a nucleic acid molecule encoding Cas9, but altering the corresponding codon(s) responsible for the mutation(s) or modification(s) of the protein so that the modified or mutated Cas9 is expressed, for example, using a vector expression system such as a bacterial expression system or a viral vector expression system. The modified or mutated Cas9 expressed in this manner can then be readily purified. The modified or mutated Cas9 of the present invention can be used in CRISPR-Cas systems and in any application of CRISPR-Cas systems; and preferably have the advantage of reduced or virtually absent or essentially absent or absent off-target effects and/or increased on-target effects. Accordingly, the Cas9 of the present invention or the CRISPR-Cas system of the present invention with the Cas9 of the present invention can be delivered using a delivery system, which can be one or more vectors, including the one discussed herein.)

System for testing the activity of modified Cas9

Modified Cas9 enzymes were tested by co-transfection of a plasmid encoding the mutant Cas9 and a plasmid encoding the sgRNA (target only) into HEK293T or HEK293FT cells. Cells were transfected at 90-95% confluence using Lipofectamine 2000, grown at 37°C and 5% _CO2 for ∼72 h, and harvested. Target and off-target genomic loci were amplified by PCR and analyzed using next-generation sequencing (NGS). % insertions/deletions for target and off-target loci were calculated from the sequencing data. SpCas9 mutants were tested with the genomic loci shown in Table A. SaCas9 mutants were tested by NGS using the EMX101 guide and OT1 - OT3 from Ran at al. 2015. Biochemical or SURVEYOR nuclease assays were not performed (all data were obtained from NGS; cf. Sidi-Chen, "Genome-wide CRISPR Screen in a Mouse Model of Tumor Growth and Metastasis," Cell 160(6): 1246–1260, DOI: http://dx.doi.org/10.1016/j.cell.2015.02.038, 12 March 2015).

Insertion/deletion analysis

Insertion/deletion analyses were performed by targeted deep sequencing and analyzed as described previously (Hsu, P. D. et al. (2013) DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832). Cells were harvested approximately 3 days post-transfection. Genomic DNA was extracted using the QuickExtract DNA Extraction Kit (Epicentre) by resuspending the pelleted cells in QuickExtract (80 μl/well in a 24-well plate or 20 μl/well in a 96-well plate), followed by incubation at 65°C for 15 min, 68°C for 15 min, and 98°C for 10–15 min. PCR fragments for NGS analysis were generated in two-step PCR reactions. Briefly, second-round PCR backbone primers were used to amplify genomic regions of interest (Table 2), followed by overlapping PCR to attach Illumina P5 adaptors as well as unique sample-specific barcodes to the first-round PCR product.

Example 2. Initial analysis of SpCas9 with single mutations (EMX1 and VEGFA target sequences).

49 original SpCas9 with single point mutations were generated and tested for insertion/deletion formation. The target sequences in this example were sequences from the EMX1 and VEGFA genes. Both target and off-target sequences are shown in Table A below, with PAM sequences indicated. For off-target sequences, mismatches to the target sequence are shown in bold and underlined. The results are shown in Figures 2A and 2B.

The following mutants showed reduced activity at off-target sites compared to the wild-type enzyme (see Figures 2A and 2B).

R63A

H415A

H447A

R778A

R780A

Q807A

K810A

R832A

K848A

K855A

K968A

R976A

H982A

K1000A

K1003A

K1047A

R1060A

K1107A

R1114A

K1118A

K1200A

Example 3. Further analysis of SpCas9 with single mutations

Several modified SpCas9 enzymes with single point mutations identified in Example 2 were further tested for insertion/deletion formation using a third guide sequence and additional off-target loci. The target and off-target sequences are shown in Table A below, with PAM sequences indicated. In the case of an off-target sequence, mismatches to the target sequence are shown underlined and in bold. The results are shown in Figure 3. The modified SpCas9 enzymes tested in this Example were as follows:

R780A

K810A

K848A

K855A

R976A

H982A

K1003A

R1060A

As shown in Figure 3, all eight modified enzymes showed reduced activity at off-target sites compared to the unmodified (wild-type) enzyme (SpCas9).

Example 4. Further analysis of SpCas9 with single mutations (target sequence VEGFA1).

Several modified SpCas9 enzymes with single point mutations, including the mutants described in Example 2, were tested for the formation of insertions/deletions for a second distinct target sequence, in this case VEGFA1 is the VEGFA gene sequence. The modified SpCas9 enzymes tested in this example were as follows:

R780A

K810A

K848A

K855A

R976A

H982A

K1003A

R1060A

H1240A

H1311A

The target and non-target sequences are shown in Table A below, with PAM sequences indicated. In the case of a non-target sequence, mismatches to the target sequence are shown underlined and in bold. The results are shown in Figure 3.

Example 5. Analysis of combination mutants of SpCas9

Twenty-four modified SpCas9 enzymes with double point mutations and 14 modified enzymes with triple point mutations were prepared and tested for insertion/deletion formation at two different target sequences, in this case the EMX1 and VEGFA gene sequences (VEGFA3 is a sequence in VEGFA). The target and off-target sequences are shown in Table A below, with PAM sequences indicated. In the case of an off-target sequence, mismatches to the target sequence are shown in bold and underlined. The mutants tested and the results are shown in Figures 4 and 5; an asterisk in Figures 4 and 5 indicates an embodiment that is considered advantageous in the present invention. As shown in Figures 4 and 5, all mutants showed a significant reduction in activity at the off-target loci OT 46, OT4 and OT18 compared to the wild-type enzyme. Several combination mutants additionally showed reduced activity at all four non-target loci while maintaining target activity similar to WT; these were as follows.

Mutant Remainder Remainder Remainder 1 K810A K848A 2 K848A K855A 3 R780A K1003A 4 K810A K1003A 5 R780A R1060A 6 K810A R1060A 7 K855A R1060A 8 H982A K1003A K1129E 9 R780A K1003A R1060A 10 K810A K1003A R1060A 11 K848A K1003A R1060A 12 K855A K1003A R1060A 13 K855A K1003A E610G

Table A. SpCas9 Guides (SpCas9 target and off-target loci; red indicates mismatches in off-target sequences; vertical line | indicates SpCas9 cut site; off-target sequences are excluded by mutation/modification according to the present invention)

Table B. Disclosed mutations in SpCas9 (the cited documents are incorporated by reference; the right to expressly disclaim any of these mutations individually is reserved, and it should be noted that none of the known mutations are disclosed or suggested to provide a reduced off-target effect and/or an increased ability to modify one or more target loci compared to the unmodified enzyme, with the caveat that K1107A, KES→GG and KES→KG of Anders et al may provide specificity for mismatches at positions 1 and 2 of sgRNA BUT at the cost of moderately reduced target cleavage efficiency, and they also do not provide a more detailed description of the specificity characteristics, so the general statement that these known mutants are not disclosed or suggested remains valid for the present invention; and it is stipulated that the present invention may include any mutation/modification from the table below in combination with a modification/mutation that provides reduced off-target effect, provided that the addition of one or more of the mutations/modifications listed below does not adversely affect the effect of reduced ability to modify off-target loci and/or increased ability to modify one or more target loci achieved in the present invention):

Remainder Position New remainder Domain Effect Source D 10 A RuvC-I Nikaza Cong et al., Science (2013) S 15 A RuvC-I Decreased activity Nishimasu et al., Cell (2014) R 63 A BH WT Nishimasu et al., Cell (2014) R 66 A BH Bridged helix; "significantly reduced DNA cleavage activity" Nishimasu et al., Cell (2014) R 69 A BH "reduced DNA cleavage activity" Nishimasu et al., Cell (2014) R 70 A BH Lack of activity Nishimasu et al., Cell (2014) R 74 A BH Bridged helix; "greatly reduced DNA cleavage activity" Nishimasu et al., Cell (2014) R 75 A BH "reduced DNA cleavage activity" Nishimasu et al., Cell (2014) K 76 A BH Significantly reduced activity Hemphill et al., JACS (2015) R 78 A BH "moderately reduced activity" Nishimasu et al., Cell (2014) C 80 L BH WT Nishimasu et al., Cell (2014) 97-150 Deletion Lack of activity Nishimasu et al., Cell (2014) K 163 A RecI Decreased activity Hemphill et al., JACS (2015) K 163 A RecI "reduced DNA cleavage activity" Nishimasu et al., Cell (2014) R 165 A RecI WT Nishimasu et al., Cell (2014) 175-307 Deletion RecII REC2 domain; moderately reduced activity Nishimasu et al., Cell (2014) 312-409 Deletion Lack of activity Nishimasu et al., Cell (2014) PWN 475-477 AAA RecI "a subtle but reproducible decrease in activity" Jinek et al., Science (2014) K 510 A RecI Decreased activity Hemphill et al., JACS (2015) K 510 A RecI WT Nishimasu et al., Cell (2014) C 574 E RecI WT Nishimasu et al., Cell (2014) K 742 A RuvC-II WT Hemphill et al., JACS (2015) E 762 A RuvC-II Nikaza Nishimasu et al., Cell (2014) H 840 A HNH Nikaza Ran et al., Cell (2014) N 854 A HNH Decreased activity Nishimasu et al., Cell (2014) N 863 A HNH Nikaza Nishimasu et al., Cell (2014) K 866 A HNH Lack of activity Hemphill et al., JACS (2015) H 982 A HNH Decreased activity Nishimasu et al., Cell (2014) H 983 A RuvC-III Nikaza Nishimasu et al., Cell (2014) D 986 A RuvC-III Nikaza Nishimasu et al., Cell (2014) K 1107 A PI Moderately reduced activity; provides specificity for positions 1 and 2 Anders et al., Nature (2014) E.S. 1108-1109 G PI Moderately reduced activity; provides specificity for positions 1 and 2 Anders et al., Nature (2014) KES 1107-1109 GG PI Moderately reduced activity; provides specificity for positions 1 and 2 Anders et al., Nature (2014) DWD 1125-1127 AAA PI WT Jinek et al., Science (2014) R 1333 A PI PAM recognition; "nearly abolished cleavage of linearized plasmid DNA" Anders et al., Nature (2014) R 1333 E PI "did not result in a switch in specificity towards alanine-rich PAMs" Anders et al., Nature (2014) R 1335 A PI PAM recognition; "nearly abolished cleavage of linearized plasmid DNA" Anders et al., Nature (2014) R 1335 E PI "did not result in a switch in specificity towards alanine-rich PAMs" Anders et al., Nature (2014) 1099-1368 Deletion PI Lack of activity Nishimasu et al., Cell (2014)

Example 6. Analysis of additional SpCas9 mutants (VEGFA3 target sequence)

Several additional modified SpCas9 enzymes with single point mutations were generated and tested for insertion/deletion formation using the VEGFA3 target sequence, which is the VEGFA gene sequence. The modified SpCas9 enzymes tested in this example were as follows:

Mutant Remainder 1 R403A 2 R63A 3 K782A 4 K890A 5 K1107A 6 R778A 7 K1200A 8 K1114A 9 K1118A 10 K890A 11 H415A

As shown in Figure 2, several mutants showed significant reductions in activity at both the OT1 and OT4 off-target loci compared to the wild-type enzyme. Several mutants additionally showed reductions in activity at all three off-target loci compared to the wild-type enzyme, these were as follows:

Mutant Remainder 1 R403A 2 R63A 3 K782A 5 K1107A 6 R778A 7 K1200A 11 H415A

Example 7. Analysis of SaCas9 mutants (EMX101 target sequence)

Several modified SaCas9 enzymes with single point mutations were generated and tested for insertion/deletion formation using a target sequence, which is a sequence from the EMX101 gene. The target and off-target sequences are shown in Table C below, with the PAM sequence indicated. For the off-target sequence, mismatches to the target sequence are shown in bold and underlined. The modified SaCas9 enzymes tested in this example were the alanine mutants shown below:

K518A

K523A

K525A

H557A

R561A

K572A

R686A

K687A

K692A

R694A

H700A

K751A

As shown in Figure 6, several mutants showed significant reductions in activity at both the OT2 and OT3 off-target sequences compared to the wild-type enzyme. Several mutants additionally showed reductions in activity at all three off-target loci compared to the wild-type enzyme, these were as follows:

K523A

K525A

R561A

K572A

R694A

H700A

Table C. Sequence information, including PAMs, for guides used to validate SaCas9 mutations (mismatches in off-target sequences are indicated in red; off-target sequences are excluded by the mutation/modification of the present invention)

Figure 7 provides additional data regarding the following SaCas9 mutants disclosed herein.

Mutant Remainder 1 R245A 2 R480A 3 R497A 4 R499A 5 R617A 6 R630A 7 R634A 8 R644A 9 R650A 10 R654A 11 K736A

Mutants R245A, R480A, R499A, R650A and R654A were effective in reducing off-target effects, with R480A, R499A and R245A being particularly effective.

Example 8. List of modified Cas9 enzymes

With respect to SpCas9, the single and combination mutants listed herein, including the above-mentioned examples, are currently considered advantageous because they have demonstrated a preferential improvement in specificity. SpCas9 and SaCas9 mutants, including those tested and otherwise mentioned in the present invention, are listed below in Tables 1-7.

Table 1. List of quadruple mutants of SpCas9

Mutant Remainder Remainder Remainder Remainder QM1 R63A K855A R1060A E610G QM2 R63A H982A K1003A K1129E QM3 R63A K810A K1003A R1060A

Table 2. List of single mutants of SpCas9

Mutant The remainder and
replacement 1 R63A 2 H415A 3 H447A 4 R778A 5 R780A 6 R783A 7 Q807A 8 K810A 9 R832A 10 K848A 11 K855A 12 K968A 13 R976A 14 H982A 15 K1000A 16 K1003A 17 K1047A 18 R1060A 19 K1107A 20 R1114A 21 K1118A 22 R403A 23 K1200A

Table 3. List of double and triple mutants of SpCas9

Mutant Remainder and replacement 1 R780A R1060A 2 R780A K1003A 3 K810A K848A 4 K810A K855A 5 K848A K855A 6 K855A R1060A 7 R780A K1003A R1060A 8 K855A K1003A R1060A 9 H982A K1003A K1129E 10 K810A K1003A R1060A

Table 4. List of single mutants of SaCas9

Mutant Remainder 1 H700 2 R694 3 K692 4 R686 5 K687 6 K751 7 R561 8 H557 9 K572 10 K523 11 K518 12 K525

Table 5. List of single mutants of SaCas9

Mutant Remainder 2 R245 3 R480 4 R497 5 R499 6 R617 7 R630 8 R634 9 R644 10 R650 11 R654 12 K736

Typical examples of SpCas9 mutants are listed in Table 6 below.

Table 6. List of single mutants of SpCas9

Mutant The remainder and
replacement 1 N14K 2 N776L 3 E781L 4 E809K 5 L813R 6 S845K 7 L847R 8 D849A 9 I852K 10 D859A 11 S964K 12 V975K 13 E977K 14 N978K

Table 7 below shows mutants exemplified in the present invention, including those mentioned in the examples.

Table 7. Typical mutants within the scope of the present invention Single mutants Mutant Remainder Plot Mutant Remainder Plot SM1 K775A Groove SM32 K1107A PL SM2 R780A Groove SM33 E1108A PL SM3 R780A Groove SM34 S1109A PL SM4 K810A Groove SM35 ΔK1107 PL SM5 R832A Groove SM36 ΔE1108 PL SM6 K848A Groove SM37 ΔS1109 PL SM7 K855A Groove SM38 ES_G PL SM8 R859A Groove SM39 KES_GG PL SM9 K862A Groove SM40 R778A DNA SM10 K866A Groove SM41 K782A DNA SM11 K961A Groove SM42 R783A DNA SM12 K968A Groove SM43 K789A DNA SM13 K974A Groove SM44 K797A DNA SM14 R976A Groove SM45 K890A DNA SM15 H982A Groove SM46 R1114A cDNA SM16 H983A Groove SM47 K1118A cDNA SM17 K1014A Groove SM48 K1200A cDNA SM18 K1047A Groove SM49 R63A sgRNA SM19 K1059A Groove SM50 K163A sgRNA SM20 R1060A Groove SM51 R165A sgRNA SM21 K1003A Groove SM52 R403A sgRNA SM22 H1240A Groove SM53 H415A sgRNA SM23 K1244A Groove SM54 R447A sgRNA SM24 K1289A Groove SM55 K1000A Groove SM25 K1296A Groove SM26 H1297A Groove SM27 R1298A Groove SM28 K1300A Groove SM29 R1303A Groove SM30 H1311A Groove SM31 K1325A Groove

Triple mutants

Example 9. Modification of Cas9 to enhance target activity

Initially, the inventors performed modifications to uncharged amino acids located in the groove between the RuvC-III and HNH domains of SpCas9. The amino acids to be modified included amino acids with uncharged side chains, including serine, threonine, asparagine, and glutamine. Some amino acids were changed to positively charged amino acids, such as arginine or lysine. The effect of such single amino acid changes in SpCas9 on the formation of insertions/deletions at target loci was assessed using next-generation sequencing as described above. Favorable mutations with increased/enhanced target activity compared to the unmodified enzyme were selected. The inventors evaluated double and triple mutations as described above. Particularly favorable mutations with enhanced target activity were selected.

The inventors evaluated such mutations in SaCas9 in the same way as described for SpCas9. Particularly favorable mutations with enhanced target activity were selected.

The inventors extended the modification zone to uncharged amino acids located near the groove between the RuvC-III and HNH domains of SpCas9 and beyond. Again, the effect of these changes on the formation of insertions/deletions at the target loci was assessed using the SURVEYOR nuclease assay as described above. Favorable mutations with increased/enhanced target activity compared to the unmodified enzyme were selected. Similar analyses were performed for SaCas9.

The inventors combined SpCas9 mutations that showed enhanced on-target activity with mutations that showed reduced off-target activity. Again, the effect of these changes on insertion/deletion formation at target loci was assessed using the SURVEYOR nuclease assay described above. Particularly favorable mutations with increased/enhanced on-target activity and reduced off-target activity compared to the unmodified enzyme were selected. Similar assays were performed for SpCas9.

Example 10. Analysis of SpCas9 mutants (multiple target sequences)

Three mutants, K855A (single mutation) and TM14 and TM15 (both triple mutants), were tested for insertion/deletion formation using the target sequences of EMX101, EMX1.1, EMX1.2, EMX1.3, EMX1.8, EMX1.10, DNMT1.1, DNMT1.2, DNMT1.4, DNMT1.7, VEGFA4, VEGFA5, and VEGFA3. As shown in Figure 10, all three mutants showed on-target activity and low off-target activity against OT4.

An expanded panel of single, double, and triple mutants were tested for insertion/deletion formation using target sequences of EMX101, EMX1.1, EMX1.2, EMX1.3, EMX1.8, EMX1.10, DNMT1.1, DNMT1.2, DNMT1.4, DNMT1.7, VEGFA4, VEGFA5, and VEGFA3. Mutants included E779L, R780A, K810A, K848A, K855A, R976A, H982A, DM11, DM17, DM19, DM20, DM23, DM24, DM25, DM35, DM40, TM14, TM15, and TM16. Figure 11 summarizes the on-target and off-target activity for OT4. Thus, decreased activity was observed at nearly all off-target loci assessed in the genome, as well as at a large panel of mismatched guides.

Example 11. Analysis of SpCas9 mutants (VEGFA3 target sequence and non-target sequences)

Several mutant modified SpCas9 enzymes were generated and tested for insertion/deletion formation using a target sequence that is a sequence from the VEGFA3 gene. The modified SpCas9 enzymes tested in this example included:

R780A

K848A

K1000A

K848A R1060A

R780A R1114A

H982A K1003A R1060A

R63A K848A R1060A

R63A K855A R1060A E610G

K1107A

T13I R63A K810A

R63A K855A

R63A H982A

G12D R63A R1060A

H415A K848A

H415A K848A

R780A R1114A

K848A K1107A

K848A E1108A

S1109A

R63A E610G K855A R1060A

R63A K848A R1060A

As shown in Figures 12 and 13, several mutants showed significant reductions in activity at one or more of the three off-target loci OT1, OT4, and OT18 compared to the wild-type enzyme. Several mutants additionally showed reductions in activity at all three off-target loci compared to the wild-type enzyme. These included:

R780A R1114A

H982A K1003A K1129E

R63A K855A R1060A E610G

K1107A

R63A K855A

R63A H982A

K848A K1107A

R63A E610G K855A R1060A

R63A K848A R1060A

Example 12. Analysis of SpCas9 mutants (EMX1.3 target sequence and non-target sequences)

Several mutant modified SpCas9 enzymes were tested for insertion/deletion formation using the target sequence, which is the EMX1.3 sequence. The modified SpCas9 enzymes tested in this example included:

N14K

E779L

E809K

L813R

S845K

L847R

D849A

D861K

E977K

I978K

N979L

N980K

As shown in Figure 14, certain mutants showed high target activity, and among them, differences in specificity for the off-target sequences OT14, OT23, OT35, OT46, and OT53. Certain mutants showed higher specificity than the wild type, while others showed high activity for the off-target sequences.

Example 13. Analysis of SpCas9 mutants (EMX1.3 target sequence)

Several mutant modified SpCas9 enzymes were tested for insertion/deletion formation using mismatch guides, with the target sequence being the EMX1.3 sequence. The three modified SpCas9 enzymes tested in this example were:

K855A

K810A, K1003A, R1060A

K848A, K1003A, R1060A

The results are shown in Figure 15.

Example 14. Enhanced Cas9 mutants are characterized by high activity and specificity

Six of the 29 point mutants had at least 10-fold reduced off-target activity compared to wild-type (WT) SpCas9 while maintaining on-target cleavage efficiency, and 6 others had improved specificity by 2-5-fold. These mutants also exhibited enhanced specificity when tested at a second locus, VEGFA(1) (Fig. 15D). Although some point mutants were more specific than WT SpCas9 in targeting EMX1(1) and VEGFA(1), off-target insertions/deletions were still detected (~0.1%) (Fig. 15D). To further improve specificity, we performed combinatorial mutagenesis using the best point mutants identified in the initial screen. Eight of the 35 combination mutants retained wild-type on-target activity and exhibited undetectable levels of off-target insertions/deletions in EMX1(1) OT1, VEGFA(1) OT1, and VEGFA(2) OT2 (Fig. 15E). To ensure that the observed increase in specificity was not due to reduced on-target activity, we measured on-target insertion/deletion formation at 10 target loci using the top 16 mutants (Fig. 15F), determined by a combination of on-target and off-target activity. The applicants observed high efficiency and specificity in the case of three mutants: SpCas9(K855A), SpCas9(K810A/K1003A/R1060A) (also called eSpCas9(1.0)) and SpCas9(K848A/K1003A/R1060A) (also called eSpCas9(1.1)). These three variants were selected for further analysis.

To assess whether SpCas9(K855A), eSpCas9(1.0), and eSpCas9(1.1) generally retained efficient nuclease activity, we measured the formation of targeted insertions/deletions at 24 target sites spanning 10 different genomic loci (Figure 16A). All three mutants produced insertion/deletion levels similar to SpCas9 WT at most target sites (Figure 16B). To test whether the improvements in specificity could be explained by reduced Cas9 expression, we performed Western blotting on SpCas9 and found that all three mutants were expressed at levels equivalent to or higher than SpCas9 WT (Figure 16C). This demonstrated that the improvements in specificity were not due to reduced protein expression levels.

We then compared the specificity of the three mutants with WT SpCas9 using truncated guide sequences (18 nt for EMX1(1) and 17 nt for VEGFA(1)), which have been shown to reduce the formation of off-target insertions/deletions. All three mutants showed reduced cleavage at all off-target sites assessed. Moreover, eSpCas9(1.0) and eSpCas9(1.1) resulted in the exclusion of 20 of 24 of these sites. In contrast, WT SpCas9 with truncated guides resulted in the exclusion of 14 of 24 sites, but also increased off-target activity at 5 sites compared to WT SpCas9 with full-length guides.

To assess the tolerance of SpCas9(K855A), eCas9(1.0), and eCas9(1.1) to mismatch target sites, we systematically mutated the VEGFA(1) guide sequence to introduce single and double base mismatches at different positions (Fig. 17A-C). Compared to SpCas9 WT, all three mutants induced lower insertion/deletion rates when using mismatch guides. Notably, eSpCas9(1.0) and eSpCas9(1.1) induced lower insertion/deletion rates even at single base mismatches located outside the 7-12 bp of the seed sequence. Given that the applicants did not observe any differences between eSpCas9(1.0) and eSpCas9(1.1) in terms of specificity, SpCas9(K855A) and eSpCas9(1.1) were selected for further analysis based on target potency.

The specificity of whole-genome editing by SpCas9(K855A) and eSpCas9(1.1) was assessed using BLESS (direct in situ break labeling, streptavidin enrichment, and next-generation sequencing), which quantifies DNA double-strand breaks (DSBs) throughout the genome (Fig. 17A). Cells were harvested approximately 24 h post-transfection and BLESS was performed. Briefly, a total of 10 million cells were fixed for nuclear isolation and permeabilization and then treated with proteinase K for 4 min at 37°C before inactivation with PMSF. DSBs in stripped nuclei were labeled with 200 mM hybridizable proximal linkers overnight. Following digestion of labeled nuclei with proteinase K, chromatin was mechanically sheared with a 26G needle before sonication (BioRuptor, 20 min at high speed, 50% fill factor). A total of 20 μg of sheared chromatin was captured on streptavidin beads, washed, and ligated with 200 mM distal linker. Linker hairpins were then cleaved by I-SceI digestion for 4 h at 37°C, and products were enriched by PCR for 18 cycles before proceeding to library preparation using the TruSeq Nano LT kit (Illumina). As a negative control, cells were mock transfected with Lipofectamine 2000 and pUC19 DNA and processed in parallel throughout the assay.

Calculation of DSB score to separate background DSBs from true Cas9-induced breaks was performed as described previously (Ran et al, Nature 2015), and sorting of loci based on DSB score identified top off-target sites that had been previously identified for these sgRNA targets. To provide additional detection capability beyond these top off-target loci, based on preliminary Cas9-BLESS data, we found that a homology search algorithm could further identify true Cas9-induced DSBs. The homology search algorithm searched for the best-matching guide sequence within a 50-nt genomic region on either side of the middle of the DSB cluster identified in BLESS for all PAM sequences representing NGG and NAG. The homology-based score was calculated using the following weighting values: a match between sgRNA and genomic sequence gives a score of +3, a mismatch is -1, while an insertion or deletion between sgRNA and genomic sequence is scored as -5. Therefore, a target sequence with a full 20 bp guide length + PAM would have a score of 69. The final homology score for a DSB cluster was set as the maximum score for all possible sequences. By applying these weighting values, it was empirically found that true off-target loci (for which insertions/deletions were identified by targeted deep sequencing) and background DSBs were completely separated when a cutoff value of >50 was applied for the homology score. Applying this homology criterion to the top 200 DSB loci identified by BLESS allowed further discrimination of off-target loci from background DSBs.

Applicants analyzed the effects of both mutants on the EMX1(1) and VEGFA(1) targets and compared these results with SpCas9 WT (Fig. 17B). Both SpCas9(K855A) and eSpCas9(1.1) exhibited a reduction in off-target whole-genome cleavage and did not result in the formation of any new off-target sites (Fig. 17C-D).

Example 15. Targeting mechanism and nuclease activity of Cas9

Off-target cleavage occurs when the binding strength of Cas9 to the non-targeted DNA strand exceeds the DNA rehybridization forces. According to this model, mutations designed to weaken interactions between Cas9 and the non-complementary DNA strand result in significant improvements in specificity. The model also suggests that, conversely, specificity can be reduced by strengthening interactions between Cas9 and the non-targeted strand. Two mutants, S845K and L847R, were generated according to this model, each of which exhibited reduced specificity (Fig. 24).

Example 16. Specificity of Staphylococcus aureus Cas9 (SaCas9)

Similar strategies can also be applied to other Cas9 family proteins. A specificity-enhanced version of Staphylococcus aureus Cas9 (SaCas9) was generated in a manner similar to eSpCas9. Single and double amino acid residue mutants in the groove between the RuvC and HNH domains were screened for reduced off-target cleavage. The specificity-enhanced mutants were combined to generate a SaCas9 variant that retained on-target cleavage of EMX site 7 and had significantly reduced off-target cleavage (Fig. 25). The crystal structure of SaCas9 shows the groove between the HNH and RuvC domains mutated to construct nucleases with improved specificity.

Example 17. Activation of HBG1

Complexes containing spCas9 mutants or spCas9 with different guide RNAs were tested for HBG1 activation. Figure 31 shows activation by complexes containing Cas9 molecules lacking nuclease activity (e.g., dCas9, R780A/K810A, and R780A/K855A; see also Figure 4) or by complexes with nuclease-competent Cas9 (e.g., unmutated spCas9(px165), R780A, K810A, or K848A with truncated (i.e., "15 bp long") guide RNAs. The R780A mutant is exceptional in that it shows activation with all three guides tested.

Example 18. Particle-mediated delivery of CRISPR-Cas9 components to hematopoietic stem cells (HSC)

Applicants have demonstrated that Cas9 can be delivered into cells via particles. Many nucleic acid-based therapeutics may require the simultaneous delivery of both one or more sgRNAs and the Cas9 nuclease. Accordingly, applicants have demonstrated the feasibility of delivering a complex of modified Cas9 enzyme and sgRNA in this manner.

The modified Cas9 enzyme was tested for co-delivery with one or more guide RNAs into cells via particles. sgRNA targeting the EMX1 gene was mixed with eSpCas9(1.1) (K848A, K1003A, R1060A) at a 1:1 molar ratio at room temperature for 30 min in sterile, nuclease-free 1X PBS. The control was the same sgRNA mixed with SpCas9. DOTAP, DMPC, PEG, and cholesterol were dissolved separately in 100% ethanol. The two solutions were mixed together to form particles containing Cas9-sgRNA complexes. After particle formation, HSCs in 96-well plates were transfected with 15 μg Cas9 protein per well. Three days after transfection, HSCs were harvested and the specificity of whole-genome editing was assessed using BLESS, and off-target loci were identified using a homology search algorithm. The number of on-target insertions and deletions (I/D) at the EMX1 locus and I/D at multiple off-target sites were quantified. eSpCas9(1.1) exhibited reduced whole-genome off-target cleavage and no new off-target sites.

Example 19. Particle-mediated delivery of CRISPR-Cas9 components to hematopoietic stem cells (HSCs) and HBB repair

Two sgRNAs targeting sequences on either side of the common GAG->GTG point mutation in the beta globin (HBB) gene were mixed with eSpCas9(1.1) (K848A, K1003A, R1060A) at a 1:1 molar ratio of sgRNA to enzyme at room temperature for 30 min in sterile nuclease-free 1X PBS. The control was the same sgRNA mixed with SpCas9. DOTAP, DMPC, PEG, and cholesterol were separately dissolved in 100% ethanol. These two solutions and the template nucleic acid to correct the GAG->GTG point mutation were mixed to form particles containing Cas9-sgRNA complexes and template. Following particle formation, HSCs in 96-well plates were transfected with 15 μg Cas9 protein per well. Three days post-transfection, HSCs were harvested and tested for GAG->GTG point mutation repair. Corrected cells were then assessed for whole-genome editing specificity using BLESS and off-target loci were identified using a homology search algorithm. Insertions/deletions at multiple off-target sites were quantified. eSpCas9(1.1) exhibited reduced whole-genome off-target cleavage and no new off-target sites.

Table of primers for obtaining mutations Primer name for SpCas9 Subsequence SPCAS9-N ATGGTCTCACCGGTGCCACCATGGACTATAAG K775A_F ATGGTCTCAGGCGAACAGCCGCGAGAGAATGAAGCGGAT R780A_F ATGGTCTCCAGGCGATGAAGCGGATCGAAGAGGGCATCA Q807A_F ATGGTCTCAGGCGAACGAGAAGCTGTACCTGTACTACCTG K810A_F ATGGTCTCAGGCGCTGTACCTGTACTACCTGCAGAATGG R832A_F ATGGTCTCCAGCCTGTCCGACTACGATGTGGACCATATC K848A_F ATGGTCTCAGCCGACGACTCCATCGACAACAAGGTGCTGACC K855A_F ATGGTCTCCAGCAGTGCTGACCAGAAGCGACAAGAACGGGG R859_F ATGGTCTCACGCAAGCGACAAGAACCGGGGCAAGAG K862_F ATGGTCTCACGCGAACCGGGGCAAGAGCGACAAC K866_F ATGGTCTCACGCGAGCGACAACGTGCCCTCCGAA K961_F ATGGTCTCAGCGCTGGTGTCCGATTTCCGGAAGGATTTC K968A_F ATGGTCTCAGCGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAAC K974A_F ATGGTCTCACGCAGTGCGCGAGATCAACAACTACCACCA R976A_F ATGGTCTCATGGCCGAGATCAACAACTACCACCACGCC H982A_F ATGGTCTCACGCCCACGCCCACGACGCCTAC H983A_F ATGGTCTCACGCCGCCCACGACGCCTACCTGAA K1014A_F ATGGTCTCCAGCGGTGTACGACGTGCGGAAGATGATCG K1047A_F ATGGTCTCACGCGACCGAGATTACCCTGGCCAACG K1059A_F ATGGTCTCAGCGCGGCCTCTGATCGAGACAAAACGG R1060A_F ATGGTCTCAGGCGCCTCTGATCGAGACAAAACGGCG K1003A_F ATGGTCTCAGCGCTGGAAAGCGAGTTCGTGTACGGC H1240A_F ATGGTCTCACGCCTATGAGAAGCTGAAGGGCTCCCC K1244A_F ATGGTCTCAGGCGCTGAAGGGCTCCCCCGAG K1289A_F ATGGTCTCACGCAGTGCTGTCCGCCTACAACAAGCAC K1296A_F ATGGTCTCACGCGCACCGGGATAAGCCCATCAGAG H1297A_F ATGGTCTCAAGGCCCGGGATAAGCCCATCAGAGAGC R1298A_F ATGGTCTCAGCGGATAAGCCCATCAGAGAGCAGGCC K1300A_F ATGGTCTCAGCGCCCATCAGAGAGCAGGCCGAG R1303A_F ATGGTCTCACGCAGAGCAGGCCGAGAATTCATCACC H1311A_F ATGGTCTCACGCCCTGTTTACCCTGACCAATCTGGGAG K1325A_F ATGGTCTCACGCGTACTTTGACACCACCATCGACCGG K1107A_F ATGGTCTCACGCCGAGTCTATCCTGCCCAAGAGGAACAG E1108A_F ATGGTCTCAAGCCTCTATCCTGCCCAAGAGGAACAGCGA S1109A_F ATGGTCTCAGGCCATCCTGCCCAAGAGGAACAGCGATAA ΔK1107_F ATGGTCTCACGAGTCTATCCTGCCCAAGAGGAACAGCGA ΔE1108_F ATGGTCTCAATCTATCCTGCCCAAGAGGAACAGCGATAA ΔS1109_F ATGGTCTCAGATCCTGCCCAAGAGGAACAGCGATAAGCT KES_KG_F ATGGTCTCAAGGCATCCTGCCCAAGAGGAACAGCGATAA KES_GG_F ATGGTCTCACGGCATCCTGCCCAAGAGGAACAGCGATAA R778A_F ATGGTCTCACGCCGAGAGAATGAAGCGGATCGAAGAGGG K782A_F ATGGTCTCCAGGCCCGGATCGAAGAGGGCATCAAAGAGCT R783A_F ATGGTCTCAGGCCATCGAAGAGGGCATCAAAGAGCTGGG K789A_F ATGGTCTCACGCCGAGCTGGGCAGCCAGATCCTGAAAGA K797A_F ATGGTCTCAGGCCGAACACCCCGTGGAAAACACCCAGCT K890A_F ATGGTCTCACGCCCTGATTACCCAGAGAAAGTTCGACAA R1114A_F ATGGTCTCAGGCCAACAGCGATAAGCTGATCGCCAGAAA K1118A_F ATGGTCTCATGCCCTGATCGCCAGAAAGAAGGACTGGGA K1200A_F ATGGTCTCATGCCTACTCCCTGTTCGAGCTGGAAAACGG R63A_F ATGGTCTCACGCCCTGAAGAGAACCGCCAGAAGAAGATA K163A_F ATGGTCTCACGCCTTCGGGGCCACTTCCTGATCGAGGG R165A_F ATGGTCTCACGCCGGCCACTTCCTGATCGAGGGCGACCT R403A_F ATGGTCTCAGGCCACCTTCGACAACGGCAGCATCCCCCA H415A_F ATGGTCTCACGCCCTGGGAGAGCTGCACGCCATTCTGCG R447A_F ATGGTCTCACGCCATCCCCTACTACGTGGGCCCTCTGGC K1000A_F ATGGTCTCAAGCCTACCCTAAGCTGGAAAGCGAGTTCGT SPCAS9-C ATGGTCTCAAAATTCTTACTTTTTCTTTTTTTGCCTGGCC K775A_R ATGGTCTCACGCCTGTCCCTTCTGGGTGGTCTGG R780A_R ATGGTCTCACGCCTCGCGGCTGTTCTTCTGTCCCT Q807A_R ATGGTCTCACGCCAGCTGGGTGTTTTCCACGGGG K810A_R ATGGTCTCACGCCTCGTTCTGCAGCTGGGTGTTTTCCA R832A_R ATGGTCTCAGGGCGTTGATGTCCAGTTCCCTGGTCCAC K848A_R ATGGTCTCACGGCCAGAAAGCTCTGAGGCACGATTGGTCCAC K855A_R ATGGTCTCACTGCGTTGTCGATGGAGTCGTCCTTCAGAAAGCTCTG R859_R ATGGTCTCATGCGGTCAGCACCTTGTTGTCGATGGAGTC K862_R ATGGTCTCACGCGTCGCTTCTGGTCAGCACCTTGTTG K866_R ATGGTCTCACGCGCCCCGGTTCTTGTCGCTTCTG K961_R ATGGTCTCCAGCGCGGACTTCAGGGTGATCACTTTCACTTC K968A_R ATGGTCTCACCCGCCCGGAAATCGGACACCAGCTTG K974A_R ATGGTCTCATGCGTAAAACTGGAAATCCTTCCGAAATCGGACAC R976A_R ATGGTCTCAGCCACTTTGTAAAACTGGAAATCCTTCCGAAATCGG H982A_R ATGGTCTCAGGCGTAGTTGTTGATCTCGCGCACTTTGTAAAACTG H983A_R ATGGTCTCAGGCGTGGTAGTTGTTGATCTCGCGCACTTTG K1014A_R ATGGTCTCACCGCGTAGTCGCCGTACACGAACTCG K1047A_R ATGGTCTCACGCGAAAAAGTTCATGATGTTGCTGTAGAAGAAGTACTTGG K1059A_R ATGGTCTCAGCGCCCGGATCTCGCCGTTGGC R1060A_R ATGGTCTCACGCCTTCCGGATCTCGCCGTTGGC K1003A_R ATGGTCTCAGCGCAGGGTACTTTTTGATCAGGGCGGTTC H1240A_R ATGGTCTCAGGCGCTGGCCAGGTACAGGAAGTTCAC K1244A_R ATGGTCTCACGCCTCATAGTGGCTGGCCAGGTACAG K1289A_R ATGGTCTCATGCGTCCAGATTAGCGTCGGCCAGGATC K1296A_R ATGGTCTCACGCGTTGTAGGCGGACAGCACTTTGTCC H1297A_R ATGGTCTCCAGCCTTGTTGTAGGCGGACAGCACTTTGTCC R1298A_R ATGGTCTCACCGCGTGCTTGTTGTAGGCGGACAGC K1300A_R ATGGTCTCAGCGCATCCCGGTGCTTGTTGTAGGCG R1303A_R ATGGTCTCATGCGATGGGCTTATCCCGGTGCTTGTTGTAG H1311A_R ATGGTCTCAGGCGATGATATTCTCGGCCTGCTCTCTGATG K1325A_R ATGGTCTCACGCGAAGGCGGCAGGGGCTCC K1107A_R ATGGTCTCAGGCGCTGAAGCCGCCTGTCTGCACCTCGGT E1108A_R ATGGTCTCAGGCTTTGCTGAAGCCGCCTGTCTGCACCTC S1109A_R ATGGTCTCAGGCCTCTTTGCTGAAGCCGCCTGTCTGCAC ΔK1107_R ATGGTCTCACTCGCTGAAGCCGCCTGTCTGCACCTCGGT ΔE1108_R ATGGTCTCAAGATTTGCTGAAGCCGCCTGTCTGCACCTC ΔS1109_R ATGGTCTCAGATCTCTTTGCTGAAGCCGCCTGTCTGCAC KES_KG_R ATGGTCTCAGCCTTTGCTGAAGCCGCCTGTCTGCACCTC KES_GG_R ATGGTCTCAGCCGCCGCTGAAGCCGCCTGTCTGCACCTC R778A_R ATGGTCTCAGGCGCTGTTCTTCTGTCCCTTCTGGGTGGT K782A_R ATGGTCTCAGGCCATTCTCTCGCGGCTGTTCTTCTGTCC R783A_R ATGGTCTCAGGCTTCATTCTCTCGCGGCTGTTCTTCTG K789A_R ATGGTCTCAGGCGATGCCCTCTTCGATCCGCTTCATTCT K797A_R ATGGTCTCAGGCCAGGATCTGGCTGCCCAGCTCTTTTGAT K890A_R ATGGTCTCAGGCGGCGTTCAGCAGCTGCCGCCAGTAGTT R1114A_R ATGGTCTCAGGCCTTGGGCAGGATAGACTCTTTGCTGAA K1118A_R ATGGTCTCAGGCATCGCTGTTCCTCTTGGGCAGGATAGA K1200A_R ATGGTCTCAGGCAGGCAGCTTGATGATCAGGTCCTTTTT R63A_R ATGGTCTCAGGCGGTGGCCTCGGCTGTTTCGCCGCTGTC K163A_R ATGGTCTCAGGCGATCATGTGGGCCAGGGCCAGATAGAT R165A_R ATGGTCTCAGGCGAACTTGATCATGTGGGCCAGGGCCAG R403A_R ATGGTCTCAGGCCTGCTTCCGCAGCAGGTCCTCTCTGTT H415A_R ATGGTCTCAGGCGATCTGTGGGGGATGCCTGCCGTTGTC R447A_R ATGGTCTCAGGCGAAGGTCAGGATCTTCTCGATCTTTTC K1000A_R ATGGTCTCAGGCTTTGATCAGGGCGGTTCCCACGACGGC Primer name for SaCas9 Subsequence SACAS9-N ATGAAGACTACCGGTGCCACCATGGCCC K518A_F ATGAAGACTAGCGTACCTGATCGAGAAGATCAAGCTGCA K523A_F ATGAAGACTAGCGATCAAGCTGCACGACATGCAGGA K525A_F ATGAAGACTAGCGCTGCACGACATGCAGGAAGGC H557A_F ATGAAGACTAGCCATCATCCCCAGAAGCGTGTCCTTC R561A_F ATGAAGACTAGCAAGCGTGTCCTTCGACAACAGCTTC K572A_F ATGAAGACTAGCGGTGCTCGTGAAGCAGGAAGAAAACA R686A_F ATGAAGACTAGCGAAGTGGAAGTTTAAGAAAGAGCGGAACAA K692A_F ATGAAGACTAGCAGAGCGGAAACAAGGGGTACAAGCAC R694A_F ATGAAGACTAGCGAACAAGGGTACAAGCACCACGC H700A_F ATGAAGACTAGCCCACGCCGAGGACGCCCTGA K751A_F ATGAAGACTAGCAGAGATCTTCATCACCCCCCCACCAG R497A_F ATGAAGACTAGCCAACCGGCAGACCAACGAGCG R499A;Q500K_F ATGAAGACTAGCAAAGACCAACGAGCGGATCGAGG R634A_F ATGAAGACTAGCGTTCTCCGTGCAGAAAGACTTCATCAAC R654A;G655R_F ATGAAGACTAGCCCGCCTGATGAACCTGCTGCGG SACAS9-C ATGAAGACTAAATTCTTAAGCGTAATCTGGAACATCGTATGG K518A_R ATGAAGACTAACGGCGGCGTTCTCTTTTGCCGGTGG K523A_R ATGAAGACTATCGCCTCGATCAGGTACTTGGCGTTCTCTT K525A_R ATGAAGACTAGCGCGATCTTCTCGATCAGGTACTTGGCGT H557A_R ATGAAGACTATGGCGTCCACCTCATAGTTGAAGGGGTTGT R561A_R ATGAAGACTATTGCGGGGATGATGTGGTCCACCTCATA K572A_R ATGAAGACTACCGCGTTGTTGAAGCTGTTGTCGAAGGACA R686A_R ATGAAGACTATCGCCCGCAGAAAGCTGGTGAAGCC K692A_R ATGAAGACTACTGCCTTAAACTTCCACTTCCGCCGCA R694A_R ATGAAGACTATCGCCTCTTTCTTAAACTTCCACTTCCGCC H700A_R ATGAAGACTAGGGCCTTGTACCCCTTGTTCCGCTCTTTC K751A_R ATGAAGACTACTGCGTACTCCTGCTCGGTTTCGATCTCG R497A_R ATGAAGACTATGGCCTTCTGCATCTCGTTGATCATTTTCTG R499A;Q500K_R ATGAAGACTATTGCGTTCCGCTTCTGCATCTCGTTGA R634A_R ATGAAGACTAACGCGTTGATGTCCCGTTCTTCCAGCA R654A;G655R_R ATGAAGACTAGGGCGGTGGCGTATCTGGTATCCACCA

Table of guide sequences and primers for NGS

SpCas9 wild type

ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCC CAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGAT ACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGG CACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCC CACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGC CAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGG GCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTT CTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTG CTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCC CATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCA TTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGAAACAGCAGATTCGCCTGGATG ACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCC AAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTT CAAGACCAACCGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCT GCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAAACCTATGCCC ACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGT CCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCC GGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAA GGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACC TGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAA GCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCC GAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGAC AAGCTGATCCGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGC CGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAA GTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGG ATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGA AAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGAT CACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCG GAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGC AGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACC GGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAG GTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGTAA

>K855A

ATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACA GCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCC CTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACG ACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTAGAGA AAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGC TGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAG CTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACG ACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTG AGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCT ACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGAC AACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCGCATCCCCTACT ACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATG ACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCC TGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAA GATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACA GAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAG CAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGCG ATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAA ATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGC AGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCC ATCGACAACGCGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAA AGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAAC ACTAAGTACGACGAGAATGACAAGCTGATCCGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCC ACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATC GGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAAACGGCGAAACCGGGGAGATCGTGT GGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAG CTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGC TGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAA AACGGCCGGAAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATA ATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAG CACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAG AGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGTAA

eSpCas9(1.0)

ATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACA GCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGC CCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGAC GACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAG AAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAG CTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCA GCTGCCCGGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGAC GACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCT GAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGC TACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCG ACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTA CTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGA TGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTT CCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGG AAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGA CAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACA AGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGG CGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATC GAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGC TGCAGAACGAGGCCCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGAC TCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGA GAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATG AACACTAAGTACGACGAGAATGACAAGCTGATCCGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACG CCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTGCGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAA ATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGGCGCCTCTGATCGAGACAAAACGGCGAAACCGGGGAGATCG TGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGAT AAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAG AGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTG GAAAACGGCCGGAAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGG ATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAAC AAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCA AAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAG

eSpCas9(1.1)

ATGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACA GCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCC CTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACG ACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTAGAGA AAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGC TGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAG CTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACG ACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTG AGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCT ACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGAC AACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCGCATCCCCTACT ACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATG ACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCC TGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAA GATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACA GAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAG CAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGCG ATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAA ATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGC AGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGGCGGACGACTCC ATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAA AGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAAC ACTAAGTACGACGAGAATGACAAGCTGATCCGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCC ACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTGCGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATC GGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGGCGCCTCTGATCGAGACAAAACGGCGAAACCGGGGAGATCGTGT GGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAG CTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGC TGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAA AACGGCCGGAAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATA ATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAG CACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAG AGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGTAA

Having thus described in detail the preferred embodiments of the present invention, it is to be understood that the present invention, as defined in the foregoing sections, should not be limited to the specific details set forth in the foregoing description, since many obvious variations thereof are possible without departing from the spirit or scope of the present invention.

--->

SEQUENCE LIST

<110> THE BROAD INSTITUTE, INC.

MASSACHUSETTS INSTITUTE OF TECHNOLOGY

<120> CRISPR MUTATIONS THAT REDUCE OFF-TARGET EFFECTS

<130> 47627.99.2017

<140>

<141>

<150> 62/269,876

<151> 2015-12-18

<150> 62/255,256

<151> 2015-11-13

<150> 62/237,360

<151> 2015-10-05

<150> 62/207,312

<151> 2015-08-19

<150> 62/181,453

<151> 2015-06-18

<160> 547

<170> PatentIn version 3.5

<210> 1

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic peptide"

<400> 1

Gly Gly Gly Ser

1

<210> 2

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic peptide"

<400> 2

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 3

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic polypeptide"

<400> 3

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> 4

<211> 45

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic polypeptide"

<400> 4

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> 5

<211> 60

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic polypeptide"

<400> 5

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> 6

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (1)..(20)

<223> a, c, t or g

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 6

nnnnnnnnnn nnnnnnnnnn ngg

23

<210> 7

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (1)..(12)

<223> a, c, t or g

<220>

<221> modified_base

<222> (13)..(13)

<223> a, c, t, g, unknown or other

<400> 7

nnnnnnnnnn nnngg

15

<210> 8

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (1)..(20)

<223> a, c, t or g

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 8

nnnnnnnnnn nnnnnnnnnn ngg

23

<210> 9

<211> 14

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (1)..(11)

<223> a, c, t or g

<220>

<221> modified_base

<222> (12)..(12)

<223> a, c, t, g, unknown or other

<400> 9

nnnnnnnnnn nngg

14

<210> 10

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (1)..(20)

<223> a, c, t or g

<220>

<221> modified_base

<222> (21)..(22)

<223> a, c, t, g, unknown or other

<400> 10

nnnnnnnnnn nnnnnnnnnn nnagaaw

27

<210> 11

<211> 19

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (1)..(12)

<223> a, c, t or g

<220>

<221> modified_base

<222> (13)..(14)

<223> a, c, t, g, unknown or other

<400> 11

nnnnnnnnnn nnnnagaaw

19

<210> 12

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (1)..(20)

<223> a, c, t or g

<220>

<221> modified_base

<222> (21)..(22)

<223> a, c, t, g, unknown or other

<400> 12

nnnnnnnnnn nnnnnnnnnn nnagaaw

27

<210> 13

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (1)..(11)

<223> a, c, t or g

<220>

<221> modified_base

<222> (12)..(13)

<223> a, c, t, g, unknown or other

<400> 13

nnnnnnnnnn nnnagaaw

18

<210> 14

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (1)..(20)

<223> a, c, t or g

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<220>

<221> modified_base

<222> (24)..(24)

<223> a, c, t, g, unknown or other

<400> 14

nnnnnnnnnn nnnnnnnnnn nggng

25

<210> 15

<211> 17

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (1)..(12)

<223> a, c, t or g

<220>

<221> modified_base

<222> (13)..(13)

<223> a, c, t, g, unknown or other

<220>

<221> modified_base

<222> (16)..(16)

<223> a, c, t, g, unknown or other

<400> 15

nnnnnnnnnn nnnggng

17

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (1)..(20)

<223> a, c, t or g

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<220>

<221> modified_base

<222> (24)..(24)

<223> a, c, t, g, unknown or other

<400> 16

nnnnnnnnnn nnnnnnnnnn nggng

25

<210> 17

<211> 16

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (1)..(11)

<223> a, c, t or g

<220>

<221> modified_base

<222> (12)..(12)

<223> a, c, t, g, unknown or other

<220>

<221> modified_base

<222> (15)..(15)

<223> a, c, t, g, unknown or other

<400> 17

nnnnnnnnnn nnggng

16

<210> 18

<211> 137

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic polynucleotide"

<220>

<221> modified_base

<222> (1)..(20)

<223> a, c, t, g, unknown or other

<400> 18

nnnnnnnnnn nnnnnnnnnn gtttttgtac tctcaagatt tagaaataaa tcttgcagaa

60

gctacaaaga taaggcttca tgccgaaatc aacaccctgt cattttatgg cagggtgttt

120

tcgttattta atttttt

137

<210> 19

<211> 123

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic polynucleotide"

<220>

<221> modified_base

<222> (1)..(20)

<223> a, c, t, g, unknown or other

<400> 19

nnnnnnnnnn nnnnnnnnnn gtttttgtac tctcagaaat gcagaagcta caaagataag

60

gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg gtgttttcgt tatttaattt

120

ttt

123

<210> 20

<211> 110

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic polynucleotide"

<220>

<221> modified_base

<222> (1)..(20)

<223> a, c, t, g, unknown or other

<400> 20

nnnnnnnnnn nnnnnnnnnn gtttttgtac tctcagaaat gcagaagcta caaagataag

60

gcttcatgcc gaaatcaaca ccctgtcatt ttatggcagg gtgttttttt

110

<210> 21

<211> 102

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic polynucleotide"

<220>

<221> modified_base

<222> (1)..(20)

<223> a, c, t, g, unknown or other

<400> 21

nnnnnnnnnn nnnnnnnnnn gttttagagc tagaaatagc aagttaaaat aaggctagtc

60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt tt

102

<210> 22

<211> 88

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (1)..(20)

<223> a, c, t, g, unknown or other

<400> 22

nnnnnnnnnn nnnnnnnnnn gttttagagc tagaaatagc aagttaaaat aaggctagtc

60

cgttatcaac ttgaaaaagt gttttttt

88

<210> 23

<211> 76

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (1)..(20)

<223> a, c, t, g, unknown or other

<400> 23

nnnnnnnnnn nnnnnnnnnn gttttagagc tagaaatagc aagttaaaat aaggctagtc

60

cgttatcatt tttttt

76

<210> 24

<211> 7

<212> PROTEIN

<213> Monkey virus 40

<400> 24

Pro Lys Lys Lys Arg Lys Val

1 5

<210> 25

<211> 16

<212> PROTEIN

<213> Unspecified

<220>

<221> source

<223> /note="Description of the unknown: Sequence

"two-part NLS from nucleoplasmin"

<400> 25

Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys

1 5 10 15

<210> 26

<211> 9

<212> PROTEIN

<213> Unspecified

<220>

<221> source

<223> /note="Description of unknown: NLS sequence from

C-myc"

<400> 26

Pro Ala Ala Lys Arg Val Lys Leu Asp

1 5

<210> 27

<211> 11

<212> PROTEIN

<213> Unspecified

<220>

<221> source

<223> /note="Description of unknown: NLS sequence from

C-myc"

<400> 27

Arg Gln Arg Arg Asn Glu Leu Lys Arg Ser Pro

1 5 10

<210> 28

<211> 38

<212> PROTEIN

<213> Homo sapiens

<400> 28

Asn Gln Ser Ser Asn Phe Gly Pro Met Lys Gly Gly Asn Phe Gly Gly

1 5 10 15

Arg Ser Ser Gly Pro Tyr Gly Gly Gly Gly Gln Tyr Phe Ala Lys Pro

20 25 30

Arg Asn Gln Gly Gly Tyr

35

<210> 29

<211> 42

<212> PROTEIN

<213> Unspecified

<220>

<221> source

<223> /note="Description of unknown: Domain sequence

IBB from importin-alpha"

<400> 29

Arg Met Arg Ile Glx Phe Lys Asn Lys Gly Lys Asp Thr Ala Glu Leu

1 5 10 15

Arg Arg Arg Arg Val Glu Val Ser Val Glu Leu Arg Lys Ala Lys Lys

20 25 30

Asp Glu Gln Ile Leu Lys Arg Arg Asn Val

35 40

<210> 30

<211> 8

<212> PROTEIN

<213> Unspecified

<220>

<221> source

<223> /note="Description of the unknown: Sequence of

T-protein myoma"

<400> 30

Val Ser Arg Lys Arg Pro Arg Pro

1 5

<210> 31

<211> 8

<212> PROTEIN

<213> Unspecified

<220>

<221> source

<223> /note="Description of the unknown: Sequence of

T-protein myoma"

<400> 31

Pro Pro Lys Lys Ala Arg Glu Asp

1 5

<210> 32

<211> 8

<212> PROTEIN

<213> Homo sapiens

<400> 32

Pro Gln Pro Lys Lys Lys Pro Leu

1 5

<210> 33

<211> 12

<212> PROTEIN

<213> Mus musculus

<400> 33

Ser Ala Leu Ile Lys Lys Lys Lys Lys Met Ala Pro

1 5 10

<210> 34

<211> 5

<212> PROTEIN

<213> Influenza virus

<400> 34

Asp Arg Leu Arg Arg

1 5

<210> 35

<211> 7

<212> PROTEIN

<213> Influenza virus

<400> 35

Pro Lys Gln Lys Lys Arg Lys

1 5

<210> 36

<211> 10

<212> PROTEIN

<213> Hepatitis delta virus

<400> 36

Arg Lys Leu Lys Lys Lys Ile Lys Lys Leu

1 5 10

<210> 37

<211> 10

<212> PROTEIN

<213> Mus musculus

<400> 37

Arg Glu Lys Lys Lys Phe Leu Lys Arg Arg

1 5 10

<210> 38

<211> 20

<212> PROTEIN

<213> Homo sapiens

<400> 38

Lys Arg Lys Gly Asp Glu Val Asp Gly Val Asp Glu Val Ala Lys Lys

1 5 10 15

Lys Ser Lys Lys

20

<210> 39

<211> 17

<212> PROTEIN

<213> Homo sapiens

<400> 39

Arg Lys Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys

1 5 10 15

Lys

<210> 40

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic peptide"

<400> 40

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10

<210> 41

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic peptide"

<400> 41

Ala Glu Ala Ala Ala Lys Ala

1 5

<210> 42

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic peptide"

<400> 42

Gly Gly Gly Gly Ser

1 5

<210> 43

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic peptide"

<400> 43

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 44

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic peptide"

<400> 44

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 45

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic peptide"

<400> 45

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> 46

<211> 35

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic polypeptide"

<400> 46

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> 47

<211> 40

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic polypeptide"

<400> 47

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> 48

<211> 50

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic polypeptide"

<400> 48

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

50

<210> 49

<211> 55

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic polypeptide"

<400> 49

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

GlySer GlyGlyGlyGlySer

50 55

<210> 50

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic peptide"

<220>

<221> MODIFIED_RESIDUE

<222> (2)..(2)

<223> Ahh

<220>

<221> MODIFIED_RESIDUE

<222> (5)..(5)

<223> Ahh

<220>

<221> MODIFIED_RESIDUE

<222> (8)..(8)

<223> Ahh

<220>

<221> MODIFIED_RESIDUE

<222> (11)..(11)

<223> Ahh

<400> 50

Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg Arg Xaa Arg

1 5 10

<210> 51

<211> 20

<212> DNA

<213> Homo sapiens

<400> 51

gagtccgagc agaagaagaa

20

<210> 52

<211> 20

<212> DNA

<213> Homo sapiens

<400> 52

gagtcctagc aggagaagaa

20

<210> 53

<211> 20

<212> DNA

<213> Homo sapiens

<400> 53

gagtctaagc agaagaagaa

20

<210> 54

<211> 12

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 54

guuuuagagc ua

12

<210> 55

<211> 9

<212> PROTEIN

<213> Unspecified

<220>

<221> source

<223> /note="Description of unknown: Peptide related to

family of peptides containing the LAGLIDADG motif"

<400> 55

Leu Ala Gly Leu Ile Asp Ala Asp Gly

1 5

<210> 56

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 56

gagtccgagc agaagaagaa ggg

23

<210> 57

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 57

gagtctaagc agaagaagaa gag

23

<210> 58

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 58

gagttagagc agaagaagaa agg

23

<210> 59

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 59

gggtgggggg agtttgctcc tgg

23

<210> 60

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 60

ggggagggtgg agtttgctcc tgg

23

<210> 61

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 61

cggggggaggg agtttgctcc tgg

23

<210> 62

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 62

ggtgagtgag tgtgtgcgtg tgg

23

<210> 63

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 63

ggtgagtgag tgtgtgtgtg agg

23

<210> 64

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 64

agtgagtgag tgtgtgtgtg ggg

23

<210> 65

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 65

gctgagtgag tgtatgcgtg tgg

23

<210> 66

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 66

gttgagtgaa tgtgtgcgtg agg

23

<210> 67

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 67

tgtgggtgag tgtgtgcgtg agg

23

<210> 68

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 68

ggcctcccca aagcctggcc agggagt

27

<210> 69

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 69

gacctcccca tagcctggcc agggagg

27

<210> 70

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 70

ggcctgccca aggcctgacc aagggaa

27

<210> 71

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 71

ggcctcccaa agccaggcca ggggga

26

<210> 72

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 72

atggtctcac cggtgccacc atggactata ag

32

<210> 73

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 73

atggtctcag gcgaacagcc gcgagagaat gaagcggat

39

<210> 74

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 74

atggtctcag gcgatgaagc ggatcgaaga gggcatca

38

<210> 75

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 75

atggtctcag gcgaacgaga agctgtacct gtactacctg

40

<210> 76

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 76

atggtctcag gcgctgtacc tgtactacct gcagaatgg

39

<210> 77

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 77

atggtctcag ccctgtccga ctacgatgtg gaccatatc

39

<210> 78

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 78

atggtctcag ccgacgactc catcgacaac aaggtgctga cc

42

<210> 79

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 79

atggtctcag cagtgctgac cagaagcgac aagaaccggg

40

<210> 80

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 80

atggtctcac gcaagcgaca agaaccgggg caagag

36

<210> 81

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 81

atggtctcac gcgaaccggg gcaagagcga caac

34

<210> 82

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 82

atggtctcac gcgagcgaca acgtgccctc cgaa

34

<210> 83

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 83

atggtctcag cgctggtgtc cgatttccgg aaggatttc

39

<210> 84

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 84

atggtctcag cggatttcca gttttacaaa gtgcgcgaga tcaacaac

48

<210> 85

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 85

atggtctcac gcagtgcgcg agatcaacaa ctaccacca

39

<210> 86

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 86

atggtctcat ggccgagatc aacaactacc accacgcc

38

<210> 87

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 87

atggtctcac gcccacgccc acgacgccta c

31

<210> 88

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 88

atggtctcac gccgcccacg acgcctacct gaa

33

<210> 89

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 89

atggtctcag cggtgtacga cgtgcggaag atgatcg

37

<210> 90

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 90

atggtctcac gcgaccgaga ttaccctggc caacg

35

<210> 91

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 91

atggtctcag cgcggcctct gatcgagaca aacgg

35

<210> 92

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 92

atggtctcag gcgcctctga tcgagacaaa cggcg

35

<210> 93

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 93

atggtctcag cgctggaaag cgagttcgtg tacggc

36

<210> 94

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 94

atggtctcac gcctatgaga agctgaaggg ctcccc

36

<210> 95

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 95

atggtctcag gcgctgaagg gctcccccga g

31

<210> 96

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 96

atggtctcac gcagtgctgt ccgcctacaa caagcac

37

<210> 97

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 97

atggtctcac gcgcaccggg ataagcccat cagag

35

<210> 98

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 98

atggtctcaa ggcccgggat aagcccatca gagagc

36

<210> 99

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 99

atggtctcag cggataagcc catcagagag caggcc

36

<210> 100

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 100

atggtctcag cgcccatcag agagcaggcc gag

33

<210> 101

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 101

atggtctcac gcagagcagg ccgagaatat catccacc

38

<210> 102

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 102

atggtctcac gccctgttta ccctgaccaa tctggggag

38

<210> 103

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 103

atggtctcac gcgtactttg acaccaccat cgaccgg

37

<210> 104

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 104

atggtctcac gccgagtcta tcctgcccaa gaggaacag

39

<210> 105

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 105

atggtctcaa gcctctatcc tgcccaagag gaacagcga

39

<210> 106

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 106

atggtctcag gccatcctgc ccaagaggaa cagcgataa

39

<210> 107

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 107

atggtctcac gagtctatcc tgcccaagag gaacagcga

39

<210> 108

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 108

atggtctcaa tctatcctgc ccaagaggaa cagcgataa

39

<210> 109

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 109

atggtctcag atcctgccca agaggaacag cgataagct

39

<210> 110

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 110

atggtctcaa ggcatcctgc ccaagaggaa cagcgataa

39

<210> 111

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 111

atggtctcac ggcatcctgc ccaagaggaa cagcgataa

39

<210> 112

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 112

atggtctcac gccgagagaa tgaagcggat cgaagaggg

39

<210> 113

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 113

atggtctcag gcccggatcg aagagggcat caaagagct

39

<210> 114

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 114

atggtctcag gccatcgaag agggcatcaa agagctggg

39

<210> 115

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 115

atggtctcac gccgagctgg gcagccagat cctgaaaga

39

<210> 116

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 116

atggtctcag gccgaacacc ccgtggaaaa cacccagct

39

<210> 117

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 117

atggtctcac gccctgatta cccagagaaa gttcgacaa

39

<210> 118

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 118

atggtctcag gccaacagcg ataagctgat cgccagaaa

39

<210> 119

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 119

atggtctcat gccctgatcg ccagaaagaa ggactggga

39

<210> 120

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 120

atggtctcat gcctactccc tgttcgagct ggaaaacgg

39

<210> 121

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 121

atggtctcac gccctgaaga gaaccgccag aagaagata

39

<210> 122

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 122

atggtctcac gccttccggg gccacttcct gatcgaggg

39

<210> 123

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 123

atggtctcac gccggccact tcctgatcga gggcgacct

39

<210> 124

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 124

atggtctcag gccaccttcg acaacggcag catccccca

39

<210> 125

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 125

atggtctcac gccctgggag agctgcacgc cattctgcg

39

<210> 126

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 126

atggtctcac gccatcccct actacgtggg ccctctggc

39

<210> 127

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 127

atggtctcaa gcctacccta agctggaaag cgagttcgt

39

<210> 128

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 128

atggtctcaa attcttactt tttctttttt gcctggcc

38

<210> 129

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 129

atggtctcac gcctgtccct tctgggtggt ctgg

34

<210> 130

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 130

atggtctcac gcctcgcggc tgttcttctg tccct

35

<210> 131

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 131

atggtctcac gccagctggg tgttttccac gggg

34

<210> 132

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 132

atggtctcac gcctcgttct gcagctgggt gttttcca

38

<210> 133

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 133

atggtctcag ggcgttgatg tccagttcct ggtccac

37

<210> 134

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 134

atggtctcac ggccagaaag ctctgaggca cgatatggtc cac

43

<210> 135

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 135

atggtctcac tgcgttgtcg atggagtcgt ccttcagaaa gctctg

46

<210> 136

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 136

atggtctcat gcggtcagca ccttgttgtc gatggagtc

39

<210> 137

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 137

atggtctcac gcgtcgcttc tggtcagcac cttgttg

37

<210> 138

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 138

atggtctcac gcgccccggt tcttgtcgct tctg

34

<210> 139

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 139

atggtctcag cgcggacttc agggtgatca ctttcacttc

40

<210> 140

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 140

atggtctcac cgcccggaaa tcggacacca gcttg

35

<210> 141

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 141

atggtctcat gcgtaaaact ggaaatcctt ccggaaatcg gacac

45

<210> 142

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 142

atggtctcag ccactttgta aaactggaaa tccttccgga aatcgg

46

<210> 143

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 143

atggtctcag gcgtagttgt tgatctcgcg cactttgtaa aactg

45

<210> 144

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 144

atggtctcag gcgtggtagt tgttgatctc gcgcactttg

40

<210> 145

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 145

atggtctcac cgcgtagtcg ccgtacacga actcg

35

<210> 146

<211> 50

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 146

atggtctcac gcgaaaaagt tcatgatgtt gctgtagaag aagtacttgg

50

<210> 147

<211> 31

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 147

atggtctcag cgcccggatc tcgccgttgg c

31

<210> 148

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 148

atggtctcac gccttccgga tctcgccgtt ggc

33

<210> 149

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 149

atggtctcag cgcagggtac tttttgatca gggcggttc

39

<210> 150

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 150

atggtctcag gcgctggcca ggtacaggaa gttcac

36

<210> 151

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 151

atggtctcac gcctcatagt ggctggccag gtacag

36

<210> 152

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 152

atggtctcat gcgtccagat tagcgtcggc caggatc

37

<210> 153

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 153

atggtctcac gcgttgtagg cggacagcac tttgtcc

37

<210> 154

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 154

atggtctcag ccttgttgta ggcggacagc actttgtcc

39

<210> 155

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 155

atggtctcac cgcgtgcttg ttgtaggcgg acagc

35

<210> 156

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 156

atggtctcag cgcatcccgg tgcttgttgt aggcg

35

<210> 157

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 157

atggtctcat gcgatgggct tatcccggtg cttgttgtag

40

<210> 158

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 158

atggtctcag gcgatgatat tctcggcctg ctctctgatg

40

<210> 159

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 159

atggtctcac gcgaaggcgg caggggctcc

30

<210> 160

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 160

atggtctcag gcgctgaagc cgcctgtctg cacctcggt

39

<210> 161

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 161

atggtctcag gctttgctga agccgcctgt ctgcacctc

39

<210> 162

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 162

atggtctcag gcctctttgc tgaagccgcc tgtctgcac

39

<210> 163

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 163

atggtctcac tcgctgaagc cgcctgtctg cacctcggt

39

<210> 164

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 164

atggtctcaa gatttgctga agccgcctgt ctgcacctc

39

<210> 165

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 165

atggtctcag atctctttgc tgaagccgcc tgtctgcac

39

<210> 166

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 166

atggtctcag cctttgctga agccgcctgt ctgcacctc

39

<210> 167

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 167

atggtctcag ccgccgctga agccgcctgt ctgcacctc

39

<210> 168

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 168

atggtctcag gcgctgttct tctgtccctt ctgggtggt

39

<210> 169

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 169

atggtctcag gccattctct cgcggctgtt cttctgtcc

39

<210> 170

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 170

atggtctcag gccttcattc tctcgcggct gttcttctg

39

<210> 171

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 171

atggtctcag gcgatgccct cttcgatccg cttcattct

39

<210> 172

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 172

atggtctcag gccaggatct ggctgcccag ctctttgat

39

<210> 173

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 173

atggtctcag gcggcgttca gcagctgccg ccagtagtt

39

<210> 174

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 174

atggtctcag gccttgggca ggatagactc tttgctgaa

39

<210> 175

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 175

atggtctcag gcatcgctgt tcctcttggg caggataga

39

<210> 176

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 176

atggtctcag gcaggcagct tgatgatcag gtccttttt

39

<210> 177

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 177

atggtctcag gcggtggcct cggctgtttc gccgctgtc

39

<210> 178

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 178

atggtctcag gcgatcatgt gggccagggc cagatagat

39

<210> 179

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 179

atggtctcag gcgaacttga tcatgtgggc cagggccag

39

<210> 180

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 180

atggtctcag gcctgcttcc gcagcaggtc ctctctgtt

39

<210> 181

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 181

atggtctcag gcgatctggt gggggatgct gccgttgtc

39

<210> 182

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 182

atggtctcag gcgaaggtca ggatcttctc gatcttttc

39

<210> 183

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 183

atggtctcag gctttgatca gggcggttcc cacgacggc

39

<210> 184

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 184

atgaagacta ccggtgccac catggccc

28

<210> 185

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 185

atgaagacta gcgtacctga tcgagaagat caagctgca

39

<210> 186

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 186

atgaagacta gcgatcaagc tgcacgacat gcagga

36

<210> 187

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 187

atgaagacta gcgctgcacg acatgcagga aggc

34

<210> 188

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 188

atgaagacta gccatcatcc ccagaagcgt gtccttc

37

<210> 189

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 189

atgaagacta gcaagcgtgt ccttcgacaa cagcttc

37

<210> 190

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 190

atgaagacta gcggtgctcg tgaagcagga agaaaaca

38

<210> 191

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 191

atgaagacta gcgaagtgga agtttaagaa agagcggaac aa

42

<210> 192

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 192

atgaagacta gcagagcgga acaaggggta caagcac

37

<210> 193

<211> 36

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 193

atgaagacta gcgaacaagg ggtacaagca ccacgc

36

<210> 194

<211> 32

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 194

atgaagacta gcccacgccg aggacgccct ga

32

<210> 195

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 195

atgaagacta gcagagatct tcatcacccc ccaccag

37

<210> 196

<211> 33

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 196

atgaagacta gccaaccggc agaccaacga gcg

33

<210> 197

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 197

atgaagacta gcaaagacca acgagcggat cgagg

35

<210> 198

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 198

atgaagacta gcgttctccg tgcagaaaga cttcatcaac

40

<210> 199

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 199

atgaagacta gcccgcctga tgaacctgct gcgg

34

<210> 200

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 200

atgaagacta aattcttaag cgtaatctgg aacatcgtat gg

42

<210> 201

<211> 34

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 201

atgaagacta acgcggcgtt ctctttgccg gtgg

34

<210> 202

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 202

atgaagacta tcgcctcgat caggtacttg gcgttctctt

40

<210> 203

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 203

atgaagacta gcgcgatctt ctcgatcagg tacttggcgt

40

<210> 204

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 204

atgaagacta tggcgtccac ctcatagttg aaggggttgt

40

<210> 205

<211> 38

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 205

atgaagacta ttgcggggat gatgtggtcc acctcata

38

<210> 206

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 206

atgaagacta ccgcgttgtt gaagctgttg tcgaaggaca

40

<210> 207

<211> 35

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 207

atgaagacta tcgcccgcag aaagctggtg aagcc

35

<210> 208

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 208

atgaagacta ctgccttaaa cttccacttc cgccgca

37

<210> 209

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 209

atgaagacta tcgcctcttt cttaaacttc cacttccgcc

40

<210> 210

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 210

atgaagacta gggccttgta ccccttgttc cgctctttc

39

<210> 211

<211> 39

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 211

atgaagacta ctgcgtactc ctgctcggtt tcgatctcg

39

<210> 212

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 212

atgaagacta tggccttctg catctcgttg atcattttct g

41

<210> 213

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 213

atgaagacta ttgcgttccg cttctgcatc tcgttga

37

<210> 214

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 214

atgaagacta acgcgttgat gtcccgttct tccagca

37

<210> 215

<211> 37

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 215

atgaagacta gggcggtggc gtatctggta tccacca

37

<210> 216

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 216

ggtgagtgag tgtgtgcgtg ngg

23

<210> 217

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 217

ggtgagtgag tgtgtgtgtg ngg

23

<210> 218

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 218

gctgagtgag tgtatgcgtg ngg

23

<210> 219

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 219

ggtgagtgag tgcgtgcggg ngg

23

<210> 220

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 220

tgtgggtgag tgtgtgcgtg ngg

23

<210> 221

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 221

agtgaatgag tgtgtgtgtg ngg

23

<210> 222

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 222

tgtgagtaag tgtgtgtgtg ngg

23

<210> 223

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 223

actgtgtgag tgtgtgcgtg ngg

23

<210> 224

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 224

agcgagtggg tgtgtgcgtg ngg

23

<210> 225

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 225

agtgtgtgag tgtgtgcgtg ngg

23

<210> 226

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 226

tgtgggtgag tgtgtgcgtg nga

23

<210> 227

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 227

agcgagtgag tgtgtgtgtg ngg

23

<210> 228

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 228

gtagagtgag tgtgtgtgtg ngg

23

<210> 229

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 229

tgagtgtgag tgtgtgcgtg ngg

23

<210> 230

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 230

agagagtgag tgtgtgcatg ngg

23

<210> 231

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 231

gttgagtgaa tgtgtgcgtg ngg

23

<210> 232

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 232

cgtgagtgag tgtgtacctg ngg

23

<210> 233

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 233

gggtgggggg agtttgctcc ngg

23

<210> 234

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 234

ggggagggtgg agtttgctcc ngg

23

<210> 235

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 235

cggggggaggg agtttgctcc ngg

23

<210> 236

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 236

tagtggaggg agcttgctcc ngg

23

<210> 237

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 237

gcgtgggggg tgtttgctcc ngg

23

<210> 238

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 238

ttggggggggc agtttgctcc ngg

23

<210> 239

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 239

gagtccgagc agaagaagaa ngg

23

<210> 240

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 240

gagttagagc agaagaagaa ngg

23

<210> 241

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 241

gagtctaagc agaagaagaa nag

23

<210> 242

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 242

gaggccgagc agaaaaagga ngg

23

<210> 243

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 243

aagtctgagc acaagaagaa ngg

23

<210> 244

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 244

gagtcctagc aggagaagaa nag

23

<210> 245

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 245

acgtctgagc agaagaagaa ngg

23

<210> 246

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 246

gtcacctcca atgactaggg ngg

23

<210> 247

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 247

gggcaaccac aaacccacga ngg

23

<210> 248

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 248

gcttgtccct ctgtcaatgg ngg

23

<210> 249

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 249

gcgccaccgg ttgatgtgat ngg

23

<210> 250

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 250

gacatcgatg tcctccccat ngg

23

<210> 251

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 251

gcctccccaa agcctggcca ngg

23

<210> 252

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 252

gccccgggct tcaagccctg ngg

23

<210> 253

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 253

ggcagagtgc tgcttgctgc ngg

23

<210> 254

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 254

gctaaagagg gaatgggctt ngg

23

<210> 255

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 255

gtttgggagg tcagaaatag ngg

23

<210> 256

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 256

gttggagcgg ggagaaggcc ngg

23

<210> 257

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 257

gaggctgggg tggaggtgtt ngg

23

<210> 258

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 258

gtgggtgagt gagtgcgtgc ngg

23

<210> 259

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 259

gattcctggt gccagaaaca ngg

23

<210> 260

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 260

gggcagtttg ctcctggcac ngg

23

<210> 261

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 261

ggagagaggc tcccatcacg ngg

23

<210> 262

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 262

gagaagagaa gtggggtggg ngg

23

<210> 263

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 263

gtgtgtgtgt gagggtgtaa ngg

23

<210> 264

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 264

ggtgagtgag tgtgtgtgtg ngg

23

<210> 265

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (21)..(21)

<223> a, c, t, g, unknown or other

<400> 265

gaagaatgga cagaactctg ngg

23

<210> 266

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 266

ccatctcatc cctgcgtgtc tccgcgtcct tcgagagtga ggac

44

<210> 267

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 267

ccatctcatc cctgcgtgtc tccagggacc cctctgacag act

43

<210> 268

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 268

ccatctcatc cctgcgtgtc tccgcccatt tctcctttga ggt

43

<210> 269

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 269

ccatctcatc cctgcgtgtc tcccctccca caggaatttg aag

43

<210> 270

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 270

ccatctcatc cctgcgtgtc tcctgtcacc acacagttac cacct

45

<210> 271

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 271

ccatctcatc cctgcgtgtc tccataaggg gcaagttctg ggctat

46

<210> 272

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 272

ccatctcatc cctgcgtgtc tcctgatgaa gctgcctttc ctaagc

46

<210> 273

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 273

ccatctcatc cctgcgtgtc tcctctgcca gatccttagg cg

42

<210> 274

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 274

ccatctcatc cctgcgtgtc tccgacgtct gggtcccgag c

41

<210> 275

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 275

ccatctcatc cctgcgtgtc tcctcctgtg gaacaaccag acacc

45

<210> 276

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 276

ccatctcatc cctgcgtgtc tccaagctgc tggctttcct aag

43

<210> 277

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 277

ccatctcatc cctgcgtgtc tccaggaccc aggtttgcac t

41

<210> 278

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 278

ccatctcatc cctgcgtgtc tccatgatta gaaacctgca ctcccag

47

<210> 279

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 279

ccatctcatc cctgcgtgtc tccgtgggca ccaggagcgt ag

42

<210> 280

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 280

ccatctcatc cctgcgtgtc tccggcctcg ggaaacttac aat

43

<210> 281

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 281

ccatctcatc cctgcgtgtc tccagtgcct tgcacaaata ggc

43

<210> 282

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 282

ccatctcatc cctgcgtgtc tccctgccat tgtgaacagt gct

43

<210> 283

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 283

ccatctcatc cctgcgtgtc tccaagcaac tccagtccca aat

43

<210> 284

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 284

ccatctcatc cctgcgtgtc tcccctgcag gtgtctcctt ttc

43

<210> 285

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 285

ccatctcatc cctgcgtgtc tccacttctt gggcagtgat gga

43

<210> 286

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 286

ccatctcatc cctgcgtgtc tcctgcaaag ctaagcagag atgc

44

<210> 287

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 287

ccatctcatc cctgcgtgtc tccgcagaga tgcctatgcc tacat

45

<210> 288

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 288

ccatctcatc cctgcgtgtc tccacatgcg attctgcagg gaa

43

<210> 289

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 289

ccatctcatc cctgcgtgtc tcccaaagta caaacggcag aagc

44

<210> 290

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 290

ccatctcatc cctgcgtgtc tccttctgag ggctgctacc tgt

43

<210> 291

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 291

ccatctcatc cctgcgtgtc tcccacggcc tttgcaaata gag

43

<210> 292

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 292

ccatctcatc cctgcgtgtc tcctgggaga gagacccctt ctt

43

<210> 293

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 293

ccatctcatc cctgcgtgtc tccgttctga cattcctcct gaggga

46

<210> 294

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 294

ccatctcatc cctgcgtgtc tccccagact cagtaaagcc tgga

44

<210> 295

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 295

ccatctcatc cctgcgtgtc tccggccctt cctctgtact ctatac

46

<210> 296

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 296

ccatctcatc cctgcgtgtc tccccaatgg ggaggacatc gat

43

<210> 297

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 297

ccatctcatc cctgcgtgtc tccccaatgg ggaggacatc gat

43

<210> 298

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 298

ccatctcatc cctgcgtgtc tccaacccac gagggcagag t

41

<210> 299

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 299

ccatctcatc cctgcgtgtc tcccaaagta caaacggcag aagc

44

<210> 300

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 300

ccatctcatc cctgcgtgtc tcccaaagta caaacggcag aagc

44

<210> 301

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 301

ccatctcatc cctgcgtgtc tccaacccac gagggcagag t

41

<210> 302

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 302

ccatctcatc cctgcgtgtc tccaacccac gagggcagag t

41

<210> 303

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 303

ccatctcatc cctgcgtgtc tccccaatgg ggaggacatc gat

43

<210> 304

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 304

ccatctcatc cctgcgtgtc tccaagcaac tccagtccca aat

43

<210> 305

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 305

ccatctcatc cctgcgtgtc tccaagcaac tccagtccca aat

43

<210> 306

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 306

ccatctcatc cctgcgtgtc tccgcgtctt cgagagtgag gac

43

<210> 307

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 307

ccatctcatc cctgcgtgtc tcccctccca caggaatttg aag

43

<210> 308

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 308

ccatctcatc cctgcgtgtc tcccctccca caggaatttg aag

43

<210> 309

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 309

ccatctcatc cctgcgtgtc tcctgttaaa aacacaacat cagtgcat

48

<210> 310

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 310

ccatctcatc cctgcgtgtc tccacatgcg attctgcagg gaa

43

<210> 311

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 311

ccatctcatc cctgcgtgtc tccacttctt gggcagtgat gga

43

<210> 312

<211> 41

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 312

ccatctcatc cctgcgtgtc tccaggaccc aggtttgcac t

41

<210> 313

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 313

ccatctcatc cctgcgtgtc tccagggacc cctctgacag act

43

<210> 314

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 314

ccatctcatc cctgcgtgtc tccagggacc cctctgacag act

43

<210> 315

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 315

ccatctcatc cctgcgtgtc tccggccctt cctctgtact ctatac

46

<210> 316

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 316

cctctctatg ggcagtcggt gatgggggag agggacacac agat

44

<210> 317

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 317

cctctctatg ggcagtcggt gatgcacacc cacaccctca taca

44

<210> 318

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 318

cctctctatg ggcagtcggt gatgagccac agaggtggag actg

44

<210> 319

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 319

cctctctatg ggcagtcggt gatggcaccc caacacctac atct

44

<210> 320

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 320

cctctctatg ggcagtcggt gatggggaat ctaatgtatg gcatgg

46

<210> 321

<211> 46

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 321

cctctctatg ggcagtcggt gatgtgtgac ccaaaagatt cccacc

46

<210> 322

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 322

cctctctatg ggcagtcggt gatgcacagg cactaacttc ttcagccta

49

<210> 323

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 323

cctctctatg ggcagtcggt gatgccccag caaaacgcac tg

42

<210> 324

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 324

cctctctatg ggcagtcggt gatgccacac acagcgtctt ccg

43

<210> 325

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 325

cctctctatg ggcagtcggt gatgtcaaag ctgtatcccc attgccta

48

<210> 326

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 326

cctctctatg ggcagtcggt gatgagcaac gagacgttaa ccc

43

<210> 327

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 327

cctctctatg ggcagtcggt gatgttctgc cactggctta gctt

44

<210> 328

<211> 49

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 328

cctctctatg ggcagtcggt gatggtaagt gaatctctgt ctgtctcat

49

<210> 329

<211> 47

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 329

cctctctatg ggcagtcggt gatgcaggag gttaaatccc tcctcca

47

<210> 330

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 330

cctctctatg ggcagtcggt gatggtttcc cccatgcttt tctt

44

<210> 331

<211> 42

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 331

cctctctatg ggcagtcggt gatggaaggg ttggtttgga ag

42

<210> 332

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 332

cctctctatg ggcagtcggt gatgaggcat gagccactga gact

44

<210> 333

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 333

cctctctatg ggcagtcggt gatgccctag tgactgccgt ctg

43

<210> 334

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 334

cctctctatg ggcagtcggt gatggccaca gtcgtgtcat cttg

44

<210> 335

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 335

cctctctatg ggcagtcggt gatgtacaag gtgagcctgg gtct

44

<210> 336

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 336

cctctctatg ggcagtcggt gatggaaaga aagccccacc ctcg

44

<210> 337

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 337

cctctctatg ggcagtcggt gatgcaccct cgctctttta gtctc

45

<210> 338

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 338

cctctctatg ggcagtcggt gatgtcagag ggtgctgtct gtct

44

<210> 339

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 339

cctctctatg ggcagtcggt gatggttgcc caccctagtc attg

44

<210> 340

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 340

cctctctatg ggcagtcggt gatggcccaa tcattgatgc tttt

44

<210> 341

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 341

cctctctatg ggcagtcggt gatgggcttt cacaaggatg cagt

44

<210> 342

<211> 48

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 342

cctctctatg ggcagtcggt gatgtcctgc tctcacttag actttctc

48

<210> 343

<211> 58

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 343

cctctctatg ggcagtcggt gatgatggct tacatattta ttagataaaa tgtattcc

58

<210> 344

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 344

cctctctatg ggcagtcggt gatgtggccc cagtctctct tcta

44

<210> 345

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 345

cctctctatg ggcagtcggt gatgtgccag tgcctcaaga atgtc

45

<210> 346

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 346

cctctctatg ggcagtcggt gatgtccagc ttgggcccac

40

<210> 347

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 347

cctctctatg ggcagtcggt gatgtccagc ttgggcccac

40

<210> 348

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 348

cctctctatg ggcagtcggt gatggaggag aaggccaagt ggtc

44

<210> 349

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 349

cctctctatg ggcagtcggt gatggttgcc caccctagtc attg

44

<210> 350

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 350

cctctctatg ggcagtcggt gatggttgcc caccctagtc attg

44

<210> 351

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 351

cctctctatg ggcagtcggt gatggaggag aaggccaagt ggtc

44

<210> 352

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 352

cctctctatg ggcagtcggt gatggaggag aaggccaagt ggtc

44

<210> 353

<211> 40

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 353

cctctctatg ggcagtcggt gatgtccagc ttgggcccac

40

<210> 354

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 354

cctctctatg ggcagtcggt gatgccctag tgactgccgt ctg

43

<210> 355

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 355

cctctctatg ggcagtcggt gatgccctag tgactgccgt ctg

43

<210> 356

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 356

cctctctatg ggcagtcggt gatgggggag agggacacac agat

44

<210> 357

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 357

cctctctatg ggcagtcggt gatggcaccc caacacctac atct

44

<210> 358

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 358

cctctctatg ggcagtcggt gatggcaccc caacacctac atct

44

<210> 359

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 359

cctctctatg ggcagtcggt gatgcgtgtt ccccagagtg actt

44

<210> 360

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 360

cctctctatg ggcagtcggt gatgtcagag ggtgctgtct gtct

44

<210> 361

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 361

cctctctatg ggcagtcggt gatgtacaag gtgagcctgg gtct

44

<210> 362

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 362

cctctctatg ggcagtcggt gatgttctgc cactggctta gctt

44

<210> 363

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 363

cctctctatg ggcagtcggt gatgcacacc cacaccctca taca

44

<210> 364

<211> 44

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 364

cctctctatg ggcagtcggt gatgcacacc cacaccctca taca

44

<210> 365

<211> 45

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic primer"

<400> 365

cctctctatg ggcagtcggt gatgtgccag tgcctcaaga atgtc

45

<210> 366

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 366

ggtgagtgag tgtgtgcgtg tgg

23

<210> 367

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 367

gctgagtgag tgtatgcgtg tgg

23

<210> 368

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 368

tgtgggtgag tgtgtgcgtg agg

23

<210> 369

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 369

agagagtgag tgtgtgcatg agg

23

<210> 370

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 370

agtgagtgag tgtgtgtgtg ggg

23

<210> 371

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 371

ggtgagtgag tgtgtgtgtg agg

23

<210> 372

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 372

ggtgagtgag tgcgtgcggg tgg

23

<210> 373

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 373

agtgtgtgag tgtgtgcgtg tgg

23

<210> 374

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 374

tgtgagtaag tgtgtgtgtg tgg

23

<210> 375

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 375

ggtgagtgtg tgtgtgcatg tgg

23

<210> 376

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 376

gttgagtgaa tgtgtgcgtg agg

23

<210> 377

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 377

gtgagtgagt gtgtgtgtgt gag

23

<210> 378

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 378

agcgagtggg tgtgtgcgtg ggg

23

<210> 379

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 379

ggtgagtgag tgtgtgcgtg tgg

23

<210> 380

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 380

gctgagtgag tgtatgcgtg tgg

23

<210> 381

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 381

tgtgggtgag tgtgtgcgtg agg

23

<210> 382

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 382

agagagtgag tgtgtgcatg agg

23

<210> 383

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 383

agtgagtgag tgtgtgtgtg ggg

23

<210> 384

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 384

ggtgagtgag tgtgtgtgtg agg

23

<210> 385

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 385

ggtgagtgag tgcgtgcggg tgg

23

<210> 386

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 386

agtgtgtgag tgtgtgcgtg tgg

23

<210> 387

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 387

tgtgagtaag tgtgtgtgtg tgg

23

<210> 388

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 388

ggtgagtgtg tgtgtgcatg tgg

23

<210> 389

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 389

gttgagtgaa tgtgtgcgtg agg

23

<210> 390

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 390

gtgagtgagt gtgtgtgtgt gag

23

<210> 391

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 391

agcgagtggg tgtgtgcgtg ggg

23

<210> 392

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 392

ggtgagtgag tgtgtgcgtg tgg

23

<210> 393

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 393

gctgagtgag tgtatgcgtg tgg

23

<210> 394

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 394

tgtgggtgag tgtgtgcgtg agg

23

<210> 395

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 395

agagagtgag tgtgtgcatg agg

23

<210> 396

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 396

agtgagtgag tgtgtgtgtg ggg

23

<210> 397

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 397

ggtgagtgag tgtgtgtgtg agg

23

<210> 398

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 398

ggtgagtgag tgcgtgcggg tgg

23

<210> 399

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 399

agtgtgtgag tgtgtgcgtg tgg

23

<210> 400

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 400

tgtgagtaag tgtgtgtgtg tgg

23

<210> 401

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 401

ggtgagtgtg tgtgtgcatg tgg

23

<210> 402

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 402

gttgagtgaa tgtgtgcgtg agg

23

<210> 403

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 403

gtgagtgagt gtgtgtgtgt gag

23

<210> 404

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 404

agcgagtggg tgtgtgcgtg ggg

23

<210> 405

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 405

gagtccgagc agaagaagaa ggg

23

<210> 406

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 406

gagttagagc agaagaagaa agg

23

<210> 407

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 407

gagtctaagc agaagaagaa gag

23

<210> 408

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 408

aagtctgagc acaagaagaa tgg

23

<210> 409

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 409

gagtcctagc aggagaagaa gag

23

<210> 410

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 410

gagtccgagc agaagaagaa ggg

23

<210> 411

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 411

gagttagagc agaagaagaa agg

23

<210> 412

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 412

gagtctaagc agaagaagaa gag

23

<210> 413

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 413

aagtctgagc acaagaagaa tgg

23

<210> 414

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 414

gagtcctagc aggagaagaa gag

23

<210> 415

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 415

gagtccgagc agaagaagaa ggg

23

<210> 416

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 416

gagttagagc agaagaagaa agg

23

<210> 417

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 417

gagtctaagc agaagaagaa gag

23

<210> 418

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 418

aagtctgagc acaagaagaa tgg

23

<210> 419

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 419

gagtcctagc aggagaagaa gag

23

<210> 420

<211> 4203

<212> DNA

<213> Staphylococcus aureus

<400> 420

atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag

60

tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag

120

tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag

180

aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg

240

aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag

300

atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc

360

ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac

420

gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac

480

agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc

540

cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg

600

ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc

660

ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat

720

ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg

780

agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg

840

cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac

900

cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac

960

atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga

1020

tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct

1080

gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac

1140

ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac

1200

ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc

1260

ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg

1320

cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg

1380

accttccgca tcccctacta cgtgggccct ctggccagg gaaacagcag attcgcctgg

1440

atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag

1500

ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac

1560

gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg

1620

accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag

1680

aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg

1740

aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa

1800

gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag

1860

gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca

1920

ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac

1980

gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg

2040

aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag

2100

tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt

2160

aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt

2220

gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg

2280

gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc

2340

agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc

2400

gaagaggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc

2460

cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg

2520

gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag

2580

agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg

2640

ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg

2700

cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag

2760

agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc

2820

cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac

2880

gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc

2940

gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc

3000

cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg

3060

gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag

3120

agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac

3180

tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag

3240

acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg

3300

aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc

3360

ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag

3420

gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg

3480

gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg

3540

gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc

3600

aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc

3660

gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac

3720

gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag

3780

ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac

3840

tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac

3900

gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag

3960

caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc

4020

aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac

4080

gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag

4140

ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg gccaggcaaa aaagaaaaag

4200

taa

4203

<210> 421

<211> 4272

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic polynucleotide"

<400> 421

atggactata aggaccacga cggagactac aaggatcatg atattgatta caaagacgat

60

gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc

120

gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc

180

accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac

240

agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc

300

acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat

360

ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg

420

gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac

480

atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa

540

ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg

600

atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg

660

gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc

720

aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg

780

ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg

840

attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat

900

gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag

960

atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg

1020

ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg

1080

atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag

1140

cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc

1200

tacattgacg gcggagccag ccadgaagag ttctacaagt tcatcaagcc catcctggaa

1260

aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag

1320

cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc

1380

attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag

1440

aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga

1500

ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg

1560

gtggacaagg gcgcttccgc cccagcttc atcgagcgga tgaccaactt cgataagaac

1620

ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat

1680

aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc

1740

ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg

1800

aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc

1860

ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc

1920

aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg

1980

accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac

2040

ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg

2100

ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat

2160

ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc

2220

ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac

2280

gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg

2340

aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc

2400

gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg

2460

aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg

2520

gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat

2580

atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc

2640

gtgcctcaga gctttctgaa ggacgactcc atcgacaacg cggtgctgac cagaagcgac

2700

aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac

2760

tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc

2820

aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg

2880

gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact

2940

aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag

3000

ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac

3060

caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac

3120

cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg

3180

atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac

3240

atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct

3300

ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc

3360

accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag

3420

acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc

3480

agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat

3540

tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa

3600

gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt

3660

ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac

3720

tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag

3780

aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac

3840

tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag

3900

cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc

3960

ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc

4020

atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct

4080

gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag

4140

gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac

4200

ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa

4260

aagaaaaagt aa

4272

<210> 422

<211> 4269

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic polynucleotide"

<400> 422

atggactata aggaccacga cggagactac aaggatcatg atattgatta caaagacgat

60

gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc

120

gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc

180

accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac

240

agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc

300

acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat

360

ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg

420

gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac

480

atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa

540

ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg

600

atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg

660

gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc

720

aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg

780

ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg

840

attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat

900

gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag

960

atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg

1020

ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg

1080

atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag

1140

cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc

1200

tacattgacg gcggagccag ccadgaagag ttctacaagt tcatcaagcc catcctggaa

1260

aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag

1320

cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc

1380

attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag

1440

aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga

1500

ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg

1560

gtggacaagg gcgcttccgc cccagcttc atcgagcgga tgaccaactt cgataagaac

1620

ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat

1680

aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc

1740

ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg

1800

aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc

1860

ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc

1920

aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg

1980

accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac

2040

ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg

2100

ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat

2160

ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc

2220

ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac

2280

gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg

2340

aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc

2400

gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg

2460

aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg

2520

gaaaacaccc agctgcagaa cgaggccctg tacctgtact acctgcagaa tgggcgggat

2580

atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc

2640

gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac

2700

aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac

2760

tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc

2820

aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg

2880

gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact

2940

aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag

3000

ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac

3060

caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac

3120

cctgcgctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg

3180

atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac

3240

atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaaggcgcct

3300

ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc

3360

accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag

3420

acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc

3480

agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat

3540

tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa

3600

gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt

3660

ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac

3720

tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag

3780

aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac

3840

tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag

3900

cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc

3960

ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc

4020

atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct

4080

gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag

4140

gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac

4200

ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa

4260

aagaaaaag

4269

<210> 423

<211> 4272

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

synthetic polynucleotide"

<400> 423

atggactata aggaccacga cggagactac aaggatcatg atattgatta caaagacgat

60

gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc

120

gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc

180

accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac

240

agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc

300

acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat

360

ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg

420

gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac

480

atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa

540

ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg

600

atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg

660

gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc

720

aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg

780

ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg

840

attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat

900

gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag

960

atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg

1020

ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg

1080

atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag

1140

cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc

1200

tacattgacg gcggagccag ccadgaagag ttctacaagt tcatcaagcc catcctggaa

1260

aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag

1320

cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc

1380

attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag

1440

aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga

1500

ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg

1560

gtggacaagg gcgcttccgc cccagcttc atcgagcgga tgaccaactt cgataagaac

1620

ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat

1680

aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc

1740

ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg

1800

aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc

1860

ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc

1920

aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg

1980

accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac

2040

ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg

2100

ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat

2160

ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc

2220

ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac

2280

gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg

2340

aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc

2400

gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg

2460

aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg

2520

gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat

2580

atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc

2640

gtgcctcaga gctttctggc ggacgactcc atcgacaaca aggtgctgac cagaagcgac

2700

aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac

2760

tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc

2820

aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg

2880

gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact

2940

aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag

3000

ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac

3060

caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac

3120

cctgcgctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg

3180

atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac

3240

atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaaggcgcct

3300

ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc

3360

accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag

3420

acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc

3480

agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat

3540

tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa

3600

gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt

3660

ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac

3720

tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag

3780

aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac

3840

tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag

3900

cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc

3960

ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc

4020

atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct

4080

gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag

4140

gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac

4200

ctgtctcagc tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa

4260

aagaaaaagt aa

4272

<210> 424

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 424

gagtccgagc agaagaagaa ggg

23

<210> 425

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 425

gtcacctcca atgactaggg tgg

23

<210> 426

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 426

gacatcgatg tcctccccat tgg

23

<210> 427

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 427

gcctccccaa agcctggcca ggg

23

<210> 428

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 428

gccccgggct tcaagccctg tgg

23

<210> 429

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 429

ggcagagtgc tgcttgctgc tgg

23

<210> 430

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 430

gattcctggt gccagaaaca ggg

23

<210> 431

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 431

ggtgagtgag tgtgtgcgtg tgg

23

<210> 432

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 432

gctaaagagg gaatgggctt tgg

23

<210> 433

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 433

gtttgggagg tcagaaatag ggg

23

<210> 434

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 434

gagtccgagc agaagaagaa ggg

23

<210> 435

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 435

gtcacctcca atgactaggg tgg

23

<210> 436

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 436

gggcaaccac aaacccacga ggg

23

<210> 437

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 437

gcttgtccct ctgtcaatgg cgg

23

<210> 438

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 438

gcgccaccgg ttgatgtgat ggg

23

<210> 439

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 439

gacatcgatg tcctccccat tgg

23

<210> 440

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 440

gcctccccaa agcctggcca ggg

23

<210> 441

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 441

gccccgggct tcaagccctg tgg

23

<210> 442

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 442

ggcagagtgc tgcttgctgc tgg

23

<210> 443

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 443

ggtgagtgag tgtgtgcgtg tgg

23

<210> 444

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 444

gctaaagagg gaatgggctt tgg

23

<210> 445

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 445

gtttgggagg tcagaaatag ggg

23

<210> 446

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 446

gttggagcgg ggagaaggcc agg

23

<210> 447

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 447

gggtgggggg agtttgctcc tgg

23

<210> 448

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 448

gtagagtgag tgtgtgtgtg tgg

23

<210> 449

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 449

gaagaatgga cagaactctg agg

23

<210> 450

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 450

gattcctggt gccagaaaca ggg

23

<210> 451

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 451

gtgggtgagt gagtgcgtgc ggg

23

<210> 452

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 452

gaggctgggg tggaggtgtt ggg

23

<210> 453

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 453

ggtgagtgag tgtgtgtgtg agg

23

<210> 454

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 454

gtgtgtgtgt gagggtgtaa ggg

23

<210> 455

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 455

gggcagtttg ctcctggcac agg

23

<210> 456

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 456

ggagagaggc tcccatcacg ggg

23

<210> 457

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 457

gagaagagaa gtggggtggg ggg

23

<210> 458

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 458

gtgtgtctgt gtgggtgagt gagtgtgtgc gtgtggggtt gag

43

<210> 459

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 459

ctcaacccca cacgcacaca ctcactcacc cacacagaca cac

43

<210> 460

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 460

ggugagugag ugugugcgug

20

<210> 461

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (20)..(20)

<223> a, c, u, g, unknown or other

<400> 461

ggugagugag ugugugcgun

20

<210> 462

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (19)..(19)

<223> a, c, u, g, unknown or other

<400> 462

ggugagugag ugugugcgng

20

<210> 463

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (18)..(18)

<223> a, c, u, g, unknown or other

<400> 463

ggugagugag ugugugcnug

20

<210> 464

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (17)..(17)

<223> a, c, u, g, unknown or other

<400> 464

ggugagugag ugugugngug

20

<210> 465

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (16)..(16)

<223> a, c, u, g, unknown or other

<400> 465

ggugagugag uguguncgug

20

<210> 466

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (15)..(15)

<223> a, c, u, g, unknown or other

<400> 466

ggugagugag ugugngcgug

20

<210> 467

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (14)..(14)

<223> a, c, u, g, unknown or other

<400> 467

ggugagugag ugunugcgug

20

<210> 468

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (13)..(13)

<223> a, c, u, g, unknown or other

<400> 468

ggugagugag ugngugcgug

20

<210> 469

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (12)..(12)

<223> a, c, u, g, unknown or other

<400> 469

ggugagugag unugugcgug

20

<210> 470

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (11)..(11)

<223> a, c, u, g, unknown or other

<400> 470

ggugagugag ngugugcgug

20

<210> 471

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (10)..(10)

<223> a, c, u, g, unknown or other

<400> 471

ggugagugan ugugugcgug

20

<210> 472

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (9)..(9)

<223> a, c, u, g, unknown or other

<400> 472

ggugagugng ugugugcgug

20

<210> 473

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (8)..(8)

<223> a, c, u, g, unknown or other

<400> 473

ggugagunag ugugugcgug

20

<210> 474

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (7)..(7)

<223> a, c, u, g, unknown or other

<400> 474

ggugagngag ugugugcgug

20

<210> 475

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (6)..(6)

<223> a, c, u, g, unknown or other

<400> 475

gguganugag ugugugcgug

20

<210> 476

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (5)..(5)

<223> a, c, u, g, unknown or other

<400> 476

ggugngugag ugugugcgug

20

<210> 477

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (4)..(4)

<223> a, c, u, g, unknown or other

<400> 477

ggunagugag ugugugcgug

20

<210> 478

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (3)..(3)

<223> a, c, u, g, unknown or other

<400> 478

ggngngagugag ugugugcgug

20

<210> 479

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (2)..(2)

<223> a, c, u, g, unknown or other

<400> 479

gnugugugag ugugugcgug

20

<210> 480

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<220>

<221> modified_base

<222> (1)..(1)

<223> a, c, u, g, unknown or other

<400> 480

nggagugag ugugugcgug

20

<210> 481

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 481

ggugagugag ugugugcgug

20

<210> 482

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 482

ggugagugag ugugugcgug

20

<210> 483

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 483

ggugagugag ugugugcgug

20

<210> 484

<211> 20

<212> RNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 484

ggugagugag ugugugcgug

20

<210> 485

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 485

ggtgagtgag tgtgtgcgtg

20

<210> 486

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 486

gacgagtgag tgtgtgcgtg

20

<210> 487

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 487

ggcaagtgag tgtgtgcgtg

20

<210> 488

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 488

ggtaggtgag tgtgtgcgtg

20

<210> 489

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 489

ggtggatgag tgtgtgcgtg

20

<210> 490

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 490

ggtgaacgag tgtgtgcgtg

20

<210> 491

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 491

ggtgagcaag tgtgtgcgtg

20

<210> 492

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 492

ggtgagtagg tgtgtgcgtg

20

<210> 493

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 493

ggtgagtgga tgtgtgcgtg

20

<210> 494

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 494

ggtgagtgaa cgtgtgcgtg

20

<210> 495

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 495

ggtgagtgag catgtgcgtg

20

<210> 496

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 496

ggtgagtgag tacgtgcgtg

20

<210> 497

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 497

ggtgagtgag tgcatgcgtg

20

<210> 498

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 498

ggtgagtgag tgtacgcgtg

20

<210> 499

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 499

ggtgagtgag tgtgcacgtg

20

<210> 500

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 500

ggtgagtgag tgtgtatgtg

20

<210> 501

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 501

ggtgagtgag tgtgtgtatg

20

<210> 502

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 502

ggtgagtgag tgtgtgcacg

20

<210> 503

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 503

ggtgagtgag tgtgtgcgca

20

<210> 504

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 504

gagtccgagc agaagaagaa ggg

23

<210> 505

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 505

gagttagagc agaagaagaa agg

23

<210> 506

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 506

gagtctaagc agaagaagaa gag

23

<210> 507

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 507

aagtctgagc acaagaagaa tgg

23

<210> 508

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 508

gagtcctagc aggagaagaa gag

23

<210> 509

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 509

ggtgagtgag tgtgtgcgtg tgg

23

<210> 510

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 510

gctgagtgag tgtatgcgtg tgg

23

<210> 511

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 511

tgtgggtgag tgtgtgcgtg agg

23

<210> 512

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 512

agagagtgag tgtgtgcatg agg

23

<210> 513

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 513

ggtgagtgag tgtgtgtgtg agg

23

<210> 514

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 514

ggtgagtgag tgcgtgcggg tgg

23

<210> 515

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 515

agtgtgtgag tgtgtgcgtg tgg

23

<210> 516

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 516

tgtgagtaag tgtgtgtgtg tgg

23

<210> 517

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 517

tgtgagtgag tgtgtgtgtg tga

23

<210> 518

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 518

agcgagtggg tgtgtgcgtg ggg

23

<210> 519

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 519

cgtgagtgag tgtgtacctg ggg

23

<210> 520

<211> 1443

<212> PROTEIN

<213> Staphylococcus aureus

<400> 520

Ala Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val

1 5 10 15

Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe

20 25 30

Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile

35 40 45

Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu

50 55 60

Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys

65 70 75 80

Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser

85 90 95

Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys

100 105 110

His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr

115 120 125

His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp

130 135 140

Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His

145 150 155 160

Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro

165 170 175

Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr

180 185 190

Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala

195 200 205

Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn

210 215 220

Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn

225 230 235 240

Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe

245 250 255

Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp

260 265 270

Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp

275 280 285

Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp

290 295 300

Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser

305 310 315 320

Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys

325 330 335

Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe

340 345 350

Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser

355 360 365

Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp

370 375 380

Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg

385 390 395 400

Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu

405 410 415

Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe

420 425 430

Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile

435 440 445

Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp

450 455 460

Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu

465 470 475 480

Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr

485 490 495

Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser

500 505 510

Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys

515 520 525

Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln

530 535 540

Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr

545 550 555 560

Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp

565 570 575

Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly

580 585 590

Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp

595 600 605

Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr

610 615 620

Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala

625 630 635 640

His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr

645 650 655

Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp

660 665 670

Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe

675 680 685

Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe

690 695 700

Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu

705 710 715 720

His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly

725 730 735

Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly

740 745 750

Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln

755 760 765

Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile

770 775 780

Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro

785 790 795 800

Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu

805 810 815

Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg

820 825 830

Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys

835 840 845

Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg

850 855 860

Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys

865 870 875 880

Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys

885 890 895

Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp

900 905 910

Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr

915 920 925

Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp

930 935 940

Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser

945 950 955 960

Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Thr

965 970 975

Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe Ile

980 985 990

Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala Gln

995 1000 1005

Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys

1010 1015 1020

Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val

1025 1030 1035

Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile

1040 1045 1050

Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val

1055 1060 1065

Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe

1070 1075 1080

Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala

1085 1090 1095

Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe

1100 1105 1110

Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala

1115 1120 1125

Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu

1130 1135 1140

Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val

1145 1150 1155

Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr

1160 1165 1170

Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys

1175 1180 1185

Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro

1190 1195 1200

Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val

1205 1210 1215

Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys

1220 1225 1230

Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser

1235 1240 1245

Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys

1250 1255 1260

Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu

1265 1270 1275

Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly

1280 1285 1290

Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val

1295 1300 1305

Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser

1310 1315 1320

Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys

1325 1330 1335

His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys

1340 1345 1350

Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala

1355 1360 1365

Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn

1370 1375 1380

Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala

1385 1390 1395

Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser

1400 1405 1410

Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr

1415 1420 1425

Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp

1430 1435 1440

<210> 521

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 521

gagtccgagc agaagaagaa ggg

23

<210> 522

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 522

gagttagagc agaagaagaa agg

23

<210> 523

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 523

gagtctaagc agaagaagaa gag

23

<210> 524

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 524

gagtcctagc aggagaagaa tag

23

<210> 525

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 525

gaggccgagc agaagaagaa ggg

23

<210> 526

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 526

aagtctgagc acaagaagaa cgg

23

<210> 527

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 527

acgtctgagc agaagaagaa tgg

23

<210> 528

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 528

gggtgggggg agtttgctcc tgg

23

<210> 529

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 529

ggggagggtgg agtttgctcc tgg

23

<210> 530

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 530

gcgtgggggg tgtttgctcc cgg

23

<210> 531

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 531

cggggggaggg agtttgctcc tgg

23

<210> 532

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 532

tagtggaggg agcttgctcc tgg

23

<210> 533

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 533

ttggggggggc agtttgctcc tgg

23

<210> 534

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 534

ggtgagtgag tgtgtgcgtg tgg

23

<210> 535

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 535

ggtgagtgag tgtgtgtgtg agg

23

<210> 536

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 536

tgtgggtgag tgtgtgcgtg agg

23

<210> 537

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 537

gctgagtgag tgtatgcgtg tgg

23

<210> 538

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 538

ggtgagtgag tgcgtgcggg tgg

23

<210> 539

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 539

agtgtgtgag tgtgtgcgtg tgg

23

<210> 540

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 540

tgtgggtgag tgtgtgcgtg aga

23

<210> 541

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 541

agtgaatgag tgtgtgtgtg tgg

23

<210> 542

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 542

agcgagtgag tgtgtgtgtg ggg

23

<210> 543

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 543

tgtgagtaag tgtgtgtgtg tgg

23

<210> 544

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 544

agcgagtggg tgtgtgcgtg ggg

23

<210> 545

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 545

gtagagtgag tgtgtgtgtg tgg

23

<210> 546

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 546

actgtgtgag tgtgtgcgtg agg

23

<210> 547

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of artificial sequence:

Synthetic oligonucleotide"

<400> 547

tgagtgtgag tgtgtgcgtg ggg

23

<---

Claims (31)

1. An engineered Cas9 protein comprising at least one modification compared to a wild-type Cas9 protein, wherein said modification comprises an amino acid substitution with a neutral or negatively charged amino acid residue at position 13, 63, 415, 610, 775, 779, 799, 780, 810, 832, 848, 855, 861, 862, 866, 961, 968, 974, 976, 1000, 1003, 1014, 1047, 1060, 1107, 1108, 1109, 1114, 1129, 1240, 1289, 1296, 1297, 1300, 1311 or 1325 according to the amino acid position numbering of Cas9 from Streptococcus pyogenes (SpCas9), and the said engineered Cas9 protein has reduced off-target site activity compared to the wild-type Cas9 protein. 2. The engineered Cas9 protein of claim 1, wherein the modification comprises a substitution of Lys, His, Arg, Glu, Asp, Ser, Gly or Thr. 3. The engineered Cas9 protein of claim 1, wherein the modification comprises a substitution with Gly, Ala, Ile, Glu or Asp. 4. The engineered Cas9 protein of claim 1, wherein the modification comprises an amino acid substitution in the binding groove between the RuvC and HNH domains. 5. The engineered Cas9 protein of claim 1, wherein the modification comprises a mutation in the RuvCI, RuvCIII, RuvCIII or HNH domain. 6. The engineered Cas9 protein of claim 1, wherein the modification at position 63, 415, 775, 779, 780, 810, 832, 848, 855, 861, 862, 866, 961, 968, 974, 976, 1000, 1003, 1014, 1047, 1060, 1107, 1108, 1109, 1114, 1129, 1240, 1289, 1296, 1297, 1300, 1311 or 1325 comprises alanine substitution. 7. The engineered Cas9 protein of claim 1, wherein the modification at position 13 comprises an isoleucine substitution, the modification at position 610 comprises a glycine substitution, the modification at position 799 comprises a leucine substitution, and/or wherein the modification at position 1129 comprises a glutamic acid substitution. 8. The engineered Cas9 protein according to claim 1, characterized in that the modification comprises K775A, E779L, R780A, K810A, R832A, K848A, K855A, K862A, K961A, K968A, K974A, R976A, K1000A, K1014A, K1047A, R1060A, K1003A, S1109A, H1240A, K1289A, K1296A, H1297A, K1300A, H1311A or K1325A. 9. The engineered Cas9 protein of claim 1, wherein the modification comprises R780A and K810A, or R780A and K855A, or R780A and R976A, or K848A and R976A, or K855A and R976A, or R780A and K848A, or K810A and K848A, or K848A and K855A, or K810A and K855A, or K1003A and R1060A, or R780A and K1003A, or K810A and R1003A, or K848A and K1003A, or R780A and R1060A, or K810A and R1060A, or K848A and R1060A, or R780A and R1114A or K848A and R1114A or R63A and K855A or H415A and R780A or H415A and K848A or K848A and E1108A or K810A and K1003A or R780A and R1060A or K810A and R1060A or K848A and R1060A. 10. The engineered Cas9 protein of claim 1, wherein the modification comprises R780A, K1003A, and R1060A, or K810A, K1003A, and R1060A, or K848A, K1003A, and R1060A, or K855A, K1003A, and R1060A, or R63A, K848A, and R1060A, or T13I, R63A, and K810A. 11. The engineered Cas9 protein of claim 1, wherein the modification comprises R63A, E610G, K855A and R1060A; or R63A, K855A, R1060A and E610G. 12. The engineered Cas9 protein of claim 1, wherein the engineered Cas9 protein is a mutant of a Cas9 protein from a genus comprising Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, or Corynebacter. 13. The engineered Cas9 protein of claim 1, wherein the engineered Cas9 protein comprises one or more nuclear localization signal (NLS) domains. 14. The engineered Cas9 protein of claim 1, wherein the engineered Cas9 protein comprises at least two or more NLSs. 15. The engineered Cas9 protein of claim 1, wherein the engineered Cas9 protein comprises one or more heterologous functional domains. 16. The engineered Cas9 protein of claim 15, wherein one or more heterologous functional domains comprise one or more transcriptional activation domains. 17. The engineered Cas9 protein of claim 16, wherein the transcriptional activation domain comprises VP64. 18. The engineered Cas9 protein of claim 15, wherein one or more heterologous functional domains comprise one or more transcriptional repression domains. 19. The engineered Cas9 protein of claim 18, wherein the transcriptional repression domain comprises a KRAB domain or a SID domain. 20. The engineered Cas9 protein of claim 15, wherein one or more heterologous functional domains comprise one or more nuclease domains. 21. The engineered Cas9 protein of claim 20, wherein the nuclease domain comprises Fok1. 22. The engineered Cas9 protein of claim 15, wherein the one or more heterologous functional domains have one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor 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. 23. A nucleic acid molecule encoding an engineered Cas9 protein according to claim 1, wherein said nucleic acid molecule comprises a nucleotide sequence encoding an engineered Cas9 protein according to claim 1, wherein the nucleotide sequence is codon optimized for expression in a eukaryote. 24. A composition for modifying an organism by manipulating a target sequence in a genomic locus of interest, comprising the engineered Cas9 protein of claim 1 and one or more guide polynucleotides comprising a guide sequence, a paired tracr sequence and a tracr sequence, wherein said modification does not include modification of the genetic integrity of the human germline and the use of human embryos for industrial and commercial purposes. 25. A CRISPR-Cas9 system for modifying an organism by manipulating a target sequence at a genomic locus of interest, comprising an engineered Cas9 protein according to claim 1 in complex with a guide polynucleotide comprising a guide sequence, a tracr paired sequence and a tracr sequence, wherein said modification does not include modification of the genetic integrity of the human germline and the use of human embryos for industrial and commercial purposes. 26. A vector system for delivering a CRISPR-Cas9 system according to claim 25, comprising one or more vectors, wherein the one or more vectors comprise: a) a first regulatory element operably linked to a nucleotide sequence encoding the engineered Cas9 protein of claim 1; and b) a second regulatory element operably linked to one or more nucleotide sequences encoding one or more nucleic acid molecules comprising a targeting polynucleotide comprising a targeting sequence, a tracr sequence and a tracr mate sequence, where components (a) and (b) are located on the same or different vectors. 27. An isolated eukaryotic cell for obtaining a cell containing a modified target locus, containing the vector system according to paragraph 26. 28. A method for modifying a target locus of interest, comprising delivering to said locus an engineered Cas9 protein according to claim 1 and one or more targeting polynucleotides, wherein the engineered Cas9 protein forms a complex with the one or more targeting polynucleotides and upon binding of the complex to the target locus of interest, the Cas9 protein induces modification of the target locus of interest, wherein said modification does not include modification of the genetic integrity of the human germline and the use of human embryos for industrial and commercial purposes. 29. An isolated host cell for producing the engineered Cas9 protein according to claim 1, wherein said host cell comprises a nucleic acid molecule according to claim 23 and a regulatory element operably linked to the nucleotide sequence of this nucleic acid molecule according to claim 23.