WO2023138617A1 - Engineered casx nuclease, effector protein and use thereof - Google Patents

Abstract

Provided is an engineered CasX nuclease. The CasX nuclease relates to substituting positively charged amino acids, which are specifically: T26R, K610R, K640, S759R, and K808R, for one or more amino acids interacting with PAM in a wild-type PlmCasX nuclease (SEQ ID NO: 1).

Description

Engineered CasX nucleases, effector proteins and uses thereof technical field

This application belongs to the field of biotechnology. More specifically, the present application relates to CasX nucleases, effector proteins, and uses thereof having enhanced catalytic activity (eg, gene editing activity).

Background technique

Genome editing is an important and useful technique in genome research. Several systems are available for genome editing, including clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems, transcription activator-like effector nuclease (TALEN) systems, and zinc finger nuclease (ZFN) systems.

The CRISPR-Cas system is an efficient and cost-effective genome editing technology that can be widely applied in a range of eukaryotes from yeast and plants to zebrafish and humans (see review: Van der Oost 2013, Science 339:768-770, and Charpentier and Doudna, 2013, Nature 495:50-51). The CRISPR-Cas system provides adaptive immunity in archaea and bacteria by combining CasX effector proteins and CRISPR RNA (crRNA). To date, two classes (classes 1 and 2) of CRISPR-Cas systems including six (types I–VI) have been characterized based on the system's outstanding functional and evolutionary modularity. In the second category of CRISPR-Cas systems, type II Cas9 system and type V-A/B/E/J Cas12a/Cas12b/Cas12e/Cas12j system have been exploited for genome editing and offer broad prospects for biomedical research.

However, current CRISPR-Cas systems have several limitations, including limited gene editing efficiency. Therefore, improved methods and systems are needed for efficient genome editing across multiple loci.

Contents of the invention

The application provides the following technical solutions:

1. An engineered CasX nuclease; It comprises one or more mutations based on the reference CasX nuclease, the mutation is: the amino acid that interacts with the nucleic acid in the reference CasX nuclease is replaced with a positively charged amino acid; the reference CasX nuclease is a natural wild-type CasX nuclease. Preferably, the nucleic acid is a PAM.

2. The engineered CasX nuclease as described in item 1, wherein, the CasX nuclease is selected from Any one of the following group: PlmCasX, DpbCasX.

3. The engineered CasX nuclease as described in item 2, wherein, the one or more amino acids interacting with the nucleic acid are amino acids within 9 angstroms in the three-dimensional structure, especially one or more amino acids at the following positions: 26, 27, 29, 105, 195, 198, 204, 222, 230, 512, 564, 565, 610, 640; wherein, the amino acid position numbering is as shown in SEQ ID NO.1 define.

4. The engineered CasX nuclease as described in item 3, wherein the positively charged amino acid is R or K; preferably the positively charged amino acid is R.

5. The engineered CasX nuclease as described in item 4, wherein, the replacement of one or more amino acids interacting with nucleic acid in the reference CasX nuclease with a positively charged amino acid refers to one or more of the following replacements: T26R, K610R, K640, S759R, K808R; 640R; (4) S759R; (5) K808R; (6) T26R and K610R; (7) T26R and K610R and K808R.

6. An engineered CasX nuclease comprising an amino acid sequence as shown in any one of SEQ ID NO.2～8 or having at least 95% or more identity with the amino acid sequence shown in any one of SEQ ID NO.2～8.

7. An engineered CasX effector protein, comprising the engineered CasX nuclease or a functional derivative thereof according to any one of items 1-6.

8. The engineered CasX effector protein as described in item 7, wherein said effector protein can induce double-strand breaks or single-strand breaks in DNA molecules.

9. The engineered CasX effector protein as described in item 7, wherein the functional derivative of the engineered CasX nuclease is an enzyme inactive mutant.

10. The engineered CasX effector protein according to any one of items 7 to 9, which further comprises a functional domain fused with the engineered CasX nuclease.

11. The engineered CasX effector protein as described in item 10, wherein said functional domain is selected from the group consisting of translation initiation domain, transcriptional repression domain, transactivation domain, epigenetic modification domain, nucleobase editing domain, reverse transcriptase domain, reporter molecular domain and nuclease domain.

12. The engineered CasX effector protein according to any one of items 7 to 11, comprising a first polypeptide and a second polypeptide, the first polypeptide comprising amino acid residues 1 to X of the N-terminal part of the engineered CasX nuclease described in any one of items 1-6, and the second polypeptide comprising the engineered CasX nuclease described in any one of items 1-6 Amino acid residue X+1 of the CasX nuclease of UL to the C-terminus of the CasX nuclease, wherein the first polypeptide and the second polypeptide are capable of associating with each other in the presence of a guide RNA comprising a guide sequence to form a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) complex specifically binding to a target nucleic acid comprising a target sequence complementary to the guide sequence.

13. An engineered CRISPR-CasX system comprising:

(a) the engineered CasX effector protein of any one of items 7-12; and

(b) a guide RNA comprising a guide sequence complementary to a target sequence, or one or more nucleic acids encoding said guide RNA,

Wherein the engineered CasX effector protein and the guide RNA are capable of forming a CRISPR complex that specifically binds to a target nucleic acid comprising the target sequence and induces modification of the target nucleic acid.

14. A method for detecting target nucleic acid in a sample, comprising:

(a) contacting the sample with the engineered CRISPR-CasX system of item 13 and a tagged detection nucleic acid that is single-stranded and does not hybridize to the guide sequence of the guide RNA; and

(b) measuring a detectable signal generated by cleavage of the tagged detection nucleic acid by the engineered CasX effector protein, thereby detecting the target nucleic acid.

15. Use of the engineered CRISPR-CasX system as described in item 13 in the preparation of a drug for treating a disease or a disease associated with a target nucleic acid in an individual's cells; preferably, the disease or disease is selected from the group consisting of cancer, cardiovascular disease, hereditary disease, autoimmune disease, metabolic disease, neurodegenerative disease, eye disease, bacterial infection and viral infection.

16. A method of modifying a target nucleic acid comprising a target sequence, comprising contacting said target nucleic acid with the engineered CRISPR-CasX system of item 13.

17. A kit comprising: one or more AAV vectors encoding any one of the engineered CasX nuclease according to any one of items 1-6, the engineered CasX effector protein according to items 7-12 and the engineered CRISPR-CasX system according to item 13.

Beneficial effects obtained by the technical solution of the present application

The engineered CasX nuclease and its effector proteins in this application have higher activity, such as catalytic efficiency of cutting nucleic acid substrates and gene editing efficiency in cells. The engineered Plm CasX nuclease in this application has superior gene editing efficiency in mammalian cells (such as human cells) than existing conventional Cas gene editing tools; the gene editing efficiency in human cells has reached about 80%. At the same time, the engineered CasX nuclease and its effector protein of the present application also have the following advantages: small protein, crRNA The components are simple, the PAM sequence is simple, and the protein itself can process the precursor crRNA. These advantages make the highly efficient engineered CasX nuclease and its effector protein of the present application very suitable for gene editing or gene regulation in vivo.

Description of drawings

Figure 1: Comparison of gene editing efficiencies of 14 engineered CRISPR-PlmCasXs with single point mutations at two loci (AAVS1-2 and CCR5-2) in human cells (with each other and with wild-type PlmCasX enzymes).

Figure 2: In human cells, at four gene loci (AAVS1-2, AAVS1-7, CCR5-2 and CD34-1), the gene editing efficiency comparison of three kinds of engineered CRISPR-PlmCasX with single point mutation and two point mutations (compared with each other and compared with wild-type PlmCasX enzyme).

Figure 3: Comparison of gene editing efficiencies of 16 engineered CRISPR-PlmCasX with single point mutations at two loci (AAVS1-2 and CCR5-2) in human cells (compared to each other and to wild-type PlmCasX enzymes).

Figure 4: In human cells, at four gene loci (AAVS1-2, AAVS1-7, CCR5-2 and CD34-1), the gene editing efficiency comparison of three kinds of engineered CRISPR-PlmCasX with single point mutation and multiple point mutation (compared with each other and compared with wild-type PlmCasX enzyme).

Detailed ways

It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art should understand that they may use different terms to refer to the same component. The specification and claims do not use differences in nouns as a way of distinguishing components, but use differences in functions of components as a criterion for distinguishing. "Includes" or "comprises" mentioned throughout the specification and claims is an open term, so it should be interpreted as "including but not limited to". The subsequent description in the specification is a preferred implementation mode for implementing the present invention, but the description is for the purpose of the general principles of the specification, and is not intended to limit the scope of the present invention. The scope of protection of the present invention should be defined by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

the term

As used herein, "effector protein" refers to a protein having an activity such as site-specific binding activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity, single-strand RNA cleavage activity or transcription regulation activity. As used herein, "guide RNA" and "gRNA" are used interchangeably herein to refer to an RNA capable of forming a complex with a CasX effector protein and a target nucleic acid (eg, double-stranded DNA). CasX This paper also considers CasX, an array of precursor guide RNAs that can be processed into multiple crRNAs. "crRNA" or "CRISPR RNA" comprises a guide sequence with sufficient complementarity to the target sequence of a target nucleic acid (e.g., double-stranded DNA) that directs sequence-specific binding of the CRISPR complex to the target nucleic acid.

As used herein, the terms "nucleic acid," "polynucleotide," and "nucleotide sequence" are used interchangeably to refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogs thereof. "Oligonucleotide" and "oligonucleotide" are used interchangeably to refer to short polynucleotides having no more than about 50 nucleotides.

As used herein, "complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid through conventional Watson-Crick base pairing. Percent complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (i.e., Watson-Crick base pairing) with a second nucleic acid (e.g., 5, 6, 7, 8, 9, 10 out of 10, about 50%, 60%, 70%, 80%, 90% and 100% complementary, respectively). "Perfectly complementary" means that all contiguous residues of a nucleic acid sequence form hydrogen bonds with the same number of contiguous residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100 over a region of about 40, 50, 60, 70, 80, 100, 150, 200, 250 or more nucleotides, or to two nucleic acids that hybridize under stringent conditions.

As used herein, "stringent conditions" for hybridization refers to conditions under which a nucleic acid having complementarity to a target sequence primarily hybridizes to the target sequence and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on many factors. In general, the longer the sequence, the higher the temperature at which the sequence will specifically hybridize 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, Chapter 2 "Overview of principles of hybridization and the strategy of nuclear acid probe assay," Else vier, N, Y.

"Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized by hydrogen bonding between the bases of the nucleotide residues. Hydrogen bonding can occur through Watson-Crick base pairing, Hoogstein bonding, or in any other sequence-specific manner. A sequence that is capable of hybridizing to a given sequence is called the "complement" of that given sequence.

"Percent (%) sequence identity" for a nucleic acid sequence is defined as the percentage of nucleotides in a candidate sequence that are identical to nucleotides in a specific nucleic acid sequence after aligning the sequences by allowing gaps, if necessary, to achieve the maximum percent sequence identity. "Percent sequence identity (%)" for a peptide, polypeptide or protein sequence is the percentage of amino acid residues in a candidate sequence that are identical to those in a particular peptide or amino acid sequence after aligning the sequences by allowing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN ^™ (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

The terms "polypeptide" and "peptide" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. A protein can have one or more polypeptides. The term also encompasses amino acid polymers that have been modified; eg, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (such as conjugation with a labeling component).

As used herein, "variant" is interpreted as a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties. A typical variant of a polynucleotide differs from the nucleic acid sequence of another reference polynucleotide. Changes in the nucleic acid sequence of a variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as described below. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Usually, the differences are limited such that the sequences of the reference polypeptide and the variant are very similar overall and identical in many regions. The amino acid sequence of a variant and reference polypeptide may differ by any combination of one or more substitutions, additions, deletions. The substituted or inserted amino acid residues may or may not be those encoded by the genetic code. Variants of a polynucleotide or polypeptide may be naturally occurring (such as allelic variants), or may be variants that are not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides can be prepared by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those skilled in the art.

As used herein, the term "wild-type" has the meaning commonly understood by those skilled in the art, meaning a typical form of an organism, strain, gene or characteristic that distinguishes it from a mutant or variant as it exists in nature. It can be isolated from resources in nature and has not been deliberately modified.

As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably to refer to the intervention of man. These terms, when used to describe a nucleic acid molecule or polypeptide, 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 or occurs in nature.

As used herein, the term "orthologue/ortholog" has the meaning commonly understood by those of ordinary skill in the art. As a further guidance, an "ortholog" of a protein as referred to herein refers to a protein belonging to a different species that performs the same or a similar function as the protein that is its ortholog.

As used herein, the term "identity" is used to denote a sequence match between two polypeptides or between two nucleic acids. When a position in two compared sequences is occupied by the same base or subunit of an amino acid monomer (for example, a position in each of two DNA molecules is occupied by an adenine, or a position in each of two polypeptides is occupied by a lysine), then each molecule is identical at that position. The "percent identity" between these two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions being compared x 100. For example, two sequences are 60% identical if 6 out of 10 positions of the two sequences match. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of a total of 6 positions match). Typically, such comparisons are made when two sequences are aligned to yield maximum identity. Such alignment can be achieved, for example, by the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by a computer program such as the Align program (DNAstar, Inc.). The PAM 120 weighted residue table can also be used, integrated into the ALIGN program (version 2.0) using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)). A gap length penalty of 12 and a gap penalty of 4 are used to determine the percent identity between two amino acid sequences. In addition, the Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) algorithm integrated into the GAP program of the GCG software package (available from www.gcg.com) can be used to determine the distance between two amino acid sequences using the Blossum 62 matrix or the PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6. percent identity.

"Cell" as used herein is understood not only to refer to a specific single cell, but also to the progeny or potential progeny of that cell. Because certain modifications may have occurred in the progeny, due to mutations or environmental influences, such progeny may in fact differ from the parental cells and still be included within the scope of the term herein.

As used herein, the terms "transduction" and "transfection" include methods known in the art to introduce DNA into cells to express a protein or molecule of interest using an infectious agent such as a virus or otherwise. In addition to viruses or virus-like reagents, there are chemical-based transfection methods such as the use of calcium phosphate, dendrimers non-chemical methods such as electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, plasmid delivery or transposons; particle-based methods such as the use of gene guns, magnetofection or magnet-assisted transfection, particle bombardment; and hybridization methods such as nucleofection ).

As used herein, the term "transfected", "transformed" or "transduced" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected", "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid.

The term "in vivo" refers to the organism from which cells are obtained. "Ex vivo" or "in vitro" means outside the organism from which cells are obtained.

As used herein, "treatment/treating" is a method used to obtain beneficial or desired results, including clinical results. For the purposes of this invention, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: alleviation of one or more symptoms caused by the disease, reduction of the extent of the disease, stabilization of the disease (e.g., prevention or delay of progression of the disease), prevention or delay of spread of the disease (e.g., metastasis), prevention or delay of recurrence of the disease, reduction of the rate of recurrence of the disease, delay or slowing of the progression of the disease, amelioration of the disease state, provision of remission (partial or total) of the disease, reduction in the dose of one or more other drugs required to treat the disease, delay of the progression of the disease, improvement of life quality, and/or prolong survival. "Treatment" also includes reducing the pathological consequences of a disorder, condition or disease. The methods of the invention contemplate any one or more of these aspects of treatment.

As used herein, the term "effective amount" refers to an amount of a compound or composition sufficient to treat a particular disorder, condition or disease, eg, ameliorate, alleviate, lessen and/or delay one or more symptoms thereof. As understood in the art, an "effective amount" may be administered in one or more doses, ie, a single dose or multiple doses may be required to achieve the desired therapeutic endpoint.

"Subject," "individual," or "patient" are used interchangeably herein for purposes of treatment and refer to any animal classified as a mammal, including humans, livestock, and farm animals, as well as zoo, farm, or pet animals such as dogs, horses, cats, cows, etc. In some embodiments, the individual is a human individual.

It is to be understood that embodiments of the invention described herein include "consisting of" and/or "consisting essentially of" embodiments. Reference herein to "about" a value or parameter includes (and describes) variations for that value or parameter itself. For example, a description referring to "about X" includes description of "X".

As used herein, a reference to a "not" value or parameter generally means and describes an "except" value or parameter. For example, the method is not used to treat type X cancer, meaning that the method is used to treat cancer other than type X.

As used herein, the term "about X-Y" has the same meaning as "about X to about Y".

As used herein and in the appended claims, the singular forms "a/an" and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any optional elements. This statement is therefore intended to serve as an antecedent basis for the use of exclusive terms such as "only," "only," etc., or limitations on the use of "no" in conjunction with the recitation of claim elements.

As used herein, the term "and/or" in words such as "A and/or B" is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, as used herein, the term "and/or" in words such as "A, B and/or C" is intended to include each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

The application provides engineered CasX nuclease in a first aspect

In some embodiments, an engineered CasX nuclease is provided; it comprises one or more mutations based on the reference CasX nuclease, the mutation is: one or more amino acids in the reference CasX nuclease that interact with nucleic acid are replaced with positively charged amino acids; the reference CasX nuclease is a natural wild-type CasX nuclease. Preferably, the natural wild-type CasX nuclease is selected from any one of the following groups: PlmCasX, DpbCasX.

The present application provides methods for engineering enzymes by introducing amino acid mutations based on the engineering principles described above, which lead to increased enzyme activity in vitro and in vivo. The engineered CasX nuclease contains one or more specific mutations as described below. In some embodiments, any one or more of the mutations described herein can be combined with existing CasX mutations to provide an engineered CasX nuclease with higher activity.

In a specific embodiment, an engineered CasX nuclease is provided; the engineered CasX nuclease comprises a mutation that replaces one or more amino acids interacting with nucleic acid in the reference CasX nuclease with a positively charged amino acid. Wherein, the one or more amino acids interacting with the nucleic acid are amino acids within 9 angstroms in the three-dimensional structure, especially one or more amino acids at the following positions: 26, 27, 29, 105, 195, 198, 204, 222, 230, 512, 564, 565, 610, 640; wherein, the amino acid position numbers are as defined in SEQ ID NO.1.

In a specific embodiment, the positively charged amino acid is R or K; preferably positively charged Amino acid is R.

In a specific embodiment, the replacement of one or more amino acids interacting with nucleic acid in the reference CasX nuclease with a positively charged amino acid refers to one or more of the following replacements: T26R, K610R, K640, S759R, K808R; preferably, the engineered CasX nuclease comprises any mutation or combination of mutations in the following group: (1) T26R; (2) K610R; (3) K640R; (4) S75 9R; (5) K808R; (6) T26R and K610R; (7) T26R and K610R and K808R; wherein, the amino acid position numbering is as defined in SEQ ID NO.1.

In yet another specific embodiment, the engineered CasX nuclease of the amino acid sequence shown in any one of SEQ ID NO.2～8 is provided.

In the context of this specification, the meaning of T26R is; in the cited amino acid sequence, the No. 26 amino acid T (threonine) is replaced by R (arginine); here, common amino acids and their three-letter and single-letter abbreviations are listed as follows:
Alanine AlaA; Arginine Arg R;
Aspartate Asp D; Cysteine Cys C;
Glutamine Gln Q; Glutamate Glu E;
Histidine His H; Isoleucine Ile I;
Glycine Gly G; Asparagine AsnN;
Leucine Leu L; Lysine Lys K;
Methionine MetM; Phenylalanine Phe F;
Proline Pro P; Serine Ser S;
Threonine ThrT; Tryptophan Trp W;
Tyrosine TyrY; Valine ValV.

As used herein, "the amino acid is at position X, wherein said amino acid position numbering is as defined in SEQ ID NO.1" means: the amino acid residue is located at a certain position of the reference enzyme CasX, which is equivalent to the position X of SEQ ID NO:1, and the amino acid sequence of the reference enzyme CasX is aligned with the amino acid sequence of SEQ ID NO:1 based on sequence homology. For example, Figure 6 shows the homology alignment of the amino acid sequences of CasX2 (SEQ ID NO.1) and CasX1 (SEQ ID NO.13). Those skilled in the art can use software commonly used in the art, such as Clustal Omega, to compare and align the amino acid sequence of any reference CasX nuclease with SEQ ID NO.1 for sequence identity, and then obtain the amino acid position in the reference CasX nuclease corresponding to the amino acid position defined based on SEQ ID NO.1 described in this application.

In some embodiments, the reference PlmCasX (Planctomycetes CasX) nuclease is native PlmCasX. In some embodiments, the reference PlmCasX nuclease is an engineered PlmCasX nuclease. CasX is a new type of CRISPR-Cas gene editing system excavated from metagenomics, which was first reported in 2017 (article: New CRISPR–Cas systems from uncultivated microbes). According to the latest CRISPR system classification criteria, CasX belongs to the V-E subclass. In 2019, a study (article: CasX enzymes comprise a distinct family of RNA-guided genome editors) reported that CasX (DpbCasX) from Deltaproteobacteria and CasX (PlmCasX) from Planctomycetes can perform gene editing in mammalian cells. However, this study showed that CasX was inefficient in mammalian cells, and the article did not explore the editing efficiency of PlmCasX at endogenous genomic loci.

In some embodiments, the engineered CasX of the present application is an endonuclease, which binds to a specific site of a target sequence and cuts under the guidance of a guide RNA, and has DNA and RNA endonuclease activity. In some embodiments, the CasX is capable of autonomous crRNA biogenesis by processing a precursor crRNA array. Autonomous pre-crRNA processing facilitates CasX delivery, enabling double-nicking applications, as two separate genomic sites can be targeted by a single crRNA transcript. The CasX protein then processes the CRISPR array into two homologous crRNAs, forming a paired nicking complex. In some embodiments, the guide RNA comprises a precursor crRNA expressed by a CRISPR array consisting of target sequences interleaved with unprocessed DR sequences, repeated by intrinsic precursor crRNA processing of the effector protein to enable simultaneous targeting of one, two or more sites. CasX nucleases from a variety of organisms can be used as the reference CasX nucleases to provide the engineered CasX nucleases and effector proteins of the present application. In some embodiments, the CasX reference CasX nuclease has enzymatic activity. In some embodiments, the reference CasX is a nuclease, ie, cleaves both strands of a target duplex nucleic acid (eg, duplex DNA). In some embodiments, the reference CasX is a nickase, ie, cleaves a single strand of a target duplex nucleic acid (eg, duplex DNA). In some embodiments, the CasX reference CasX nuclease is enzymatically inactive. Orthologs having a certain sequence identity (such as at least about 60%, 70%, 80%, 85%, 90%, 95%, 98% or more) with CasX or its functional derivatives can be used as the basis for designing the engineered CasX nuclease or effector protein of the present application.

In some embodiments, the engineered CasX nuclease is based on a functional variant of a naturally occurring CasX nuclease. In some embodiments, the functional variant has one or more mutations, such as amino acid substitutions, insertions, and deletions. For example, the functional variant may comprise any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions compared to a wild-type naturally occurring CasX nuclease. In some embodiments, the one or more substitutions are conservative substitutions. In some embodiments, the functional variant has all domains of a naturally occurring CasX nuclease. In some embodiments, the functional variant does not possess one or more domains of a naturally occurring CasX nuclease.

The present application provides engineered CasX effector proteins in a second aspect.

In a specific embodiment, the aforementioned engineered CasX nuclease or a functional derivative thereof is provided.

In a specific embodiment, the CasX nuclease is selected from any one of the following groups: PlmCasX, DpbCasX.

In yet another embodiment there is provided an engineered CasX effector protein, wherein the effector protein is capable of inducing double-strand breaks or single-strand breaks in DNA molecules.

In yet another specific embodiment, an engineered CasX effector protein is provided, wherein the functional derivative of the engineered CasX nuclease is an enzyme inactive mutant.

In yet another specific embodiment, an engineered CasX effector protein is provided, which further comprises a functional domain fused with the engineered CasX nuclease.

An engineered CasX effector protein is provided in a specific embodiment, wherein the functional domain is selected from the group consisting of a translation initiation domain, a transcriptional repression domain, a transactivation domain, an epigenetic modification domain, a nucleobase editing domain, a reverse transcriptase domain, a reporter domain and a nuclease domain.

In yet another specific embodiment, an engineered CasX effector protein is provided, comprising a first polypeptide and a second polypeptide, the first polypeptide comprising amino acid residues 1 to X of the N-terminal part of the aforementioned engineered CasX nuclease, and the second polypeptide comprising amino acid residues X+1 of the aforementioned engineered CasX nuclease to the C-terminal of the CasX nuclease, wherein the first polypeptide and the second polypeptide can associate with each other in the presence of a guide RNA comprising a guide sequence to form a clustered regularly interspaced short palindromic repeat sequence (CRISPR) that specifically binds to a target nucleic acid ) complex, the target nucleic acid comprising a target sequence complementary to the guide sequence.

In a specific embodiment, an engineered CasX effector protein based on any of the engineered CasX nucleases described herein is provided. In some embodiments, the engineered CasX Effector proteins have enzymatic activity. In some embodiments, the engineered CasX effector protein is a nuclease that cleaves both strands of a target duplex nucleic acid (eg, duplex DNA). In some embodiments, the engineered CasX effector protein is a nickase, ie, cleaves a single strand of a target duplex nucleic acid (eg, duplex DNA). In some embodiments, the engineered CasX effector protein comprises an enzyme-inactive mutant of the engineered CasX nuclease. Mutations of one or more amino acid residues in the active site of the CasX nuclease result in an inactive CasX. In some embodiments, the engineered CasX enzymes provided herein can be modified to have reduced nuclease activity, e.g., the nuclease is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% inactive compared to a wild-type CasX enzyme. The nuclease activity can be reduced by several methods, for example, introducing mutations into the nucleic acid interaction domain of the nuclease or CasX enzyme. In some embodiments, catalytic residues for nuclease activity are identified, and these amino acid residues can be replaced with different amino acid residues (eg, glycine or alanine) to reduce the nuclease activity.

The engineered CasX nuclease has increased activity compared to the reference CasX nuclease. In some embodiments, the activity is target DNA binding activity. In some embodiments, the activity is a site-specific nuclease activity. In some embodiments, the activity is double-stranded DNA cleavage activity. In some embodiments, the activity is single-stranded DNA cleavage activity, including, for example, site-specific DNA cleavage activity or non-specific DNA cleavage activity. In some embodiments, the activity is single-stranded RNA cleavage activity, eg, site-specific RNA cleavage activity or non-specific RNA cleavage activity. In some embodiments, the activity is measured in vitro. In some embodiments, the activity is measured in cells, such as bacterial cells, plant cells, or eukaryotic cells. In some embodiments, the activity is measured in mammalian cells, such as rodent cells or human cells. In some embodiments, the activity is measured in human cells, such as 293T cells. In some embodiments, the activity is measured in mouse cells, eg, Hepal-6 cells. In some embodiments, the engineered CasX nuclease has at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more increased site-specific nuclease activity compared to a reference CasX nuclease. The site-specific nuclease activity of the engineered CasX nuclease can be measured using methods known in the art, including, for example, gel shift assays, agarose gel electrophoresis-based in vitro cleavage assays as described in the Examples provided herein.

In some embodiments, the activity is gene editing activity in the cell. In some embodiments, the cells are bacterial cells, plant cells, or eukaryotic cells. In some embodiments, the cells are mammalian cells such as rodent cells or human cells. In some embodiments, the cells are 293T cells. In some embodiments, the activity is measured in mouse cells, eg, Hepal-6 cells. In some embodiments, the activity is an indel forming activity at a target genomic site in the cell, such as site-specific cleavage of a target nucleic acid by the engineered CasX nuclease and DNA repair by a non-homologous end joining (NHEJ) mechanism. In some embodiments, the activity is insertion of an exogenous nucleic acid sequence at a target genomic site in the cell, such as site-specific cleavage of the target nucleic acid by the engineered CasX nuclease and DNA repair by a homologous recombination (HR) mechanism. In some embodiments the engineered CasX nuclease increases the gene editing (e.g., indel formation) activity of any of at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more at a genomic locus of a cell (e.g., a human cell such as a 293T cell or a mouse Hepa1-6 cell) compared to a reference CasX nuclease. In some embodiments, the engineered CasX nuclease is at least about 20%, 30%, 40%, 60%, 70%, 80%, 90%, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold increased at multiple (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) genomic sites in a cell (e.g., a human cell such as a 293T cell or a mouse Hepa1-6 cell) compared to a reference CasX2 nuclease The gene editing (eg, indel formation) activity of any of 10-fold, 10-fold, or more. In some embodiments, the engineered CasX nuclease is capable of editing a greater number of genomic sites than the reference CasX nuclease. In some embodiments, the consensus nucleic acid sequence of the engineered CasX nuclease is identical to the reference CasX nuclease.

The gene editing efficiency of an engineered CasX nuclease in a cell can be determined using methods known in the art, including, for example, a T7 endonuclease 1 (T7E1 ) assay, sequencing of target DNA (including, for example, Sanger sequences, and next-generation sequencing), tracking indels by decomposition (TIDE) assays, or indel detection by amplicon analysis (IDAA) assays. See, eg, Sentmanat MF et al., "A survey of validation strategies for CRISPR-Cas9 editing," Scientific Reports, 2018, 8, article number 888, which is hereby incorporated by reference in its entirety. In some embodiments, the gene editing efficiency of the engineered CasX nuclease in cells is measured using targeted next generation sequencing (NGS), eg, as described in the Examples herein. Exemplary genomic sites for determining the gene editing efficiency of the engineered CasX nuclease include, but are not limited to, AAVS1, CCR5, CD34, and the like. In some embodiments, the gene editing efficiency (eg indel rate) of the engineered CasX nuclease At least 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or more.

The present application provides engineered CasX effector proteins with improved activities, such as target binding, double-strand cleavage activity, nickase activity, and/or gene editing activity. In some embodiments, an engineered CasX effector protein (e.g., a CasX nuclease, a CasX nickase, a CasX fusion effector protein, or a split CasX effector protein) comprising any of the engineered CasX nucleases described herein or a functional derivative thereof is provided.

The present application provides engineered CasX effector proteins comprising functional variants of the engineered CasX nucleases described herein. In some embodiments, the amino acid sequence of the functional variant differs by at least one amino acid residue (eg, with a deletion, insertion, substitution, and/or fusion) when compared to the amino acid sequence of a corresponding engineered CasX nuclease. In some embodiments, the functional variant has one or more mutations, such as amino acid substitutions, insertions and/or deletions. For example, the functional variant may comprise any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions compared to the engineered CasX nuclease. In some embodiments, the one or more substitutions are conservative substitutions. In some embodiments, the functional variant has all domains of the engineered CasX nuclease. In some embodiments, the functional variant does not have one or more domains of the engineered CasX nuclease.

For any of the CasX variant proteins described herein (e.g., a nickase CasX protein, an inactivated or catalytically inactive CasX (dCasX), a fusion CasX), the CasX variant can include a CasX protein sequence having the same parameters described above (e.g., domains present, percent identity, etc.).

In some embodiments, the functional variant has a different catalytic activity than the non-mutated form of its engineered CasX nuclease. In some embodiments, the mutation (eg, amino acid substitution, insertion, and/or deletion) is in the catalytic domain (eg, RuvC domain) of the CasX effector protein. In some embodiments, the variant comprises mutations in multiple catalytic domains. A CasX effector protein that cleaves one strand of a double-stranded target nucleic acid but not the other is referred to herein as a "nickase" (eg, a "CasX nickase"). CasX proteins that are substantially devoid of nuclease activity are referred to herein as inactive CasX proteins ("dCasX") (disclaimer: in the case of fusion CasX effector proteins, a heterologous polypeptide (fusion partner) can provide nuclease activity, which will be described in detail below). In some embodiments, a CasX effector protein is considered to lack substantially all DNA cleavage activity when the mutant enzyme has less than about 25%, 10%, 5%, 1%, 0.1%, 0.01% or less DNA cleavage activity relative to its non-mutated form.

The present application also relates to an engineered CasX effector protein according to the foregoing, which comprises a first polypeptide and a second polypeptide, the first polypeptide comprising amino acid residues 1 to X of the N-terminal part of the aforementioned engineered CasX nuclease, and the second polypeptide comprising amino acid residues X+1 of the aforementioned engineered CasX nuclease to the C-terminus of the CasX nuclease, wherein the first polypeptide and the second polypeptide can associate with each other in the presence of a guide RNA comprising a guide sequence to form a clustered regularly interspaced short palindromic repeat (CRISPR) complex that specifically binds to a target nucleic acid A target nucleic acid comprising a target sequence complementary to said guide sequence.

The application also provides split CasX effector proteins based on any of the engineered CasX effector proteins described herein. Split CasX effectors may be advantageous for delivery. In some embodiments, the engineered CasX effector protein is split into two parts of the enzyme, which can be reconstituted together to provide a substantially functioning CasX effector protein. CasX effector proteins can be provided using known methods, for example, fragmented forms of the Cas12 and Cas9 proteins have been described in, for example, WO2016/112242, WO2016/205749 and PCT/CN 2020/111057, which are incorporated herein by reference in their entirety.

In some embodiments, a split-type CasX effector protein is provided, comprising a first polypeptide and a second polypeptide, the first polypeptide comprising an N-terminal portion of any one of the engineered CasX nucleases described herein or a functional derivative thereof, the second polypeptide comprising a C-terminal portion of the engineered CasX nuclease or a functional derivative thereof, wherein the first polypeptide and the second polypeptide can associate with each other in the presence of a guide RNA comprising a guide sequence to form a CRISPR complex specifically binding to a target nucleic acid, the target nucleic acid comprising a target sequence complementary to the guide sequence. In some embodiments, the first polypeptide and the second polypeptide each comprise a dimerization domain. In some embodiments, the first dimerization domain and the second dimerization domain associate with each other in the presence of an inducing agent (eg, rapamycin). In some embodiments, the first polypeptide and the second polypeptide do not comprise a dimerization domain. In some embodiments, the segmented CasX effector protein is autoinducible.

Splitting can be done in a way that does not affect the catalytic domain. CasX effector proteins can act as nucleases (including nicking enzymes) or can be inactivated enzymes, which are essentially RNA-guided DNA-binding proteins with little or no catalytic activity (eg, due to mutations in their catalytic domain).

In some embodiments, the nuclease lobe and the α-helical lobe of the CasX protein are expressed as separate polypeptides. Although the leaves do not interact on their own, RNA guide sequences recruit them into a complex that recapitulates the activity of the full-length CasX enzyme and catalyzes site-specific DNA cleavage. In some embodiments, modified RNA guide sequences can be used to prevent dimerization by Instead, the activity of the split-type enzyme is eliminated, allowing the development of an inducible dimerization system. Such split-type enzymes are described, for example, in Wright, Addison V., et al. "Rational design of a split-Cas9 enzyme complex," Proc. Nat'l. Acad. Sci., 112.10(2015):2984-2989, which is incorporated herein by reference in its entirety.

The split CasX effector protein portion described herein can be designed to be split in half by splitting (i.e., splitting) a reference engineered CasX effector protein (e.g., full-length engineered CasX) at a split position, which is the point at which the N-terminal portion of the reference CasX effector protein is separated from the C-terminal portion. In some embodiments, the N-terminal portion comprises amino acid residues 1 to X, and the C-terminal portion comprises amino acid residues X+1 to the C-terminus of the reference CasX effector protein. In this example, the numbering is sequential, but this is not required, as it is also contemplated that amino acids (or nucleotides encoding them) may be trimmed from either split ends and/or mutations (e.g., insertions, deletions, and substitutions) in the interior region of the polypeptide chain, provided that the reconstituted CasX effector protein retains sufficient DNA binding activity (if desired), DNA nickase or cleavage activity, e.g., at least 40%, 50%, 60%, 70%, 80%, 90% compared to the reference CasX effector protein % or 95% active.

Cutpoints can be designed in silico and cloned into constructs. During this process, mutations can be introduced into segmented CasX effector proteins and non-functional domains can be removed. In some embodiments, the two parts or fragments (i.e., N-terminal and C-terminal fragments) of the split CasX effector protein can form a complete CasX effector protein comprising, for example, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the complete CasX effector protein sequence.

The segmented CasX effector proteins may each comprise one or more dimerization domains. In some embodiments, the first polypeptide comprises a first dimerization domain fused to a first segmented CasX effector protein portion, and the second polypeptide comprises a second dimerization domain fused to a second segmented CasX effector protein portion. The dimerization domain can be fused to the segmented CasX effector protein portion by a peptide linker (eg, a flexible peptide linker such as a GS linker) or a chemical bond. In some embodiments, the dimerization domain is fused to the N-terminus of the segmented CasX effector protein portion. In some embodiments, the dimerization domain is fused to the C-terminus of the segmented CasX effector portion. In some embodiments the segmented CasX effector protein does not comprise any dimerization domains.

In some embodiments, the dimerization domain facilitates the association of two segmented CasX effector protein moieties. In some embodiments, the segmented CasX effector portion is induced by an inducer to associate or dimerize into a functional CasX effector. In some embodiments, the Cut-type CasX effector proteins contain an inducible dimerization domain. In some embodiments, the dimerization domain is not an inducible dimerization domain, ie, the dimerization domain dimerizes in the absence of an inducing agent.

An inducing agent can be an inducing energy source or an inducing molecule other than a guide RNA (eg, sgRNA). The inducer partially remodels the two segmented CasX effector proteins into a functional CasX effector protein through the induced dimerization of the dimerization domain. In some embodiments, the inducing agent brings together the two segmented CasX effector protein moieties by inducing association of the inducible dimerization domain. In some embodiments, the two segmented CasX effector protein moieties do not associate with each other to remodel into a functional CasX effector protein in the absence of an inducing agent. In some embodiments, in the absence of an inducing agent, two separate CasX effector protein moieties can associate with each other in the presence of a guide RNA (eg, crRNA) to reconstitute into a functional CasX effector protein.

The inducer of the present application may be heat, ultrasound, electromagnetic energy or chemical compounds. In some embodiments, the inducing agent is an antibiotic, small molecule, hormone, hormone derivative, steroid, or steroid derivative. In some embodiments, the inducer is abscisic acid (ABA), doxycycline (DOX), cumate, rapamycin, 4-hydroxytamoxifen (4OHT), estrogen, or ecdysone. In some embodiments, the segmented CasX effector system is an inducer-controlled system selected from the group consisting of an antibiotic-based induction system, an electromagnetic energy-based induction system, a small molecule-based induction system, a nuclear receptor-based induction system, and a hormone-based induction system. In some embodiments, the segmented CasX effector system is an inducer-controlled system selected from the group consisting of a tetracycline (Tet)/DOX inducible system, a light inducible system, an ABA inducible system, a cumate repressor/operator system, a 4OHT/estrogen inducible system, an ecdysone-based inducible system, and a FKBP12/FRAP (FKBP12-rapamycin complex) inducible system. Such inducers are also discussed herein and in PCT/US2013/051418, which is hereby incorporated by reference in its entirety. The FRB/FKBP/rapamycin system has been described in Paulmurugan and Gambhir, Cancer Res, August 15, 2005 65; 7413, and Crabtree et al., Chemistry & Biology 13, 99-107, Jan 2006, which are hereby incorporated by reference in their entirety.

In some embodiments, pairs of split CasX effector proteins are separated and inactive until dimerization of the dimerization domains (eg, FRB and FKBP) is induced, which dimerization results in reassembly of functional CasX effector protein nucleases. In some embodiments, the first split CasX effector protein comprising the first half of an inducible dimer (e.g., FRB) is separated delivered, and/or in a position separate from the second split CasX effector protein comprising the second half of the inducible dimer (eg, FKBP).

Other exemplary FKBP-based induction systems that can be used in the inducer-controlled split CasX effector systems described herein include, but are not limited to: FKBP that dimerizes with calcineurin (CNA) in the presence of FK506; FKBP that dimerizes with CyP-Fas in the presence of FKCsA; FKBP that dimerizes with FRB in the presence of rapamycin; GyrB that dimerizes with GryB in the presence of coumarycin; GAI that dimerizes with GID1 in the presence of GA; or Snap-tag that dimerizes with HaloTag in the presence of HaXS.

Alternatives within the FKBP family itself were also considered. For example, FKBPs homodimerize (ie, one FKBP dimerizes with another FKBP) in the presence of FK1012.

In some embodiments, the dimerization domain is FKBP and the inducer is FK1012. In some embodiments, the dimerization domain is GryB and the inducer is Coumarin. In some embodiments, the dimerization domain is ABA and the inducing agent is gibberellin.

In some embodiments, the segmented CasX effector portion can be autoinduced (ie, autoactivated or autoinduced) in the absence of an inducing agent to associate/dimerize into a functional CasX effector. Without being bound by any theory or hypothesis, the auto-induction of the segmented CasX effector portion may be mediated by binding to a guide RNA such as crRNA. In some embodiments, the first polypeptide and the second polypeptide do not comprise a dimerization domain. In some embodiments, the first polypeptide and the second polypeptide comprise a dimerization domain.

In some embodiments, the reconstituted CasX effector protein of the split CasX effector system described herein (including inducer-controlled and auto-inducible systems) has an editing efficiency of at least 70% (such as at least about any of 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more efficiency, or 100% efficiency) relative to a reference CasX effector protein editing efficiency.

In some embodiments, the reconstituted CasX effector protein of the inducer-controlled split CasX effector system described herein has an editing efficiency of no more than 50% (such as no more than any of about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or less efficiency, or 0% efficiency) relative to the editing efficiency of a reference CasX effector protein in the absence of the inducer (i.e., due to auto-induction).

The present application also provides engineered CasX effector proteins comprising additional protein domains and/or components, such as linkers, nuclear localization/export sequences, functional domains and/or reporter proteins.

In some embodiments, the engineered CasX effector protein is a protein complex comprising one or more heterologous protein domains (e.g., about or greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains) and the nucleic acid targeting domain of the engineered CasX nuclease or a functional derivative thereof. In some embodiments, the engineered CasX effector protein is a fusion protein comprising one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains) fused to the engineered CasX nuclease.

In some embodiments, the engineered CasX effector protein of the present application may comprise (for example, through a fusion protein, such as through one or more peptide linkers, such as a GS peptide linker, etc.) one or more functional domains or associate (for example, through co-expression of multiple proteins) with it. In some embodiments, the one or more functional domains are enzymatic domains. These functional domains can have various activities, such as DNA and/or RNA methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and switching activity (e.g., light-induced). In some embodiments, the one or more functional domains are transcriptional activation domains (ie, transactivation domains) or repressor domains. In some embodiments, the one or more functional domains are histone modification domains. In some embodiments, the one or more functional domains are transposase domains, HR (homologous recombination) machinery domains, recombinase domains, and/or integrase domains. In some embodiments, the functional domain is Krüppel-associated box (KRAB), VP64, VP16, Fok1, P65, HSF1, MyoD1, Biotin-APEX, APOBEC1, AID, PmCDA1, Tad1, and M-MLV reverse transcriptase. In some embodiments, the functional domain is selected from the group consisting of a translation initiation domain, a transcriptional repression domain, a transactivation domain, an epigenetic modification domain, a nucleobase editing domain (eg, a CBE or ABE domain), a reverse transcriptase domain, a reporter domain (eg, a fluorescent domain), and a nuclease domain.

In some embodiments, the positioning of one or more functional domains in the engineered CasX effector protein allows correct spatial orientation of the functional domains to affect the target with the conferred functional effect. For example, if the functional domain is a transcriptional activator (eg, VP16, VP64, or p65), the transcriptional activator is placed in a spatial orientation that enables it to affect the transcription of the target. Likewise, a transcriptional repressor is positioned to affect the transcription of a target, and a nuclease (eg, Fok1 ) is positioned to cleave or partially cleave the target. In some embodiments, the functional domain is located at the N-terminus of the engineered CasX effector protein. In some embodiments, the functional domain is located at the C-terminus of the engineered CasX effector protein. In some embodiments, the engineered CasX effector The response protein contains a first functional domain at the N-terminus and a second functional domain at the C-terminus. In some embodiments, the engineered CasX effector protein comprises a catalytically inactive mutant of any of the engineered CasX nucleases described herein fused to one or more functional domains.

In some embodiments, the engineered CasX effector protein is a transcriptional activator. In some embodiments, the engineered CasX effector protein comprises an enzyme-inactive variant of any of the engineered CasX nucleases described herein fused to a transactivation domain. In some embodiments, the transactivation domain is selected from the group consisting of VP64, p65, HSF1, VP16, MyoD1, HSF1, RTA, SET7/9, and combinations thereof. In some embodiments, the transactivation domain comprises VP64, p65, and HSF1. In some embodiments, the engineered CasX effector protein comprises two split CasX effector polypeptides, each fused to a transactivation domain.

In some embodiments, the engineered CasX effector protein is a transcriptional repressor. In some embodiments, the engineered CasX effector protein comprises an enzyme-inactive variant of any of the engineered CasX nucleases described herein fused to a transcriptional repression domain. In some embodiments, the transcriptional repressor domain is selected from the group consisting of Krüppel-associated box (KRAB), EnR, NuE, NcoR, SID, SID4X, and combinations thereof. In some embodiments, the engineered CasX effector protein comprises two split CasX effector polypeptides, each fused to a transcriptional repression domain.

In some embodiments, the engineered CasX effector protein is a base editor, such as a cytosine editor or an adenosine editor. In some embodiments, the engineered CasX effector protein comprises an enzyme-inactive variant of any of the engineered CasX nucleases described herein fused to a nucleobase editing domain, such as a cytosine base editing (CBE) domain or an adenosine base editing (ABE) domain. In some embodiments, the nucleobase editing domain is a DNA editing domain. In some embodiments, the nucleobase editing domain has deaminase activity. In some embodiments, the nucleobase editing domain is a cytosine deaminase domain. In some embodiments, the nucleobase editing domain is an adenosine deaminase domain. Exemplary base editors based on Cas nucleases are described, eg, in WO2018/165629A1 and WO2019/226953A1, which are incorporated herein by reference in their entirety. Exemplary CBE domains include, but are not limited to, activation-induced cytidine deaminase or AID (e.g., hAID), apolipoprotein B mRNA editing complex or APOBEC (e.g., rat APOBEC1, hAPOBEC3 A/B/C/D/E/F/G), and PmCDA1. Exemplary ABE domains include, but are not limited to: TadA, ABE8, and variants thereof (see, eg, Gaudelli et al., 2017, Nature 551:464-471; and Richter et al., 2020, Nature Biotechnology 38:883-891). In some embodiments, the functional domain is APOBEC1 domain, such as a rat APOBEC1 domain. In some embodiments, the functional domain is a TadA domain, such as an E. coli TadA domain. In some embodiments the engineered CasX effector protein further comprises one or more nuclear localization sequences.

In some embodiments, the engineered CasX effector protein is a master editor. Cas9-based master editors are described, e.g., in A. Anzalone et al., Nature, 2019, 576(7785):149-157, which is hereby incorporated by reference in its entirety. In some embodiments, the engineered CasX effector protein comprises a nickase variant of any one of the engineered CasX nucleases described herein fused to a reverse transcriptase domain. In some embodiments, the functional domain is a reverse transcriptase domain. In some embodiments, the reverse transcriptase domain is M-MLV reverse transcriptase or a variant thereof, such as an M-MLV reverse transcriptase having one or more mutations of D200N, T306K, W313F, T330P, and L603W. In some embodiments, an engineered CRISPR/CasX system comprising said master editor is provided. In some embodiments, the engineered CRISPR/CasX system further comprises a second CasX nickase, eg, based on the same engineered CasX nuclease as the primary editor. In some embodiments, the engineered CRISPR/CasX system comprises a master editor guide RNA (pegRNA) comprising a primer binding site and a reverse transcriptase (RT) template sequence.

In some embodiments, the application provides segmented CasX effector systems having one or more (e.g., 1, 2, 3, 4, 5, 6 or more) functional domains associated with (i.e., bound or fused to) one or both of the segmented CasX effector protein moieties. Said functional domain may be provided as part of said first and/or second segmented CasX effector protein, as a fusion within the construct. The functional domain is usually fused to other parts of the segmented CasX effector protein (eg, segmented CasX effector protein portion) via a peptide linker (such as a GS linker). These functional domains can be used to reprogram the function of this segmented CasX effector system based on catalytically inactive CasX effector proteins.

In some embodiments, the engineered CasX effector protein comprises one or more nuclear localization sequences (NLS) and/or one or more nuclear export sequences (NES). Exemplary NLS sequences include, eg, PKKKRKVPG and ASPKKKRKV. NLS and/or NES can be operably linked to the N-terminus and/or C-terminus of the engineered CasX effector protein or a polypeptide chain in the engineered CasX effector protein.

In some embodiments, the engineered CasX effector protein can encode additional components, such as a reporter protein. In some embodiments, the engineered CasX effector protein comprises a fluorescent protein, such as GFP. Such systems may allow imaging of genomic loci (see, for example, "Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System" Chen B et al. Cell 2013). In some embodiments the engineered CasX effector protein is an inducible segmentable CasX effector system that can be used to image genomic loci.

In yet another specific embodiment, an engineered CasX effector protein is provided, wherein the effector protein is capable of inducing double-strand breaks or single-strand breaks in DNA molecules.

In yet another specific embodiment, an engineered CasX effector protein is provided, wherein the functional derivative of the engineered CasX nuclease is an enzyme inactive mutant.

In yet another specific embodiment, an engineered CasX effector protein is provided, which further comprises a functional domain fused with the engineered CasX nuclease.

In yet another specific embodiment, an engineered CasX effector protein is provided, wherein the functional domain is selected from the group consisting of a translation initiation domain, a transcriptional repression domain, a transactivation domain, an epigenetic modification domain, a nucleobase editing domain, a reverse transcriptase domain, a reporter domain and a nuclease domain.

The present application provides an engineered CRISPR-CasX system in a third aspect

In a specific embodiment, an engineered CRISPR-CasX system is provided, comprising: (a) the aforementioned engineered CasX effector protein; and (b) a guide RNA comprising a guide sequence complementary to a target sequence, or one or more nucleic acids encoding the guide RNA, wherein the engineered CasX effector protein and the guide RNA can form a CRISPR complex, and the CRISPR complex specifically binds to the target nucleic acid comprising the target sequence and induces modification of the target nucleic acid.

In a specific embodiment, the CasX nuclease is selected from any one of the following groups: PlmCasX, DpbCasX.

In some embodiments, the engineered CRISPR-CasX system comprises: (a) any of the engineered CasX effector proteins described herein (e.g., an engineered CasX nuclease, nickase, segmented CasX, transcription repressor, transcription activator, base editor, or master editor); and (b) a guide RNA comprising a guide sequence complementary to a target sequence, or one or more nucleic acids encoding the guide RNA, wherein the engineered CasX effector protein and the guide RNA are capable of forming a CRISPR complex, the The CRISPR complex specifically binds to a target nucleic acid comprising the target sequence and induces modification of the target nucleic acid. In some embodiments, the engineered CRISPR-CasX system comprises one or more nucleic acids encoding the engineered CasX effector protein and/or the guide RNA. In some embodiments, the engineered CRISPR-CasX The system comprises an array of precursor guide RNAs that can be processed into multiple crRNAs, eg, by the engineered CasX effector proteins. In some embodiments, the engineered CRISPR-CasX system comprises one or more vectors encoding the engineered CasX effector protein and/or the guide RNA. In some embodiments, the engineered CRISPR-CasX system comprises a ribonucleoprotein (RNP) complex comprising the engineered CasX effector protein bound to the guide RNA.

The engineered CRISPR-CasX system of the present application can comprise any suitable guide RNA. A guide RNA (gRNA) may comprise a guide sequence capable of hybridizing to a target sequence in a target nucleic acid of interest, such as a genomic site of interest in a cell. In some embodiments, the gRNA comprises a CRISPR RNA (crRNA) sequence comprising the guide sequence.

Typically, the crRNAs described herein include direct repeat sequences and spacer sequences. In certain embodiments, the crRNA comprises, consists essentially of, or consists of a direct repeat sequence linked to a guide sequence or a spacer sequence. In some embodiments, the crRNA includes a direct repeat sequence, a spacer sequence, and a direct repeat sequence (DR-spacer sequence-DR), which are typical features of a precursor crRNA (pre-crRNA) configuration. In some embodiments, the crRNA includes truncated direct repeat and spacer sequences, which are typical features of processed or mature crRNA. In some embodiments, the CRISPR-CasX effector protein forms a complex with an RNA guide sequence, and the spacer sequence directs the complex to sequence-specific binding to a target nucleic acid that is complementary to the spacer sequence.

In some embodiments, the guide RNA is a crRNA comprising a guide sequence. In some embodiments, the engineered CRISPR-CasX system comprises an array of precursor guide RNAs encoding a plurality of crRNAs. In some embodiments, the CasX effector protein cleaves the array of precursor guide RNAs to generate a plurality of crRNAs. In some embodiments, the engineered CRISPR-CasX system comprises an array of precursor guide RNAs encoding multiple crRNAs, wherein each crRNA comprises a different guide sequence.

The guide sequence can be of suitable length. In some embodiments, the guide sequence is between about 18 and about 35 nucleotides, including, for example, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides. The guide sequence may be at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% complementary to the target sequence of the target nucleic acid.

Also provided herein are constructs, vectors, and expression systems encoding any of the engineered CasX effector proteins (eg, engineered CasX nucleases) described herein. In some embodiments, the construct, vector or expression system further comprises one or more gRNA or crRNA arrays.

A "vector" is a composition of matter comprising an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. In general, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites and one or more selectable markers. The term "vector" should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like.

In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof. In some embodiments, the vector is a phage vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), among other handbooks of virology and molecular biology.

A number of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The heterologous nucleic acid can be inserted into a vector and packaged in retroviral particles using techniques known in the art. Recombinant virus can then be isolated and delivered to the engineered mammalian cells in vitro or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, a self-inactivating lentiviral vector is used.

In certain embodiments, the vector is an adeno-associated virus (AAV) vector, such as AAV2, AAV8, or AAV9, which can administer the adenovirus or adeno-associated virus in a single dose comprising at least 1 x ¹⁰⁵ particles (also known as particle units, pu). In some embodiments, the administered amount is at least about 1×10 ⁶ particles, at least about 1×10 ⁷ particles, at least about 1×10 ⁸ particles, or at least about 1×10 ⁹ particles of adeno-associated virus. Delivery methods and dosages are described, for example, in WO 2016205764 and US Patent No. 8,454,972, which are incorporated herein by reference in their entirety.

In some embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector. For example, in some embodiments, modified AAV vectors can be used for delivery. Modified AAV vectors can to be based on one or more of several capsid types, including AAV1, AAV2, AAV5, AAV6, AAV8, AAV8.2, AAV9, AAV rh10, modified AAV vectors (e.g. modified AAV2, modified AAV3, modified AAV6) and pseudotyped AAV (e.g. AAV2/8, AAV2/5 and AAV2/6). Exemplary AAV vectors and techniques that can be used to generate rAAV particles are known in the art (see, e.g., Aponte-Ubillus et al. (2018) Appl. Microbiol. Biotechnol. 102(3):1045-54; Zhong et al. (2012) J. Genet. Syndr. Gene Ther. 160:38-47 (1987); Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-60; US Patent Nos. 4,797,368 and 5,173,414; International Publication Nos. WO2015/054653 and WO93/24641, each of which is incorporated herein by reference).

Any known AAV vector used to deliver Cas9 and other Cas proteins can be used to deliver the engineered CasX system of the present application.

Methods for introducing vectors into mammalian cells are known in the art. Vectors can be transferred into host cells by physical, chemical or biological means.

Physical methods used to introduce vectors into host cells include: calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. In some embodiments, the vector is introduced into the cells by electroporation.

Biological methods for introducing heterologous nucleic acids into host cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, eg human, cells.

Chemical methods for introducing vectors into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro delivery vehicle is a liposome (eg, an artificial membrane vesicle). In some embodiments, the engineered CRISPR-CasX system is delivered in nanoparticles as RNPs.

In some embodiments, the vector or expression system encoding the CRISPR-CasX system or components thereof comprises one or more selectable or detectable markers that provide a means to isolate or efficiently select cells that contain and/or have been modified (e.g., at an early stage and at a large scale) by the CRISPR-CasX system.

Reporter genes can be used to identify potentially transfected cells and assess the function of regulatory sequences. Typically, a reporter gene is a gene that is absent or not expressed in the recipient organism or tissue, and the expression of the polypeptide it encodes Reaching is evidenced by some readily detectable property, such as enzymatic activity. Expression of the reporter gene is measured at an appropriate time after introduction of the DNA into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (eg, Ui-Tei et al. FEBS Letters 479:79-82 (2000)).

Other methods of confirming the presence of heterologous nucleic acid in host cells include, for example, molecular biological assays, such as Southern and Northern blots, RT-PCR and PCR, well known to those skilled in the art; biochemical assays, such as detection of the presence or absence of specific peptides by immunological methods such as ELISA and Western blotting.

In some embodiments, the nucleic acid sequence encoding the engineered CasX effector protein and/or the guide RNA is operably linked to a promoter. In some embodiments, the promoter is an endogenous promoter relative to the cell engineered using the engineered CRISPR-CasX system. For example, the nucleic acid encoding the engineered CasX effector protein can be knocked into the genome of an engineered mammalian cell downstream of an endogenous promoter using any method known in the art. In some embodiments, the endogenous promoter is the promoter of an abundant protein such as β-actin. In some embodiments, the endogenous promoter is an inducible promoter, eg, inducible by an endogenous activation signal of the engineered mammalian cell. In some embodiments, wherein the engineered mammalian cell is a T cell, the promoter is a T cell activation dependent promoter (such as the IL-2 promoter, NFAT promoter or NFκB promoter).

In some embodiments, the promoter is a heterologous promoter relative to the cell engineered using the engineered CRISPR-CasX system. A variety of promoters have been explored for expression of genes in mammalian cells, and any promoter known in the art may be used in this application. Promoters can be broadly classified as constitutive promoters or regulated promoters, such as inducible promoters.

In some embodiments, the nucleic acid sequence encoding the engineered CasX effector protein and/or the guide RNA is operably linked to a constitutive promoter. Constitutive promoters allow the constitutive expression of heterologous genes (also known as transgenes) in host cells. Exemplary constitutive promoters contemplated herein include, but are not limited to: cytomegalovirus (CMV) promoter, human elongation factor-1α (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerol kinase promoter (PGK), Simian virus 40 early promoter (SV40), and chicken β-actin promoter coupled to CMV early enhancer (CAG). In some embodiments, the promoter is a CAG promoter comprising a cytomegalovirus (CMV) early enhancer element, Promoter, first exon and first intron of chicken β-actin gene, and splice acceptor of rabbit β-globin gene.

In some embodiments, the nucleic acid sequence encoding the engineered CRISPR-CasX effector protein and/or the guide RNA is operably linked to an inducible promoter. Inducible promoters are a type of regulated promoter. The inducible promoter can be induced by one or more conditions, such as physical conditions, the microenvironment or the physiological state of the host cell, an inducer (ie, an inducer), or a combination thereof. In some embodiments, the inducing conditions are selected from the group consisting of inducing agent, irradiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox state, tumor environment, and activation state of the cells to be engineered by the engineered CRISPR-CasX system. In some embodiments, the promoter is inducible by a small molecule inducing agent such as a chemical compound. In some embodiments, the small molecule is selected from the group consisting of doxycycline, tetracycline, alcohol, metal, or steroid. Chemically inducible promoters have been most extensively studied. Such promoters include promoters whose transcriptional activity is regulated by the presence or absence of small molecule chemicals such as doxycycline, tetracycline, alcohols, steroids, metals and other compounds. The doxycycline-inducible system with retrotetracycline-controlled transactivator (rtTA) and tetracycline-responsive element promoter (TRE) is currently the most mature system. WO9429442 describes the tight control of gene expression in eukaryotic cells by tetracycline-responsive promoters. WO9601313 discloses tetracycline regulated transcriptional regulators. Additionally, Tet technologies such as the Tet-on system have been described at, for example, the TetSystems.com website. In this application, any known chemically regulated promoter can be used to drive the expression encoding the engineered CRISPR-CasX protein and/or the guide RNA.

In some embodiments, the nucleic acid sequence encoding the engineered CasX effector protein is codon optimized.

In some embodiments, an expression construct is provided comprising a codon-optimized sequence encoding the engineered CasX effector protein ligated to a BPK2104-ccdB vector. In some embodiments, the expression construct encodes a tag (eg, a 10xHis tag) operably linked to the C-terminus of the engineered CasX effector protein.

In some embodiments, each engineered split CasX construct encodes a fluorescent protein such as GFP or RFP. The reporter protein can be used to assess colocalization and/or dimerization of the engineered CasX protein, for example using microscopy. Nucleic acid sequences encoding engineered CasX effector proteins can be fused to nucleic acid sequences encoding additional components using sequences encoding self-cleaving peptides such as T2A, P2A, E2A or F2A peptides.

In some embodiments, expression constructs for use in mammalian cells (eg, human cells) are provided, said constructs comprising a nucleic acid sequence encoding said engineered CasX effector protein. In some embodiments, the expression construct comprises a codon optimized sequence encoding the engineered CasX effector protein inserted into the pCAG-2A-eGFP vector such that the CasX protein is operably linked to eGFP. In some embodiments, a second vector is provided for expression of a guide RNA (eg, crRNA or pre-crRNA array) in a mammalian cell (eg, a human cell). In some embodiments, the sequence encoding the guide RNA is expressed in the pUC19-U6-i2-cr RNA vector backbone.

The present application provides a method for detecting target nucleic acid in a sample in a fourth aspect

In a specific embodiment, a method for detecting a target nucleic acid in a sample is provided, comprising: (a) contacting the sample with the aforementioned engineered CRISPR-CasX system and a tagged detection nucleic acid, the detection nucleic acid is single-stranded and does not hybridize with the guide sequence of the guide RNA; and (b) measuring a detectable signal generated by cutting the tagged detection nucleic acid with the engineered CasX effector protein, thereby detecting the target nucleic acid.

In a specific embodiment, the CasX nuclease is selected from any one of the following groups: PlmCasX, DpbCasX.

The present application also provides methods for detecting target nucleic acids using any of the engineered CasX effector proteins or CRISPR-CasX systems with improved activity. The use of CasX effector proteins as detection reagents can take advantage of the discovery that, once activated by detection of target DNA, type V CRISPR/Cas proteins (e.g., CasX) can promiscuously cleave non-targeted single-stranded DNA (ssDNA or RNA, i.e., single-stranded nucleic acid to which the guide sequence of a guide RNA does not hybridize). Thus, when target DNA (double-stranded or single-stranded) is present in the sample (e.g., in some cases above a threshold amount), the result is cleavage of the single-stranded nucleic acid in the sample, which can be detected using any convenient detection method (e.g., using tagged single-stranded detection nucleic acids such as DNA or RNA). CasX can cleave ssDNA and ssRNA. For example methods using Cas proteins as detection reagents are described in US10253365 and WO2020/056924, which are hereby incorporated by reference in their entirety.

In some embodiments, there is provided a method of detecting target DNA (e.g., double-stranded or single-stranded) in a sample comprising: (a) contacting the sample with: (i) any of the engineered CasX effector proteins described herein; (ii) a guide RNA comprising a guide sequence that hybridizes to the target DNA; and (b) measuring a detectable signal produced by cleavage of the single-stranded detection nucleic acid by the engineered CasX effector protein. In some cases, the single-stranded detection nucleic acid includes a fluorescence emitting dye pair (eg, the fluorescence emitting dye pair is a fluorescence resonance energy transfer (FRET) pair, a quencher/fluorescent pair). In certain instances, the target DNA is viral DNA (eg, papillomavirus, hepadnavirus, herpesvirus, adenovirus, poxvirus, parvovirus, etc.). In some embodiments, the single-stranded detection nucleic acid is DNA. In some embodiments, the single-stranded detection nucleic acid is RNA.

The method for detecting target DNA (single-stranded or double-stranded) in a sample of the present disclosure can detect target DNA with high sensitivity.在一些情况下，本公开的方法可以用于检测存在于包含多个DNA(包括所述靶DNA和多个非靶DNA)的样品中的靶DNA，其中所述靶DNA以每10 ⁷个非靶DNA中的一个或更多个拷贝存在(例如，每10 ⁶个非靶DNA中的一个或更多个拷贝，每10 ⁵个非靶DNA中的一个或更多个拷贝，每10 ⁴个非靶DNA中的一个或更多个拷贝，每10 ³个非靶DNA中的一个或更多个拷贝，每10 ²个非靶DNA中的一个或更多个拷贝，每50个非靶DNA中的一个或更多个拷贝，每20个非靶DNA中的一个或更多个拷贝，每10个非靶DNA中的一个或更多个拷贝或每5个非靶DNA中的一个或更多个拷贝)。 In some embodiments, the engineered CasX effector protein described herein can detect target DNA with higher sensitivity than the reference CasX nuclease. In some embodiments, the engineered CasX effector protein can detect target DNA with a sensitivity of 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher compared to the reference CasX nuclease.

In a fifth aspect, the present application relates to a method of modifying a target nucleic acid comprising a target sequence

In a specific embodiment, there is provided a method of modifying a target nucleic acid comprising a target sequence, comprising contacting the target nucleic acid with the aforementioned engineered CRISPR-CasX system.

In a specific embodiment, the CasX nuclease is selected from any one of the following groups: PlmCasX, DpbCasX.

In some embodiments, the present application provides methods of modifying a target nucleic acid comprising a target sequence comprising contacting the target nucleic acid with any of the engineered CRISPR-CasX systems described herein. In some embodiments, the method is performed in vitro. In some embodiments, the target nucleic acid is present in a cell. In some embodiments, the cells are bacterial cells, yeast cells, Mammalian cells, plant cells or animal cells. In some embodiments, the method is performed ex vivo. In some embodiments, the method is performed in vivo.

In some embodiments, the target nucleic acid is cleaved or a target sequence in the target nucleic acid is altered by the engineered CRISPR-CasX system. In some embodiments, expression of the target nucleic acid is altered by the engineered CRISPR-CasX system. In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the target sequence is associated with a disease or condition. In some embodiments, the engineered CRISPR-CasX system comprises an array of precursor guide RNAs encoding multiple crRNAs, wherein each crRNA comprises a different guide sequence.

In some embodiments, the present application provides methods of treating a disease or condition associated with a target nucleic acid in a cell of an individual comprising using any of the methods described herein to modify the target nucleic acid in the cell of the individual, thereby treating the disease or condition. In some embodiments, the disease or condition is selected from the group consisting of cancer, cardiovascular disease, genetic disease, autoimmune disease, metabolic disease, neurodegenerative disease, eye disease, bacterial infection, and viral infection.

The engineered CRISPR-CasX systems described herein can modify target nucleic acids in cells in a variety of ways, depending on the type of CasX effector protein engineered in the CRISPR-CasX system. In some embodiments, the methods induce site-specific cleavage in the target nucleic acid. In some embodiments, the methods cleave genomic DNA in cells such as bacterial cells, plant cells, or animal cells (eg, mammalian cells). In some embodiments, the method kills the cell by cleavage of genomic DNA in the cell. In some embodiments the methods cleave viral nucleic acid in the cell.

In some embodiments, the methods alter (eg, increase or decrease) the expression level of the target nucleic acid in a cell. In some embodiments, the method uses an engineered CasX effector protein to increase the expression level of the target nucleic acid in a cell, eg, based on an enzymatically inactive CasX protein fused to a transactivation domain. In some embodiments, the method uses an engineered CasX effector protein to reduce the expression level of the target nucleic acid in a cell, eg, based on an enzymatically inactive CasX protein fused to a transcriptional repression domain. In some embodiments, the method uses an engineered CasX effector protein to introduce an epigenetic modification into the target nucleic acid in a cell, eg, based on an enzymatically inactive CasX protein fused to an epigenetic modification domain. The engineered CasX system described herein can be used to introduce other modifications into the target nucleic acid, depending on the functional domains comprised by the engineered CasX effector protein.

In some embodiments, the method alters a target sequence in the target nucleic acid in a cell. In some embodiments, the method introduces a mutation into the target nucleic acid in a cell. In some embodiments, the methods use one or more endogenous DNA repair pathways, such as non-homologous end joining (NHEJ) or homology-directed recombination (HDR), to repair double-strand breaks induced in the target DNA in the cell as a result of sequence-specific cleavage by the CRISPR complex. Exemplary mutations include, but are not limited to, insertions, deletions, substitutions, and frameshifts. In some embodiments, the method inserts donor DNA at the target site. In some embodiments, insertion of donor DNA results in the introduction of a selectable marker or reporter protein into the cell. In some embodiments, insertion of donor DNA results in knock-in of the gene. In some embodiments, insertion of donor DNA results in a knockout mutation. In some embodiments, insertion of donor DNA results in substitutional mutations such as single nucleotide substitutions. In some embodiments, the method induces a phenotypic change in the cell.

In some embodiments, the engineered CRISPR-CasX system is used as part of a genetic circuit, or to insert a genetic circuit into the genomic DNA of a cell. The inducer-controlled engineered segmented CasX effector proteins described herein are particularly useful as components of genetic circuits. Genetic circuits can be used in gene therapy. Methods and techniques for designing and using genetic circuits are known in the art. Further reference can be made to eg Brophy, Jennifer AN, and Christopher A. Voigt."Principles of genetic circuit design." Nature methods 11.5(2014):508.

The engineered CRISPR-CasX systems described herein can be used to modify a variety of target nucleic acids. In some embodiments, the target nucleic acid is in a cell. In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the target nucleic acid is extrachromosomal DNA. In some embodiments, the target nucleic acid is foreign to the cell. In some embodiments, the target nucleic acid is viral nucleic acid such as viral DNA. In some embodiments, the target nucleic acid is a plasmid in a cell. In some embodiments, the target nucleic acid is a horizontally transferred plasmid. In some embodiments, the target nucleic acid is RNA.

In some embodiments, the target nucleic acid is an isolated nucleic acid such as isolated DNA. In some embodiments, the target nucleic acid is present in a cell-free environment. In some embodiments, the target nucleic acid is an isolated vector such as a plasmid. In some embodiments, the target nucleic acid is an isolated linear DNA fragment.

The methods described herein are applicable to any suitable cell type. In some embodiments, the cells are bacteria, yeast cells, fungal cells, algal cells, plant cells, or animal cells (eg, mammalian cells, such as human cells). In some embodiments, the cells are naturally derived from Such as cells isolated from a tissue biopsy. In some embodiments, the cells are cells isolated from cell lines cultured in vitro. In some embodiments, the cells are from a primary cell line. In some embodiments, the cells are from an immortalized cell line. In some embodiments, the cells are genetically engineered cells.

In some embodiments, the cell is an animal cell of an organism selected from the group consisting of cattle, sheep, goats, horses, pigs, deer, chickens, ducks, geese, rabbits, and fish.

In some embodiments, the cell is a plant cell of an organism selected from the group consisting of corn, wheat, barley, oat, rice, soybean, oil palm, safflower, sesame, tobacco, flax, cotton, sunflower, pearl millet, millet, sorghum, canola, hemp, vegetable crops, forage crops, industrial crops, tree crops, and biomass crops.

In some embodiments, the cells are mammalian cells. In some embodiments, the cells are human cells. In some embodiments, the human cells are human embryonic kidney 293T (HEK293T or 293T) cells or HeLa cells. In some embodiments, the cells are human embryonic kidney (HEK293T) cells. In some embodiments, the cells are mouse Hepal-6 cells. In some embodiments, the mammalian cells are selected from the group consisting of immune cells, liver cells, tumor cells, stem cells, zygotes, muscle cells, and skin cells.

In some embodiments, the cell is an immune cell selected from the group consisting of cytotoxic T cells, helper T cells, natural killer (NK) T cells, iNK-T cells, NK-T-like cells, γδ T cells, tumor infiltrating T cells, and dendritic cell (DC) activated T cells. In some embodiments, the methods generate modified immune cells, such as CAR-T cells or TCR-T cells.

In some embodiments, the cells are embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, progenitor cells of gametes, cells in gametes, zygotes, or embryos.

The methods described herein can be used to modify target cells in vivo, ex vivo, or in vitro, and can be done in such a way that the cells are altered such that, once modified, progeny or cell lines of the modified cells retain the altered phenotype. The modified cells and progeny may be part of a multicellular organism such as a plant or animal with ex vivo or in vivo applications such as genome editing and gene therapy.

In some embodiments, the method is performed ex vivo. In some embodiments, after introducing the engineered CRISPR-CasX system into the cells, the modified cells (eg, mammalian cells) are propagated ex vivo. In some embodiments, the modified cells are cultured to propagate for at least about any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days. In some embodiments, the modified cells are cultured for no more than about 1 day, 2 days, 3 days Any of days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days or 14 days. In some embodiments, the modified cells are further evaluated or screened to select cells with one or more desired phenotypes or properties.

In some embodiments, the target sequence is a sequence associated with a disease or condition. Exemplary diseases or conditions include, but are not limited to, cancer, cardiovascular disease, genetic disease, autoimmune disease, metabolic disease, neurodegenerative disease, eye disease, bacterial infection, and viral infection. In some embodiments, the disease or condition is a genetic disease. In some embodiments, the disease or condition is a monogenic disease or condition. In some embodiments, the disease or condition is a polygenic disease or condition.

In some embodiments, the target sequence has a mutation compared to the wild-type sequence. In some embodiments, the target sequence has a single nucleotide polymorphism (SNP) associated with a disease or condition.

In some embodiments, the donor DNA inserted into the target nucleic acid encodes a biological product selected from the group consisting of reporter proteins, antigen-specific receptors, therapeutic proteins, antibiotic resistance proteins, RNAi molecules, cytokines, kinases, antigens, antigen-specific receptors, cytokine receptors, and suicide polypeptides. In some embodiments, the donor DNA encodes a therapeutic protein. In some embodiments, the donor DNA encodes a therapeutic protein that can be used in gene therapy. In some embodiments, the donor DNA encodes a therapeutic antibody. In some embodiments, the donor DNA encodes an engineered receptor, such as a chimeric antigen receptor (CAR) or an engineered TCR. In some embodiments, the donor DNA encodes a therapeutic RNA, such as a small RNA (eg, siRNA, shRNA, or miRNA) or a long non-coding RNA (lincRNA).

The methods described herein can be used for multiplex gene editing or modulation at two or more (eg, 2, 3, 4, 5, 6, 8, 10 or more) different target sites. In some embodiments, the methods detect or modify multiple target nucleic acids or target nucleic acid sequences. In some embodiments, the method comprises contacting a target nucleic acid with a guide RNA comprising a plurality (eg, 2, 3, 4, 5, 6, 8, 10 or more) of crRNA sequences, wherein each crRNA comprises a different target sequence.

Also provided are engineered cells comprising a modified target nucleic acid produced using any of the methods described herein. The engineered cells can be used in cell therapy. Autologous or allogeneic cells can be used to prepare engineered cells using the methods described herein for cell therapy.

The methods described herein can also be used to generate isogenic lines of cells (eg, mammalian cells) to study genetic variants.

Also provided are engineered non-human animals comprising engineered cells described herein. In some embodiments, the engineered non-human animal is a genome-edited non-human animal. The engineered non-human animals can be used as disease models.

Techniques for generating non-human genome edited or transgenic animals are well known in the art and include, but are not limited to: pronuclear microinjection, viral infection, transformation of embryonic stem cells and induced pluripotent stem (iPS) cells. Detailed methods that can be used include, but are not limited to, those described by Sundberg and Ichiki (2006, Genetically Engineered Mice Handbook, CRC Press) and those described by Gibson (2004, A Primer Of Genome Science 2nd ed. Sunderland, Mass.: Sinauer).

The engineered animal can be of any suitable species including, but not limited to: cattle, horses, sheep, dogs, deer, felines, goats, pigs, primates, and lesser known mammals such as elephants, deer, zebras, or camels.

In a sixth aspect, the present application relates to the use of CRISPR-CasX system to target nuclear cells in cells for the preparation of treatments and individuals. Use in medicine for an acid-related disease or condition

In some embodiments, a use of the aforementioned engineered CRISPR-CasX system in the preparation of a medicament for treating a disease or disorder associated with a target nucleic acid in a cell of an individual is provided; preferably, the disease or disorder is selected from the group consisting of cancer, cardiovascular disease, genetic disease, autoimmune disease, metabolic disease, neurodegenerative disease, eye disease, bacterial infection, and viral infection.

In a specific embodiment, the CasX nuclease is selected from any one of the following groups: PlmCasX, DpbCasX.

Further provided is a method of treatment using any of the methods of modifying a target nucleic acid in a cell according to the herein described. In some embodiments, the present application provides a method of treating a disease or condition associated with a target nucleic acid in a cell of an individual comprising contacting the target nucleic acid with any of the engineered CRISPR-CasX systems described herein, wherein the guide sequence of the guide RNA is complementary to the target sequence of the target nucleic acid, wherein the engineered CasX effector protein and the guide RNA associate with each other to bind to the target nucleic acid to modify the target nucleic acid such that the disease or condition is treated. In some embodiments, mutations (eg, knockout or knockin mutations) are introduced into the target nucleic acid. In some embodiments, expression of the target nucleic acid is enhanced. In some embodiments, expression of the target nucleic acid is inhibited. In some embodiments, the present application provides methods of treating a disease or condition in an individual comprising administering to the individual an effective amount of any of the engineered CRISPR-CasX systems described herein and donor DNA encoding a therapeutic agent, wherein the guide sequence of the guide RNA is Complementary to a target sequence of a target nucleic acid of the individual, wherein the engineered CasX effector protein and the guide RNA bind to each other to bind to the target nucleic acid and insert donor DNA into the target sequence, thereby allowing the disease or condition to be treated.

In some embodiments, the present application provides a method of treating a disease or condition in an individual comprising administering to the individual an effective amount of an engineered cell comprising a modified target nucleic acid, wherein the engineered cell is prepared by contacting the cell with any of the engineered CRISPR-CasX systems described herein, wherein the guide sequence of the guide RNA is complementary to the target sequence of the target nucleic acid, wherein the engineered CasX effector protein and the guide RNA associate with each other to bind to the target nucleic acid to modify the target nucleic acid. In some embodiments, the engineered cells are immune cells. In some embodiments, the individual is a human. In some embodiments, the individual is an animal, eg, a model animal such as a rodent, pet, or farm animal. In some embodiments, the individual is a mammal.

In some embodiments, the disease or condition is selected from the group consisting of cancer, cardiovascular disease, genetic disease, autoimmune disease, metabolic disease, neurodegenerative disease, eye disease, bacterial infection, and viral infection. In some embodiments, the target nucleic acid is PCSK9. In some embodiments, the disease or condition is cardiovascular disease. In some embodiments, the disease or condition is coronary artery disease. In some embodiments, the method reduces cholesterol levels in the individual. In some embodiments, the method treats diabetes in the individual.

The present application relates to a delivery method in the seventh aspect

In some embodiments, the engineered CRISPR-CasX system described herein, or components thereof, nucleic acid molecules thereof, or nucleic acid molecules encoding or providing components thereof, can be delivered to a host cell by a variety of delivery systems such as plasmids or viruses (e.g., any of the vectors described in the "Constructs and Vectors" section above). In some embodiments or methods, the engineered CRISPR-CasX system can be delivered by other methods, such as nucleofection or electroporation of a ribonucleoprotein complex consisting of the engineered CasX effector protein and its one or more cognate RNA guide sequences.

In some embodiments, delivery is via nanoparticles or exosomes.

In some embodiments, paired CasX nickase complexes can be delivered directly using nanoparticles or other direct protein delivery methods such that complexes comprising two paired crRNA elements are co-delivered. In addition, proteins can be delivered to cells via viral vectors or directly followed by CRISPR arrays containing two paired spacers for double nicks. In some cases, the For direct RNA delivery, the RNA can be conjugated to at least one sugar moiety such as N-acetylgalactosamine (GalNAc) (particularly triantennary GalNAc).

In a specific embodiment, the CasX nuclease is selected from any one of the following groups: PlmCasX, DpbCasX.

The application relates to kits and articles of manufacture in an eighth aspect

A kit is provided in a specific embodiment, comprising: one or more AAV vectors encoding any of the aforementioned engineered CasX nuclease, the aforementioned engineered CasX effector protein, and the aforementioned engineered CRISPR-CasX system.

In a specific embodiment, the CasX nuclease is selected from any one of the following groups: PlmCasX, DpbCasX.

A "vector" is a composition of matter comprising an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. In general, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites and one or more selectable markers. The term "vector" should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like.

In some embodiments, the vector is a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex virus vectors, and derivatives thereof. In some embodiments, the vector is a phage vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), among other handbooks of virology and molecular biology.

A number of virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The heterologous nucleic acid can be inserted into a vector and packaged in retroviral particles using techniques known in the art. Recombinant virus can then be isolated and delivered to the engineered mammalian cells in vitro or ex vivo. Many retroviral systems are known in the art. In some embodiments, an adenoviral vector is used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. In some embodiments, a self-inactivating lentiviral vector is used.

In certain embodiments, the vector is an adeno-associated virus (AAV) vector, such as AAV2, AAV8, or AAV9, which can administer the adenovirus or adeno-associated virus in a single dose comprising at least 1 x ¹⁰⁵ particles (also known as particle units, pu). In some embodiments, the administered amount is at least about 1×10 ⁶ particles, at least about 1×10 ⁷ particles, at least about 1×10 ⁸ particles, or at least about 1×10 ⁹ particles of adeno-associated virus. Delivery methods and dosages are described, for example, in WO 2016205764 and US Patent No. 8,454,972, which are incorporated herein by reference in their entirety.

In some embodiments, the vector is a recombinant adeno-associated virus (rAAV) vector. For example, in some embodiments, modified AAV vectors can be used for delivery. Modified AAV vectors can be based on one or more of several capsid types, including AAV1, AAV2, AAV5, AAV6, AAV8, AAV8.2, AAV9, AAV rh10, modified AAV vectors (e.g., modified AAV2, modified AAV3, modified AAV6) and pseudotyped AAV (e.g., AAV2/8, AAV2/5, and AAV2/6). Exemplary AAV vectors and techniques that can be used to generate rAAV particles are known in the art (see, e.g., Aponte-Ubillus et al. (2018) Appl. Microbiol. Biotechnol. 102(3): 1045-54; Zhong et al. (2012) J. Genet. Syndr. Gene Ther. 160:38-47 (1987); Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-60; U.S. Patent Nos. 4,797,368 and 5,173,414; International Publication Nos. WO2015/054653 and WO93/24641, each of which is incorporated herein by reference).

Any known AAV vector used to deliver Cas9 and other Cas proteins can be used to deliver the engineered CasX system of the present application.

Also provided are compositions, kits, unit doses, and articles of manufacture comprising one or more components of any of the engineered CasX nucleases, engineered CasX effector proteins, or engineered CRISPR-CasX systems described herein.

In some embodiments, kits are provided comprising: one or more AAV vectors encoding any of the engineered CasX nucleases, engineered CasX effector proteins, or engineered CRISPR-CasX systems described herein. In some embodiments, the kit further comprises one or more guide RNAs. In some embodiments, the kit further comprises donor DNA. In some embodiments, the kit further comprises cells such as human cells.

The kit may comprise one or more additional components, such as containers, reagents, media, cytokines, buffers, antibodies, etc., to allow propagation of the engineered cells. The kit may also comprise a device for administering the composition.

The kit may also comprise instructions for using the engineered CRISPR-CasX system described herein, such as methods for detecting or modifying target nucleic acids. In some embodiments, the kit comprises instructions for treating or diagnosing the disease or condition. The instructions pertaining to the use of the kit components will generally include information on the amount to be administered, the schedule of administration and the route of administration for such deliberate treatment. The container can be a unit dose, bulk package (eg, a multi-dose package), or a subunit dose. For example, kits comprising sufficient doses of a composition disclosed herein can be provided to provide effective treatment of an individual over an extended period of time. The kit can also include a plurality of unit doses of the composition and instructions for use packaged in quantities sufficient for storage and use in a pharmacy (eg, hospital pharmacy and compounding pharmacy).

The kits of the invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (eg, sealed mylar or plastic bags), and the like. Kits may optionally provide additional components such as buffers and explanatory information. Accordingly, the present application also provides an article of manufacture including vials (eg, sealed vials), bottles, jars, flexible packaging, and the like.

The article of manufacture may comprise a container and a label or package insert on or adhered to the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container can be formed from a variety of materials such as glass or plastic. Typically, the container contains a composition effective to treat a disease or condition described herein, and may have a sterile access port (eg, the container may be a bag of intravenous solution or a vial with a stopper pierceable by a hypodermic needle). The label or package insert indicates that the composition is used to treat a particular condition in an individual. The label or package insert will further include instructions for administering the composition to the individual.

Package Insert means the instructions commonly included in commercial packages of therapeutic products that contain information on the indications, usage, dosage, administration, contraindications and/or warnings regarding the use of such therapeutic products. Additionally, the article of manufacture may also include a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. From a commercial and user perspective, it can also include other materials, including other buffers, diluents, filters, needles, and syringes.

Example part

Specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although specific embodiments of the invention are shown in the drawings, it should be understood that the invention may be embodied in various forms and is not limited to the embodiments set forth herein. Rather, these embodiments are provided for more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

Example 1: Replace the amino acid that interacts with PAM in the reference PlmCasX enzyme with a positively charged amino acid, and verify its gene editing efficiency.

Plasmid construction

We loaded the DNA sequences encoding wild-type PlmCasX and mutants into the pCAG-2A-EGFP plasmid to construct a CasX expression plasmid. A vector expressing Cas protein crRNA or sgRNA in 293T was constructed by ligating the annealed oligonucleotide containing the target sequence into the BasI-digested pUC19-U6-crRNA/sgRNA backbone.

Cell culture, transfection, and fluorescence-activated cell sorting (FACS)

HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-cell culture dishes (Corning) for 16 hours until the cell density reached 70%. By using Lipofectamine 3000 (Invitrogen), 600ng of a plasmid encoding a Cas protein and 300ng of a plasmid encoding a crRNA were transfected into cells cultured in a 24-well cell culture dish. 120 h after transfection, cells were digested with trypsin-EDTA (0.05%) (Gibco), and then subjected to fluorescence-activated cell sorting (FACS).

Targeted Deep Sequencing Analysis for Genome Modifications

FACS-sorted GFP-positive 293FT cells were lysed with buffer L and incubated at 55 °C for 3 h, then at 95 °C for 10 min. dsDNA fragments containing target sites in different genomic loci were PCR amplified using the corresponding primers. For targeted deep sequencing, target loci are directly amplified by barcoded PCR using cell lysates as templates. PCR products were purified and pooled into several libraries for high-throughput sequencing. The frequency (%) of indels was analyzed using CRISPResso2 software by calculating the ratio of reads containing indels or indels. Reads with an amount less than 0.05% of complete reads were discarded.

Example 1-A selects three engineered PlmCasXs with single amino acid substitutions

According to the method described in Example 1, engineered PlmCasX enzymes with a single mutation in the amino acid sequence were respectively expressed, and the preferred amino acid replacement method and its corresponding gene editing efficiency are shown in Figure 1 and Table 1. In Figure 1, we first selected the distance between PlmCasX and PAM DNA Within 14 amino acids: T26, G27, M29, P105, D195, S198, E204, S222, S230, Q512, S564, G565, K610, E640 for arginine (R) point mutation test. By comparing these mutants with wild-type PlmCasX in 293T cells at 2 genomic loci: AAVS1-2, CCR5-2 We found that mutants with the following amino acid substitutions: T26R, K610R and E640R can effectively improve the gene editing efficiency, especially T26R, which can increase the efficiency by 36 times (this is the gene editing efficiency of the reference enzyme for AAVS1-2) and 37 times (this is the gene editing efficiency of the reference enzyme for CCR5-2). The mutants S230R, Q512R, and S564R have serious negative effects on the improvement of PlmCasX efficiency.

Table 1

Example 1-B selects engineered PlmCasX with two amino acid substitutions

According to the method described in Example 1, engineered PlmCasX enzymes with a single mutation in the amino acid sequence were respectively expressed, and the preferred amino acid replacement method and its corresponding gene editing efficiency are shown in Figure 2 and Table 2. In Figure 2, we superimposed the T26R and K610R mutants described in Example 1-A to construct a new PlmCasX mutant: T26R+K610R. By comparing these mutants with wild-type PlmCasX in 293T cells at 2 genomic loci: AAVS1-2, For the gene editing efficiency of CCR5-2, we found that the engineered PlmCasX with a double point mutation (T26R+K610R) had the highest gene editing efficiency at all four tested genomic loci relative to the engineered PlmCasX with a single point mutation.

Table 2:

Example 2: Replace the amino acids in the reference PlmCasX enzyme that interact with the ssDNA substrate with positively charged amino acids, and verify its gene editing efficiency.

Plasmid construction

We loaded the DNA sequences encoding wild-type PlmCasX and mutants into the pCAG-2A-EGFP plasmid to construct a CasX expression plasmid. A vector expressing Cas protein crRNA or sgRNA in 293T was constructed by ligating the annealed oligonucleotide containing the target sequence into the BasI-digested pUC19-U6-crRNA/sgRNA backbone.

Cell culture, transfection, and fluorescence-activated cell sorting (FACS)

HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-cell culture dishes (Corning) for 16 hours until the cell density reached 70%. By using Lipofectamine 3000 (Invitrogen), 600ng of a plasmid encoding a Cas protein and 300ng of a plasmid encoding a crRNA were transfected into cells cultured in a 24-well cell culture dish. 120 h after transfection, cells were digested with trypsin-EDTA (0.05%) (Gibco), and then subjected to fluorescence-activated cell sorting (FACS).

Targeted Deep Sequencing Analysis for Genome Modifications

FACS-sorted GFP-positive 293T cells were lysed with buffer L and incubated at 55 °C for 3 h, then at 95 °C for 10 min. dsDNA fragments containing target sites in different genomic loci were PCR amplified using the corresponding primers. For targeted deep sequencing, target loci are directly amplified by barcoded PCR using cell lysates as templates. PCR products were purified and pooled into several libraries for high-throughput sequencing. The frequency (%) of indels was analyzed using CRISPResso2 software by calculating the ratio of reads containing indels or indels. Reads with an amount less than 0.05% of complete reads were discarded.

An engineered PlmCasX with a single amino acid substitution was selected

According to the method described in Example 2, engineered PlmCasX enzymes with a single mutation in the amino acid sequence were respectively expressed, and the preferred amino acid replacement mode and its corresponding gene editing efficiency are shown in Figure 3 and Table 3. In Figure 3, we first selected the PlmCasX distance ssDNA The 16 amino acids within: G661, E662, N663, K699, N757, L758, S759, Q765, G766, E773, T806, K808, H903, Q907, E908, F958 were tested for point mutations of arginine (R). By comparing the gene editing efficiency of these mutants and wild-type PlmCasX at two genomic loci: AAVS1-2, CCR5-2 in 293T cells, we found that the mutants with the following amino acid substitutions: S759R and K808R can effectively improve gene editing efficiency. However, multiple mutants had severe negative effects on the improvement of PlmCasX efficiency.

table 3

Example 3: Combine some of the amino acid mutants obtained from the screening in Examples 1-2 that can improve the gene editing efficiency of the wild-type PlmCasX, and verify their gene editing efficiency.

Plasmid construction

We loaded the DNA sequences encoding wild-type PlmCasX and mutants into the pCAG-2A-EGFP plasmid to construct a CasX expression plasmid. A vector expressing Cas protein crRNA or sgRNA in 293T was constructed by ligating the annealed oligonucleotide containing the target sequence into the BasI-digested pUC19-U6-crRNA/sgRNA backbone.

Cell culture, transfection, and fluorescence-activated cell sorting (FACS)

HEK293T cells were cultured in DMEM (Gibco) containing 1% penicillin-streptomycin (Gibco) and 10% fetal bovine serum (Gibco). Cells were seeded in 24-cell culture dishes (Corning) for 16 hours until the cell density reached 70%. By using Lipofectamine 3000 (Invitrogen), 600ng of a plasmid encoding a Cas protein and 300ng of a plasmid encoding a crRNA were transfected into cells cultured in a 24-well cell culture dish. 120 h after transfection, cells were digested with trypsin-EDTA (0.05%) (Gibco), and then subjected to fluorescence-activated cell sorting (FACS).

Targeted Deep Sequencing Analysis for Genome Modifications

FACS-sorted GFP-positive 293FT cells were lysed with buffer L and incubated at 55 °C for 3 h, then at 95 °C for 10 min. dsDNA fragments containing target sites in different genomic loci were PCR amplified using the corresponding primers. For targeted deep sequencing, target loci are directly amplified by barcoded PCR using cell lysates as templates. PCR products were purified and pooled into several libraries for high-throughput sequencing. The frequency (%) of indels was analyzed using CRISPResso2 software by calculating the ratio of reads containing indels or indels. Reads with an amount less than 0.05% of complete reads were discarded.

An engineered PlmCasX with optimal amino acid substitutions was selected

According to the method described in Example 3, the engineered PlmCasX enzymes with single and multiple mutations in the amino acid sequence were respectively expressed, and the preferred amino acid replacement method and its corresponding gene editing efficiency are shown in Figure 4 and Table 4. In Figure 4, we further combined the optimal point mutation combination T26R+K610R obtained in Example 1-B and the optimal point mutation K808R in Example 2 into a protein, By comparing the gene editing efficiency of this new mutant (T26R+K610R+K808R) and wild-type PlmCasX at four genomic loci: AAVS1-2, AAVS1-7, CCR5-2 and CD34-1 in 293T cells, we found that the mutant T26R+K610R+K808R had the highest gene editing efficiency at all four tested genomic loci. Compared with wild-type PlmCasX, the gene editing efficiency of T26R+K610R+K808R was increased by 474 times (this is the gene editing efficiency of the reference enzyme for AAVS1-2), 16 times (this is the gene editing efficiency of the reference enzyme for AAVS1-7), 152 times (this is the gene editing efficiency of the reference enzyme for CCR5-2) and 287 times (this is the gene editing efficiency of the reference enzyme for CD34-1 efficiency).

Table 4

Although the embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to the above-mentioned specific embodiments and application fields, and the above-mentioned specific embodiments are only illustrative and instructive, rather than restrictive. Under the enlightenment of this specification and without departing from the protection scope of the claims of the present invention, those skilled in the art can also make many forms, which all belong to the protection of the present invention.

sequence listing

Claims (17)

An engineered CasX nuclease; it comprises one or more mutations based on the reference CasX nuclease, the mutation is: the amino acid in the reference CasX nuclease that interacts with nucleic acid is replaced with a positively charged amino acid; the reference CasX nuclease is a natural wild-type CasX nuclease. The engineered CasX nuclease of claim 1, wherein the natural wild-type CasX nuclease is selected from any one of the following groups: PlmCasX, DpbCasX. The engineered CasX nuclease according to claim 2, wherein the one or more amino acids interacting with the nucleic acid are amino acids within 9 angstroms in the three-dimensional structure, especially one or more amino acids at the following positions: 26, 27, 29, 105, 195, 198, 204, 222, 230, 512, 564, 565, 610, 640; wherein the amino acid position numbering is as defined in SEQ ID NO.1. The engineered CasX nuclease according to claim 3, wherein the positively charged amino acid is R or K; preferably the positively charged amino acid is R. The engineered CasX nuclease according to claim 4, wherein, the replacement of one or more amino acids interacting with PAM in the reference CasX nuclease with a positively charged amino acid refers to one or more of the following replacements: T26R, K610R, K640, S759R, K808R; preferably, the engineered CasX nuclease comprises any one of the following mutations or mutation combinations: (1) T26R; (2) K610R; (3) K6 40R; (4) S759R; (5) K808R; (6) T26R and K610R; (7) T26R and K610R and K808R. Comprising an amino acid sequence as shown in any one of SEQ ID NO.2-8 or an engineered CasX nuclease having at least 95% identity with the amino acid sequence shown in any one of SEQ ID NO.2-8. An engineered CasX effector protein comprising the engineered CasX nuclease or functional derivative thereof according to any one of claims 1-6. The engineered CasX effector protein of claim 7, wherein said effector protein is capable of inducing double-strand breaks or single-strand breaks in DNA molecules. The engineered CasX effector protein of claim 7, wherein the functional derivative of the engineered CasX nuclease is an enzyme inactive mutant. The engineered CasX effector protein according to any one of claims 7 to 9, further comprising a functional domain fused with the engineered CasX nuclease. The engineered CasX effector protein as claimed in claim 10, wherein said functional domain is selected from the group consisting of translation initiation domain, transcription repression domain, transactivation domain, epigenetic modification domain, nucleobase editing domain, reverse transcriptase domain, reporter domain and nuclease domain. The engineered CasX effector protein according to any one of claims 7 to 11, comprising a first polypeptide and a second polypeptide, the first polypeptide comprising amino acid residues 1 to X of the N-terminal portion of the engineered CasX nuclease described in any one of claims 1-6, the second polypeptide comprising amino acid residues X+1 to the C-terminal of the CasX nuclease engineered in any one of claims 1-6, wherein said first polypeptide and said second polypeptide can be associated with each other in the presence of a guide RNA comprising a guide sequence to form an A Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) complex specifically binds a target nucleic acid comprising a target sequence complementary to the guide sequence. An engineered CRISPR-CasX system comprising: (a) the engineered CasX effector protein of any one of claims 7-12; and (b) a guide RNA comprising a guide sequence complementary to a target sequence, or one or more nucleic acids encoding said guide RNA, Wherein the engineered CasX effector protein and the guide RNA are capable of forming a CRISPR complex that specifically binds to a target nucleic acid comprising the target sequence and induces modification of the target nucleic acid. A method for detecting target nucleic acid in a sample, comprising: (a) contacting the sample with the engineered CRISPR-CasX system of claim 13 and a tagged detection nucleic acid that is single stranded and does not hybridize to the guide sequence of the guide RNA; and (b) measuring a detectable signal generated by cleavage of the tagged detection nucleic acid by the engineered CasX effector protein, thereby detecting the target nucleic acid. Use of the engineered CRISPR-CasX system as claimed in claim 13 in the preparation of a medicament for treating a disease or a disease associated with a target nucleic acid in an individual's cells; preferably, the disease or disease is selected from the group consisting of cancer, cardiovascular disease, genetic disease, autoimmune disease, metabolic disease, neurodegenerative disease, eye disease, bacterial infection, and viral infection. A method of modifying a target nucleic acid comprising a target sequence comprising contacting the target nucleic acid with the engineered CRISPR-CasX system of claim 13. A kit comprising: one or more AAV vectors encoding any one of the engineered CasX nuclease according to any one of claims 1-6, the engineered CasX effector protein according to claims 7-12 and any one of the engineered CRISPR-CasX system according to claim 13.