Improved activity cas proteins and uses thereof

Abstract

The present application belongs to the field of nucleic acid editing, in particular, the field of clustered regularly interspaced short palindromic repeats (CRISPR) technology. Specifically, the present application provides a Cas mutant protein with improved activity and increased editing efficiency. Compared with the wild-type parent Cas protein, the Cas mutant protein of the present application has significantly improved activity and has a wide application prospect.

Description

[0001] This application claims priority to Chinese patent application CN202311600750.8, filed on November 28, 2023. This application incorporates the entirety of the aforementioned Chinese patent application. TECHNICAL FIELD

[0002] The present application relates to the field of gene editing, in particular the field of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. Specifically, the present application relates to a mutant Cas protein with improved activity and editing efficiency, and applications thereof. BACKGROUND

[0003] CRISPR / Cas technology is a widely used gene editing technology that uses RNA to guide specific binding to target sequences on the genome and cleave DNA to produce double-strand breaks, and uses biological non-homologous end joining or homologous recombination for site-directed gene editing.

[0004] The CRISPR / Cas9 system is the most commonly used Type II CRISPR system, which recognizes the PAM motif of 3’-NGG and performs blunt-end cleavage on target sequences. The CRISPR / Cas Type V system is a newly discovered CRISPR system that recognizes the motif of 5’-TTN and performs sticky-end cleavage on target sequences, such as Cpf1, C2c1, CasX, and CasY. However, the different CRISPR / Cas systems currently available each have different advantages and disadvantages. For example, Cas9, C2c1, and CasX all require two RNA guide RNAs, while Cpf1 only requires one guide RNA and can be used for multiplex gene editing. CasX has a size of 980 amino acids, while common Cas9, C2c1, CasY, and Cpf1 typically have a size of about 1300 amino acids. In addition, the PAM sequences of Cas9, Cpf1, CasX, and CasY are complex and diverse, while C2c1 recognizes the stringent 5’-TTN, so its target sites are easier to predict than other systems, thereby reducing potential off-target effects.

[0005] Chinese patent CN111757889B discloses a Cas protein Cas12f.4, and also discloses that the protein can perform gene editing in eukaryotic cells. However, the editing activity of the protein is not high. In order to improve the editing efficiency of the protein, the present application optimizes the protein and improves its editing efficiency in eukaryotic cells. SUMMARY

[0006] The inventors of the present application have improved the editing activity of Cas12f.4 (referred to as Cas12i3 or Cas12i.3 in the present application) protein through site-directed mutagenesis, and expanded its application range.

[0007] Cas effector proteins

[0008] In one aspect, the present application provides a Cas mutant protein with improved activity or increased activity, which has a mutation at any one or any number of the following amino acid positions corresponding to the amino acid sequence shown in SEQ ID No. 1: position 60, position 82, position 107, position 349, position 561, position 719 or position 747, compared with the amino acid sequence of the parent Cas protein.

[0009] The above-mentioned amino acid positions refer to the positions from the N-terminus of SEQ ID No. 1.

[0010] In one embodiment, the 60th amino acid is mutated to an amino acid other than K, for example, A, V, G, L, Q, F, W, Y, D, N, E, S, M, T, C, P, H, R, I; preferably, it is mutated to R.

[0011] In one embodiment, the 82nd amino acid or the 349th amino acid is mutated to an amino acid other than S, for example, A, V, G, L, Q, F, W, Y, D, N, E, K, M, T, C, P, H, R, I; preferably, the 82nd amino acid or the 349th amino acid is mutated to T.

[0012] In one embodiment, the 107th amino acid is mutated to an amino acid other than I, for example, A, V, G, L, Q, P, W, Y, D, S, E, K, M, T, C, N, H, R, F; preferably, it is mutated to V.

[0013] In one embodiment, the 561st amino acid or the 747th amino acid is mutated to an amino acid other than L, for example, A, V, G, N, Q, F, W, Y, D, S, E, K, M, T, C, P, H, R, I; preferably, the 561st amino acid or the 747th amino acid is mutated to F.

[0014] In one embodiment, the 719th amino acid is mutated to an amino acid other than A, for example, F, V, G, L, Q, P, W, Y, D, S, E, K, M, T, C, N, H, R, I; preferably, it is mutated to V.

[0015] In one embodiment, the amino acid sequence of the parent Cas protein has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID No. 1.

[0016] In one embodiment, the parent Cas protein is a Cas protein of the Cas12 family, preferably a Cas protein of the Cas12i family, e.g., Cas12il, Cas12i2, Cas12i3, etc.

[0017] It is clear to the person skilled in the art that the structure of a protein can be changed without adversely affecting its activity and functionality, e.g., one or more conservative amino acid substitutions can be introduced into the amino acid sequence of a protein without adversely affecting the activity and / or three-dimensional structure of the protein molecule. Examples of conservative amino acid substitutions and embodiments are clear to the person skilled in the art. Specifically, an amino acid residue can be replaced with another amino acid residue belonging to the same group, i.e., a non-polar amino acid residue is replaced with another non-polar amino acid residue, a polar uncharged amino acid residue is replaced with another polar uncharged amino acid residue, a basic amino acid residue is replaced with another basic amino acid residue, and an acidic amino acid residue is replaced with another acidic amino acid residue. Such substituted amino acid residues can or can not be encoded by the genetic code. A conservative substitution in which one amino acid is replaced with another amino acid belonging to the same group falls within the scope of the present application, provided that the substitution does not result in the inactivation of the biological activity of the protein. Thus, the proteins of the present application can comprise one or more conservative substitutions in the amino acid sequence, which are preferably replaced with substitutions resulting from Table 1. In addition, the present application also encompasses proteins comprising one or more other non-conservative substitutions, provided that the non-conservative substitution does not significantly affect the desired functions and biological activities of the proteins of the present application.

[0018] Conservative amino acid substitutions can be made at one or more predicted nonessential amino acid residues. A "nonessential" amino acid residue is a residue that can be altered (deleted, substituted, or replaced) without altering the biological activity of the protein, while an "essential" amino acid residue is required for biological activity. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Amino acid substitutions can be made in non-conserved regions of the Cas mutant proteins described above. In general, such substitutions are not made in conserved amino acid residues, or in active site residues located within conserved motifs, where such residues are required for protein activity. However, it will be appreciated by one of skill in the art that functional variants can have fewer conserved or non-conserved alterations in conserved regions.

[0019] Table 1

[0020]

[0021]

[0022] It is well understood in the art that one or more amino acid residues can be altered (substituted, deleted, truncated, or inserted) from the N- and / or C-terminus of a protein while still retaining its functional activity. Thus, proteins having one or more amino acid residues altered from the N- and / or C-terminus of a Cas protein while retaining its desired functional activity are also within the scope of the present application. These alterations can include alterations introduced by modern molecular methods, such as PCR, including PCR amplification of a protein coding sequence by inclusion of an amino acid encoding sequence in the oligonucleotides used in the PCR amplification.

[0023] It is recognized that proteins can be altered in a variety of ways, including amino acid substitutions, deletions, truncations, and insertions, and methods for such manipulations are generally known in the art. For example, amino acid sequence variants of the above-described proteins can be prepared by mutations of the DNA. They can also be accomplished by other forms of mutagenesis and / or by directed evolution, e.g., using known mutagenesis, recombination, and / or shuffling methods, in combination with relevant screening methods, to make single or multiple amino acid substitutions, deletions, and / or insertions.

[0024] It will be appreciated by one of skill in the art that these minor amino acid changes in the Cas proteins of the present application can occur (e.g., naturally-occurring mutations) or be produced (e.g., using r-DNA technology) without loss of protein function or activity. If the mutations occur in the catalytic domain, active site, or other functional domain of the protein, the properties of the polypeptide can change, but the polypeptide can retain its activity. If the mutations are not near the catalytic domain, active site, or other functional domain, less impact can be expected.

[0025] The essential amino acids of the Cas mutant proteins of the application can be determined by those of skill in the art, according to methods known in the art, such as site-directed mutagenesis or protein evolution or bioinformatic analysis. The catalytic domain, active site, or other functional domains of a protein can also be determined by physical analysis of the structure, such as by nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutagenesis of putative key site amino acids.

[0026] In the present application, the amino acid residues can be represented by single letter or three letter codes, such as: alanine (Ala, A), valine (Val, V), glycine (Gly, G), leucine (Leu, L), glutamine (Gln, Q), phenylalanine (Phe, F), tryptophan (Trp, W), tyrosine (Tyr, Y), aspartic acid (Asp, D), asparagine (Asn, N), glutamic acid (Glu, E), lysine (Lys, K), methionine (Met, M), serine (Ser, S), threonine (Thr, T), cysteine (Cys, C), proline (Pro, P), isoleucine (lie, I), histidine (His, H), arginine (Arg, R).

[0027] The term "AxxB" means that the amino acid A at position xx is changed to amino acid B, such as K60R, which means that the K at position 60 is changed to R. When multiple amino acid positions are changed simultaneously, the changes can be represented in a similar fashion, such as K60R-S82T, which means that the K at position 60 is changed to R and the S at position 82 is changed to T.

[0028] The specific amino acid positions (numbering) within the protein according to the present application are determined by aligning the amino acid sequence of the protein of interest with SEQ ID No. 1 using standard sequence alignment tools, such as aligning the two sequences using the Smith-Waterman algorithm or using the CLUSTALW2 algorithm, wherein the sequences are considered aligned when the alignment score is highest. The alignment score can be calculated according to the method described in Wilbur, W. J. and Lipman, D. J. (1983) Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA, 80:726-730. In the ClustalW2 (1.82) algorithm it is preferred to use the default parameters: Protein gap open penalty = 10.0; Protein gap extension penalty = 0.2; Protein matrix = Gonnet; Protein / DNA end gap = -1; Protein / DNA GAP DIST = 4. It is preferred to use the AlignX program (part of the vector NTI suite) to determine the positions of the specific amino acids within the protein according to the present application by aligning the amino acid sequence of the protein with SEQ ID No. 1 using the default parameters for multiple alignment (gap open penalty: 10 gap extension penalty 0.05).

[0029] In one embodiment, the amino acid sequence of the parent Cas protein has at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1 %, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to SEQ ID No. 1.

[0030] In some embodiments, the parent Cas protein is a naturally occurring wild-type Cas protein; in other embodiments, the parent Cas protein is an engineered Cas protein.

[0031] In one embodiment, the parent Cas protein is a Cas protein of the Cas12 family, preferably a Cas protein of the Cas12i family, for example, Cas12il, Cas12i2, Cas12i3.

[0032] Cas proteins or Casl2i proteins from a variety of organisms can be used as a parent Cas protein, which in some embodiments has nuclease activity. In some embodiments, the parent Cas protein is a nuclease, i.e., cleaves both strands of a target double-helical nucleic acid (e.g., double-helical DNA). In some embodiments, the parent Cas protein is a nickase, i.e., cleaves a single strand of a target double-helical nucleic acid (e.g., double-helical DNA).

[0033] In one embodiment, the Cas mutant protein is selected from any one of the following groups I-III:

[0034] I, a Cas mutant protein resulting from a mutation of the amino acid sequence set forth in SEQ ID No. 1 at any one or any number of the following amino acid positions: position 60, position 82, position 107, position 349, position 561, position 719, or position 747;

[0035] II, a Cas mutant protein having the mutation positions described in I; and having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the Cas mutant protein described in I;

[0036] III, a Cas mutant protein having the mutation positions described in I; and having a sequence with substitution, deletion, or addition of one or more amino acids compared to the Cas mutant protein described in I; the one or more amino acids include substitution, deletion, or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.

[0037] Biological functions of the Cas protein include, but are not limited to, an activity of binding to a guide RNA, an endonuclease activity, an activity of binding to a specific site of a target sequence and cleaving under the guidance of a guide RNA, including but not limited to Cis cleavage activity and Trans cleavage activity.

[0038] In the present application, the Cas mutant protein can also be referred to as a mutant Cas protein, or a Cas protein variant.

[0039] The present application also provides a fusion protein comprising a Cas mutant protein as described above and other modified moieties.

[0040] In one embodiment, the modified moiety is selected from another protein or polypeptide, a detectable label, or any combination thereof.

[0041] In one embodiment, the modification moiety is selected from the group consisting of an epitope tag, a reporter gene sequence, a nuclear localization signal (NLS) sequence, a targeting moiety, a transcriptional activation domain (e.g., VP64), a transcriptional repression domain (e.g., KRAB domain or SID domain), a nuclease domain (e.g., Fokl), and a domain having an activity selected from the group consisting of a nucleotide deaminase, a methylase activity, a demethylase, a transcriptional activation activity, a transcriptional repression activity, a transcriptional release factor activity, a histone modification activity, a nuclease activity, a single-stranded RNA cleavage activity, a double-stranded RNA cleavage activity, a single-stranded DNA cleavage activity, a double-stranded DNA cleavage activity, and a nucleic acid binding activity; and any combination thereof. The NLS sequence is well known to one of skill in the art, examples of which include but are not limited to the SV40 large T antigen, EGL-13, c-Myc, and TUS proteins.

[0042] In one embodiment, the NLS sequence is located at, near, or proximal to the terminus (e.g., N-terminus, C-terminus, or both) of the Cas protein of the application.

[0043] The epitope tag is well known to one of skill in the art, including but not limited to His, V5, FLAG, HA, Myc, VSV-G, Trx, and the like, and one of skill in the art can select other suitable epitope tags (e.g., for purification, detection, or tracking).

[0044] The reporter gene sequence is well known to one of skill in the art, examples of which include but are not limited to GST, HRP, CAT, GFP, HcRed, DsRed, CFP, YFP, BFP, and the like.

[0045] In one embodiment, the fusion protein of the application comprises a domain capable of binding to a DNA molecule or an intracellular molecule, such as maltose binding protein (MBP), the DNA binding domain (DBD) of Lex A, the DBD of GAL4, and the like.

[0046] In one embodiment, the fusion protein of the application comprises a detectable label, such as a fluorescent dye, e.g., FITC or DAPI.

[0047] In one embodiment, the Cas protein of the application is optionally coupled, conjugated, or fused to the modification moiety via a linker.

[0048] In one embodiment, the modification moiety is directly linked to the N-terminus or C-terminus of the Cas protein of the application.

[0049] In one embodiment, the modification moiety is linked to the N- or C-terminus of the Cas protein of the present application via a linker. Such linkers are well known in the art, examples of which include, but are not limited to, linkers comprising one or more (e.g., 1, 2, 3, 4, or 5) amino acids (e.g., Glu or Ser) or amino acid derivatives (e.g., Ahx, β-Ala, GABA, or Ava), or PEG, etc.

[0050] The Cas protein, protein derivative, or fusion protein of the present application is not limited by the way it is produced, for example, it can be produced by a genetic engineering method (recombinant technology), or by a chemical synthesis method.

[0051] Nucleic acids of Cas proteins

[0052] In another aspect, the present application provides an isolated polynucleotide comprising:

[0053] (a) a polynucleotide sequence encoding the Cas mutant protein or fusion protein of the present application;

[0054] or a polynucleotide complementary to the polynucleotide of (a).

[0055] In one embodiment, the nucleotide sequence is codon-optimized for expression in a prokaryotic cell. In one embodiment, the nucleotide sequence is codon-optimized for expression in a eukaryotic cell.

[0056] In one embodiment, the cell is an animal cell, for example, a mammalian cell.

[0057] In one embodiment, the cell is a human cell.

[0058] In one embodiment, the cell is a plant cell, for example, a cell of a cultivated plant (e.g., cassava, corn, sorghum, wheat, or rice), an alga, a tree, or a vegetable.

[0059] In one embodiment, the polynucleotide is preferably single-stranded or double-stranded.

[0060] Guide RNA (gRNA)

[0061] In another aspect, the present application provides a gRNA, which comprises a first segment and a second segment; the first segment is also known as a "scaffold region", a "protein-binding segment", a "protein-binding sequence", or a "direct repeat sequence"; the second segment is also known as a "targeting sequence of a targeting nucleic acid" or a "targeting segment of a targeting nucleic acid", or a "guide sequence targeting a target sequence".

[0062] The first segment of the gRNA is capable of interacting with the Cas protein of the present application, thereby allowing the Cas protein and the gRNA to form a complex.

[0063] In a preferred embodiment, the first segment is a direct repeat sequence as described above.

[0064] The targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid of the present application comprises a nucleotide sequence that is complementary to a sequence in the target nucleic acid. In other words, the targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid of the present application interacts with the target nucleic acid in a sequence-specific manner via hybridization (i.e., base pairing). Thus, the targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid can be altered, or can be modified to hybridize to any desired sequence within the target nucleic acid. The nucleic acid is selected from DNA or RNA.

[0065] The percentage of complementarity between the targeting sequence of the targeting nucleic acid or the targeting segment of the targeting nucleic acid and the target sequence of the target nucleic acid can be at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%).

[0066] The "scaffold region", "protein-binding segment", "protein-binding sequence", or "direct repeat sequence" of the gRNA of the present application can interact with the CRISPR protein (or, Cas protein). The gRNA of the present application, through the targeting sequence of the targeting nucleic acid, directs the Cas protein it interacts with to a specific nucleotide sequence within the target nucleic acid.

[0067] Preferably, the guide RNA comprises a first segment and a second segment from 5' to 3' direction.

[0068] In the present application, the second segment can also be understood as a guide sequence that hybridizes to the target sequence.

[0069] The gRNA of the present application is capable of forming a complex with the Cas protein.

[0070] Vectors

[0071] The present application also provides a vector comprising the Cas mutant protein, the isolated nucleic acid molecule or the polynucleotide as described above; preferably, it further comprises a regulatory element operably linked thereto.

[0072] In one embodiment, the regulatory element is selected from one or more of the group consisting of enhancer, transposon, promoter, terminator, leader sequence, polyadenylation sequence, marker gene.

[0073] In one embodiment, the vector comprises a cloning vector, an expression vector, a shuttle vector, an integrating vector.

[0074] In some embodiments, the vectors included in the system are viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors), and can also be plasmids, viruses, cosmids, bacteriophages, and the like, which are well known to those skilled in the art.

[0075] CRISPR system

[0076] The present application provides an engineered non-naturally occurring vector system, or a CRISPR-Cas system, comprising a Cas mutant protein or a nucleic acid sequence encoding the Cas mutant protein and a nucleic acid encoding one or more guide RNAs.

[0077] In one embodiment, the nucleic acid sequence encoding the Cas mutant protein and the nucleic acid encoding one or more guide RNAs are artificially synthesized.

[0078] In one embodiment, the nucleic acid sequence encoding the Cas mutant protein and the nucleic acid encoding one or more guide RNAs are not naturally co-existing.

[0079] The one or more guide RNAs target one or more target sequences in a cell. The one or more target sequences hybridize to a genomic locus of a DNA molecule encoding one or more gene products, and direct the Cas protein to the genomic locus of the DNA molecule encoding the one or more gene products, where the Cas protein modifies, edits, or cleaves the target sequence, whereby the expression of the one or more gene products is altered or modified.

[0080] The cell of the present application comprises one or more of an animal, a plant, or a microorganism.

[0081] In some embodiments, the Cas protein is codon-optimized for expression in a cell.

[0082] In some embodiments, the Cas protein directs cleavage of one or two strands at the location of the target sequence.

[0083] The present application also provides an engineered non-naturally occurring vector system, which can comprise one or more vectors, the one or more vectors comprising:

[0084] a) a first regulatory element operably linked to a gRNA,

[0085] b) a second regulatory element operably linked to the Cas protein;

[0086] wherein components (a) and (b) are located on the same or different vectors of the system.

[0087] The first and second regulatory elements include promoters (e.g., constitutive promoters or inducible promoters), enhancers (e.g., 35S promoter or 35S enhanced promoter), internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals such as polyadenylation signals and poly-U sequences).

[0088] In some embodiments, the vectors in the system are viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors), and can also be plasmids, viruses, cosmids, bacteriophages, and the like, which are well known to those skilled in the art.

[0089] In some embodiments, the system provided herein is in a delivery system. In some embodiments, the delivery system is a nanoparticle, a liposome, an exosome, a microvesicle, and a gene gun.

[0090] In one embodiment, the target sequence is a DNA or RNA sequence from a prokaryotic cell or a eukaryotic cell. In one embodiment, the target sequence is a non-naturally occurring DNA or RNA sequence.

[0091] In one embodiment, the target sequence is present within a cell. In one embodiment, the target sequence is present within a nucleus or within a cytoplasm (e.g., organelle). In one embodiment, the cell is a eukaryotic cell. In other embodiments, the cell is a prokaryotic cell.

[0092] In one embodiment, the Cas protein is linked to one or more NLS sequences. In one embodiment, the fusion protein comprises one or more NLS sequences. In one embodiment, the NLS sequence is linked to the N-terminus or C-terminus of the protein. In one embodiment, the NLS sequence is fused to the N-terminus or C-terminus of the protein.

[0093] In another aspect, the present application relates to an engineered CRISPR system comprising the above-mentioned Cas protein and one or more guide RNAs, wherein the guide RNA comprises a direct repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid, and the Cas protein is capable of binding the guide RNA and targeting a target nucleic acid sequence complementary to the spacer sequence.

[0094] Protein-nucleic acid complexes / compositions

[0095] In another aspect, the present application provides a complex or composition comprising:

[0096] (i) a protein component selected from the group consisting of: a Cas protein, a derivatized protein or a fusion protein, and any combination thereof; and

[0097] (ii) a nucleic acid component comprising (a) a guide sequence capable of hybridizing to a target sequence; and (b) a direct repeat sequence capable of binding to a Cas protein of the present application.

[0098] The protein component and the nucleic acid component are associated with each other to form a complex.

[0099] In one embodiment, the nucleic acid component is a guide RNA in a CRISPR-Cas system.

[0100] In one embodiment, the complex or composition is non-naturally occurring or modified. In one embodiment, at least one component in the complex or composition is non-naturally occurring or modified. In one embodiment, the first component is non-naturally occurring or modified; and / or, the second component is non-naturally occurring or modified.

[0101] Activated CRISPR complexes

[0102] In another aspect, the present application also provides an activated CRISPR complex comprising: (1) a protein component selected from the group consisting of: a Cas protein, a derivatized protein or a fusion protein, and any combination thereof; (2) a gRNA comprising (a) a guide sequence capable of hybridizing to a target sequence; and (b) a direct repeat sequence capable of binding to a Cas protein of the present application; and (3) a target sequence bound to the gRNA. Preferably, the binding is by the target sequence of the targeting nucleic acid on the gRNA to the target nucleic acid.

[0103] The term "activated CRISPR complex", "activated complex" or "ternary complex" as used herein refers to a complex of a Cas protein, a gRNA and a target nucleic acid in a CRISPR system after binding or modification.

[0104] The Cas protein and the gRNA of the present application can form a binary complex that is activated upon binding to a nucleic acid substrate that is complementary to the spacer sequence in the gRNA (or alternatively, to the guide sequence that hybridizes to the target nucleic acid). In some embodiments, the spacer sequence of the gRNA is perfectly matched to the target substrate. In other embodiments, the spacer sequence of the gRNA is partially (contiguously or non-contiguously) matched to the target substrate.

[0105] In preferred embodiments, the activated CRISPR complex can exhibit collateral nuclease cleavage activity, which refers to the non-specific cleavage activity or the staggered cleavage activity exhibited by the activated CRISPR complex to single-stranded nucleic acids, also known in the art as trans cleavage activity.

[0106] Delivery and delivery compositions

[0107] The Cas proteins, gRNAs, fusion proteins, nucleic acid molecules, vectors, systems, complexes, and compositions of the application can be delivered by any method known in the art. Such methods include, but are not limited to, electroporation, lipofection, nucleofection, microinjection, sonoporation, biolistics, calcium phosphate-mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection, magnetofection, lipofection, perforation transfection, optical transfection, reagent-enhanced nucleic acid uptake, and delivery via liposomes, immunoliposomes, viral particles, artificial virions, and the like.

[0108] Accordingly, in another aspect, the application provides a delivery composition comprising a delivery vehicle, and one or any number of the following: a Cas protein, fusion protein, nucleic acid molecule, vector, system, complex, and composition of the application.

[0109] In one embodiment, the delivery vehicle is a particle.

[0110] In one embodiment, the delivery vehicle is selected from the group consisting of a lipid particle, a sugar particle, a metal particle, a protein particle, a liposome, an exosome, a microvesicle, a gene gun, or a viral vector (e.g., a replication-defective retrovirus, a lentivirus, an adenovirus, or an adeno-associated virus).

[0111] Host cells

[0112] The application also relates to a cell or cell line, or progeny thereof, in vitro, ex vivo, or in vivo, comprising a Cas protein, fusion protein, nucleic acid molecule, protein-nucleic acid complex, activated CRISPR complex, vector, delivery composition of the application.

[0113] In certain embodiments, the cell is a prokaryotic cell.

[0114] In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human cell. In certain embodiments, the cell is a non-human mammalian cell, e.g., a cell of a non-human primate, a bovine, an ovine, a porcine, a canine, a monkey, a rabbit, a rodent (e.g., a rat or a mouse). In certain embodiments, the cell is a non-mammalian eukaryotic cell, e.g., a cell of an avian bird (e.g., a chicken), a fish, or a crustacean (e.g., a clam, a shrimp). In certain embodiments, the cell is a plant cell, e.g., a cell of a monocotyledonous or dicotyledonous plant or a cell of a cultivated plant or a food crop such as cassava, corn, sorghum, soybean, wheat, oat, or rice, e.g., a cell of an alga, a tree, or a plant that produces a fruit or a vegetable (e.g., a tree such as a citrus tree, a nut tree; a solanaceous plant, cotton, tobacco, tomato, grape, coffee, cacao, etc.).

[0115] In certain embodiments, the cell is a stem cell or a stem cell line.

[0116] In certain instances, the host cell of the present application comprises a modification of a gene or genome that is not present in its wild type.

[0117] Methods and uses of gene editing

[0118] The Cas mutant protein, nucleic acid, composition, CRISPR / Cas system, vector system, delivery composition, or activated CRISPR complex, or host cell of the present application can be used for any one or any number of the following uses: targeting and / or editing a target nucleic acid; cleaving double-stranded DNA, single-stranded DNA, or single-stranded RNA; non-specifically cleaving and / or degrading a collateral nucleic acid; non-specifically cleaving a single-stranded nucleic acid; nucleic acid detection; detecting a nucleic acid in a sample of interest; specifically editing a double-stranded nucleic acid; base editing a double-stranded nucleic acid; base editing a single-stranded nucleic acid. In other embodiments, it can also be used for preparing a reagent or a kit for any one or any number of the above uses.

[0119] The present application also provides the use of the above-mentioned Cas protein, nucleic acid, composition, CRISPR / Cas system, vector system, delivery composition, or activated CRISPR complex in gene editing, gene targeting, or gene cleavage; or, in the preparation of a reagent or a kit for gene editing, gene targeting, or gene cleavage.

[0120] In one embodiment, the gene editing, gene targeting, or gene cleavage is performed in a cell and / or outside of a cell.

[0121] The present application also provides a method of editing, targeting or cleaving a target nucleic acid, comprising contacting the target nucleic acid with the above-mentioned Cas protein, nucleic acid, above-mentioned composition, above-mentioned CIRSPR / Cas system, above-mentioned vector system, above-mentioned delivery composition or above-mentioned activated CRISPR complex. In one embodiment, the method is editing, targeting or cleaving a target nucleic acid in or outside a cell.

[0122] The gene editing or editing a target nucleic acid includes modifying a gene, knocking out a gene, altering expression of a gene product, repairing a mutation, and / or inserting a polynucleotide, mutating a gene.

[0123] The editing can be performed in prokaryotic cells and / or eukaryotic cells.

[0124] In another aspect, the present application also provides use of the above-mentioned Cas protein, nucleic acid, above-mentioned composition, above-mentioned CIRSPR / Cas system, above-mentioned vector system, above-mentioned delivery composition or above-mentioned activated CRISPR complex in nucleic acid detection, or in the preparation of a reagent or kit for nucleic acid detection.

[0125] In another aspect, the present application also provides a method of cleaving a single-stranded nucleic acid, comprising contacting a population of nucleic acids with the above-mentioned Cas protein and gRNA, wherein the population of nucleic acids comprises a target nucleic acid and a plurality of non-target single-stranded nucleic acids, and the Cas protein cleaves the plurality of non-target single-stranded nucleic acids.

[0126] The gRNA is capable of binding to the Cas protein.

[0127] The gRNA is capable of targeting the target nucleic acid.

[0128] The contacting can be in vitro, ex vivo or inside a cell in vivo.

[0129] Preferably, the cleaving a single-stranded nucleic acid is non-specific cleaving.

[0130] In another aspect, the present application also provides use of the above-mentioned Cas protein, nucleic acid, above-mentioned composition, above-mentioned CIRSPR / Cas system, above-mentioned vector system, above-mentioned delivery composition or above-mentioned activated CRISPR complex in non-specific cleaving a single-stranded nucleic acid, or in the preparation of a reagent or kit for non-specific cleaving a single-stranded nucleic acid.

[0131] In another aspect, the present application also provides a kit for gene editing, gene targeting or gene cleaving, comprising the above-mentioned Cas protein, gRNA, nucleic acid, above-mentioned composition, above-mentioned CIRSPR / Cas system, above-mentioned vector system, above-mentioned delivery composition, above-mentioned activated CRISPR complex or above-mentioned host cell.

[0132] In another aspect, the present application also provides a kit for detecting a target nucleic acid in a sample, the kit comprising: (a) a Cas protein, or a nucleic acid encoding the Cas protein; (b) a guide RNA, or a nucleic acid encoding the guide RNA, or a precursor RNA comprising the guide RNA, or a nucleic acid encoding the precursor RNA; and (c) a single-stranded nucleic acid detector that is single-stranded and does not hybridize to the guide RNA.

[0133] It is known in the art that the precursor RNA can be cleaved or processed into the above-mentioned mature guide RNA.

[0134] In another aspect, the present application provides use of the above-mentioned Cas protein, nucleic acid, above-mentioned composition, above-mentioned CIRSPR / Cas system, above-mentioned vector system, above-mentioned delivery composition, above-mentioned activated CRISPR complex, or above-mentioned host cell in the preparation of a medicament or a kit for:

[0135] (i) gene or genome editing;

[0136] (ii) target nucleic acid detection and / or diagnosis;

[0137] (iii) modifying an organism or a non-human organism by editing a target sequence in a target locus;

[0138] (iv) treatment of a disease;

[0139] (iv) targeting a target gene.

[0140] Preferably, the above-mentioned gene or genome editing is performed in or outside a cell.

[0141] Preferably, the above-mentioned target nucleic acid detection and / or diagnosis is performed in vitro.

[0142] Preferably, the above-mentioned treatment of a disease is treatment of a disorder caused by a defect in a target sequence in a target locus.

[0143] In another aspect, the present application provides a method of detecting a target nucleic acid in a sample, the method comprising contacting the sample with a Cas protein, a gRNA (guide RNA) and a single-stranded nucleic acid detector, the gRNA comprising a region that binds to the Cas protein and a guide sequence that hybridizes to the target nucleic acid; detecting a detectable signal generated by cleavage of the single-stranded nucleic acid detector by the Cas protein, thereby detecting the target nucleic acid; the single-stranded nucleic acid detector does not hybridize to the gRNA.

[0144] Methods of specifically modifying target nucleic acids

[0145] In another aspect, the present application also provides a method of specifically modifying a target nucleic acid, the method comprising: contacting the target nucleic acid with the above-mentioned Cas protein, nucleic acid, above-mentioned composition, above-mentioned CIRSPR / Cas system, above-mentioned vector system, above-mentioned delivery composition, or above-mentioned activated CRISPR complex.

[0146] The specific modification can occur in vivo or in vitro.

[0147] The specific modification can occur in vivo or in vitro.

[0148] In some cases, the cell is selected from a prokaryotic cell or a eukaryotic cell, for example, an animal cell, a plant cell, or a microbial cell.

[0149] In one embodiment, the modification refers to a break of the target sequence, such as a single / double strand break of DNA, or a single strand break of RNA.

[0150] In some cases, the method further comprises contacting the target nucleic acid with a donor polynucleotide, wherein the donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of the copy of the donor polynucleotide is integrated into the target nucleic acid.

[0151] In one embodiment, the modification further comprises inserting an editing template (e.g., an exogenous nucleic acid) into the break.

[0152] In one embodiment, the method further comprises: contacting an editing template with the target nucleic acid, or delivering into a cell comprising the target nucleic acid. In this embodiment, the method repairs the broken target gene by homologous recombination with the exogenous template polynucleotide; in some embodiments, the repair results in a mutation, including an insertion, a deletion, or a substitution of one or more nucleotides of the target gene, and in other embodiments, the mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence.

[0153] Detection (non-specific cleavage)

[0154] In another aspect, the present application provides a method of detecting a target nucleic acid in a sample, the method comprising: contacting the sample with the above-mentioned Cas protein, nucleic acid, above-mentioned composition, above-mentioned CIRSPR / Cas system, above-mentioned vector system, above-mentioned delivery composition, or above-mentioned activated CRISPR complex and a single-stranded nucleic acid detector; detecting a detectable signal resulting from cleavage of the single-stranded nucleic acid detector by the Cas protein, thereby detecting the target nucleic acid.

[0155] In the present application, the target nucleic acid comprises ribonucleotides or deoxyribonucleotides; and includes single-stranded nucleic acids, double-stranded nucleic acids, such as single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA.

[0156] In one embodiment, the target nucleic acid is derived from a sample of virus, bacteria, microorganism, soil, water source, human, animal, plant, etc. Preferably, the target nucleic acid is the product of PCR, NASBA, RPA, SDA, LAMP, HAD, NEAR, MDA, RCA, LCR, RAM, etc.

[0157] In one embodiment, the target nucleic acid is viral nucleic acid, bacterial nucleic acid, specific nucleic acid associated with disease, such as specific mutation site or SNP site or nucleic acid different from control; preferably, the virus is plant virus or animal virus, for example, papillomavirus, hepadnavirus, herpesvirus, adenovirus, poxvirus, parvovirus, coronavirus; preferably, the virus is coronavirus, preferably, SARS, SARS-CoV2 (COVID-19), HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, Mers-Cov.

[0158] In the present application, the gRNA has at least 50% matching degree with the target sequence on the target nucleic acid, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90%.

[0159] In one embodiment, when the target sequence contains one or more characteristic sites (such as specific mutation site or SNP), the characteristic site is completely matched with the gRNA.

[0160] In one embodiment, the detection method can contain one or more gRNAs with different guide sequences targeting different target sequences.

[0161] In the present application, the single-stranded nucleic acid detector includes but is not limited to single-stranded DNA, single-stranded RNA, DNA-RNA hybrid, nucleic acid analog, base modifier, and single-stranded nucleic acid detector containing abasic spacer, etc.; "nucleic acid analog" includes but is not limited to: locked nucleic acid, bridged nucleic acid, morpholino nucleic acid, glycol nucleic acid, hexitol nucleic acid, threose nucleic acid, arabino nucleic acid, 2'oxymethyl RNA, 2'methoxyacetyl RNA, 2'fluoro RNA, 2'amino RNA, 4'sulfur RNA and combinations thereof, including optional ribonucleotide or deoxyribonucleotide residues.

[0162] In the present application, the detectable signal is realized by the following ways: visual detection, sensor-based detection, color detection, fluorescence signal-based detection, gold nanoparticle-based detection, fluorescence polarization, colloidal phase change / dispersion, electrochemical detection and semiconductor-based detection.

[0163] In the present application, preferably, the single-stranded nucleic acid detector is provided with a fluorescent group and a quencher group at its two ends, respectively, and can exhibit a detectable fluorescent signal after the single-stranded nucleic acid detector is cleaved. The fluorescent group is selected from one or any of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red, or LC RED460; and the quencher group is selected from one or any of BHQ1, BHQ2, BHQ3, Dabcy1, or Tamra.

[0164] In other embodiments, the 5' end and the 3' end of the single-stranded nucleic acid detector are respectively provided with different labeling molecules, and the colloidal gold detection method is used to detect the colloidal gold test results of the single-stranded nucleic acid detector before and after being cleaved by the Cas protein; the single-stranded nucleic acid detector before and after being cleaved by the Cas protein will exhibit different color development results on the colloidal gold detection line and the quality control line.

[0165] In some embodiments, the method of detecting a target nucleic acid can further include comparing the level of the detectable signal to a reference signal level, and determining the amount of the target nucleic acid in the sample based on the level of the detectable signal.

[0166] In some embodiments, the method of detecting a target nucleic acid can further include using RNA reporter nucleic acids and DNA reporter nucleic acids (e.g., fluorescent colors) on different channels, and determining the level of the detectable signal by measuring the signal level of the RNA and DNA reporter molecules, and by measuring the amount of the target nucleic acid in the RNA and DNA reporter molecules, based on the combination (e.g., using the minimum or product) of the level of the detectable signal to sample.

[0167] In one embodiment, the target gene is present in a cell.

[0168] In one embodiment, the cell is a prokaryotic cell.

[0169] In one embodiment, the cell is a eukaryotic cell.

[0170] In one embodiment, the cell is an animal cell.

[0171] In one embodiment, the cell is a human cell.

[0172] In one embodiment, the cell is a plant cell, such as a cell of a cultivated plant (e.g., cassava, corn, sorghum, wheat, or rice), an alga, a tree, or a vegetable.

[0173] In one embodiment, the target gene is present in a nucleic acid molecule (e.g., a plasmid) outside the cell.

[0174] In one embodiment, the target gene is present in a plasmid.

[0175] Definitions of terms

[0176] In the present application, the scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise indicated. Also, the molecular genetic, nucleic acid chemical, chemical, molecular biological, biochemical, cell culture, microbiological, cell biological, genomic, and recombinant DNA, among other, operational steps described herein are well-known and routine procedures in the corresponding fields. Also, for better understanding of the present application, the definitions and explanations of the relevant terms are provided below.

[0177] Nucleic acid cleavage or cleaving a nucleic acid herein includes: a DNA or RNA break in a target nucleic acid produced by a Cas enzyme described herein (Cis cleavage), a DNA or RNA break in a side branch nucleic acid substrate (single stranded nucleic acid substrate) (i.e., non-specific or non-targeted, Trans cleavage). In some embodiments, the cleavage is a double stranded DNA break. In some embodiments, the cleavage is a single stranded DNA break or a single stranded RNA break.

[0178] CRISPR system

[0179] As used herein, the term "Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) system" or "CRISPR system" are used interchangeably and have the meaning commonly understood by one of ordinary skill in the art, which generally includes a transcription product or other element related to the expression of a CRISPR-associated ("Cas") gene, or a transcription product or other element capable of directing the activity of the Cas gene.

[0180] CRISPR / Cas complex

[0181] As used herein, the term "CRISPR / Cas complex" refers to a complex formed by the binding of a guide RNA or mature crRNA to a Cas protein, which includes a direct repeat sequence that is hybridized to a guide sequence of a target sequence and bound to a Cas protein, the complex being capable of recognizing and cleaving a polynucleotide that can hybridize to the guide RNA or mature crRNA.

[0182] Guide RNA (gRNA)

[0183] As used herein, the terms "guide RNA" (gRNA), "mature crRNA," "guide sequence" are used interchangeably and have the meaning generally understood by those skilled in the art. Generally, a guide RNA can comprise or essentially consist of or consist of a direct repeat sequence and a guide sequence.

[0184] In certain instances, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target sequence to hybridize to the target sequence and direct specific binding of a CRISPR / Cas complex to the target sequence. In one embodiment, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned, is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. Determining optimal alignment is within the capabilities of a person of ordinary skill in the art. For example, there are published and commercially available alignment algorithms and programs such as, but not limited to, ClustalW, Smith-Waterman in matlab, Bowtie, Geneious, Biopython, and SeqMan.

[0185] Target sequence

[0186] A "target sequence" refers to a polynucleotide targeted by a guide sequence in a gRNA, e.g., a sequence that has complementarity to the guide sequence, wherein hybridization between the target sequence and the guide sequence will facilitate formation of a CRISPR / Cas complex (including a Cas protein and a gRNA). Perfect complementarity is not required, so long as there is sufficient complementarity to cause hybridization and facilitate formation of a CRISPR / Cas complex.

[0187] A target sequence can comprise any polynucleotide, such as DNA or RNA. In certain instances, the target sequence is located within a cell or outside of a cell. In certain instances, the target sequence is located in the nucleus or cytoplasm of a cell. In certain instances, the target sequence can be located within an organelle of a eukaryotic cell, e.g., a mitochondrion or a chloroplast. A sequence or template that can be used for recombination into a target locus comprising the target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence." In one embodiment, the editing template is an exogenous nucleic acid. In one embodiment, the recombination is homologous recombination.

[0188] In the present application, "target sequence" or "target polynucleotide" or "target nucleic acid" can be any endogenous or exogenous polynucleotide to a cell (e.g., a eukaryotic cell). For example, the target polynucleotide can be a polynucleotide present in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or junk DNA). In some cases, the target sequence should be associated with a protospacer adjacent motif (PAM).

[0189] Single-stranded nucleic acid detector

[0190] The single-stranded nucleic acid detector of the present application refers to a sequence containing 2-200 nucleotides, preferably, 2-150 nucleotides, preferably, 3-100 nucleotides, preferably, 3-30 nucleotides, preferably, 4-20 nucleotides, more preferably, 5-15 nucleotides. Preferably, it is a single-stranded DNA molecule, a single-stranded RNA molecule or a single-stranded DNA-RNA hybrid.

[0191] The single-stranded nucleic acid detector comprises different reporter groups or labeling molecules at both ends, which do not exhibit a reporter signal when in the initial state (i.e., in the uncut state), and exhibit a detectable signal after the single-stranded nucleic acid detector is cut, i.e., exhibit a detectable difference before and after cutting.

[0192] In one embodiment, the reporter group or labeling molecule comprises a fluorescent group selected from one or any of FAM, FITC, VIC, JOE, TET, CY3, CY5, ROX, Texas Red or LC RED460; and a quenching group selected from one or any of BHQ1, BHQ2, BHQ3, Dabcy1 or Tamra.

[0193] In one embodiment, the single stranded nucleic acid detector has a first molecule (e.g. FAM or FITC) attached to the 5' end and a second molecule (e.g. biotin) attached to the 3' end. The reaction system containing the single stranded nucleic acid detector is used in conjunction with a flow strip to detect the target nucleic acid (preferably, a colloidal gold detection method). The flow strip is designed to have two capture lines, with an antibody to the first molecule (i.e. first molecule antibody) at the sample contact end (colloidal gold), an antibody to the first molecule antibody at the first line (control line), and an antibody to the second molecule (i.e. second molecule antibody, e.g. avidin) at the second line (test line). As the reaction flows along the strip, the first molecule antibody binds to the first molecule carrying the cleaved or uncleaved oligonucleotide to the capture lines, the cleaved reporter will bind to the antibody to the first molecule antibody at the first capture line, and the uncleaved reporter will bind to the second molecule antibody at the second capture line. The binding of the reporter group at each line will result in a strong readout / signal (e.g. color). As more reporter is cleaved, more signal will accumulate at the first capture line, and less signal will appear at the second line. In certain aspects, the present application relates to the use of a flow strip as described herein for detecting a nucleic acid. In certain aspects, the present application relates to a method of detecting a nucleic acid using a flow strip as defined herein, e.g. a (lateral) flow test or a (lateral) flow immuno-chromatographic assay. In certain aspects, the molecules in the single stranded nucleic acid detector can be replaced by each other, or the position of the molecules can be changed, as long as the reporting principle is the same or similar to the present application, and the improved ways are also encompassed in the present application.

[0194] The detection method described in the present application can be used for quantitative detection of the target nucleic acid to be detected. The quantitative detection index can be quantified according to the signal strength of the reporter group, such as the luminescence intensity of the fluorescent group, or the width of the color developing strip, etc.

[0195] Wild type

[0196] As used herein, the term "wild type" has the meaning generally understood by those skilled in the art, which indicates the typical form of an organism, strain, gene or characteristic that distinguishes it from a mutant or variant form when it exists in nature, which can be isolated from a source in nature and has not been intentionally modified by man.

[0197] Derivatization

[0198] As used herein, the term "derivatized" refers to a chemical modification of an amino acid, polypeptide, or protein, in which one or more substituents have been covalently attached to the amino acid, polypeptide, or protein. The substituents can also be referred to as side chains.

[0199] A derivatized protein is a derivative of the protein, typically, derivatization of the protein does not adversely affect the desired activity of the protein (e.g., activity of binding to a guide RNA, endonuclease activity, activity of binding to and cleaving a target sequence at a specific site under the guidance of a guide RNA), that is, the derivative of the protein has the same activity as the protein.

[0200] Derivatized protein

[0201] Also referred to as a "protein derivative," refers to a modified form of a protein, e.g., in which one or more amino acids of the protein can be deleted, inserted, modified and / or substituted.

[0202] Non-naturally occurring

[0203] As used herein, the terms "non-naturally occurring" or "engineered" are used interchangeably and indicate artificial involvement. When these terms are used to describe a nucleic acid molecule or polypeptide, it indicates that the nucleic acid molecule or polypeptide is at least substantially isolated from at least another component with which it is associated in nature or as found in nature.

[0204] Orthologue

[0205] As used herein, the term "orthologue" has the meaning generally understood by those skilled in the art. As a further guide, an "orthologue" of a protein as described herein refers to a protein belonging to a different species which performs the same or a similar function as the protein of which it is an orthologue.

[0206] Identity

[0207] As used herein, the term "identity" is used in reference to the matching of sequences between two polypeptides or between two nucleic acids. When a position in each of two sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage of identity" between two sequences is a function of the number of matching positions shared by the sequences divided by the number of positions compared x 100. For example, if 6 of 10 positions in two sequences are matched then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of 6 positions are matched). Typically, the comparison is made over the length of the sequences being compared, after aligning the two sequences to produce maximum identity. Such alignment can be achieved by methods such as that of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed by computer program such as the Align program (DNAstar, Inc.). Percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) as integrated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, percentage identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J MoI Biol. 48:444-453 (1970)) as implemented in the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a gap length weight of 1, 2, 3, 4, 5, or 6.

[0208] Vectors

[0209] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular), nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of nucleic acids known in the art. A vector can be introduced into a host cell by transformation, transduction, or transfection, and the resulting genetically modified host cell can express the genetic material elements carried by the vector. A vector can be introduced into a host cell to thereby produce a transcript, protein, or peptide, including a protein, fusion protein, isolated nucleic acid molecule, etc. (e.g., a CRISPR transcript, such as a nucleic acid transcript, protein, or enzyme) as described herein. A vector can contain a variety of control elements, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector can contain a replication origin.

[0210] One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.

[0211] Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication-defective retroviruses, adenoviruses, replication-defective adenoviruses, and adeno-associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors) are capable of autonomous replication in a host cell into which they are introduced.

[0212] Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0213] Host cells

[0214] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, and eukaryotic cells such as microbial cells, fungal cells, animal cells, and plant cells.

[0215] Those of skill in the art will appreciate that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of the desired gene, etc.

[0216] Regulatory elements

[0217] As used herein, the term "regulatory element" is intended to include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences), which are described in detail in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, CA (1990). In certain instances, regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells as well as those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Tissue-specific promoters can direct expression primarily in a desired tissue of interest, such as muscle, neuronal, bone, skin, blood, a particular organ (e.g., liver, pancreas), or a particular cell type (e.g., lymphocytes). In certain instances, regulatory elements can also direct expression in a temporal-dependent manner, such as in a cell cycle-dependent or developmental stage-dependent manner, which can or can not be tissue- or cell type-specific. In certain instances, the term "regulatory element" encompasses enhancer elements, such as the WPRE; the CMV enhancer; the R-U5' segment in the LTR of HTLV-I ((Mol. Cell. Biol., vol. 8(1), pp. 466-472, 1988); the SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit beta-globin (Proc. Natl. Acad. Sci. USA., vol. 78(3), pp. 1527-31, 1981).

[0218] Promoter

[0219] As used herein, the term "promoter" has its art-understood meaning and refers to a non-coding nucleotide sequence located upstream of a gene that initiates expression of the downstream gene. A constitutive promoter is a nucleotide sequence that, when operably linked with a polynucleotide encoding or specifying a gene product, results in production of the gene product in a cell under most or all physiological conditions of the cell. An inducible promoter is a nucleotide sequence that, when operably linked with a polynucleotide encoding or specifying a gene product, results in production of the gene product in a cell essentially only when an inducer corresponding to the promoter is present in the cell. A tissue-specific promoter is a nucleotide sequence that, when operably linked with a polynucleotide encoding or specifying a gene product, results in production of the gene product in a cell essentially only when the cell is of the tissue type to which the promoter corresponds.

[0220] NLS

[0221] A "nuclear localization signal" or "nuclear localization sequence" (NLS) is an amino acid sequence that "tags" a protein for import into the nucleus by nuclear transport, i.e., a protein with an NLS is transported to the nucleus. Typically, an NLS comprises positively charged Lys or Arg residues that are exposed on the surface of the protein. Exemplary nuclear localization sequences include, but are not limited to, NLS from SV40 large T antigen, EGL-13, c-Myc, and TUS protein. In some embodiments, the NLS comprises a PKKKRKV sequence. In some embodiments, the NLS comprises an AVKRPAATKKAGQAKKKKLD sequence. In some embodiments, the NLS comprises a PAAKRVKLD sequence. In some embodiments, the NLS comprises a MSRRRKANPTKLSENAKKLAKEVEN sequence. In some embodiments, the NLS comprises a KLKIKRPVK sequence. Other nuclear localization sequences include, but are not limited to, the acidic M9 domain of hnRNP Al, the sequence KIPIK in the yeast transcriptional repressor Matα2, and PY-NLS.

[0222] Operably linked

[0223] As used herein, the term "operably linked" is intended to mean that a nucleotide sequence of interest is linked to the one or more regulatory elements in a manner that allows expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or, when the vector is introduced into a host cell, in the host cell).

[0224] Complementarity

[0225] As used herein, the term "complementarity" refers to the ability of a nucleic acid to form one or more hydrogen bonds with another nucleic acid sequence by virtue of traditional Watson-Crick or other non-traditional types of bonding. Percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 is 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfect complementarity" indicates that all consecutive residues of a nucleic acid sequence form hydrogen bonds with the same number of consecutive residues in a second nucleic acid sequence. "Substantially complementary" as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to hybridization under stringent conditions of two nucleic acids.

[0226] Stringent conditions

[0227] As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence will hybridize primarily to that target sequence and not to non-target sequences. Stringent conditions are often sequence- dependent, and are varied 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.

[0228] Hybridization

[0229] The terms "hybridize" or "complementary" or "substantially complementary" refer to the non-covalent binding of nucleotides of a nucleic acid (e.g., RNA, DNA) to another nucleic acid by means of complementary base pairing and / or G / U base pairing, "annealing" or "hybridizing" in a sequence-specific, anti-parallel fashion (i.e., nucleic acid specifically binds to complementary nucleic acid).

[0230] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases can occur. Suitable conditions for hybridization between two nucleic acids depend on the length and complementarity of the nucleic acids, which are variables known in the art. Typically, the length of the hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).

[0231] It is understood that the sequence of a polynucleotide need not be 100% complementary to the sequence of its target nucleic acid to hybridize specifically thereto. A polynucleotide can comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% complementarity to the sequence of the target region in the sequence of the target nucleic acid to which it hybridizes.

[0232] Hybridization of a target sequence to a gRNA means that at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleic acid sequences of the target sequence and the gRNA can hybridize, forming a complex; or that at least 12, 15, 16, 17, 18, 19, 20, 21, 22, or more bases of the nucleic acid sequences of the target sequence and the gRNA can base pair, hybridizing to form a complex.

[0233] Expression

[0234] As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed from a DNA template (e.g., transcribed into mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. Transcripts and encoded polypeptides can be collectively referred to as "gene product." If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell.

[0235] Linker

[0236] As used herein, the term "linker" refers to a linear polypeptide formed by the linkage of a plurality of amino acid residues via peptide bonds. The linkers of the present application can be artificial synthetic amino acid sequences, or naturally occurring polypeptide sequences, such as polypeptides having a hinge region function. Such linker polypeptides are well known in the art (see, e.g., Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J. et al. (1994) Structure 2:1121-1123).

[0237] Treatment

[0238] As used herein, the term "treatment" refers to the treatment or cure of a disorder, the delay of onset of symptoms of a disorder, and / or the delay of progression of a disorder.

[0239] Subject

[0240] As used herein, the term "subject" includes, but is not limited to, various animals, plants, and microorganisms.

[0241] Animal

[0242] For example, a mammal, for example, a bovine, equine, ovine, porcine, canine, feline, leporid, rodent (e.g., a mouse or rat), non-human primate (e.g., a macaque or cynomolgus monkey), or a human. In certain embodiments, the subject (e.g., a human) has a disorder (e.g., a disorder resulting from a disease-associated gene defect).

[0243] Plant

[0244] The term "plant" is to be understood as any differentiated multicellular organism capable of performing photosynthesis, including crop plants at any stage of maturation or development, in particular monocotyledonous or dicotyledonous plants, vegetable crops, including artichokes, Brussels sprouts, cress, leeks, asparagus, lettuce (e.g., iceberg lettuce, leaf lettuce, long-leaf lettuce), bok choy, yellow fleshed yam, melons (e.g., muskmelons, watermelons, crenshaw melons, cantaloupes, Roman melons), oilseed crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, collard greens, curly kale, Chinese cabbage, bok choy), artichokes, radishes, napa, okra, onions, celery, parsley, chickpeas, parsnips, chicory, peppers, potatoes, gourds (e.g., zucchini, cucumbers, crookneck squash, calabaza, pumpkin), radishes, dry globe onions, turnip greens, eggplants (also known as aubergines), burdock, endive, green onions, endive, garlic, spinach, green onions, zucchini, greens, sugar beets (sugar beets and fodder beets), sweet potatoes, Swiss chard, wasabi, tomatoes, turnips, and spices; fruits and / or vine crops, such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, aronia, almonds, chestnuts, hazelnuts, pecans, pistachios, walnuts, citrus, blueberries, boysenberries, cranberries, currants, goji berries, raspberries, strawberries, blackberries, grapes, avocados, bananas, kiwis, persimmons, pomegranates, pineapples, tropical fruits, pomes, melons, mangoes, papayas, and lychees; field crops, such as clover, alfalfa, milkweed, meadow grass, corn / maize (fodder corn, sweet corn, popcorn), hops, jojoba, peanuts, rice, safflower, small grain crops (barley, oats, rye, wheat, etc.), sorghum, tobacco, kapok, legumes (beans, lentils, peas, soybeans), oil plants (rape, mustard, poppy, olive, sunflower, coconut, castor oil plant, cocoa, groundnuts), Arabidopsis, fiber plants (cotton, flax, jute), lauraceae (cinnamon, camphor), or a plant such as coffee, sugar cane, tea, and natural rubber plants; and / or bedding plants, such as flowering plants, cacti, succulents and / or ornamental plants, and trees such as forests (broad-leaved trees and evergreens, such as conifers), fruit trees, ornamental trees, and nut-bearing trees, and shrubs and other young plants.

[0245] Beneficial effects of the invention

[0246] The present application improves the activity of Cas12i3 protein by mutation, which has wide application prospects.

[0247] Embodiments of the present application will now be described in detail with reference to the accompanying drawings and examples, but as the skilled person will appreciate, the following drawings and examples are provided by way of illustration only and are not limiting of the scope of the present application. Various objects and advantageous aspects of the present application will become apparent to the skilled person in the art from the following detailed description of the preferred embodiments in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0248] Figure 1 . Schematic diagram of vector and plasmid construction, Figure 1 A is a protein expression vector map, Figure 1 B is a plasmid map of lethal genes.

[0249] Figure 2 . Schematic diagram of plasmid library screening process.

[0250] Figure 3 . Verification results of editing efficiency of single-site amino acid mutation Cas protein. DETAILED DESCRIPTION

[0251] The following examples merely illustrate the application and are not intended to limit the application in any way. The experiments and methods described in the examples were performed essentially as described in the art and as described in various references unless otherwise indicated. For example, the general techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, among others, used in the present application can be found in Sambrook, Fritsch, and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed. (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames, and G. R. Taylor eds. (1995), Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, eds. (1988); and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)).

[0252] In addition, where particular conditions are not specified in the examples, those conditions were performed under routine conditions or as suggested by the manufacturer. Where the manufacturer of reagents or instruments is not indicated, it is intended that the reagents or instruments used were of a routine variety available from a commercial vendor. Those skilled in the art will recognize that the examples describe the present application in a manner sufficient to allow for the practice thereof, and are not intended to limit the scope of the application as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

[0253] Example 1. Obtaining Cas mutant proteins

[0254] Cas12i3 library construction: For the known Cas protein (Cas12f.4 in CN111757889B, which is referred to as Cas12i3 in this embodiment), the library was constructed by error-prone PCR library kit (GeneMorph II, Agilent) (according to the operation of the library kit instruction). After the PCR product was recovered by running the gel, it was connected to the protein expression vector pET28a-Trc-Cas, and the protein expression vector pET28a-Trc-Cas map is as follows Figure 1 A part is shown, wherein Trc is a promoter, Cas12i is a Cas12i3 protein, and crRNA is a guide RNA. The ligation product was transformed into DH5a competent cells, and then the library plasmid was extracted by using a kit. The plasmid map of the expressed lethal gene is as follows Figure 1 B part is shown. The amino acid sequence of the wild type Cas12i3 is shown as SEQ ID No. 1, and the nucleic acid sequence is shown as SEQ ID No. 2.

[0255] SEQ ID No. 1:

[0256]

[0257] SEQ ID No. 2:

[0258]

[0259] Screening and transformation of library plasmid: the ccdB lethal gene plasmid was transformed into BW25141 E. coli, screened by Amp antibiotic, and single colonies were selected to make competent cells. The library plasmid of Cas12i3 was transformed into BW25141 competent cells containing lethal genes by 1 ug of electroporation, and after 1.5 hours of recovery in a 30°C 250 rpm shaker, 1 mM IPTG was added to induce culture for 2 hours, and then the bacterial solution was spread on the kan and 2% arabinose medium and incubated overnight. Then, the revived single colonies were selected for sequencing to identify the corresponding variants (experimental method reference: Zhang, Liyang, et al. "AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines." Nature communications 12.1 (2021): 3908), and the schematic diagram of plasmid library screening process is shown in Figure 2

[0260] The amino acid sites of the Cas12i3 mutations involved in the present embodiment include K60R, S82T, I107V, S349T, L553G, L561F, A719V, and L747F. Based on the above mutations, the Cas mutant proteins with single-site mutations in the above amino acids (named by mutation type) were obtained based on SEQ ID No. 1: K60R, S82T, I107V, S349T, L553G, L561F, A719V, and L747F. The above mutation sites are the mutation of the 60th amino acid from the N terminus of SEQ ID No. 1 to R, the mutation of the 82nd amino acid to T, the mutation of the 107th amino acid to V, the mutation of the 349th amino acid to T, the mutation of the 553rd amino acid to G, the mutation of the 561st amino acid to F, the mutation of the 719th amino acid to V, or the mutation of the 747th amino acid to F.

[0261] Example 2. Verification of editing activity of Cas mutant proteins

[0262] ​A fluorescent reporter system suitable for verifying the cleavage of Cas12i3 was constructed according to the reference (Yang, Yi, et al. "Highly efficient and rapid detection of the cleavage activity of Cas9 / gRNA via a fluorescent reporter." Applied biochemistry and biotechnology 180.4 (2016): 655-667.). The fluorescent reporter vector has RFP and non-luminous GFFP fluorescent proteins, and the Cas vector has CFP fluorescent protein. The target site on the GFFP sequence was selected for testing, and the underlined position is the PAM sequence. After transfecting CHO cells for 48 h, the editing efficiency can be determined by the ratio of CFP and RFP GFP fluorescence by flow sorting. Different mutants constructed in Example 1 were selected for testing, and the results are shown in TTT FIG. 2. Taking the editing efficiency of the WT wild-type Cas12i3 (SEQ ID No. 1) as the benchmark, the editing efficiency of the mutants K60R, S82T, I107V, S349T, L561F, A719V, and L747F of Example 1 tested relative to the WT wild-type Cas12i3 was significantly improved, with an improvement of more than 50%; the editing efficiency of the mutant L553G relative to the WT wild-type Cas12i3 was reduced. Figure 3

[0263] The above results reflect that the 60th, 82nd, 107th, 349th, 561st, 719th, or 747th amino acid from the N terminus of SEQ ID No. 1 is a key site for Cas12i3 to exert activity, and the editing efficiency of Cas12i3 can be greatly improved by mutation of the above-mentioned amino acid sites.

[0264] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details according to all the teachings disclosed, and these changes are within the scope of protection of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.​

Claims

1. A Cas mutant protein, wherein the mutant protein, compared with the amino acid sequence of the parental Cas protein, has a mutation at any of the following amino acid sites corresponding to the amino acid sequence shown in SEQ ID No. 1: position 60, position 82, position 107, position 349, position 561, position 719, or position 747; wherein the amino acid at position 60 is mutated to R, the amino acid at position 82 is mutated to T, the amino acid at position 107 is mutated to V, the amino acid at position 349 is mutated to T, the amino acid at position 561 is mutated to F, the amino acid at position 719 is mutated to V, and the amino acid at position 747 is mutated to F.

2. The Cas mutant protein according to claim 1, characterized in that, The parental Cas protein is the Cas12i protein.

3. A fusion protein comprising the Cas mutant protein of any one of claims 1-2 and other modified portions.

4. An isolated polynucleotide, characterized in that, The polynucleotide is a polynucleotide sequence encoding any one of the Cas mutant proteins of claims 1-2, or a polynucleotide sequence encoding the fusion protein of claim 3.

5. A carrier, characterized in that, The vector comprises the polynucleotide of claim 4 and a regulatory element operatively linked thereto.

6. A CRISPR-Cas system, characterized in that, The system comprises the Cas mutant protein according to any one of claims 1-2 and at least one gRNA; The gRNA can bind to the Cas mutant protein as described in any one of claims 1-2.

7. A composition, characterized in that, The composition comprises: (i) A protein component selected from: the Cas mutant protein of any one of claims 1-2 or the fusion protein of claim 3; (ii) A nucleic acid component, which is gRNA, said gRNA being capable of binding the Cas mutant protein of any one of claims 1-2; The protein components and nucleic acid components combine to form a complex.

8. An engineered host cell, characterized in that, The host cell comprises the Cas mutant protein of any one of claims 1-2, or the fusion protein of claim 3, or the polynucleotide of claim 4, or the vector of claim 5, or the CRISPR-Cas system of claim 6, or the composition of claim 7.

9. The application of the Cas mutant protein of any one of claims 1-2, or the fusion protein of claim 3, or the polynucleotide of claim 4, or the vector of claim 5, or the CRISPR-Cas system of claim 6, or the composition of claim 7, or the host cell of claim 8 in gene editing, wherein the application is for purposes other than disease diagnosis and treatment. Alternatively, in the preparation of a formulation or kit for use in gene editing.

10. The use of the Cas mutant protein of any one of claims 1-2, or the fusion protein of claim 3, or the polynucleotide of claim 4, or the vector of claim 5, or the CRISPR-Cas system of claim 6, or the composition of claim 7, or the host cell of claim 8 in any one or more of the following, wherein the use is for non-disease diagnosis and treatment purposes: gene targeting, gene cleavage; Alternatively, in the preparation of a formulation or kit for: gene targeting, gene cleavage.

11. The use of the Cas mutant protein of any one of claims 1-2, or the fusion protein of claim 3, or the polynucleotide of claim 4, or the vector of claim 5, or the CRISPR-Cas system of claim 6, or the composition of claim 7, or the host cell of claim 8 in any one or more of the following applications, said applications being for non-disease diagnosis and treatment purposes: cutting double-stranded DNA, single-stranded DNA, or single-stranded RNA, specifically editing double-stranded nucleic acids; Alternatively, in the preparation of formulations or kits for use in: editing target sequences in target loci to modify organisms.

12. The use of the Cas mutant protein of any one of claims 1-2, or the fusion protein of claim 3, or the polynucleotide of claim 4, or the vector of claim 5, or the CRISPR-Cas system of claim 6, or the composition of claim 7, or the host cell of claim 8 in any one or more of the following, wherein the use is for non-disease diagnosis and treatment purposes: base editing of double-stranded nucleic acid, base editing of single-stranded nucleic acid; Alternatively, in the preparation of a formulation or kit for the treatment of a disease or for targeting a gene.

13. A method for editing a target nucleic acid, the method being a method for non-disease diagnosis and treatment purposes, the method comprising contacting the target nucleic acid with a Cas mutant protein of any one of claims 1-2, or a fusion protein of claim 3, or a polynucleotide of claim 4, or a vector of claim 5, or a CRISPR-Cas system of claim 6, or a composition of claim 7, or a host cell of claim 8.

14. A method for targeting or cleaving a target nucleic acid, the method being a method for non-disease diagnosis and treatment purposes, the method comprising contacting the target nucleic acid with a Cas mutant protein of any one of claims 1-2, or a fusion protein of claim 3, or a polynucleotide of claim 4, or a vector of claim 5, or a CRISPR-Cas system of claim 6, or a composition of claim 7, or a host cell of claim 8.

15. A kit for gene editing, the kit comprising the Cas mutant protein of any one of claims 1-2, or the fusion protein of claim 3, or the polynucleotide of claim 4, or the vector of claim 5, or the CRISPR-Cas system of claim 6, or the composition of claim 7, or the host cell of claim 8.

16. A kit for gene targeting or gene cutting, the kit comprising the Cas mutant protein of any one of claims 1-2, or the fusion protein of claim 3, or the polynucleotide of claim 4, or the vector of claim 5, or the CRISPR-Cas system of claim 6, or the composition of claim 7, or the host cell of claim 8.

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