Proteins, polynucleotides, vectors, vector systems, compositions, kits, cells, methods for modifying target DNA, and methods of production

Abstract

A novel protein that can be used to modify target DNA is provided. The protein of the present disclosure is a protein represented by the following formula (a1), (a2), or (a3): (a1) a protein consisting of any one of a plurality of specific amino acid sequences; (a2) a protein having nuclease activity, which consists of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted, or added in any of the amino acid sequences of the multiple specific sequences; (a3) A protein having an amino acid sequence having 80% or more identity to any one of the amino acid sequences of the multiple specific sequences, and having nuclease activity.

Description

[Technical field]

[0001] The present disclosure relates to proteins, polynucleotides, vectors, vector systems, compositions, kits, cells, methods for modifying target DNA, and methods of production. [Background technology]

[0002] In recent years, genome editing using the CRISPR / Cas9 (Clustered Regularly Interspaced Palindromic Repeats / CRISPR-associated protein 9) system and the like has been widely used. In addition to Cas9, other Cas proteins such as Cas12 have been explored, and modified versions of Cas proteins such as MAD7 have been created (Patent Document 1). [Prior art documents] [Non-patent literature]

[0003] [Patent Document 1] International Publication No. 2020 / 086475 Summary of the Invention [Problem to be solved by the invention]

[0004] Therefore, an object of the present disclosure is to provide a new protein that can be used to modify target DNA. [Means for solving the problem]

[0005] To achieve the above object, the protein of the present disclosure includes the following protein (a1), (a2), or (a3): (a1) a protein consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 19; (a2) a protein having nuclease activity, which consists of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted, or added in any of the amino acid sequences of SEQ ID NOs: 1 to 19; (a3) A protein having an amino acid sequence having 80% or more identity to any of the amino acid sequences of SEQ ID NOs: 1 to 19, and having nuclease activity.

[0006] The polynucleotides of the present disclosure encode the proteins of the present disclosure.

[0007] A vector of the present disclosure comprises a polynucleotide of the present disclosure.

[0008] The vector system of the present disclosure comprises a first vector comprising a polynucleotide of the present disclosure; and a second vector comprising a polynucleotide encoding a guide RNA for the target site.

[0009] The composition of the present disclosure comprises a protein of the present disclosure, a polynucleotide of the present disclosure, and / or a vector of the present disclosure, It includes a guide RNA for the target site or a polynucleotide encoding the same.

[0010] The kit of the present disclosure comprises a protein of the present disclosure, a polynucleotide of the present disclosure, and / or a vector of the present disclosure, It includes a guide RNA for the target site or a polynucleotide encoding the same.

[0011] A cell of the present disclosure comprises a protein of the present disclosure, a polynucleotide of the present disclosure, a vector of the present disclosure, and / or a vector system of the present disclosure.

[0012] The method for modifying a target DNA of the present disclosure includes a modification step of modifying the target DNA in a cell by contacting the target DNA with a protein of the present disclosure and a guide RNA for a target site in the target DNA.

[0013] The method of the present disclosure is a method of producing a cell or a non-human animal in which target DNA is modified, comprising the steps of: The method includes an introduction step of introducing a protein of the present disclosure, a polynucleotide of the present disclosure, and / or a vector of the present disclosure and a guide RNA for the target site into a cell or a non-human animal. Effect of the Invention

[0014] The present disclosure makes it possible to provide a new protein that can be used to modify target DNA. [Brief description of the drawings]

[0015]

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Figure 8

Figure 9

[0016] <Definition> As used herein, "protein" or "peptide" refers to a polymer composed of unmodified (naturally occurring), modified, and / or artificial amino acids.

[0017] As used herein, "nuclease" refers to an enzyme capable of cleaving phosphodiester bonds between nucleotides of nucleic acids. Examples of the nuclease include exonucleases that digest nucleic acids from their ends and endonucleases that cleave the middle of a nucleotide chain. The nucleases can be classified into DNA nucleases that act on DNA and RNA nucleases that act on RNA.

[0018] As used herein, the term "protospacer adjacent motif (PAM) sequence" refers to a sequence adjacent to a sequence targeted by a Cas nuclease in the CRISPR bacterial adaptive immune system. The length and base sequence of the PAM sequence vary depending on the type of Cas nuclease.

[0019] As used herein, "coding sequence" refers to a nucleic acid (RNA or DNA molecule) that comprises a nucleotide sequence that encodes a protein. The coding sequence can be codon optimized.

[0020] As used herein, "polynucleotide" refers to a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and / or modified nucleotides. When "polynucleotide" is used in combination with a specific protein, the "polynucleotide" refers to a polymer of nucleotides that encodes the amino acid sequence of the protein. Examples of the polynucleotide include genomic DNA, cDNA, and mRNA. The polynucleotide may be, for example, single-stranded or double-stranded. The polynucleotide can be interchangeably referred to as "nucleic acid" or "oligonucleotide."

[0021] As used herein, "target site" refers to a region or base sequence of a polynucleotide that is targeted by a complex of a protein and a guide RNA of the present disclosure.

[0022] As used herein, "nucleic acid modification activity" refers to an activity capable of modifying bases (e.g., A, T, C, G, or U) in a nucleic acid sequence such as DNA or RNA. The "nucleic acid modification" refers to the substitution, conversion, deletion, insertion, addition, etc. of a base in a nucleic acid sequence.

[0023] As used herein, "guide RNA" refers to an RNA that can target the protein of the present disclosure to a target DNA sequence. The guide RNA includes a crRNA (CRISPR RNA) that binds to a target site, and optionally a tracrRNA (trans activating crRNA) that is involved in the activity of the CRISPR-Cas system depending on the type of the Cas protein. The guide RNA may be, for example, a single-stranded RNA in which the crRNA and tracrRNA are directly or indirectly linked, or may be two RNAs, the crRNA and the tracrRNA.

[0024] As used herein, "crRNA" refers to an RNA having a base sequence (hereinafter also referred to as "sequence") complementary to a target DNA sequence. The crRNA is an RNA that constitutes the CRISPR / Cas system, and is known to function to recognize a target DNA sequence. The crRNA generally includes a spacer sequence and a repeat sequence. The spacer sequence can also be referred to as a sequence (guide sequence) that can form a double strand with the complementary strand of the target DNA sequence. The crRNA can form a double strand with, for example, either the sense strand or the antisense strand of the target DNA.

[0025] As used herein, "tracrRNA" refers to an RNA having a sequence complementary to a portion of the crRNA. The sequence complementary to a portion of the crRNA is also referred to as an anti-repeat region (sequence). The tracrRNA includes an anti-repeat region (sequence) (repeat region) followed by one or more hairpin structures, and has a sequence capable of forming a stem loop. It is known that the tracrRNA functions as a scaffold for binding the Cas protein and crRNA in some Cas proteins. The protein of the present disclosure can directly bind to crRNA, for example.

[0026] In this specification, "hybridization" means annealing with a complementary polynucleotide caused by nucleotide complementarity, specifically, by the complementarity of bases in nucleotides. That is, it means that two polynucleotides can form a non-covalent pair through hydrogen bonds. The hybridization may be, for example, the binding of two complementary base sequences, or substantially complementary base sequences with one or more mismatched base pairs.

[0027] As used herein, "complementarity" or "complementary" refers to the ability to form nucleotide pairs, i.e., base pairs, between one polynucleotide and another polynucleotide.

[0028] As used herein, a "vector" is in vitro or in vivo By "transfected" is meant a nucleic acid delivered to a host cell, or a recombinant plasmid or virus containing said nucleic acid.

[0029] As used herein, "target DNA sequence" refers to the base sequence of a target polynucleotide in a target DNA to which a guide RNA hybridizes.

[0030] As used herein, "eukaryote" refers to an organism that has a nucleus enclosed by a nuclear membrane.

[0031] As used herein, "prokaryote" refers to an organism that does not have a membrane-enclosed nucleus.

[0032] Sequence information of the proteins described herein or the nucleic acids (e.g., DNA or RNA) encoding them is available from Protein Data Bank, UniProt, Ensembl, GenBank, etc. Furthermore, the nucleic acid sequence of RNA can also be obtained from the nucleic acid sequence of the corresponding DNA using appropriate sequence conversion software or the like.

[0033] The present disclosure will be described below with examples, but the present disclosure is not limited to the following examples and can be modified as desired. In addition, each description in the present disclosure can be mutually incorporated unless otherwise specified. In this specification, when the expression "~" is used, it is used to mean including the numerical value or physical value before and after it. In this specification, the expression "A and / or B" includes "A only", "B only", and "both A and B".

[0034] <Protein> In one embodiment, the present disclosure provides a new protein that can be used to modify target DNA. The protein of the present disclosure is a protein of the following (a). The protein of the following (a) may have nuclease activity or nickase activity. (a) a protein selected from (a1), (a2), and (a3) ​​below: (a1) a protein consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 19; (a2) a protein consisting of an amino acid sequence in which one or several amino acids have been deleted, inserted, substituted, or added in any of the amino acid sequences of SEQ ID NOs: 1 to 19; (a3) A protein consisting of an amino acid sequence having 80% or more identity to any of the amino acid sequences of SEQ ID NOs: 1 to 19.

[0035] The Cas protein recognizes the PAM sequence of the target DNA and modifies the target DNA by the nuclease activity of the Cas protein. The PAM sequence that the Cas protein can recognize varies depending on the type and origin of the Cas protein. However, it is known that there is a bias in the PAM sequence recognized by the Cas protein. For example, it is known that NAAN, NAGN, or NGAN (N=A, T, G, or C; the same applies below) is difficult to recognize by the Cas protein, especially the commonly used Cas9 and Cas12. As a result of an intensive search for various Cas proteins, the present inventors found a protein capable of recognizing NAAN, NAGN, or NGAN as the PAM sequence, and completed the present disclosure. According to the present disclosure, a new protein capable of recognizing NAAN, NAGN, or NGAN as the PAM sequence can be provided. Therefore, according to the present disclosure, modification of the target DNA can be performed even for target sites having base sequences such as NAAN, NAGN, or NGAN that were difficult to modify with conventional Cas proteins.

[0036] The (a1) protein is a protein that the present inventors designed as a Cas-like protein.

[0037] ST9.5_13 (SEQ ID NO: 1) E DLNYGFKKGRFKVERQVYQKFETMLINKLNYLVFKNRGVTEDGGLLRGYQLTYIPESLKNVGRQCGCIFY VPAA YTSKIDPTTGFADIFRFKNLTVEEKRDFIRKFDYIKYDLEKEMFVFAFDFKNFVTQNVEMSKNDWCVYTNGIRVKRRYANGRFTNETDEIDINVLMKKTFERTDIDWNDGHNL IDEIIDYDLESQIVDIFKMAVQMRNSRSEAEDRDYDRLVSPVLNESGDFFDSSKVGENLPKDADANGAYCIALKGLYKVRQIQENWNEEEKFSRAKLRISNKDWFDFVQNKRYL

[0038] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 1, the "one or several" in the protein (a2) may be within a range in which the (a2) functions as a protein having nuclease activity. The "one or several" in the (a2) may be, for example, 1 to 287, 1 to 284, 1 to 215, 1 to 143, 1 to 142, 1 to 71, 1 to 57, 1 to 43, 1 to 28, 1 to 14, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 or 2 in the amino acid sequence of SEQ ID NO: 1. In the present disclosure, the numerical range of the number discloses, for example, all positive integers within that range.

[0039] In the protein (a2), the substitution is preferably a conservative substitution. The conservative substitution means a substitution of an amino acid residue with an amino acid residue having a similar side chain. Examples of the conservative substitution include substitution between amino acid residues having a basic side chain such as lysine, arginine, histidine, etc.; substitution between amino acid residues having an acidic side chain such as aspartic acid, glutamic acid, etc.; substitution between amino acid residues having a non-charged polar side chain such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, etc.; substitution between amino acid residues having a non-polar side chain such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, etc.; substitution between amino acid residues having a β-branched side chain such as threonine, valine, isoleucine, etc.; substitution between amino acid residues having an aromatic side chain such as tyrosine, phenylalanine, tryptophan, histidine, etc. (hereinafter the same).

[0040] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 1, in the proteins (a1) to (a3), for example, the underlined amino acid E (glutamic acid) at position 1,136 or an amino acid residue corresponding to the amino acid E may be substituted with G (glycine). In this case, since the proteins (a1) to (a3) ​​can retain nuclease activity, it is preferable that the 1209th amino acid A (alanine) or an amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), as shown by the underlines, and it is even more preferable that the 1207th amino acid V (valine) or an amino acid residue corresponding to the V is substituted with A (alanine), the 1208th amino acid P (proline) or an amino acid residue corresponding to the P is substituted with D (aspartic acid), the 1209th amino acid A (alanine) or an amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and the 1210th amino acid A (alanine) or an amino acid residue corresponding to the amino acid A is substituted with R (arginine).

[0041] ST9.5_14 (SEQ ID NO: 2) E DLNYGFKKGRFKVERQVYQKFETMLINKLNYLVFKNRGVTEDGGLLRGYQLTYIPESLKNVGRQCGCIFY VPAA YTSKIDPTTGFADIFRFKNLTVEEKRDFIRKFDYIKYDLEKEMFVFAFDFKNFVTQNVEMSKNDWCVYTNGIRVKRRYANGRFTNETDEIDINVLMKKTFERTDIDWNDGHNL IDEIIDYDLESQIVDIFKMAVQMRNSRSEAEDRDYDRLVSPVLNESGDFFDSSKVGENLPKDADANGAYCIALKGLYKVRQIQENWNEEEKFSRAKLRISNKDWFDFVQNKRYL

[0042] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 2, the "one or several" in the protein (a2) may be within a range in which the (a2) functions as a protein having nuclease activity. The "one or several" in the (a2) may be, for example, 1 to 286, 1 to 214, 1 to 143, 1 to 142, 1 to 71, 1 to 57, 1 to 43, 1 to 28, 1 to 14, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the amino acid sequence of SEQ ID NO: 2.

[0043] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 2, in the proteins (a1) to (a3), for example, the underlined amino acid E at position 1129 or the amino acid residue corresponding to the amino acid E may be substituted with G. In this case, since the proteins (a1) to (a3) ​​can retain nuclease activity, it is preferable that the underlined amino acid A (alanine) at position 1202 or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and it is even more preferable that the underlined amino acid V (valine) at position 1200 or the amino acid residue corresponding to the V is substituted with A (alanine), the 1201st amino acid P (proline) or the amino acid residue corresponding to the P is substituted with D (aspartic acid), the 1202nd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and the 1203rd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with R (arginine).

[0044] ST9.5_38 (SEQ ID NO: 3) E DLNYGFKKGRFKVERQVYQKFETMLINKLNYLVFKNRGVTEDGGLLRGYQLTYIPESLKNVGRQCGCIFY VPAA YTSKIDPTTGFADIFRFKNLTVEEKRDFIRKFDYIKYDLEKEMFVFAFDFKNFVTQNVEMSKNDWCVYTNGIRVKRRYANGRFTNETDEIDINVLMKKTFERTDIDWNDGHNL IDEIIDYDLESQIVDIFKMAVQMRNSRSEAEDRDYDRLVSPVLNESGDFFDSSKVGENLPKDADANGAYCIALKGLYKVRQIQENWNEEEKFSRAKLRISNKDWFDFVQNKRYL

[0045] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 3, the "one or several" in the protein (a2) may be within a range in which the (a2) functions as a protein having nuclease activity. The "one or several" in the (a2) may be, for example, 1 to 286, 1 to 214, 1 to 143, 1 to 142, 1 to 71, 1 to 57, 1 to 43, 1 to 28, 1 to 14, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the amino acid sequence of SEQ ID NO: 3.

[0046] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 3, in the proteins (a1) to (a3), for example, the underlined amino acid E at position 1129 or the amino acid residue corresponding to the amino acid E may be substituted with G. In this case, since the proteins (a1) to (a3) ​​can retain nuclease activity, it is preferable that the underlined amino acid A (alanine) at position 1202 or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and it is even more preferable that the underlined amino acid V (valine) at position 1200 or the amino acid residue corresponding to the V is substituted with A (alanine), the 1201st amino acid P (proline) or the amino acid residue corresponding to the P is substituted with D (aspartic acid), the 1202nd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and the 1203rd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with R (arginine).

[0047] ST9.5_42 (SEQ ID NO: 4) E DLNYGFKKGRFKVERQVYQKFETMLINKLNYLVFKNRGVTEDGGLLRGYQLTYIPESLKNVGRQCGCIFY VPAA YTSKIDPTTGFADIFRFKNLTVEEKRDFIRKFDYIKYDLEKEMFVFAFDFKNFVTQNVEMSKNDWCVYTNGIRVKRRYANGRFTNETDEIDINVLMKKTFERTDIDWNDGHNL IDEIIDYDLESQIVDIFKMAVQMRNSRSEAEDRDYDRLVSPVLNESGDFFDSSKVGENLPKDADANGAYCIALKGLYKVRQIQENWNEEEKFSRAKLRISNKDWFDFVQNKRYL

[0048] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 4, the "one or several" in the protein (a2) may be within a range in which the (a2) functions as a protein having nuclease activity. The "one or several" in the (a2) may be, for example, 1 to 286, 1 to 214, 1 to 143, 1 to 142, 1 to 71, 1 to 57, 1 to 43, 1 to 28, 1 to 14, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the amino acid sequence of SEQ ID NO: 4.

[0049] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 4, in the proteins (a1) to (a3), for example, the underlined amino acid E at position 1129 or the amino acid residue corresponding to the amino acid E may be substituted with G. In this case, since the proteins (a1) to (a3) ​​can retain nuclease activity, it is preferable that the underlined amino acid A (alanine) at position 1202 or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and it is even more preferable that the underlined amino acid V (valine) at position 1200 or the amino acid residue corresponding to the V is substituted with A (alanine), the 1201st amino acid P (proline) or the amino acid residue corresponding to the P is substituted with D (aspartic acid), the 1202nd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and the 1203rd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with R (arginine).

[0050] ST9.5_44 (SEQ ID NO: 5) E DLNYGFKKGRFKVERQVYQKFETMLINKLNYLVFKNRGVTEDGGLLRGYQLTYIPESLKNVGRQCGCIFY VPAA YTSKIDPTTGFADIFRFKNLTVEEKRDFIRKFDYIKYDLEKEMFVFAFDFKNFVTQNVEMSKNDWCVYTNGIRVKRRYANGRFTNETDEIDINVLMKKTFERTDIDWNDGHNL IDEIIDYDLESQIVDIFKMAVQMRNSRSEAEDRDYDRLVSPVLNESGDFFDSSKVGENLPKDADANGAYCIALKGLYKVRQIQENWNEEEKFSRAKLRISNKDWFDFVQNKRYL

[0051] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 5, the "one or several" in the protein (a2) may be within a range in which the (a2) functions as a protein having nuclease activity. The "one or several" in the (a2) may be, for example, 1 to 286, 1 to 214, 1 to 143, 1 to 142, 1 to 71, 1 to 57, 1 to 43, 1 to 28, 1 to 14, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the amino acid sequence of SEQ ID NO: 5.

[0052] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 5, in the proteins (a1) to (a3), for example, the underlined amino acid E at position 1129 or the amino acid residue corresponding to the amino acid E may be substituted with G. In this case, since the proteins (a1) to (a3) ​​can retain nuclease activity, it is preferable that the underlined amino acid A (alanine) at position 1202 or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and it is even more preferable that the underlined amino acid V (valine) at position 1200 or the amino acid residue corresponding to the V is substituted with A (alanine), the 1201st amino acid P (proline) or the amino acid residue corresponding to the P is substituted with D (aspartic acid), the 1202nd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and the 1203rd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with R (arginine).

[0053] ST9.5_58 (SEQ ID NO: 6) E DLNYGFKKGRFKVERQVYQKFETMLINKLNYLVFKNRGVTEDGGLLRGYQLTYIPESLKNVGRQCGCIFY VPAA YTSKIDPTTGFADIFRFKNLTVEEKRDFIRKFDYIKYDLEKEMFVFAFDFKNFVTQNVEMSKNDWCVYTNGIRVKRRYANGRFTNETDEIDINVLMKKTFERTDIDWNDGHNL IDEIIDYDLESQIVDIFKMAVQMRNSRSEAEDRDYDRLVSPVLNESGDFFDSSKVGENLPKDADANGAYCIALKGLYKVRQIQENWNEEEKFSRAKLRISNKDWFDFVQNKRYL

[0054] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 6, the "one or several" in the protein (a2) may be within a range in which the (a2) functions as a protein having nuclease activity. The "one or several" in the (a2) may be, for example, 1 to 287, 1 to 215, 1 to 143, 1 to 142, 1 to 71, 1 to 57, 1 to 43, 1 to 28, 1 to 14, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the amino acid sequence of SEQ ID NO: 6.

[0055] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 6, in the proteins (a1) to (a3), for example, the underlined amino acid E at position 1136 or the amino acid residue corresponding to the amino acid E may be substituted with G. In this case, since the proteins (a1) to (a3) ​​can retain nuclease activity, it is preferable that the underlined amino acid A (alanine) at position 1209 or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and it is more preferable that the underlined amino acid V (valine) at position 1207 or the amino acid residue corresponding to the V is substituted with A (alanine), the 1208th amino acid P (proline) or the amino acid residue corresponding to the P is substituted with D (aspartic acid), the 1209th amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and the 1210th amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with R (arginine).

[0056] ST9.5_2R (SEQ ID NO: 7) E DLNYGFKKGRFKVERQVYQKFETMLINKLNYLVFKNRGVTEDGGLLRGYQLTYIPESLKNVGRQCGCIFY VPAA YTSKIDPTTGFADIFRFKNLTVEEKRDFIRKFDYIKYDLEKEMFVFAFDFKNFVTQNVEMSKNDWCVYTNGIRVKRRYANGRFTNETDEIDINVLMKKTFERTDIDWNDGHNL IDEIIDYDLESQIVDIFKMAVQMRNSRSEAEDRDYDRLVSPVLNESGDFFDSSKVGENLPKDADANGAYCIALKGLYKVRQIQENWNEEEKFSRAKLRISNKDWFDFVQNKRYL

[0057] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 7, the "one or several" in the protein (a2) may be within a range in which the (a2) functions as a protein having nuclease activity. The "one or several" in the (a2) may be, for example, 1 to 285, 1 to 214, 1 to 142, 1 to 71, 1 to 57, 1 to 42, 1 to 28, 1 to 14, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the amino acid sequence of SEQ ID NO: 7.

[0058] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 7, in the proteins (a1) to (a3), for example, the underlined amino acid E at position 1127 or the amino acid residue corresponding to the amino acid E may be substituted with G. In this case, since the proteins (a1) to (a3) ​​can retain nuclease activity, it is preferable that the underlined amino acid A (alanine) at position 1200 or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and it is even more preferable that the underlined amino acid V (valine) at position 1198 or the amino acid residue corresponding to the V is substituted with A (alanine), the 1199th amino acid P (proline) or the amino acid residue corresponding to the P is substituted with D (aspartic acid), the 1200th amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and the 1201st amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with R (arginine).

[0059] ST9.5_214 (SEQ ID NO: 8) E DLNYGFKKGRFKVERQVYQKFETMLINKLNYLVFKNRGVTEDGGLLRGYQLTYIPESLKNVGRQCGCIFY VPAA YTSKIDPTTGFADIFRFKNLTVEEKRDFIRKFDYIKYDLEKEMFVFAFDFKNFVTQNVEMSKNDWCVYTNGIRVKRRYANGRFTNETDEIDINVLMKKTFERTDIDWNDGHNL IDEIIDYDLESQIVDIFKMAVQMRNSRSEAEDRDYDRLVSPVLNESGDFFDSSKVGENLPKDADANGAYCIALKGLYKVRQIQENWNEEEKFSRAKLRISNKDWFDFVQNKRYL

[0060] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 8, the "one or several" in the protein (a2) may be within a range in which the (a2) functions as a protein having nuclease activity. The "one or several" in the (a2) may be, for example, 1 to 284, 1 to 213, 1 to 142, 1 to 71, 1 to 56, 1 to 42, 1 to 28, 1 to 14, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the amino acid sequence of SEQ ID NO: 8.

[0061] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 8, in the proteins (a1) to (a3), for example, the underlined amino acid E at position 1120 or the amino acid residue corresponding to the amino acid E may be substituted with G. In this case, since the proteins (a1) to (a3) ​​can retain nuclease activity, it is preferable that the underlined amino acid A (alanine) at position 1193 or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and it is even more preferable that the underlined amino acid V (valine) at position 1191 or the amino acid residue corresponding to the V is substituted with A (alanine), the 1192nd amino acid P (proline) or the amino acid residue corresponding to the P is substituted with D (aspartic acid), the 1193rd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and the 1194th amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with R (arginine).

[0062] ST9.5_2W (SEQ ID NO: 9) E DLNYGFKKGRFKVERQVYQKFETMLINKLNYLVFKNRGVTEDGGLLRGYQLTYIPESLKNVGRQCGCIFY VPAA YTSKIDPTTGFADIFRFKNLTVEEKRDFIRKFDYIKYDLEKEMFVFAFDFKNFVTQNVEMSKNDWCVYTNGIRVKRRYANGRFTNETDEIDINVLMKKTFERTDIDWNDGHNL IDEIIDYDLESQIVDIFKMAVQMRNSRSEAEDRDYDRLVSPVLNESGDFFDSSKVGENLPKDADANGAYCIALKGLYKVRQIQENWNEEEKFSRAKLRISNKDWFDFVQNKRYL

[0063] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 9, the "one or several" in the protein (a2) may be within a range in which the (a2) functions as a protein having nuclease activity. The "one or several" in the (a2) may be, for example, 1 to 285, 1 to 214, 1 to 142, 1 to 71, 1 to 57, 1 to 42, 1 to 28, 1 to 14, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the amino acid sequence of SEQ ID NO: 9.

[0064] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 9, in the proteins (a1) to (a3), for example, the underlined amino acid E at position 1127 or the amino acid residue corresponding to the amino acid E may be substituted with G. In this case, since the proteins (a1) to (a3) ​​can retain nuclease activity, it is preferable that the underlined amino acid A (alanine) at position 1200 or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and it is more preferable that the underlined amino acid V (valine) at position 1198 or the amino acid residue corresponding to the V is substituted with A (alanine), the 1199th amino acid P (proline) or the amino acid residue corresponding to the P is substituted with D (aspartic acid), the 1200th amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and the 1201st amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with R (arginine).

[0065] ST9.5_243 (SEQ ID NO: 10) E DLNYGFKKGRFKVERQVYQKFETMLINKLNYLVFKNRGVTEDGGLLRGYQLTYIPESLKNVGRQCGCIFY VPAA YTSKIDPTTGFADIFRFKNLTVEEKRDFIRKFDYIKYDLEKEMFVFAFDFKNFVTQNVEMSKNDWCVYTNGIRVKRRYANGRFTNETDEIDINVLMKKTFERTDIDWNDGHNL IDEIIDYDLESQIVDIFKMAVQMRNSRSEAEDRDYDRLVSPVLNESGDFFDSSKVGENLPKDADANGAYCIALKGLYKVRQIQENWNEEEKFSRAKLRISNKDWFDFVQNKRYL

[0066] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 10, the "one or several" in the protein (a2) may be within a range in which the (a2) functions as a protein having nuclease activity. The "one or several" in the (a2) may be, for example, 1 to 284, 1 to 213, 1 to 142, 1 to 71, 1 to 56, 1 to 42, 1 to 28, 1 to 14, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the amino acid sequence of SEQ ID NO: 10.

[0067] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 10, in the proteins (a1) to (a3), for example, the underlined amino acid E at position 1120 or the amino acid residue corresponding to the amino acid E may be substituted with G. In this case, since the proteins (a1) to (a3) ​​can retain nuclease activity, it is preferable that the underlined amino acid A (alanine) at position 1193 or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and it is even more preferable that the underlined amino acid V (valine) at position 1191 or the amino acid residue corresponding to the V is substituted with A (alanine), the 1192nd amino acid P (proline) or the amino acid residue corresponding to the P is substituted with D (aspartic acid), the 1193rd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and the 1194th amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with R (arginine).

[0068] ST9.5_2P (SEQ ID NO: 11) E DLNYGFKKGRFKVERQVYQKFETMLINKLNYLVFKNRGVTEDGGLLRGYQLTYIPESLKNVGRQCGCIFY VPAA YTSKIDPTTGFADIFRFKNLTVEEKRDFIRKFDYIKYDLEKEMFVFAFDFKNFVTQNVEMSKNDWCVYTNGIRVKRRYANGRFTNETDEIDINVLMKKTFERTDIDWNDGHNL IDEIIDYDLESQIVDIFKMAVQMRNSRSEAEDRDYDRLVSPVLNESGDFFDSSKVGENLPKDADANGAYCIALKGLYKVRQIQENWNEEEKFSRAKLRISNKDWFDFVQNKRYL

[0069] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 11, the "one or several" in the protein (a2) may be within a range in which the (a2) functions as a protein having nuclease activity. The "one or several" in the (a2) may be, for example, 1 to 285, 1 to 214, 1 to 142, 1 to 71, 1 to 57, 1 to 42, 1 to 28, 1 to 14, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the amino acid sequence of SEQ ID NO: 11.

[0070] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 11, in the proteins (a1) to (a3), for example, the underlined amino acid E at position 1128 or the amino acid residue corresponding to the amino acid E may be substituted with G. In this case, since the proteins (a1) to (a3) ​​can retain nuclease activity, it is preferable that the underlined amino acid A (alanine) at position 1201 or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and it is more preferable that the underlined amino acid V (valine) at position 1199 or the amino acid residue corresponding to the V is substituted with A (alanine), the 1200th amino acid P (proline) or the amino acid residue corresponding to the P is substituted with D (aspartic acid), the 1201st amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and the 1202nd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with R (arginine).

[0071] ST9.5_S3 (SEQ ID NO: 12)

[0072] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 12, the "one or several" in the protein (a2) may be within a range in which the (a2) functions as a protein having nuclease activity. The "one or several" in the (a2) may be, for example, 1 to 286, 1 to 214, 1 to 143, 1 to 142, 1 to 71, 1 to 57, 1 to 42, 1 to 28, 1 to 14, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the amino acid sequence of SEQ ID NO: 12.

[0073] ST9.5_S4 (SEQ ID NO: 13) When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 13, the "one or several" in the protein (a2) may be within a range in which the (a2) functions as a protein having nuclease activity. The "one or several" in the (a2) may be, for example, 1 to 286, 1 to 214, 1 to 143, 1 to 142, 1 to 71, 1 to 57, 1 to 42, 1 to 28, 1 to 14, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the amino acid sequence of SEQ ID NO: 13.

[0074] ST9.5_3LA1 (SEQ ID NO: 14) E DLNYGFKKGRFKVERQVYQKFETMLINKLNYLVFKNRGVTEDGGLLRGYQLTYIPESLKNVGRQCGCIFY VPAA YTSKIDPTTGFADIFRFKNLTVEEKRDFIRKFDYIKYDLEKEMFVFAFDFKNFVTQNVEMSKNDWCVYTNGIRVKRRYANGRFTNETDEIDINVLMKKTFERTDIDWNDGHNL IDEIIDYDLESQIVDIFKMAVQMRNSRSEAEDRDYDRLVSPVLNESGDFFDSSKVGENLPKDADANGAYCIALKGLYKVKQIQENWNEEEKFSRAKLRISNKDWFDFVQNKRYL

[0075] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 14, the "one or several" in the protein (a2) may be within a range in which the (a2) functions as a protein having nuclease activity. The "one or several" in the (a2) or (a3) ​​may be, for example, 1 to 286, 1 to 214, 1 to 143, 1 to 142, 1 to 71, 1 to 57, 1 to 43, 1 to 28, 1 to 14, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the amino acid sequence of SEQ ID NO: 14.

[0076] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 14, in the proteins (a1) to (a3), for example, the underlined amino acid E at position 1129 or the amino acid residue corresponding to the amino acid E may be substituted with G. In this case, since the proteins (a1) to (a3) ​​can retain nuclease activity, it is preferable that the underlined amino acid A (alanine) at position 1202 or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and it is even more preferable that the underlined amino acid V (valine) at position 1200 or the amino acid residue corresponding to the V is substituted with A (alanine), the 1201st amino acid P (proline) or the amino acid residue corresponding to the P is substituted with D (aspartic acid), the 1202nd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and the 1203rd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with R (arginine).

[0077] ST9.5_3LA4 (SEQ ID NO: 15) E DLNYGFKKGRFKVERQVYQKFETMLINKLNYLVFKNRGVTEDGGLLRGYQLTYIPESLKNVGRQCGCIFY VPAA YTSKIDPTTGFADIFRFKNLTVEEKRDFIRKFDYIKYDLEKEMFVFAFDFKNFVTQNVEMSKNDWCVYTNGIRVKRRYANGRFTNETDEIDINVLMKKTFERTDIDWNDGHNL IDEIIDYDLESQIVDIFKMAVQMRNSRSEAEDRDYDRLVSPVLNESGDFFDSSKVGENLPKDADANGAYCIALKGLYKVKQIQENWNEEEKFSRAKLRISNKDWFDFVQNKRYL

[0078] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 15, the "one or several" in the protein (a2) may be within a range in which the (a2) functions as a protein having nuclease activity. The "one or several" in the (a2) may be, for example, 1 to 286, 1 to 214, 1 to 143, 1 to 142, 1 to 71, 1 to 57, 1 to 43, 1 to 28, 1 to 14, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the amino acid sequence of SEQ ID NO: 15.

[0079] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 15, in the proteins (a1) to (a3), for example, the underlined amino acid E at position 1129 or the amino acid residue corresponding to the amino acid E may be substituted with G. In this case, since the proteins (a1) to (a3) ​​can retain nuclease activity, it is preferable that the underlined amino acid A (alanine) at position 1202 or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and it is more preferable that the underlined amino acid V (valine) at position 1200 or the amino acid residue corresponding to the V is substituted with A (alanine), the 1201st amino acid P (proline) or the amino acid residue corresponding to the P is substituted with D (aspartic acid), the 1202nd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and the 1203rd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with R (arginine).

[0080] ST9 (SEQ ID NO: 16) MGEKTNYFNNFIGISPLTKTLRNALIPTTEITQKHIIEYGIIKDDELRKENRQILKTIMDDYYRSFLTEKSAIHDIDWKPLFLEMENELRHGNNKVNLEKEQKAKRKAINKYFSEDERYKKMFSAKLLSDILPEFVIHNDEYSAEEKEEKQVIKLFSRFATSLKEYFRNRSNTFSADNISTSACHRIVNDNASIFLENVMAYRKIISSLPADELDKLESIQDKLKIKSISEVYTYDNYGKYITQEGIDLYNDICGKVNSFMNLYCQQNKENKNVYKMRKLHKQILCIAKTSYEVGEGYTSDEEVLEVFRNTLNKSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKNDAVVAIMKDLLDSVKSFENYIKAFFGEGK ETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSTEKFKLNFNSPTLARGWSKSKEYSNNAIILSKDGILYLGIFNVKNKPDKKIIEGHIRENDGDYKKMVYNLLPGANKMLPKVLISSKSGVETYKPSDYILEGYSNKHLKSSDTFDINYCHDLIDYYKDCIDIHPEWKNFDFNSDTADFEDISGFYREAVEAQGYKIDWTYISGENIEELQEKGQLFLFKIYNKDFSTKSTGTD NLHTMYLKNLFSEENLKDVVLKLNGEAEIFYRKSSIKNPIIHKKGSMLVNTYIEEEEEHDGKIVEVRKIVPEEIYKELYNHFNNNGKAELSEEAKRLESLVCHEAAKDIVKDCRYTYDKFFIHLPMTINFKASGSSSNLDMMLQYISGQNDMHIIGIDRGERNLIYVSVIDAHGNIVKQKSFNIVAGYDYQEKLKMQEGARQNARKEWKEIGKIKEIKEGYLSLVIHEIAQMVIEYNAIIAM EDLNYGFKKGRFKVERQVYQKFETMLINKLNYLVFKNRGVTEDGGLLRGYQLTYIPESLKNVGRQCGCIFY VPAA YTSKIDPTTGFADIFRFKNLTVEEKRDFIRKFDYIKYDLEKEMFVFAFDFKNFVTQNVEMSKNDWCVYTNGIRVKRRYANGRFTNETDEIDINVLMKKTFERTDIDWNDGHNL IDEIIDYDLESQIVDIFKMAVQMRNSRSEAEDRDYDRLVSPVLNESGDFFDSSKVGENLPKDADANGAYCIALKGLYKVRQIQENWNEEEKFSRAKLRISNKDWFDFVQNKRYL

[0081] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 16, the "one or several" in the protein (a2) may be within a range in which the (a2) functions as a protein having nuclease activity. The "one or several" in the (a2) may be, for example, 1 to 254, 1 to 190, 1 to 142, 1 to 127, 1 to 63, 1 to 50, 1 to 38, 1 to 25, 1 to 12, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the amino acid sequence of SEQ ID NO: 16.

[0082] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 16, in the proteins (a1) to (a3), for example, the underlined amino acid E at position 969 or the amino acid residue corresponding to the amino acid E may be substituted with G. In this case, since the proteins (a1) to (a3) ​​can retain nuclease activity, it is preferable that the underlined amino acid A (alanine) at position 1042 or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and it is more preferable that the underlined amino acid V (valine) at position 1040 or the amino acid residue corresponding to the V is substituted with A (alanine), the 1041st amino acid P (proline) or the amino acid residue corresponding to the P is substituted with D (aspartic acid), the 1042nd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and the 1043rd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with R (arginine).

[0083] ST9_2 (SEQ ID NO: 17) MGEKTNYFNNFIGISPLTKTLRNALIPTTEITQKHIIEYGIIKDDELRKENRQILKTIMDDYYRSFLTEKSAIHDIDWKPLFLEMENELRHGNNKVNLEKEQKAKRKAINKYFSEDERYKKMFSAKLLSDILPEFVIHNDEYSAEEKEEKTQVIKLFSRFATSLKEYFRNRSNTFSADNISTSACHRIVNDNASIFLENVMAYRKIISSLPADELDKLEASIQDKLKIKSISEVYTYDNYGKYITQEGIDLYNDICGKVNSFMNLYCQQNKENKNVYKMRKLHKQILCIAKTSYEVPYRFESDEELYSSVNEFLSDVEKKGIIERLQKIGENYDKYELDKVYIAGKYYETFSQKVYKEWNTINSALEIYYTNTLHGSGKSKAEKVRKAVKNDSQKSIAQINELVAEYNLCSDESVKAQNYISEITAIIGQSNMHPLEYNPEVNLVEDKGKASELKKSLDVIMNIMHWCSAFITEEIVSK DAEFYSEIEDIYDELTPVVKLYNMVRNYVTQKPYSTEKFKLNFNSPTLARGWSKSKEYSNNAIILSKDGILIYLGIFNVKNKPDKKIIEGHIRENDGDYKKMVYNLLPGANKMLPKVLISSKSGVETYKPSDYILEGYKSNKHLKSSDTFDINYCHDLIDYYKDCIDIHPEWKNFDFNFSDTADFEDISGFYREVEAQGYKIDWTYISGENIEELQEKGQLFLFKIYNKDFSTKSTGTDNLHTMYLKNLFSEENLKDVVLKLNGEAEIFYRKSSIKNPIIHKKGSMLVNKTYIEEEHDGKIVEVRKIVPEEIYKELYNHFNNNGKAELSEEAKRLESLVCHEAAKDIVKDCRYTYDKFFIHLPMTINFKASGSSSLNDMMLQYISGQNDMHIIGIDRGERNLIYVSVIDAHGNIVKQKSFNIVAGYDYQEKLKMQEGARQNARKEWKEIGKIKEIKEGYLSLVIHEIAQMVIEYNAIIAM EDLNYGFKKGRFKVERQVYQKFETMLINKLNYLVFKNRGVTEDGGLLRGYQLTYIPESLKNVGRQCGCIFY VPAA YTSKIDPTTGFADIFRFKNLTVEEKRDFIRKFDYIKYDLEKEMFVFAFDFKNFVTQNVEMSKNDWCVYTNGIRVKRRYANGRFTNETDEIDINVLMKKTFERTDIDWNDGHNL IDEIIDYDLESQIVDIFKMAVQMRNSRSEAEDRDYDRLVSPVLNESGDFFDSSKVGENLPKDADANGAYCIALKGLYKVRQIQENWNEEEKFSRAKLRISNKDWFDFVQNKRYL

[0084] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 17, the "one or several" in the protein (a2) may be within a range in which the (a2) functions as a protein having nuclease activity. The "one or several" in the (a2) may be, for example, 1 to 252, 1 to 189, 1 to 142, 1 to 126, 1 to 63, 1 to 50, 1 to 37, 1 to 25, 1 to 12, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the amino acid sequence of SEQ ID NO: 17.

[0085] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 17, in the proteins (a1) to (a3), for example, the underlined amino acid E at position 960 or the amino acid residue corresponding to the amino acid E may be substituted with G. In this case, since the proteins (a1) to (a3) ​​can retain nuclease activity, it is preferable that the underlined amino acid A (alanine) at position 1033 or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and it is more preferable that the underlined amino acid V (valine) at position 1031 or the amino acid residue corresponding to the V is substituted with A (alanine), the 1032nd amino acid P (proline) or the amino acid residue corresponding to the P is substituted with D (aspartic acid), the 1033rd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and the 1034th amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with R (arginine).

[0086] ST9_3LA (SEQ ID NO: 18) MGKNTNYFNRFIGISSSQKTLRNALIPTEITQKHIIEYGIIKDDELRKENRQILKTIMDDYYRSFLTEKSAIHDIDWKPLFLEMENELRHGNNKVNLEKEQKAKRKAINKYFSEDERYKKMFSAKLLSDILPEFVIHNDEYSAEEKEEKQVIKLFSRFATSLKEYFRNRSNTFSADNISTSACHRIVNDNASIFLENVMAYRKIISSLPADELDKLESIQDKLKIKSISEVYTYDNYGKYITQEGIDLYNDICGKVNSFMNLYCQQNKENKNVYKMRKLHKQILCIAKTSYEVGEGYTSDEEVLEVFRNTLNKSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKNDAVVAIMKDLLDSVKSFENYIKAFFGEGK ETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSTEKFKLNFNSPTLARGWSKSKEYSNNAIILSKDGILILYLGIFNVKNKPDKKIIEGHIRENDGDYKKMVYNLLSGSAKMLPKVVFAGSNEKIFGHLISKRILEIREKKLYTAAAGDRKAVAEWIDFMKSAIAIHPEWNEYFKFKFKNTAEYDNANKFYEDIDKQTYKIDWTYISGENIEELQEKGQLFLFKIYNKDFSTKSTGTDNLHTMYLKNLFSEENLKDVVLKLNGEAEIFYRKSSIKNPIIHKKGSMLVNKTYIEEEHDGKIVEVRKIVPEEIYKELYNHFNNGKAELSEEAKRLESLVCHEAAKDIVKDYRYTYDKFFIHLPMVINFKAGDSNSLNDMILQYISGQNDMHIIGIDRGERNLIYVSVIDVHGNIVKQKSFNIVGGYDYQEKLKMQEGARQNARKEWKEIGKIKEIKEGYLSLVIHEIAQMVIEYNAIIAM EDLNYGFKKGRFKVERQVYQKFETMLINKLNYLVFKNRGVTEDGGLLRGYQLTYIPESLKNVGRQCGCIFY VPAA YTSKIDPTTGFADIFRFKNLTVEEKRDFIRKFDYIKYDLEKEMFVFAFDFKNFVTQNVEMSKNDWCVYTNGIRVKRRYANGRFTNETDEIDINVLMKKTFERTDIDWNDGHNL IDEIIDYDLESQIVDIFKMAVQMRNSRSEAEDRDYDRLVSPVLNESGDFFDSSKVGENLPKDADANGAYCIALKGLYKVKQIQENWNEEEKFSRAKLRISNKDWFDFVQNKRYL

[0087] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 18, the "one or several" in the protein (a2) may be within a range in which the (a2) functions as a protein having nuclease activity. The "one or several" in the (a2) may be, for example, 1 to 254, 1 to 190, 1 to 142, 1 to 127, 1 to 63, 1 to 50, 1 to 38, 1 to 25, 1 to 12, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the amino acid sequence of SEQ ID NO: 18.

[0088] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 18, in the proteins (a1) to (a3), for example, the underlined amino acid E at position 969 or the amino acid residue corresponding to the amino acid E may be substituted with G. In this case, since the proteins (a1) to (a3) ​​can retain nuclease activity, it is preferable that the underlined amino acid A (alanine) at position 1042 or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and it is more preferable that the underlined amino acid V (valine) at position 1040 or the amino acid residue corresponding to the V is substituted with A (alanine), the 1041st amino acid P (proline) or the amino acid residue corresponding to the P is substituted with D (aspartic acid), the 1042nd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with E (glutamic acid), and the 1043rd amino acid A (alanine) or the amino acid residue corresponding to the amino acid A is substituted with R (arginine).

[0089] ST9_S (SEQ ID NO: 19)

[0090] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 19, the "one or several" in the protein (a2) may be within a range in which the (a2) functions as a protein having nuclease activity. The "one or several" in the (a2) may be, for example, 1 to 254, 1 to 190, 1 to 142, 1 to 127, 1 to 63, 1 to 50, 1 to 38, 1 to 25, 1 to 12, 1 to 10, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 or 2 in the amino acid sequence of SEQ ID NO: 19.

[0091] In the protein (a3), the "identity" may be within a range in which the protein (a3) ​​functions as a protein having nuclease activity. The "identity" of the protein (a3) ​​is, for example, 70%, 75%, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more relative to the amino acid sequence of the protein (a1). The "identity" can be calculated, for example, by using the default parameters in the homology algorithm BLAST (http: / / www.ncbi.nlm.nih.gov / BLAST / ) of the National Center for Biotechnology Information (NCBI) (hereinafter the same).

[0092] The protein of the present disclosure may recognize, for example, a protospacer adjacent motif (PAM) sequence. Examples of the PAM sequence include TTTV (SEQ ID NO: 20, V=A, G, or C; the same applies below), NAAN (SEQ ID NO: 21), NAGN (SEQ ID NO: 22), or NGAN (SEQ ID NO: 23). The NAAN (SEQ ID NO: 21), NAGN (SEQ ID NO: 22), or NGAN (SEQ ID NO: 23) are generally PAM sequences known to be difficult to recognize by Cas9 or Cas12. Examples of the NGAN (SEQ ID NO: 23) include AGAC (SEQ ID NO: 24), TGAC (SEQ ID NO: 25), GGAC (SEQ ID NO: 26), CGAC (SEQ ID NO: 27), etc. Examples of the NAGN (SEQ ID NO: 22) include AAGC (SEQ ID NO: 28), GAGC (SEQ ID NO: 29), and CAGC (SEQ ID NO: 30), and examples of the NAAN (SEQ ID NO: 21) include GAAC (SEQ ID NO: 31), CAAC (SEQ ID NO: 32), etc.

[0093] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 1, the PAM sequence of the protein (a) preferably includes, for example, GGAC (SEQ ID NO: 26), GAGC (SEQ ID NO: 29), or GAAC (SEQ ID NO: 31).

[0094] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 2, the PAM sequence of the protein (a) preferably includes, for example, TGAC (SEQ ID NO: 25) or GGAC (SEQ ID NO: 26).

[0095] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 3, the PAM sequence of the protein (a) preferably includes, for example, TGAC (SEQ ID NO: 25), GGAC (SEQ ID NO: 26), CGAC (SEQ ID NO: 27), GAGC (SEQ ID NO: 29), GAAC (SEQ ID NO: 31), or CAAC (SEQ ID NO: 32).

[0096] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 4, the PAM sequence of the protein (a) preferably includes, for example, TTTV (SEQ ID NO: 20), AGAC (SEQ ID NO: 24), TGAC (SEQ ID NO: 25), GGAC (SEQ ID NO: 26), CGAC (SEQ ID NO: 27), AAGC (SEQ ID NO: 28), GAGC (SEQ ID NO: 29), CAGC (SEQ ID NO: 30), GAAC (SEQ ID NO: 31), or CAAC (SEQ ID NO: 32).

[0097] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 5, the PAM sequence of the protein (a) preferably includes, for example, GAGC (SEQ ID NO: 29).

[0098] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 6, the PAM sequence of the protein (a) preferably includes, for example, GGAC (SEQ ID NO: 26) or GAGC (SEQ ID NO: 29).

[0099] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 7, the PAM sequence of the protein (a) preferably includes, for example, TTTV (SEQ ID NO: 20), AGAC (SEQ ID NO: 24), TGAC (SEQ ID NO: 25), GGAC (SEQ ID NO: 26), CGAC (SEQ ID NO: 27), AAGC (SEQ ID NO: 28), GAGC (SEQ ID NO: 29), GAAC (SEQ ID NO: 31), or CAAC (SEQ ID NO: 32).

[0100] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 8, the PAM sequence of the protein (a) preferably includes, for example, TTTV (SEQ ID NO: 20), AGAC (SEQ ID NO: 24), TGAC (SEQ ID NO: 25), GGAC (SEQ ID NO: 26), CGAC (SEQ ID NO: 27), GAAC (SEQ ID NO: 31), or CAAC (SEQ ID NO: 32).

[0101] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 9, the PAM sequence of the protein (a) preferably includes, for example, TGAC (SEQ ID NO: 25), AAGC (SEQ ID NO: 28), or CAAC (SEQ ID NO: 32).

[0102] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 10, the PAM sequence of the protein (a) preferably includes, for example, GGAC (SEQ ID NO: 26).

[0103] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 11, the PAM sequence of the protein (a) preferably includes, for example, GAGC (SEQ ID NO: 29).

[0104] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 12, the PAM sequence of the protein (a) preferably includes, for example, AGAC (SEQ ID NO: 24), TGAC (SEQ ID NO: 25), GGAC (SEQ ID NO: 26), CGAC (SEQ ID NO: 27), AAGC (SEQ ID NO: 28), CAGC (SEQ ID NO: 30), or GAAC (SEQ ID NO: 31).

[0105] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 13, the PAM sequence of the protein (a) preferably includes, for example, GGAC (SEQ ID NO: 26) or AAGC (SEQ ID NO: 28).

[0106] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 14, the PAM sequence of the protein (a) preferably includes, for example, GGAC (SEQ ID NO: 26).

[0107] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 15, it is preferable that the PAM sequence of the protein (a) includes, for example, TGAC (SEQ ID NO: 25), GGAC (SEQ ID NO: 26), CGAC (SEQ ID NO: 27), or GAAC (SEQ ID NO: 31).

[0108] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 16, the PAM sequence of the protein (a) preferably includes, for example, TTTV (SEQ ID NO: 20), AGAC (SEQ ID NO: 24), TGAC (SEQ ID NO: 25), GGAC (SEQ ID NO: 26), CGAC (SEQ ID NO: 27), AAGC (SEQ ID NO: 28), GAGC (SEQ ID NO: 29), CAGC (SEQ ID NO: 30), GAAC (SEQ ID NO: 31), or CAAC (SEQ ID NO: 32).

[0109] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 17, the PAM sequence of the protein (a) preferably includes, for example, TTTV (SEQ ID NO: 20), AGAC (SEQ ID NO: 24), TGAC (SEQ ID NO: 25), GGAC (SEQ ID NO: 26), CGAC (SEQ ID NO: 27), AAGC (SEQ ID NO: 28), GAGC (SEQ ID NO: 29), or CAAC (SEQ ID NO: 32).

[0110] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 18, the PAM sequence of the protein (a) preferably includes, for example, TGAC (SEQ ID NO: 25), GGAC (SEQ ID NO: 26), CGAC (SEQ ID NO: 27), or GAAC (SEQ ID NO: 31).

[0111] When the protein of the present disclosure is a protein consisting of the amino acid sequence of SEQ ID NO: 19, the PAM sequence of the protein (a) preferably includes, for example, AGAC (SEQ ID NO: 24), TGAC (SEQ ID NO: 25), GGAC (SEQ ID NO: 26), CGAC (SEQ ID NO: 27), AAGC (SEQ ID NO: 28), CAGC (SEQ ID NO: 30), or GAAC (SEQ ID NO: 31).

[0112] The protein of the present disclosure may be, for example, an RNA-dependent DNA nuclease.The protein of the present disclosure may, for example, form a complex with the guide RNA described below and function as a DNA nuclease.The nuclease may, for example, be an endonuclease.

[0113] The protein of the present disclosure may further have, for example, an enzymatic activity domain. When the protein of the present disclosure has an enzymatic activity domain, the protein of the present disclosure may not have, for example, nuclease activity. In this case, the protein of the present disclosure may or may not have, for example, nickase activity. Examples of such proteins include the following proteins (1) to (7). Examples of the enzymatic activity include nucleic acid modification activities such as methylase activity, demethylase activity, base modification activity, histone modification activity, RNA cleavage activity, and DNA cleavage activity; transcription activation activity, transcription repression activity, transcription deactivation factor activity, DNA integration activity, and nucleic acid binding activity. (1) A protein in which, in the amino acid sequence of SEQ ID NO: 1 or 6, the amino acid E at position 1136 is replaced with, for example, A other than G, and further, the amino acid A (alanine) at position 1209 or an amino acid residue corresponding to said amino acid A is preferably replaced with E (glutamic acid), the amino acid V (valine) at position 1207 or an amino acid residue corresponding to said V is replaced with A (alanine), the amino acid P (proline) at position 1208 or an amino acid residue corresponding to said P is replaced with D (aspartic acid), the amino acid A (alanine) at position 1209 or an amino acid residue corresponding to said amino acid A is replaced with E (glutamic acid), and the amino acid A (alanine) at position 1210 or an amino acid residue corresponding to said amino acid A is replaced with R (arginine), and the nuclease activity is reduced compared to that of the unsubstituted protein; (2) In the amino acid sequence of SEQ ID NO: 2 to 5, 14, or 15, the amino acid E at position 1129 is preferably replaced with, for example, A other than G, and the amino acid A (alanine) at position 1202 or an amino acid residue corresponding to the amino acid A is preferably replaced with E (glutamic acid), the amino acid V (valine) at position 1200 or an amino acid residue corresponding to the V is replaced with A (alanine), the amino acid P (proline) at position 1201 or an amino acid residue corresponding to the P is replaced with D (aspartic acid), the amino acid A (alanine) at position 1202 or an amino acid residue corresponding to the amino acid A is replaced with E (glutamic acid), and the amino acid A (alanine) at position 1203 or an amino acid residue corresponding to the amino acid A is replaced with R (arginine), and the nuclease activity is reduced compared to that of the unsubstituted protein; (3) In the amino acid sequence of SEQ ID NO: 7 or 9, the 1127th amino acid sequence E is preferably replaced with, for example, A other than G, and the 1200th amino acid A (alanine) or an amino acid residue corresponding to the amino acid A is preferably replaced with E (glutamic acid), the 1198th amino acid V (valine) or an amino acid residue corresponding to the V is replaced with A (alanine), the 1199th amino acid P (proline) or an amino acid residue corresponding to the P is replaced with D (aspartic acid), the 1200th amino acid A (alanine) or an amino acid residue corresponding to the amino acid A is replaced with E (glutamic acid), and the 1201st amino acid A (alanine) or an amino acid residue corresponding to the amino acid A is replaced with R (arginine), and the nuclease activity is reduced compared to the unsubstituted protein; (4) In the amino acid sequence of SEQ ID NO: 8 or 10, the amino acid sequence E at position 1120 is preferably replaced with, for example, A other than G, and further, the amino acid A (alanine) at position 1193 or the amino acid residue corresponding to the amino acid A is preferably replaced with E (glutamic acid), the amino acid V (valine) at position 1191 or the amino acid residue corresponding to the V is replaced with A (alanine), the amino acid P (proline) at position 1192 or the amino acid residue corresponding to the P is replaced with D (aspartic acid), the amino acid A (alanine) at position 1193 or the amino acid residue corresponding to the amino acid A is replaced with E (glutamic acid), and the amino acid A (alanine) at position 1194 or the amino acid residue corresponding to the amino acid A is replaced with R (arginine), and the nuclease activity is reduced compared to the unsubstituted protein; (5) In the amino acid sequence of SEQ ID NO: 11, the amino acid E at position 1128 is preferably replaced with, for example, A other than G, and the amino acid A (alanine) at position 1201 or the amino acid residue corresponding to the amino acid A is preferably replaced with E (glutamic acid), the amino acid V (valine) at position 1199 or the amino acid residue corresponding to the V is replaced with A (alanine), the amino acid P (proline) at position 1200 or the amino acid residue corresponding to the P is replaced with D (aspartic acid), the amino acid A (alanine) at position 1201 or the amino acid residue corresponding to the amino acid A is replaced with E (glutamic acid), and the amino acid A (alanine) at position 1202 or the amino acid residue corresponding to the amino acid A is replaced with R (arginine), and the nuclease activity is reduced compared to that of the unsubstituted protein; (6) In the amino acid sequence of SEQ ID NO: 16 or 18, the amino acid E at position 969 is preferably replaced with, for example, A other than G, and the amino acid A (alanine) at position 1042 or the amino acid residue corresponding to the amino acid A is preferably replaced with E (glutamic acid), the amino acid V (valine) at position 1040 or the amino acid residue corresponding to the V is replaced with A (alanine), the amino acid P (proline) at position 1041 or the amino acid residue corresponding to the P is replaced with D (aspartic acid), the amino acid A (alanine) at position 1042 or the amino acid residue corresponding to the amino acid A is replaced with E (glutamic acid), and the amino acid A (alanine) at position 1043 or the amino acid residue corresponding to the amino acid A is replaced with R (arginine), and the nuclease activity is reduced compared to that of the unsubstituted protein; (7) In the amino acid sequence of SEQ ID NO: 17, it is preferable that the 960th amino acid E is replaced with, for example, A other than G, and further, the 1033rd amino acid A (alanine) or an amino acid residue corresponding to the amino acid A is replaced with E (glutamic acid), the 1031st amino acid V (valine) or an amino acid residue corresponding to the V is replaced with A (alanine), the 1032nd amino acid P (proline) or an amino acid residue corresponding to the P is replaced with D (aspartic acid), the 1033rd amino acid A (alanine) or an amino acid residue corresponding to the amino acid A is replaced with E (glutamic acid), and the 1034th amino acid A (alanine) or an amino acid residue corresponding to the amino acid A is replaced with R (arginine), and the nuclease activity is reduced compared to the unsubstituted protein.

[0114] The enzyme activity domain is preferably a nucleic acid modification activity domain having a nucleic acid modification activity. The nucleic acid modification activity can be, for example, an activity that directly or indirectly causes DNA modification by modifying a nucleic acid, such as a nucleic acid decomposition activity, a nucleic acid base conversion activity, a DNA hydrolysis activity, etc. The DNA modification can be, for example, a DNA strand cleavage reaction that cleaves a DNA strand catalyzed by the nucleic acid decomposition activity, a nucleic acid base conversion reaction that converts a substituent on a purine or pyrimidine ring of a nucleic acid base to another group or the like without cleaving a DNA strand catalyzed by the nucleic acid base conversion activity, or an abasic reaction that hydrolyzes an N-glycoside bond of DNA catalyzed by the DNA glucosidase.

[0115] Examples of the nucleic acid base conversion activity include deaminase activity, etc. Examples of the deaminase activity include cytidine deaminase activity, adenosine deaminase activity, guanosine deaminase activity, etc.

[0116] Examples of the DNA glycosylase activity include thymine DNA glycosylase activity, oxoguanine glycosylase activity, alkyladenine DNA glycosylase activity, and the like.

[0117] The enzymatic activity domain is located, for example, on the 5'-end and / or 3'-end of the protein of the present disclosure.

[0118] The enzymatic activity domain is, for example, directly or indirectly linked to the protein of the present disclosure. The direct link is, for example, a link by a covalent bond, and for example, the protein of the present disclosure and the enzymatic activity domain constitute a fusion protein. The indirect link is, for example, a link via a binding tag-binding partner. The binding tag-binding partner is, for example, a combination of substances that have specific binding properties to each other. The binding tag-binding partner can be, for example, a combination of biotin and avidin or streptavidin, a combination of an SH3 domain (SH3) and an SH ligand, a combination of nickel and a His tag, a combination of an epitope tag such as flag (trademark)-tag, HA-tag, T7-tag, V5-peptide-tag, and / or Myc-tag, and an antibody against the tag, etc. In this case, the protein of the present disclosure and the enzymatic activity domain are linked (bound) via the binding tag-binding partner, for example, after transcription and translation from a polynucleotide.

[0119] In the protein of the present disclosure, for example, the amino acid at the N-terminal amino acid residue may be protected with a protecting group (eg, a C1-6 acyl group such as a C1-6 alkanoyl group such as a formyl group or an acetyl group).

[0120] In the protein of the present disclosure, for example, the N-terminal glutamine residue that may be generated by cleavage in vivo may be pyroglutamylated.

[0121] In the protein of the present disclosure, for example, substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino groups, imidazole groups, indole groups, guanidino groups, etc.) may be protected with appropriate protecting groups (e.g., C1-6 acyl groups such as C1-6 alkanoyl groups such as formyl groups and acetyl groups).

[0122] The protein of the present disclosure may be, for example, a conjugated protein such as a glycoprotein having a sugar chain bound thereto.

[0123] The protein of the present disclosure may be in the form of a salt with, for example, an acid or a base. The salt is not particularly limited, and examples thereof include acid salts and basic salts. Examples of the acid salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of the basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0124] The protein of the present disclosure can be easily produced according to known genetic engineering techniques, for example, by using PCR, restriction enzyme digestion, DNA ligation techniques, in vitro transcription / translation techniques, recombinant protein production techniques, and the like.

[0125] <Polynucleotide> In another aspect, the present disclosure provides a polynucleotide. The polynucleotide of the present disclosure comprises a coding sequence of the protein of the present disclosure. The polynucleotide of the present disclosure can be described in accordance with the description of the protein of the present disclosure.

[0126] The polynucleotide of the present disclosure can be designed by substituting corresponding codons based on the amino acid sequence of the protein of the present disclosure. The base sequence of the polynucleotide of the present disclosure may be, for example, codon-optimized.

[0127] <vector> In another aspect, the present disclosure provides a vector. The vector of the present disclosure includes the polynucleotide of the present disclosure. The vector of the present disclosure can incorporate the description of the protein and polynucleotide of the present disclosure.

[0128] The vector of the present disclosure may, for example, comprise a polynucleotide encoding a guide RNA for a target site of a target DNA. In this case, the polynucleotide encoding the guide RNA can hybridize to the target DNA, i.e., can hybridize to a target sequence in the target DNA. The polynucleotide encoding the guide RNA may further comprise other structures other than the guide RNA.

[0129] The protein of the present disclosure can, for example, form a complex with a guide RNA having a structure similar to that of the guide RNA of a Cas protein to perform modification of a target site.The protein of the present disclosure can, for example, use an RNA molecule having a structure or base sequence similar to that of the guide RNA of a Cas9 protein or the guide RNA of a Cas12 protein as the guide RNA.The protein of the present disclosure can, for example, use the guide RNA of the Cas9 protein or the guide RNA of the Cas12 protein as the guide RNA.

[0130] The guide RNA includes, for example, crRNA. Since the crRNA includes, for example, a spacer sequence and a direct repeat sequence, the guide RNA can also be said to include, for example, a spacer sequence and a direct repeat sequence. In the guide RNA, the spacer sequence hybridizes, for example, to the target nucleotide sequence of the target DNA. The spacer sequence may be, for example, completely complementary (identical) to the target nucleotide sequence of the target DNA, or may be substantially complementary (identical). The term "substantially complementary" means, for example, that the base sequence has an identity of 50% or more to the nucleotide sequence of the target DNA and can hybridize to the nucleotide sequence of the target DNA. The "identity" is, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with respect to the nucleotide sequence of the target DNA. The substantially complementary sequence is, for example, a sequence in which one or two bases of mismatched base pairs exist between the spacer sequence and the target DNA. In the guide RNA, the length of the spacer sequence is not particularly limited as long as it is a length capable of hybridizing to the target DNA, and examples thereof include 14 to 30 bases, 16 to 28 bases, 16 to 25 bases, 20 to 25 bases, 20 to 22 bases, or 21 to 23 bases.

[0131] The hybridization can be detected by various hybridization assays under stringent conditions, for example. The hybridization assay is not particularly limited, and for example, the method described in "Molecular Cloning: A Laboratory Manual 2nd Ed." edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)] can be adopted.

[0132] The "stringent conditions" may be, for example, low stringency conditions, medium stringency conditions, or high stringency conditions. "Low stringency conditions" are, for example, 5xSSC, 5xDenhardt's solution, 0.5% SDS, 50% formamide, and 32°C. "Medium stringency conditions" are, for example, 5xSSC, 5xDenhardt's solution, 0.5% SDS, 50% formamide, and 42°C. "High stringency conditions" are, for example, 5xSSC, 5xDenhardt's solution, 0.5% SDS, 50% formamide, and 50°C. Those skilled in the art can set the degree of stringency by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, and time. The "stringent conditions" may be, for example, those described in "Molecular Cloning: A Laboratory Manual 2nd Ed." edited by Sambrook et al. [Cold Spring Harbor Laboratory Press (1989)].

[0133] The spacer sequence preferably further includes a seed sequence, for example. The seed sequence is, for example, a sequence on the 5'-end side of the PAM sequence in the spacer sequence, and is important for the specificity of the guide RNA to the target DNA. In the guide RNA, the seed sequence is, for example, substantially or completely complementary to the nucleotide sequence of the target DNA, preferably the latter. When the seed sequence is substantially complementary to the nucleotide sequence of the target DNA, the substantially complementary sequence is, for example, a sequence in which there is a mismatch of 1 or 2 bases between the seed sequence and the nucleotide sequence of the target DNA.

[0134] In the guide RNA, the length of the direct repeat sequence is not particularly limited, and examples thereof include a length of 19 to 40 bases, and a length of 21 to 38 bases.

[0135] The length of the sequence of the guide RNA is set so that the protein of the present disclosure can be targeted to the target site of the target DNA. The length of the sequence of the guide RNA is not particularly limited, and examples thereof include 30 to 220 bases, 40 to 220 bases, 50 to 220 bases, 60 to 220 bases, 70 to 220 bases, 80 to 220 bases, 90 to 220 bases, 100 to 220 bases, 110 to 220 bases, 110 to 210 bases, 110 to 200 bases, 110 to 190 bases, 110 to 180 bases, 110 to 170 bases, 110 to 160 bases, and 110 to 150 bases. When the guide RNA includes crRNA and tracrRNA as separate RNAs, the length of the sequence of the guide RNA is, for example, the total length of both RNAs. When the guide RNA contains crRNA and tracrRNA in the same RNA (sgRNA, as described below), the length of the guide RNA sequence is, for example, the length of the sgRNA sequence.When the guide RNA contains crRNA, the length of the guide RNA sequence is, for example, the length of the crRNA sequence.

[0136] The guide RNA may be one type or may be multiple types. When the guide RNA is multiple types, it is preferable that the guide RNA is configured to be able to hybridize to different target DNA sequences, for example. When the guide RNA is multiple types, for example, by setting the different target DNA sequences so that the guide RNA targets the 5' side and 3' side of the target site or target region, the complex of the protein and guide RNA of the present disclosure can induce deletion of the target region between the 5' side and the 3' side (large deletion). When the guide RNA is multiple types, for example, by setting the guide RNA to target the 5' side and 3' side of the target region and introducing a donor DNA having a sequence complementary to the 5' side region (5' arm region) and the 3' side region (3' arm region), the complex of the protein and guide RNA of the present disclosure can introduce the donor DNA into the region between the 5' side and the 3' side by recombination.

[0137] The guide RNA may include only crRNA, or may include crRNA and tracrRNA. When the guide RNA includes crRNA and tracrRNA, the guide RNA may be an sgRNA (single-stranded guide strand) in which the tracrRNA and the crRNA are fused, or may not be fused, that is, may be composed of two RNAs, crRNA and tracrRNA. When the tracrRNA and the crRNA are not fused, it is preferable that the tracrRNA and the crRNA have substantially or completely complementary sequences, such as the repeat sequence and the anti-repeat region. The structure of the guide RNA (single-stranded or double-stranded) and the presence or absence of tracrRNA can be determined according to the protein of the present disclosure.

[0138] The tracrRNA includes a repeat sequence (anti-repeat region) and one or more hairpin structures. The number of the hairpin structures is not particularly limited, and is, for example, 1 to 10, 1 to 5, 1 to 3, or 2. The tracrRNA is not particularly limited as long as it can bind to the crRNA and form a complex.

[0139] The tracrRNA sequence may, for example, have nucleotide regions that have other functionalities.

[0140] The length of the tracrRNA sequence is not particularly limited and can be set according to, for example, the protein of the present disclosure. Examples of the length of the tracrRNA sequence include 10 to 200 bases, 20 to 190 bases, 30 to 180 bases, 40 to 180 bases, 50 to 180 bases, 60 to 180 bases, 70 to 180 bases, 70 to 170 bases, 70 to 160 bases, 70 to 150 bases, 70 to 140 bases, 70 to 130 bases, 70 to 120 bases, 70 to 110 bases, 70 to 100 bases, and 70 to 90 bases.

[0141] The length of the crRNA sequence is not particularly limited and can be set according to the protein of the present disclosure. The length of the tracrRNA sequence can be, for example, 25 to 80 bases, 30 to 80 bases, 35 to 80 bases, 40 to 80 bases, 49 to 80 bases, 40 to 70 bases, or 40 to 60 bases.

[0142] The crRNA sequence can be designed to target a region of the target DNA adjacent to a PAM sequence.

[0143] The base sequence of the guide RNA of the present disclosure may be, for example, the following sequence. In the base sequence of SEQ ID NO: 33 below, the underlined 36th to 56th N (N is A, T, G, or U) is a base sequence (spacer sequence) that can hybridize with the target DNA sequence in the target DNA. In the base sequence of SEQ ID NO: 33 below, N is 21 bases long, but the length of N is not limited thereto and may be within a range that allows the protein of the present disclosure to be targeted to the target site of the target DNA. The length of N is, for example, 14 to 30 bases long, 16 to 28 bases long, 16 to 25 bases long, 20 to 25 bases long, 20 to 22 bases long, or 21 to 23 bases long. Guide RNA (SEQ ID NO: 33) 5'-GUCAAAAGACCUUUGGAAUUUCUACUCUUGUAGAU NNNNNNNNNNNNNNNNNNNNN -3'

[0144] As described above, the sequence of the guide RNA is not particularly limited as long as it can hybridize to the target DNA sequence.

[0145] The polynucleotide on the vector of the present disclosure and the polynucleotide encoding the guide RNA for the target site of the target DNA are configured to be transcribed and / or translated in a host such as a cell, and to form a complex. The protein of the present disclosure and the guide RNA are, for example, bound to each other to form a complex. The complex can target the protein of the present disclosure to the target site of the target DNA, for example, depending on the sequence of the guide RNA, and modify the target DNA depending on the activity of the protein of the present disclosure. The modification can be, for example, double-strand breaks, single-strand breaks, conversion, substitution, deletion, insertion, etc. of one or more nucleotides of the target DNA. The modification can be, for example, modification of the base, sugar, or phosphate portion of the target DNA. The complex of the guide RNA and the protein of the present disclosure converts, substitutes, deletes, or inserts one or more nucleotides at the targeted site. The one or more can be, for example, 1 to 10, 1 to 5, etc.

[0146] Examples of the target DNA include chromosomal DNA, genomic DNA, mitochondrial DNA, viral DNA, binding elements, exogenous DNA, and plasmid DNA. When the target DNA encodes a gene, the target DNA can be called, for example, a target gene. The target DNA can be single-stranded (ssDNA) or double-stranded DNA (dsDNA).

[0147] The target DNA sequence to which the guide RNA can hybridize is not particularly limited and can be set, for example, according to the PAM (proto-spacer adjacent motif) sequence of the protein of the present disclosure. The length of the target DNA sequence is not particularly limited and is, for example, a length to which the guide RNA can specifically hybridize, specific examples of which include a length of 14 to 30 bases, 16 to 25 bases, etc.

[0148] The vector of the present disclosure may include, for example, a promoter sequence on the vector. The promoter can be appropriately set depending on, for example, the type of cell in which the polynucleotide encoding the protein of the present disclosure and / or the guide RNA is expressed. The promoter can be, for example, a T3 promoter, a T7 promoter, an sp6 promoter, an EF1α promoter, an SRα promoter, an SV40 (Simian virus) promoter, an LTR promoter, a CMV (Cytomegalovirus) promoter, an RSV (Respiratory syncytial virus) promoter, an HSV-tk promoter, a Cauliflower mosaic virus (CaMV) 35S promoter, an actin promoter, a heat shock promoter, a REF (Rubber Elongation Factor) promoter, polyhedrin promoter, p10 promoter, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, tac promoter, GAL1 promoter, GAL10 promoter, PH05 promoter, PGK promoter, GAP promoter, ADH promoter, SPO1 promoter, SPO2 promoter, penP promoter, gyrA-ldh promoter, pgm promoter, rrn4 promoter, P23 promoter, araBAD promoter, cat promoter, cspA promoter, EM7 promoter, J23119 promoter, T5 promoter, tac promoter, U6 promoter, TDH3 promoter, TEF1 promoter, and the like.

[0149] The vector of the present disclosure may, for example, include a polynucleotide encoding an enzymatic protein or an enzymatic domain thereof on the vector. The enzymatic activity of the enzymatic protein may, for example, be a nucleic acid modification activity such as methylase activity, demethylase activity, base modification activity, histone modification activity, RNA cleavage activity, or DNA cleavage activity; a transcription activation activity, a transcription repression activity, a transcription deactivation factor activity, a DNA integration activity, or a nucleic acid binding activity. The enzymatic protein may, for example, be a peptide fragment thereof, so long as it has catalytic activity.

[0150] The vector of the present disclosure may contain, for example, a multicloning site, an enhancer, a splicing signal, a polyA addition signal, a drug resistance gene, an auxotrophy complementation gene, a replication origin, and the like on the vector.

[0151] The vector of the present disclosure can be prepared, for example, by inserting a polynucleotide containing a coding sequence of the protein of the present disclosure into a backbone vector (hereinafter also referred to as a "basic vector"). The type of the expression vector is not particularly limited and can be appropriately determined, for example, depending on the type of the host. Specifically, when the vector is synthesized by a genetic engineering technique, the vector is first synthesized by, for example, designing and synthesizing a polynucleotide containing a coding sequence of the protein of the present disclosure. The design and synthesis can be performed, for example, by a PCR method using a vector or the like containing a polynucleotide encoding a polynucleotide containing a coding sequence of the protein of the present disclosure as a template, and a primer designed to synthesize a desired nucleic acid region. Then, a recombinant vector for protein expression (expression vector) can be obtained by linking the obtained polynucleotide to an appropriate vector, and the recombinant vector can be introduced into a host so that the target gene can be expressed to obtain a transformant (Sambrook J. et al., Molecular Cloning, A Laboratory Manual (4th edition) (Cold Spring Harbor Laboratory Press (2012)).

[0152] Examples of the vector include a phage vector, a plasmid vector, a virus vector, a retrovirus vector, a chromosomal vector, an episomal vector, and a virus-derived vector.

[0153] When the introduction method is a method for transforming a host by a heat shock method, examples of the vector include a binary vector, etc. Examples of the expression vector include pETDuet-1, pQE-80L, pUCP26Km, etc. When transforming bacteria such as Escherichia coli, examples of the expression vector include a pETDuet-1 vector (Novagen), pQE-80L (QIAGEN), pBR322, pB325, pAT153, pUC8, etc.

[0154] <Vector type> In another embodiment, the present disclosure provides a vector system.The vector system of the present disclosure comprises a first vector comprising the polynucleotide of the present disclosure and a second vector comprising the polynucleotide encoding the guide RNA for target site.The vector system of the present disclosure can be applied to the description of the protein, polynucleotide and vector of the present disclosure.

[0155] In the vector system of the present disclosure, the first vector and the second vector may be, for example, located on the same vector or on different vectors.

[0156] <Composition> In another embodiment, the present disclosure provides a composition.The composition of the present disclosure comprises the protein of the present disclosure, the polynucleotide of the present disclosure, and / or the vector of the present disclosure, and the guide RNA for the target site of the target DNA or the polynucleotide that codes for the guide RNA.The composition of the present disclosure can be applied to the description of the protein, polynucleotide, vector, and vector system of the present disclosure.

[0157] <Kit> In another embodiment, the present disclosure provides a kit that can modify target DNA.The kit of the present disclosure includes the protein of the present disclosure, the polynucleotide of the present disclosure, and / or the vector of the present disclosure, and the guide RNA for target site or the polynucleotide that codes for it.The kit of the present disclosure can be applied to the description of the protein, polynucleotide, vector, vector system, and composition of the present disclosure.

[0158] The kit of the present disclosure may include, for example, an organism. The organism may be, for example, a prokaryote or a eukaryote. The prokaryote may be, for example, a lactic acid bacterium, Bacillus subtilis var. natto ( Bacillus subtilis var. natto ), E. coli ( Escherichia coli ), Cyanobacteria ( Cyanobacteria ) and the like. Examples of the eukaryotic organisms include microorganisms, plants, animals, insects, and the like. Examples of the animals include humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, rats, and the like. The organisms may be of one type or of multiple types.

[0159] The kit of the present disclosure may include, for example, a cell. The cell may be, for example, a prokaryotic cell or a eukaryotic cell. The prokaryotic cell may be, for example, a lactic acid bacterium, a Bacillus subtilis var. natto ( Bacillus subtilis var. natto ), E. coli ( Escherichia coli ), Cyanobacteria ( Cyanobacteria ) and the like. The eukaryotic cells include, for example, microbial cells, plant cells, animal cells, and the like. The microbial cells include, for example, cells derived from microorganisms such as yeast, fungi, and protozoa. The plant cells include, for example, cells derived from plants such as seed plants, ferns, mosses, and algae. The animal cells include, for example, cells derived from animals such as vertebrates such as mammals, birds, reptiles, amphibians, and fish; arthropods such as insects; and mollusks. The cells include, in vivo Cells (in vivo cells), in vitroExamples of the cells include cells (cultured cells), primary cultured cells, etc. The cells may be of one type or of multiple types.

[0160] Kits of the present disclosure may include, for example, instructions, instructions, and the like.

[0161] The kit of the present disclosure may include, for example, a medium. Examples of the medium include YM medium, YPD medium, PD medium, DOB medium, SD medium, LB medium, NB medium, SCD medium, MRS medium, BHI medium, and the like. The medium may include, for example, a carbon source such as glucose, dextrin, soluble starch, sucrose, and the like; a nitrogen source such as ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, potato extract, and the like; inorganic substances such as calcium chloride, sodium dihydrogen phosphate, magnesium chloride, and the like; vitamins; growth promoting factors; and other additives. When the kit of the present disclosure includes the additive, the additive may be an additive to be added to the medium before the culture, or an additive to be added during the culture. The addition may be continuous or intermittent.

[0162] The kit of the present disclosure may further include, for example, a container for storing the protein of the present disclosure, the polynucleotide of the present disclosure, the vector of the present disclosure, the vector system of the present disclosure, and / or the composition of the present disclosure, and a medium.

[0163] In the kit of the present disclosure, for example, each component may be contained separately, or a part or all of the components may be contained in a mixed or unmixed state. In the kit of the present disclosure, when all the components are contained in a single container in a mixed or unmixed state, the kit of the present disclosure can be called, for example, a culture medium.

[0164] The kits of the present disclosure can also be suitably used as test kits or research kits, for example, for use in genome editing of organisms and / or cells.

[0165] <cell> In another embodiment, the present disclosure provides a cell.The cell of the present disclosure comprises the protein of the present disclosure, the polynucleotide of the present disclosure, the vector of the present disclosure, and / or the vector system of the present disclosure.The cell of the present disclosure can refer to the description of the protein, polynucleotide, vector, vector system, and kit of the present disclosure.

[0166] A cell of the present disclosure can be said to express a polynucleotide of the present disclosure when the cell comprises a polynucleotide of the present disclosure, a vector of the present disclosure, and / or a vector system of the present disclosure.

[0167] <Method of modifying target DNA> In another embodiment, the present disclosure provides a method for modifying target DNA.The method for modifying target DNA of the present disclosure comprises a step of modifying target DNA by contacting target DNA, the protein of the present disclosure, and guide RNA for a target site in the target DNA in a cell.The modification method of the present disclosure can be applied to the description of the protein, polynucleotide, vector, vector system, composition, and kit of the present disclosure.

[0168] In the modification step, a complex of the protein of the present disclosure and the guide RNA is targeted to the target DNA, and the target DNA is modified by the complex at the target site.

[0169] The modification method of the present disclosure includes, for example, an introduction step of introducing the protein of the present disclosure or a polynucleotide encoding the protein, and the guide RNA or a polynucleotide encoding the protein into the cell. The introduction can be performed by a known method capable of introducing (transfecting) an exogenous protein or exogenous DNA into a host cell, and specifically includes, for example, an introduction method using a gene gun such as a particle gun, a microinjection method, a calcium phosphate method, a polyethylene glycol method, a lipofection method using liposomes, an electroporation method, an ultrasonic nucleic acid introduction method, a DEAE-dextran method, a direct injection method using a micro glass tube, a hydrodynamic method, a cationic liposome method, a lysozyme method, a competent method, a PEG method, a method using an introduction aid, a method mediated by Agrobacterium, a protoplast method, etc. Examples of the liposome include lipofectamine and cationic liposome, etc., and examples of the introduction aid include atelocollagen, nanoparticles, and polymers, etc.

[0170] The introduction step may include a step of culturing the cells after the introduction. The culture can be performed by a known method or a modified method thereof, and under conditions, depending on the type of the cells. The medium for the culture can be the same as that described in the kit.

[0171] In the culture step, the culture pH is not particularly limited as long as it is the optimal pH for the proliferation of the cells.

[0172] In the culturing step, the culture temperature is not particularly limited as long as it is an optimal temperature for the proliferation of the cells.

[0173] In the culture step, the culture time is not particularly limited as long as it is the time during which the complex is expressed. For example, the culture time is the time during which the protein and guide RNA of the present disclosure are expressed and can modify the target DNA.

[0174] The culture may be performed, for example, by static culture or by shaking culture.

[0175] <Manufacturing method> In another embodiment, the present disclosure provides a method for producing a cell or a non-human animal in which target DNA is modified. The method for producing a cell or a non-human animal in which target DNA is modified of the present disclosure includes the introduction step of introducing the protein of the present disclosure, the polynucleotide of the present disclosure, and / or the vector of the present disclosure and the guide RNA for the target site of the target DNA into a cell or a non-human animal. The production method of the present disclosure can be applied to the description of the protein, polynucleotide, vector, vector system, composition, kit, and modification method of the present disclosure.

[0176] <Cells or non-human animals> In another aspect, the present disclosure provides a cell or a non-human animal.The cell or the non-human animal of the present disclosure is produced by the production method of the present disclosure.The cell or the non-human animal of the present disclosure can be applied to the description of the protein, the polynucleotide, the vector, the vector system, the composition, the kit, the cell, the modification method and the production method of the present disclosure. EXAMPLES

[0177] The present disclosure will be described in detail below using examples, but the present disclosure is not limited to the aspects described in the examples.

[0178] [Example 1] (1) Construction of vectors containing polynucleotides encoding each protein A vector containing a polynucleotide encoding the protein of the present disclosure (SEQ ID NO: 1 to 15) was constructed. Specifically, downstream of the CBh promoter of an expression vector into which a CAG-TurboRFP expression cassette was inserted, a coding sequence in which SV40 NLS (SEQ ID NO: 34) was added to the N-terminus of each protein of the present disclosure (SEQ ID NO: 1 to 15), nucleoplasmin NLS (SEQ ID NO: 35), a GS linker, and a 3×HA tag (SEQ ID NO: 36) were added to the C-terminus of the protein of the present disclosure, and a polynucleotide encoding a guide RNA (SEQ ID NO: 37) was inserted downstream of the U6 promoter to obtain a vector of the present disclosure. In addition, as another expression vector, an expression vector in which a polynucleotide encoding the amino acid sequence of SEQ ID NO: 16 to 19 (ST9: SEQ ID NO: 16, ST9_2: SEQ ID NO: 17, ST9_3LA: SEQ ID NO: 18, ST9_S: SEQ ID NO: 19) was inserted instead of the protein of the present disclosure was also constructed.

[0179] SV40 NLS (SEQ ID NO:34) PKKKRKV

[0180] nucleoplasmin NLS (SEQ ID NO: 35) KRPAATKKAGQAKKKK

[0181] 3x HA tag (SEQ ID NO: 36) YPYDVPDYAYPYDVPDYAYPYDVPDYA

[0182] Guide RNA (SEQ ID NO: 37) 5'-GUCAAAAGACCUUUGGAAUUUCUACUCUUGUAGAUUCUGUCCCCUCCACCCCACAG-3'

[0183] ST9 (SEQ ID NO: 16)

[0184] ST9_2 (SEQ ID NO: 17)

[0185] ST9_3LA (SEQ ID NO: 18)

[0186] ST9_S (SEQ ID NO: 19)

[0187] (2) Construction of EGxxFP assay detection vector A vector was prepared in which the EGFP amino-terminal region, the PAM sequence (SEQ ID NOs: 24 to 32), the nucleotide sequence to be cleaved, and the EGFP carboxyl-terminal region were inserted downstream of the CAG promoter in this order. The EGxxFP assay detection vector is designed so that when it is cleaved by genome editing, the EGFP sequence is repaired and green fluorescence is observed.

[0188] (3)EGxxFP assay The cleavage activity of the protein of the present disclosure against the target site was examined by EGxxFP assay. Specifically, HEK293T cells were seeded on a 24-well plate and cultured for 1.5 to 2 days at 37°C in a CO2 incubator. In the culture, DMEM (High Glucose) (Sigma-Aldrich), 10% FBS (Gibco), and 1x penicillin-streptomycin mixed solution (Nacalai Tesques) were used as the medium. After the culture, 75 μl of solution A (600 μl of Opti-MEM (Gibco), 3 μl of 2 ng / nl EGxxFP assay detection vector) was dispensed into each tube, and 0.75 μl of each expression vector constructed in Example 1 (1) at 0.4 ng / nl was mixed. After the mixing, 75 μl of solution B (600 μl of Opti-MEM (Gibco), 60 μl of PEIMax (Polysciences)) was added to prepare a mixed solution. After the preparation, the mixed solution was left to stand at room temperature (hereinafter, about 24° C.) for 20 minutes. Thereafter, the medium in the 24-well plate was replaced with a new medium, and 50 μl of the mixed solution was added to each well. After the addition, the 24-well plate was gently shaken. After the shaking, the cells were cultured for 1.5 to 2 days, and the cells were recovered by trypsin treatment. After the recovery, a diluted solution of Passive Lysis 5X Buffer (Promega) was added, and the cells were vigorously stirred to dissolve the cells. After the dissolution, centrifugation was performed at 12,000 rpm for 5 minutes, and the supernatant was recovered. 50 μl of the supernatant of each sample was added to two wells of a 96-well plate, and the fluorescence intensity of EGFP and RFP contained in the supernatant was detected using a plate reader (CYTATION (trademark) 3 imaging reader (BioTek)). In the detection, the EGFP value was normalized with the RFP value. A sample using an expression vector not containing a polynucleotide encoding the guide RNA of the present disclosure was used as a negative control, and the value of the negative control was subtracted from the value of each sample. These results are shown in Figures 1 to 4.

[0189] Figures 1 to 4 are graphs showing the results of the EGxxFP assay. In Figures 1 to 4, the vertical axis indicates the EGFP value normalized by the RFP value, and the horizontal axis indicates the type of sample. As shown in Figures 1 to 4, it was found that the protein of the present disclosure has high nuclease activity compared to the control.

[0190] [Example 2] (1) Construction of a vector containing a polynucleotide encoding a modified protein with a different amino acid having a cleavage active center A vector containing a polynucleotide encoding a protein with a modified cleavage active center was constructed. Specifically, a coding sequence in which SV40 NLS (SEQ ID NO: 34) was added to the N-terminus of a protein (CR-FD2, SEQ ID NO: 38) in which glutamic acid (E), the 969th amino acid of ST9, was replaced with glycine (G), and valine, proline, alanine, and alanine, the 1040th to 1043rd amino acids, were replaced with alanine, aspartic acid, glutamic acid, and arginine, and a nucleoplasmin NLS (SEQ ID NO: 35), a GS linker, and a 3×HA tag (SEQ ID NO: 36) were added to the C-terminus of the protein of the present disclosure was inserted downstream of the CBh promoter of an expression vector to obtain a vector. The CR-FD2 has a mutation at the site of cleavage activity and a site adjacent thereto (FIG. 5). Therefore, the CR-FD2 has a cleavage active center different from that of a protein (ST9) that does not have a mutation.

[0191] CR-FD2 (SEQ ID NO: 38) MGEKTNYFNNFIGISPLTKTLRNALIPTTEITQKHIIEYGIIKDDELRKENRQILKTIMDDYYRSFLTEKSAIHDIDWKPLFLEMENELRHGNNKVNLEKEQKAKRKAINKYFSEDERYKKMFSAKLLSDILPEFVIHNDEYSAEEKEEKQVIKLFSRFATSLKEYFRNRSNTFSADNISTSACHRIVNDNASIFLENVMAYRKIISSLPADELDKLESIQDKLKIKSISEVYTYDNYGKYITQEGIDLYNDICGKVNSFMNLYCQQNKENKNVYKMRKLHKQILCIAKTSYEVGEGYTSDEEVLEVFRNTLNKSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKNDAVVAIMKDLLDSVKSFENYIKAFFGEGK ETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSTEKFKLNFNSPTLARGWSKSKEYSNNAIILSKDGILYLGIFNVKNKPDKKIIEGHIRENDGDYKKMVYNLLPGANKMLPKVLISSKSGVETYKPSDYILEGYSNKHLKSSDTFDINYCHDLIDYYKDCIDIHPEWKNFDFNSDTADFEDISGFYREAVEAQGYKIDWTYISGENIEELQEKGQLFLFKIYNKDFSTKSTGTD NLHTMYLKNLFSEENLKDVVLKLNGEAEIFYRKSSIKNPIIHKKGSMLVNTYIEEEEEHDGKIVEVRKIVPEEIYKELYNHFNNNGKAELSEEAKRLESLVCHEAAKDIVKDCRYTYDKFFIHLPMTINFKASGSSSNLDMMLQYISGQNDMHIIGIDRGERNLIYVSVIDAHGNIVKQKSFNIVAGYDYQEKLKMQEGARQNARKEWKEIGKIKEIKEGYLSLVIHEIAQMVIEYNAIIAM GDLNYGFKKGRFKVERQVYQKFETMLINKLNYLVFKNRGVTEDGGLLRGYQLTYIPESLKNVGRQCGCIFY ADER YTSKIDPTTGFADIFRFKNLTVEEKRDFIRKFDYIKYDLEKEMFVFAFDFKNFVTQNVEMSKNDWCVYTNGIRVKRRYANGRFTNETDEIDINVLMKKTFERTDIDWNDGHNL IDEIIDYDLESQIVDIFKMAVQMRNSRSEAEDRDYDRLVSPVLNESGDFFDSSKVGENLPKDADANGAYCIALKGLYKVRQIQENWNEEEKFSRAKLRISNKDWFDFVQNKRYL

[0192] (2)EGxxFP assay The cleavage activity of the modified protein having mutations at the cleavage activity site and its adjacent sites against the target site was examined by EGxxFP assay. Specifically, the assay was carried out in the same manner as the EGxxFP assay in Example 1(3) above, except that the vector constructed in Example 2(1) above was used instead of the vector constructed in Example 1(1) above. The results are shown in FIG. 6.

[0193] Figure 6 is a graph showing the results of the EGxxFP assay. In Figure 6, the vertical axis shows the EGFP value normalized by the RFP value, and the horizontal axis shows the type of sample. As shown in Figure 6, the protein of the present disclosure with an improved cleavage active center was found to have nuclease activity.

[0194] [Example 3] (1) Construction of a vector containing a polynucleotide encoding an alanine-substituted modified protein A vector containing a polynucleotide encoding a modified protein in which the amino acid responsible for the cleavage activity center is substituted with alanine was constructed. Specifically, a coding sequence in which SV40 NLS (SEQ ID NO: 34) is added to the N-terminus of a protein (CR-FD3, SEQ ID NO: 39) in which the 969th amino acid of ST9, glutamic acid, is substituted with alanine, and the 1040th to 1043rd amino acids, valine, proline, alanine, and alanine, are substituted with alanine, aspartic acid, glutamic acid, and arginine, respectively, and a nucleoplasmin NLS (SEQ ID NO: 35), a GS linker, and a 3×HA tag (SEQ ID NO: 36) are added to the C-terminus of the protein of the present disclosure was inserted downstream of the CBh promoter of an expression vector to obtain the vector of the present disclosure. The CR-FD3 has an alanine mutation at the site of the cleavage activity (FIG. 7). Therefore, the cleavage activity center of the CR-FD3 is different from that in the case where there is no mutation (ST9). Moreover, the CR-FD3 can be said to be a protein in which a mutation has been introduced into an amino acid constituting the active center of the CR-FD2.

[0195] CR-FD3 (SEQ ID NO: 39) MGEKTNYFNNFIGISPLTKTLRNALIPTTEITQKHIIEYGIIKDDELRKENRQILKTIMDDYYRSFLTEKSAIHDIDWKPLFLEMENELRHGNNKVNLEKEQKAKRKAINKYFSEDERYKKMFSAKLLSDILPEFVIHNDEYSAEEKEEKQVIKLFSRFATSLKEYFRNRSNTFSADNISTSACHRIVNDNASIFLENVMAYRKIISSLPADELDKLESIQDKLKIKSISEVYTYDNYGKYITQEGIDLYNDICGKVNSFMNLYCQQNKENKNVYKMRKLHKQILCIAKTSYEVGEGYTSDEEVLEVFRNTLNKSEIFSSIKKLEKLFKNFDEYSSAGIFVKNGPAISTISKDIFGEWNVIRDKWNAEYDDIHLKKKAVVTEKYEDDRRKSFKKIGSFSLEQLQEYADADLSVVEKLKEIIIQKVDEIYKVYGSSEKLFDADFVLEKSLKNDAVVAIMKDLLDSVKSFENYIKAFFGEGK ETNRDESFYGDFVLAYDILLKVDHIYDAIRNYVTQKPYSTEKFKLNFNSPTLARGWSKSKEYSNNAIILSKDGILYLGIFNVKNKPDKKIIEGHIRENDGDYKKMVYNLLPGANKMLPKVLISSKSGVETYKPSDYILEGYSNKHLKSSDTFDINYCHDLIDYYKDCIDIHPEWKNFDFNSDTADFEDISGFYREAVEAQGYKIDWTYISGENIEELQEKGQLFLFKIYNKDFSTKSTGTD NLHTMYLKNLFSEENLKDVVLKLNGEAEIFYRKSSIKNPIIHKKGSMLVNTYIEEEEEHDGKIVEVRKIVPEEIYKELYNHFNNNGKAELSEEAKRLESLVCHEAAKDIVKDCRYTYDKFFIHLPMTINFKASGSSSNLDMMLQYISGQNDMHIIGIDRGERNLIYVSVIDAHGNIVKQKSFNIVAGYDYQEKLKMQEGARQNARKEWKEIGKIKEIKEGYLSLVIHEIAQMVIEYNAIIAM ADLNYGFKKGRFKVERQVYQKFETMLINKLNYLVFKNRGVTEDGGLLRGYQLTYIPESLKNVGRQCGCIFY ADER YTSKIDPTTGFADIFRFKNLTVEEKRDFIRKFDYIKYDLEKEMFVFAFDFKNFVTQNVEMSKNDWCVYTNGIRVKRRYANGRFTNETDEIDINVLMKKTFERTDIDWNDGHNL IDEIIDYDLESQIVDIFKMAVQMRNSRSEAEDRDYDRLVSPVLNESGDFFDSSKVGENLPKDADANGAYCIALKGLYKVRQIQENWNEEEKFSRAKLRISNKDWFDFVQNKRYL

[0196] (2)EGxxFP assay The cleavage activity of the modified protein having an alanine mutation at the cleavage activity site against the target site was examined by EGxxFP assay. Specifically, the assay was carried out in the same manner as the EGxxFP assay in Example 1(3) above, except that the vector constructed in Example 3(1) above was used instead of the vector constructed in Example 1(1). The results are shown in FIG.

[0197] FIG. 8 is a graph showing the results of the EGxxFP assay. In FIG. 8, the vertical axis shows the EGFP value normalized by the RFP value, and the horizontal axis shows the type of sample. As shown in FIG. 8, it was found that a protein in which the 969th amino acid, glutamic acid, was replaced with alanine, and the 1040th to 1043rd amino acids, valine, proline, alanine, and alanine, were replaced with alanine, aspartic acid, glutamic acid, and arginine, respectively, did not have nuclease activity.

[0198] [Example 4] The cleavage activity of the protein of the present disclosure against the target site when the PAM sequence is TTTC (SEQ ID NO: 40) was examined by EGxxFP assay. Specifically, the EGxxFP assay was carried out in the same manner as in Example 1(3) above, using each expression vector constructed in Example 1(1) above and the EGxxFP assay detection vector in Example 1(2) above, in which the PAM sequence is TTTC. The results are shown in FIG.

[0199] 9 is a graph showing the results of the EGxxFP assay. In FIG. 9, the vertical axis shows the EGFP value normalized by the RFP value, and the horizontal axis shows the type of sample. As shown in FIG. 9, it was found that the protein of the present disclosure recognizes the PAM sequence TTTC and exhibits high nuclease activity compared to the control.

[0200] Although the present disclosure has been described above with reference to the embodiments and examples, the present disclosure is not limited to the above-mentioned embodiments and examples. Various modifications that can be understood by a person skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0201] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety as if the contents were specifically set forth herein.

[0202] <Additional Notes> Some or all of the above-described embodiments and examples may be described as follows, but are not limited to the following: <Protein> (Appendix 1) The following protein (a1), (a2), or (a3): (a1) a protein consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 19; (a2) a protein having nuclease activity, which consists of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted, or added in any of the amino acid sequences of SEQ ID NOs: 1 to 19; (a3) A protein having an amino acid sequence having 80% or more identity to any of the amino acid sequences of SEQ ID NOs: 1 to 19, and having nuclease activity. (Appendix 2) The protein according to Appendix 1, which recognizes a protospacer adjacent motif (PAM) sequence having a base sequence selected from the group consisting of SEQ ID NOs: 20 to 32. (Appendix 3) The protein according to Appendix 1 or 2, wherein the protein (a1) is any one of the following proteins (b1) to (b6) or (b7): (b1) a protein in which the amino acid E at position 1,136 in the amino acid sequence of SEQ ID NO: 1 or 6 is substituted with G; (b2) a protein in which the amino acid E at position 1,129 in the amino acid sequence of SEQ ID NO: 2 to 5, 14, or 15 is substituted with G; (b3) a protein in which the amino acid sequence of position 1127, E, is replaced with G in the amino acid sequence of SEQ ID NO: 7 or 9; (b4) a protein in which the amino acid sequence of position 1,120, E, is replaced with G in the amino acid sequence of SEQ ID NO: 8 or 10; (b5) a protein in which the amino acid sequence of position 1128, E, is replaced with G in the amino acid sequence of SEQ ID NO: 11; (b6) a protein in which the amino acid E at position 969 in the amino acid sequence of SEQ ID NO: 16 or 18 is substituted with G; (b7) A protein in which the 960th amino acid E in the amino acid sequence of SEQ ID NO: 17 is replaced with G. (Appendix 4) The protein (a1) is a protein selected from the group consisting of (c1) to (c6) and (c7) below, (c1) a protein in which the amino acid VPAA at positions 1207 to 1210 in the amino acid sequence of SEQ ID NO: 1 or 6 is substituted with ADER; (c2) a protein in which the amino acid VPAA at positions 1200 to 1203 in the amino acid sequence of SEQ ID NO: 2 to 5, 14, or 15 is substituted with ADER; (c3) a protein in which the amino acid sequence VPAA at positions 1198 to 1201 in the amino acid sequence of SEQ ID NO: 7 or 9 is substituted with ADER; (c4) a protein in which the amino acid sequence VPAA at positions 1191 to 1194 in the amino acid sequence of SEQ ID NO: 8 or 10 is substituted with ADER; (c5) a protein in which the amino acid sequence VPAA at positions 1199 to 1202 in the amino acid sequence of SEQ ID NO: 11 is substituted with ADER; (c6) a protein in which the amino acid sequence VPAA at positions 1040 to 1043 in the amino acid sequence of SEQ ID NO: 16 or 18 is substituted with ADER; (c7) A protein in which the amino acid sequence VPAA at positions 1031 to 1034 in the amino acid sequence of SEQ ID NO: 17 is substituted with ADER. (Appendix 5) 5. A protein according to any one of appendix 1 to 4, which is an RNA-dependent DNA nuclease. (Appendix 6) A protein according to any one of claims 1 to 5, further comprising an enzymatic activity domain. (Appendix 7) 7. The protein according to any one of appendices 1 to 6, wherein the enzymatic activity domain is located on the 5'-end and / or 3'-end side of the protein according to any one of appendices 1 to 6. (Appendix 8) 8. The protein according to any one of claims 1 to 7, wherein the enzymatic activity is selected from the group consisting of a nucleic acid base conversion activity, a methylase activity, a demethylase activity, a base modification activity, a histone modification activity, an RNA cleavage activity, and a DNA cleavage activity. <Polynucleotide> (Appendix 9) A polynucleotide comprising a coding sequence for a protein according to any one of appendices 1 to 8. <vector> (Appendix 10) A vector comprising the polynucleotide described in Appendix 9. (Appendix 11) The vector of claim 10, further comprising a polynucleotide encoding a guide RNA for a target site. <Vector type> (Appendix 12) A first vector comprising the polynucleotide of appendix 9; and a second vector comprising a polynucleotide encoding a guide RNA for the target site. <Composition> (Appendix 13) A protein according to any one of appendices 1 to 8, a polynucleotide according to appendices 9, and / or a vector according to appendices 10 or 11; A composition comprising a guide RNA for a target site or a polynucleotide encoding the same. (Appendix 14) 14. The composition of claim 13, wherein the guide RNA is capable of complexing with the protein. (Appendix 15) 15. The composition of claim 13 or 14, wherein the guide RNA comprises a polynucleotide capable of hybridizing to a target site in a target DNA. (Appendix 16) 16. The composition of any of claims 13 to 15, wherein the guide RNA comprises a crRNA. <Kit> (Appendix 17) A protein according to any one of appendices 1 to 8, a polynucleotide according to appendices 9, and / or a vector according to appendices 10 or 11; A kit comprising a guide RNA for a target site or a polynucleotide encoding the same. <cell> (Appendix 18) A cell comprising a protein according to any of appendices 1 to 8, a polynucleotide according to appendices 9, a vector according to appendices 10 or 11, and / or a vector system according to appendix 12. <Method of modifying target DNA> (Appendix 19) A method for modifying a target DNA, the method comprising a modification step of modifying the target DNA by contacting the target DNA with a protein described in any one of Appendices 1 to 8 and a guide RNA for a target site in the target DNA in a cell. (Appendix 20) The modification method according to claim 19, comprising an introduction step of introducing into the cell a protein according to any one of claims 1 to 8 or a polynucleotide encoding the same, and the guide RNA or a polynucleotide encoding the same. (Appendix 21) The modification method according to claim 19 or 20, wherein in the modification step, a complex of the protein and the guide RNA is targeted to the target DNA, and the target DNA is modified by the complex at the target site. (Appendix 22) 22. The method of any one of claims 19 to 21, wherein the guide RNA comprises a polynucleotide capable of hybridizing to a target site within the target DNA. (Appendix 23) 23. The method of any one of claims 19 to 22, wherein the guide RNA comprises a crRNA. (Appendix 24) The method of any one of claims 19 to 23, wherein the modification is a double-stranded break, a single-stranded break, a conversion, a deletion and / or an insertion of one or more nucleotides in the target DNA. (Appendix 25) 25. The method of modification according to any one of claims 19 to 24, wherein the cell is an animal cell or a plant cell. (Appendix 26) 26. The method of any one of claims 19 to 25, wherein the cell is a eukaryotic cell or a prokaryotic cell. (Appendix 27) 27. The method of any one of claims 19 to 26, wherein the target DNA is chromosomal DNA. <Manufacturing method> (Appendix 28) 1. A method for producing a cell or a non-human animal having modified target DNA, comprising: A method for producing the same, comprising an introduction step of introducing a protein described in any one of Appendixes 1 to 8, a polynucleotide described in Appendix 9, and / or a vector described in Appendix 10 or 11, and a guide RNA for the target site into a cell or a non-human animal. <Cells or non-human animals> (Appendix 29) A cell or non-human animal produced by the production method of appended claim 28. [Industrial Applicability]

[0203] As described above, the protein of the present disclosure can provide a novel protein that can be used to modify target DNA. Therefore, the present invention is extremely useful in, for example, the medical, food, and agricultural fields.

Claims

1. The following protein (a1), (a2), or (a3): (a1) a protein consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 15, 18, and 19; (a2) a protein having nuclease activity, which consists of an amino acid sequence in which 1 to 143 amino acids are deleted, inserted, substituted, or added in any one of the amino acid sequences of SEQ ID NOs: 1 to 6 and 12 to 15; A protein having nuclease activity, which consists of an amino acid sequence in which 1 to 142 amino acids are deleted, inserted, substituted, or added in any of the amino acid sequences of SEQ ID NOs: 7 to 11, or A protein having nuclease activity, which consists of an amino acid sequence in which 1 to 127 amino acids are deleted, inserted, substituted, or added in the amino acid sequence of SEQ ID NO: 18 or 19; (a3) A protein having nuclease activity, which consists of an amino acid sequence having 90% or more identity to any one of the amino acid sequences of SEQ ID NOs: 1 to 15, 18, and 19.

2. The protein according to claim 1, which recognizes a protospacer adjacent motif (PAM) sequence having a base sequence selected from the group consisting of SEQ ID NOs: 20 to 32.

3. The protein according to claim 1 or 2, wherein the protein (a1) is any one of the following proteins (b1) to (b5) or (b6): (b1) a protein in which the amino acid E at position 1,136 in the amino acid sequence of SEQ ID NO: 1 or 6 is substituted with G; (b2) a protein in which the amino acid E at position 1129 in the amino acid sequence of SEQ ID NO: 2 to 5, 14, or 15 is substituted with G; (b3) a protein in which the 1127th amino acid sequence E in the amino acid sequence of SEQ ID NO: 7 or 9 is substituted with G; (b4) a protein in which the 1120th amino acid sequence E in the amino acid sequence of SEQ ID NO: 8 or 10 is substituted with G; (b5) a protein in which the 1128th amino acid sequence E in the amino acid sequence of SEQ ID NO: 11 is substituted with G; (b6) A protein in which the 969th amino acid E in the amino acid sequence of SEQ ID NO: 18 is replaced with G.

4. The protein according to claim 1 or 2, wherein the protein (a1) is any one of the following proteins (c1) to (c5) or (c6): (c1) a protein in which the amino acid VPAA at positions 1207 to 1210 in the amino acid sequence of SEQ ID NO: 1 or 6 is substituted with ADER; (c2) a protein in which the amino acid VPAA at positions 1200 to 1203 in the amino acid sequence of SEQ ID NO: 2 to 5, 14, or 15 is substituted with ADER; (c3) a protein in which the amino acid sequence VPAA at positions 1198 to 1201 in the amino acid sequence of SEQ ID NO: 7 or 9 is replaced with ADER; (c4) a protein in which the amino acid sequence VPAA at positions 1191 to 1194 in the amino acid sequence of SEQ ID NO: 8 or 10 is replaced with ADER; (c5) a protein in which the amino acid sequence VPAA at positions 1199 to 1202 in the amino acid sequence of SEQ ID NO: 11 is replaced with ADER; (c6) A protein in which the amino acid sequence VPAA at positions 1040 to 1043 in the amino acid sequence of SEQ ID NO: 18 is replaced with ADER.

5. 3. The protein of claim 1 or 2, which is an RNA-dependent DNA nuclease.

6. The protein according to claim 1 or 2, further comprising an enzymatic activity domain.

7. The protein according to claim 6, wherein the enzymatic activity domain is located on the 5'-end and / or 3'-end side of the protein according to claim 1 or 2.

8. 7. The protein of claim 6, wherein the enzymatic activity is selected from the group consisting of a nucleic acid base conversion activity, a methylase activity, a demethylase activity, a base modification activity, a histone modification activity, an RNA cleavage activity, and a DNA cleavage activity.

9. A polynucleotide comprising a coding sequence for the protein of claim 1.

10. A vector comprising the polynucleotide of claim 9.

11. The vector of claim 10 , further comprising a polynucleotide encoding a guide RNA for a target site.

12. A first vector comprising the polynucleotide of claim 9; and a second vector comprising a polynucleotide encoding a guide RNA for the target site.

13. A protein according to claim 1, a polynucleotide according to claim 9 and / or a vector according to claim 10, A composition comprising a guide RNA for a target site or a polynucleotide encoding the same.

14. 14. The composition of claim 13, wherein the guide RNA is capable of complexing with the protein.

15. 14. The composition of claim 13, wherein the guide RNA comprises a polynucleotide capable of hybridizing to a target site in a target DNA.

16. 14. The composition of claim 13, wherein the guide RNA comprises a crRNA.

17. A protein according to claim 1, a polynucleotide according to claim 9 and / or a vector according to claim 10, A kit comprising a guide RNA for a target site or a polynucleotide encoding the same.

18. A cell comprising a protein according to claim 1, a polynucleotide according to claim 9, a vector according to claim 10, and / or a vector system according to claim 12.

19. A method for modifying a target DNA, comprising a modification step of modifying the target DNA by contacting the target DNA with the protein described in claim 1 or 2 and a guide RNA for a target site in the target DNA in a cell (excluding cells in the human body).

20. The method for modification according to claim 19, comprising an introduction step of introducing the protein according to claim 1 or 2 or a polynucleotide encoding the same, and the guide RNA or a polynucleotide encoding the same into the cell.

21. The method for modification according to claim 19, wherein in the modification step, a complex of the protein and the guide RNA is targeted to the target DNA, and the target DNA is modified by the complex at the target site.

22. The method of claim 19, wherein the guide RNA comprises a polynucleotide capable of hybridizing to a target site within the target DNA.

23. The method of claim 19, wherein the guide RNA comprises a crRNA.

24. The method of claim 19, wherein the modification is a double-stranded break, a single-stranded break, a conversion, a deletion and / or an insertion of one or more nucleotides in the target DNA.

25. The method of claim 19 , wherein the cell is an animal cell or a plant cell.

26. The method of claim 19, wherein the cell is a eukaryotic or prokaryotic cell.

27. The method of claim 19, wherein the target DNA is chromosomal DNA.

28. 1. A method for producing a cell or a non-human animal having modified target DNA, comprising: A manufacturing method comprising an introduction step of introducing the protein described in claim 1, the polynucleotide described in claim 9, and / or the vector described in claim 10, and a guide RNA for a target site into a cell (excluding cells in the human body) or a non-human animal.

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