Zinc finger protein and combination thereof, zinc finger nuclease and zinc finger nuclease pair, method for editing target dna, method for producing cell having edited target dna, and kit

Abstract

This zinc finger protein is at least one selected from the group consisting of zinc finger protein I having an amino acid sequence described in SEQ ID NO: 1, zinc finger protein II having an amino acid sequence described in SEQ ID NO: 2, zinc finger protein III-1 having an amino acid sequence described in SEQ ID NO: 3, zinc finger protein III-2 having an amino acid sequence described in SEQ ID NO: 4, and zinc finger protein IV having an amino acid sequence described in SEQ ID NO: 5.

Description

Zinc finger proteins and combinations thereof, zinc finger nucleases and zinc finger nuclease pairs, methods for editing target DNA, methods for producing cells with edited target DNA, and kits

[0001] The present invention relates to zinc finger proteins and combinations thereof, zinc finger nucleases and zinc finger nuclease pairs, methods for editing target DNA, methods for producing cells with edited target DNA, and kits. More specifically, the present invention relates to zinc finger proteins and combinations thereof, zinc finger nucleases containing the zinc finger proteins and combinations (pairs) thereof, methods for editing target DNA and methods for producing cells with edited target DNA using these, and kits for use in the editing and production methods.

[0002] In recent years, attention has been focused on DNA editing techniques that utilize site-specific DNA cleavage and its repair mechanism using the CRISPR-Cas system, particularly the CRISPR-Cas9 system containing a Cas9 protein (dCas9, nCas9) that has lost some or all of its nuclease activity and its guide RNA, and various technologies have been developed to date, such as the BE (Base Editor) system and the Target-AID system. However, these technologies depend on the CRISPR-Cas system, and in order to use them to edit the genome, it is necessary to also introduce guide RNA into the cell. Therefore, a safer technology that enables genome editing using only proteins is desired.

[0003] Known technologies that enable site-specific genome editing using only proteins include TALEN (transcription activator-like effector nuclease) and ZFN (zinc finger nuclease) (Scott et al., Nat. Biotechnol., vol. 23, pp. 915-918, 2005 (Non-Patent Document 1), etc.). In these technologies, for example, the nuclease domain of the type IIS restriction enzyme FokI is used as a nuclease domain, and a fusion protein is used in which this is combined with a DNA-binding protein such as a TALE protein or a zinc finger protein. In this case, a pair of DNA-binding proteins bind to opposite strands of the target DNA, each of which sandwiches a target sequence, and the nuclease domains of FokI form a dimer, thereby exerting double-stranded DNA cleavage activity (nuclease activity) in a site-specific manner against the target sequence.

[0004] Among these, zinc finger nucleases have the advantage of having a small molecular weight. In zinc finger nucleases, in addition to the nuclease domain (wild type) of FokI derived from Flavobacterium okeanokoites, genetically engineered FokI cleavage half-domains that form heterodimers, as described in International Publication No. 2011 / 097036 (Patent Document 1), have been known. Furthermore, for example, International Publication No. 2020 / 045281 (Patent Document 2) describes the editing of target sites on target DNA using an artificial nucleic acid cleavage enzyme containing a novel nuclease domain (nuclease domain 1: ND1) and a zinc finger protein, which is different from the conventional FokI nuclease domain.

[0005] Furthermore, the optimal target sequence for site-specific genome editing is a "safe harbor" sequence, where insertion or deletion of bases has not been reported to adversely affect target cells. For example, the AAVS1 region (also known as the PPP1R2C region) on human chromosome 19 has been primarily validated as such a safe harbor because it has transcriptional activity in a variety of cells.

[0006] International Publication No. 2011 / 097036 International Publication No. 2020 / 045281

[0007] Scott et al. , Nat. Biotechnol. , vol. 23, p. 915-918, 2005

[0008] Zinc finger nucleases (ZFNs) are designed by combining a pair of zinc finger nucleases (ZFNs) that sandwich a target sequence of interest, a zinc finger protein (ZF-(L)) that recognizes the complementary sequence of the nucleotide sequence adjacent to the 5' side, and a zinc finger protein (ZF-(R)) that recognizes the nucleotide sequence adjacent to the 3' side. Each zinc finger protein typically has a structure in which 3 to 6 zinc finger modules that recognize a 3-base nucleotide sequence are linked together, thereby recognizing a 9 to 18-base nucleotide sequence (ZF recognition sequence), that is, a total of 18 to 36 bases in total on both ends of the target sequence. However, because such zinc finger proteins have a complex binding mode with the ZF recognition sequence and are difficult to prepare, a combination of zinc finger proteins with sufficient specificity for a target sequence in the AAVS1 region, and a zinc finger nuclease pair that can specifically and highly actively cleave the target sequence, i.e., that can specifically edit the AAVS1 region with a high probability, have not yet been developed.

[0009] The present invention has been made in consideration of the problems associated with the above-mentioned conventional technology, and aims to provide zinc finger proteins and combinations thereof that can be applied to zinc finger nuclease pairs to cleave and edit specific target sequences within the AAVS1 region with a high probability, zinc finger nucleases and zinc finger nuclease pairs containing the same, methods for editing target DNA using these and methods for producing cells in which target DNA has been edited, and kits for use in the editing and production methods.

[0010] To achieve the above object, the present inventors designed and tested combinations of multiple zinc finger proteins for multiple sequences within the AAVS1 region as target sequences and sequences flanking these as ZF recognition sequences. As a result, they found that only when a combination of zinc finger proteins consisting of specific amino acid sequences is applied to a zinc finger nuclease pair for two specific target sequences out of the total 10,020 bases of AAVS1, the target sequence can be cleaved with high activity and edited with a high probability, thereby completing the present invention. In their testing, the inventors found that even a shift of even a few bases between the target sequence and the ZF recognition sequence completely eliminated the cleavage activity of the target sequence, thereby providing a particularly significant technical effect.

[0011] The present invention, based on these findings, provides the following aspects: [1] A zinc finger protein, which is at least one type selected from the group consisting of zinc finger protein I consisting of the amino acid sequence set forth in SEQ ID NO: 1, zinc finger protein II consisting of the amino acid sequence set forth in SEQ ID NO: 2, zinc finger protein III-1 consisting of the amino acid sequence set forth in SEQ ID NO: 3, zinc finger protein III-2 consisting of the amino acid sequence set forth in SEQ ID NO: 4, and zinc finger protein IV consisting of the amino acid sequence set forth in SEQ ID NO: 5. [2] A combination of zinc finger proteins for a zinc finger nuclease pair, which is at least one type selected from the group consisting of a first combination consisting of zinc finger protein I consisting of the amino acid sequence set forth in SEQ ID NO: 1 and zinc finger protein II consisting of the amino acid sequence set forth in SEQ ID NO: 2, and a second combination consisting of zinc finger protein III-1 consisting of the amino acid sequence set forth in SEQ ID NO: 3 or zinc finger protein III-2 consisting of the amino acid sequence set forth in SEQ ID NO: 4, and zinc finger protein IV consisting of the amino acid sequence set forth in SEQ ID NO: 5. [3] A zinc finger nuclease that is at least one selected from the group consisting of: zinc finger nuclease I comprising a zinc finger protein I consisting of the amino acid sequence set forth in SEQ ID NO: 1 and nuclease domain I; zinc finger nuclease II comprising a zinc finger protein II consisting of the amino acid sequence set forth in SEQ ID NO: 2 and nuclease domain II; zinc finger nuclease III comprising a zinc finger protein III-1 consisting of the amino acid sequence set forth in SEQ ID NO: 3 or a zinc finger protein III-2 consisting of the amino acid sequence set forth in SEQ ID NO: 4 and nuclease domain III; and zinc finger nuclease IV comprising a zinc finger protein IV consisting of the amino acid sequence set forth in SEQ ID NO: 5 and nuclease domain IV.[4] A zinc finger nuclease pair, which is at least one selected from the group consisting of: a first zinc finger nuclease pair consisting of a zinc finger nuclease I comprising a zinc finger protein I and nuclease domain I consisting of the amino acid sequence set forth in SEQ ID NO: 1, and a zinc finger nuclease II comprising a zinc finger protein II and nuclease domain II consisting of the amino acid sequence set forth in SEQ ID NO: 2; and a second zinc finger nuclease pair consisting of a zinc finger nuclease III comprising a zinc finger protein III-1 consisting of the amino acid sequence set forth in SEQ ID NO: 3 or a zinc finger protein III-2 consisting of the amino acid sequence set forth in SEQ ID NO: 4, and a nuclease domain III, and a zinc finger nuclease IV comprising a zinc finger protein IV and nuclease domain IV consisting of the amino acid sequence set forth in SEQ ID NO: 5. [5] The zinc finger nuclease pair according to [4], wherein the pair of nuclease domain I and nuclease domain II, and the pair of nuclease domain III and nuclease domain IV are pairs that form dimers with each other. [6] The zinc finger nuclease pair according to [4] or [5], wherein the first zinc finger nuclease pair cleaves a target sequence of 5 to 7 bases between the nucleotide sequence set forth in SEQ ID NO: 6 and the nucleotide sequence set forth in SEQ ID NO: 7, and a complementary sequence thereto. [7] The zinc finger nuclease pair according to any one of [4] to [6], wherein the second zinc finger nuclease pair cleaves a target sequence of 5 to 7 bases between the nucleotide sequence set forth in SEQ ID NO: 8 and the nucleotide sequence set forth in SEQ ID NO: 9, and a complementary sequence thereto. [8] The zinc finger nuclease pair according to any one of [4] to [7], wherein the zinc finger nuclease pair targets the AAVS1 region.[9] A method for editing a target DNA, the method comprising the steps of: contacting the target DNA with the zinc finger nuclease pair according to any one of [4] to [8], and cleaving, with the first zinc finger nuclease pair, 5 to 7 bases between the nucleotide sequence set forth in SEQ ID NO: 6 and the nucleotide sequence set forth in SEQ ID NO: 7 and their complementary sequence as a target sequence, and / or cleaving, with the second zinc finger nuclease pair, 5 to 7 bases between the nucleotide sequence set forth in SEQ ID NO: 8 and the nucleotide sequence set forth in SEQ ID NO: 9 and their complementary sequence as a target sequence.

[10] The method according to [9], wherein the target DNA comprises an AAVS1 region and the method is a method for editing the AAVS1 region.

[11] A method for producing a cell in which a target DNA has been edited, the method comprising the steps of: introducing or expressing the zinc finger nuclease pair according to any one of [4] to [8] into a cell and contacting it with the target DNA, and cleaving, with the first zinc finger nuclease pair, 5 to 7 bases between the nucleotide sequence set forth in SEQ ID NO: 6 and the nucleotide sequence set forth in SEQ ID NO: 7 and their complementary sequence as a target sequence, and / or cleaving, with the second zinc finger nuclease pair, 5 to 7 bases between the nucleotide sequence set forth in SEQ ID NO: 8 and the nucleotide sequence set forth in SEQ ID NO: 9 and their complementary sequence as a target sequence.

[12] The method according to

[11] , in which the target DNA includes the AAVS1 region, is a method for producing a cell in which the AAVS1 region has been edited.

[13] A kit for use in the method according to any one of [9] to

[12] , comprising at least one zinc finger nuclease selected from the group consisting of: zinc finger nuclease I comprising zinc finger protein I and nuclease domain I consisting of the amino acid sequence set forth in SEQ ID NO: 1; zinc finger nuclease II comprising zinc finger protein II and nuclease domain II consisting of the amino acid sequence set forth in SEQ ID NO: 2; zinc finger nuclease III comprising zinc finger protein III-1 consisting of the amino acid sequence set forth in SEQ ID NO: 3 or zinc finger protein III-2 consisting of the amino acid sequence set forth in SEQ ID NO: 4 and nuclease domain III; and zinc finger nuclease IV comprising zinc finger protein IV and nuclease domain IV consisting of the amino acid sequence set forth in SEQ ID NO: 5; a polynucleotide encoding the zinc finger nuclease; and an expression vector for the zinc finger nuclease.

[0012] According to the present invention, it is possible to provide zinc finger proteins and combinations thereof that can be applied to zinc finger nuclease pairs to cleave and edit specific target sequences within the AAVS1 region with a high probability, zinc finger nucleases and zinc finger nuclease pairs containing these, methods for editing target DNA using these and methods for producing cells in which target DNA has been edited, and kits for use in the editing and production methods.

[0013] 1 is a schematic conceptual diagram showing one embodiment of the positional relationship between a zinc finger nuclease pair and target DNA of the present invention. 2 shows the appearance of an agarose gel and genome editing efficiency (Indel%) after electrophoresis in the T7E1 Assay of Test Example 1. 3 shows the appearance of an agarose gel and genome editing efficiency (Indel%) after electrophoresis in the T7E1 Assay of Test Example 2.

[0014] The present invention will be described in more detail below by taking preferred embodiments as examples, but the present invention is not limited thereto.

[0015] <Target DNA, Target Sequence> In the present invention, the nucleotide sequence to be cleaved by the zinc finger nuclease pair described below is referred to as a "target sequence," and DNA containing the target sequence is referred to as "target DNA." The target DNA according to the present invention is double-stranded DNA, and may be artificially synthesized and may contain non-natural nucleotides (artificial nucleotides, nucleotide analogs) or may be composed of natural nucleotides. Furthermore, depending on the purpose, the target DNA may be DNA present within a cell (endogenous DNA) or DNA present outside a cell. The DNA present within a cell may be endogenous DNA or exogenous DNA (e.g., DNA introduced into a cell), and when present in a eukaryotic cell, it may be present inside or outside the nucleus. The DNA present outside a cell may be DNA derived from a cell or DNA amplified and synthesized outside the cell.

[0016] Among these, the target DNA according to the present invention is preferably a DNA containing the safe harbor AAVS1 region, and more preferably human genomic DNA. The AAVS1 region is located on human chromosome 19, and the nucleotide sequence of the AAVS1 region in human genomic DNA is typically represented by Reference ID (Gene ID): 17.

[0017] The sequence on the target DNA that the zinc finger protein below recognizes and binds to is referred to as the "ZF recognition sequence," the complementary sequence of the nucleotide sequence adjacent to the 5' side of the target sequence is referred to as the "ZF recognition sequence (L)," and the nucleotide sequence adjacent to the 3' side of the target sequence is referred to as the "ZF recognition sequence (R)."

[0018] The ZF recognition sequence of the present invention is preferably such that the ZF recognition sequence (L) is a complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 6 and the ZF recognition sequence (R) is a nucleotide sequence set forth in SEQ ID NO: 7, or such that the ZF recognition sequence (L) is a complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 8 and the ZF recognition sequence (R) is a nucleotide sequence set forth in SEQ ID NO: 9.

[0019] The target sequence of the present invention is preferably 5 to 7 bases, more preferably 6 bases, sandwiched between the complementary sequence of the ZF recognition sequence (L) and the ZF recognition sequence (R) of the above combination. The target sequence of the present invention is further preferably a sequence within the AAVS1 region, and is preferably a sequence represented by 5'-GTCTCC-3' sandwiched between the nucleotide sequence set forth in SEQ ID NO: 6 (complementary sequence: ZF recognition sequence (L)) and the nucleotide sequence set forth in SEQ ID NO: 7 (ZF recognition sequence (R)), or a complementary sequence thereof, or a sequence represented by 5'-CGCCCC-3' sandwiched between the nucleotide sequence set forth in SEQ ID NO: 8 (complementary sequence: ZF recognition sequence (L)) and the nucleotide sequence set forth in SEQ ID NO: 9 (ZF recognition sequence (R)), or a complementary sequence thereof. The nucleotide sequence of the target DNA containing such a target sequence and ZF recognition sequence is particularly preferably at least one selected from the group consisting of the nucleotide sequence set forth in SEQ ID NO: 10 and its complementary sequence, and the nucleotide sequence set forth in SEQ ID NO: 11 and its complementary sequence. The nucleotide sequence set forth in SEQ ID NO: 10 is a nucleotide sequence of 42 bases from position 418 on chromosome 19 of the human genome, and the nucleotide sequence set forth in SEQ ID NO: 11 is a nucleotide sequence of 42 bases from position 490 on chromosome 19 of the human genome, both of which are located in intron 1 of the human AAVS1 region (Figure 1).

[0020] <Zinc Finger Proteins and Combinations Thereof> The zinc finger proteins of the present invention are proteins that function as DNA-binding domains in the zinc finger nucleases described below. More specifically, the zinc finger proteins of the present invention include the following five types: zinc finger protein I (herein sometimes referred to as "ZF-I") consisting of the amino acid sequence set forth in SEQ ID NO: 1, zinc finger protein II (herein sometimes referred to as "ZF-II") consisting of the amino acid sequence set forth in SEQ ID NO: 2, zinc finger protein III-1 (herein sometimes referred to as "ZF-III-1") consisting of the amino acid sequence set forth in SEQ ID NO: 3, zinc finger protein III-2 (herein sometimes referred to as "ZF-III-2") consisting of the amino acid sequence set forth in SEQ ID NO: 4, and zinc finger protein IV (herein sometimes referred to as "ZF-IV") consisting of the amino acid sequence set forth in SEQ ID NO: 5. Each of the zinc finger proteins of the present invention is composed of six zinc finger modules that recognize specific three bases in DNA, and each recognizes and binds to 18 bases as a ZF recognition sequence.

[0021] ZF-I recognizes and binds to the complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 6 (ZF recognition sequence (L)), and ZF-II recognizes and binds to the nucleotide sequence set forth in SEQ ID NO: 7 (ZF recognition sequence (R)).

[0022] A preferred combination of zinc finger proteins of the present invention is a combination of ZF-I and ZF-II (first combination). By applying this combination to the zinc finger nuclease pair described below, it is possible to specifically cleave a target sequence between the nucleotide sequence set forth in SEQ ID NO: 6 and the nucleotide sequence set forth in SEQ ID NO: 7, more preferably the sequence represented by 5'-GTCTCC-3' on the nucleotide sequence set forth in SEQ ID NO: 10 in the AAVS1 region and its complementary sequence.

[0023] ZF-III-1 and ZF-III-2 recognize and bind to the complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 8 (ZF recognition sequence (L)), and ZF-IV recognizes and binds to the nucleotide sequence set forth in SEQ ID NO: 9 (ZF recognition sequence (R)). ZF-III-2 has a skip linker consisting of the amino acid sequence set forth in SEQ ID NO: 12 introduced in place of "GE" at positions 81-82 of the amino acid sequence of ZF-III-1, and therefore recognizes the complementary sequence of the nucleotide sequence set forth in SEQ ID NO: 8, skipping "T" at the 9th base from the 3' end.

[0024] A preferred combination of zinc finger proteins of the present invention is a combination of ZF-III-1 or ZF-III-2 with ZF-IV (second combination). By applying this combination to the zinc finger nuclease pair described below, it is possible to specifically cleave a target sequence between the nucleotide sequence set forth in SEQ ID NO: 8 and the nucleotide sequence set forth in SEQ ID NO: 9, more preferably the sequence represented by 5'-CGCCCC-3' on the nucleotide sequence set forth in SEQ ID NO: 11 in the AAVS1 region and its complementary sequence.

[0025] Such zinc finger proteins can be prepared by any known method, for example, by artificial synthesis based on the amino acid sequence information. Alternatively, the zinc finger protein of the present invention can be obtained by synthesizing a polynucleotide encoding the zinc finger protein, or by linking polynucleotides encoding each zinc finger module by the Golden Gate Assembly method to form a polynucleotide encoding the zinc finger protein, inserting the polynucleotide into an expression vector, and expressing it in an appropriate host cell. The zinc finger protein of the present invention is preferably prepared in the form of a fusion protein with a nuclease domain described below as a zinc finger nuclease.

[0026] <Zinc Finger Nucleases> The zinc finger nucleases of the present invention are fusion proteins comprising a nuclease domain and a DNA-binding domain, and the DNA-binding domain comprises the zinc finger protein of the present invention. That is, the zinc finger nucleases of the present invention include the following four types: zinc finger nuclease I comprising ZF-I and nuclease domain I (herein sometimes referred to as "ZFN-I"), zinc finger nuclease II comprising ZF-II and nuclease domain II (herein sometimes referred to as "ZFN-II"), zinc finger nuclease III comprising ZF-III-1 or ZF-III-2 and nuclease domain III (herein sometimes referred to as "ZFN-III"), and zinc finger nuclease IV comprising ZF-IV and nuclease domain IV (herein sometimes referred to as "ZFN-IV").

[0027] (Nuclease Domains and Combinations Thereof) In each zinc finger nuclease of the present invention, nuclease domains I to IV may be independently the same or different from each other, as long as they can form a homodimer or a heterodimer with another nuclease domain as a zinc finger nuclease pair as described below.

[0028] Such nuclease domains I to IV (hereinafter sometimes collectively referred to as "nuclease domains") are not particularly limited as long as they form the dimer and exhibit nuclease activity (double-stranded DNA cleavage activity), and known domains can be appropriately mentioned. More specifically, for example, typically, the nuclease domain of wild-type FokI derived from Flavobacterium okeanokoites (typical amino acid sequence reference ID (Gene ID): 77466401), and the nuclease domain 1 (ND1) described in WO 2020 / 045281 (Patent Document 2) can be mentioned. Nuclease domain 1 (ND1) is a nuclease domain that has an identity of 35% to 70% with the nuclease domain of FokI. Nuclease domain 1 typically includes a polypeptide consisting of the amino acid sequence set forth in positions 391 to 585 of SEQ ID NO: 1 of Patent Document 2 (the amino acid sequence is set forth in SEQ ID NO: 45).

[0029] The above-mentioned nuclease domains also include their mutants, so long as they form the dimer and exhibit nuclease activity. Examples of the mutants include polypeptides that have one or more amino acid substitutions, deletions, insertions, and / or additions in the typical amino acid sequences of the above-mentioned nuclease domains, and that form the dimer and exhibit nuclease activity.

[0030] Here, in the amino acid sequence, "a substituted, deleted, inserted, and / or added amino acid sequence" refers to an amino acid sequence in which amino acids (amino acid residues) in the amino acid sequence have been substituted, deleted, inserted, or added, or an amino acid sequence in which two or more of these have been combined. Furthermore, "multiple" refers to an integer of 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2.

[0031] Further examples of the variants include polypeptides consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% homology to the typical amino acid sequences of each nuclease domain listed above, more preferably polypeptides consisting of an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity, and which form the dimer and exhibit nuclease activity.

[0032] Here, when referring to amino acid sequence homology, it means that when a control amino acid sequence (a typical amino acid sequence of each nuclease domain listed above) and a target amino acid sequence (an amino acid sequence of a mutant) are aligned using amino acid sequence analysis software or the like, the amino acid in the target amino acid sequence (target amino acid) at the same position as an amino acid in the control amino acid sequence (control amino acid) may be the same amino acid as the control amino acid or an amino acid with the same properties as the control amino acid. When referring to amino acid sequence identity, the target amino acid is the same amino acid as the control amino acid. Groups of amino acids with similar properties are well known in the art to which the present invention pertains, and can be classified into, for example, acidic amino acids (aspartic acid and glutamic acid); basic amino acids (lysine, arginine, histidine); neutral amino acids; and further, neutral amino acids can be classified into amino acids with hydrocarbon chains (glycine, alanine, valine, leucine, isoleucine, proline), amino acids with hydroxy groups (serine, threonine), amino acids containing sulfur (cysteine, methionine), amino acids with amide groups (asparagine, glutamine), amino acids with imino groups (proline), and amino acids with aromatic groups (phenylalanine, tyrosine, tryptophan).

[0033] Such amino acid sequence homology and identity are determined by comparing two sequences aligned to maximize sequence identity. Methods for determining sequence homology or identity (%) are known to those skilled in the art. As an algorithm for obtaining optimal alignment, homology, and identity, any algorithm known to those skilled in the art (e.g., BLAST algorithm, FASTA algorithm, etc.) can be used, and amino acid sequence homology and identity can be determined using sequence analysis software such as BLASTP and FASTA.

[0034] Further examples of the variants include polypeptides containing modified amino acids and / or unnatural amino acids. The modified amino acids are not particularly limited, but examples include methylation, esterification, amidation, acetylation, alkylation, halogenation, etc. These modified amino acids and unnatural amino acids can be introduced by known methods. Furthermore, the nuclease domain of the present invention may further include a polypeptide consisting of one amino acid residue or multiple amino acid residues, as long as it forms the dimer and exhibits nuclease activity.

[0035] Preferred mutants of the wild-type FokI nuclease domain listed above include, for example, the FokI cleavage half-domain described in WO 2011 / 097036 (Patent Document 1).

[0036] Furthermore, preferred variants of the above-mentioned exemplary nuclease domain 1 (ND1) include those in which, in the amino acid sequence of SEQ ID NO: 45, an amino acid corresponding to aspartic acid at position 99 (D99) is substituted with arginine (R99), an amino acid corresponding to arginine at position 103 (R103) is substituted with aspartic acid (D103), an amino acid corresponding to asparagine at position 112 (N112) is substituted with aspartic acid (D112), and an amino acid corresponding to asparagine at position 153 (N112) is substituted with aspartic acid (D112). and substitution of an amino acid corresponding to arginine (R153) with an amino acid corresponding to aspartic acid (N153). More preferred examples include a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 13 (R99, R103, N112, R153, referred to as "ND1RRR") and a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 14 (D99, D103, D112, N153, referred to as "ND1DDD"). In order for ND1 to exhibit the above-mentioned nuclease activity, it is necessary that at least the amino acids corresponding to positions 66 and 83 of the amino acid sequence set forth in SEQ ID NO: 45 are aspartic acid (D).

[0037] Examples of dimer combinations formed by the nuclease domain of the present invention include a homodimer of the wild-type FokI nuclease domain, a heterodimer of the wild-type FokI nuclease domain and the FokI cleavage half-domain, a homodimer of typical nuclease domain 1, and a heterodimer of ND1RRR and ND1DDD. Among these, the heterodimer of ND1RRR and ND1DDD is preferred from the viewpoint of ease of dimer formation.

[0038] The nuclease activity of the nuclease domain of the present invention when it forms the dimer is preferably equal to or greater than the nuclease activity of the nuclease domain of wild-type FokI when it forms a homodimer. For example, the nuclease activity is preferably at least 0.8-fold, at least 0.9-fold, at least 1-fold, at least 1.3-fold, at least 1.5-fold, at least 1.8-fold, at least 2-fold, at least 2.3-fold, at least 2.5-fold, at least 2.8-fold, at least 3-fold, at least 3.3-fold, at least 3.5-fold, at least 3.8-fold, or at least 4-fold greater than the nuclease activity of the nuclease domain of wild-type FokI. Such nuclease activity can be confirmed by methods known in the art. For example, it can be confirmed by incorporating the nuclease domain into a zinc finger nuclease pair and measuring double-strand DNA cleavage activity using the single-strand annealing (SSA) method or the Cel-I method.

[0039] (Linker) In the zinc finger nuclease of the present invention, the nuclease domain and the DNA-binding domain (zinc finger protein) may be linked directly or via a linker.

[0040] The linker is not particularly limited in length or type as long as it does not inhibit the effects of the present invention (such as the ability to bind to the ZF recognition sequence and the nuclease activity of the zinc finger nuclease pair), but is preferably a polypeptide consisting of 3 to 6 amino acid residues, more preferably 4 to 5 amino acid residues. An example of such a linker is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 15.

[0041] In the zinc finger nuclease of the present invention, either the nuclease domain or the DNA-binding domain (zinc finger protein) may be located at the N-terminus or the C-terminus. However, from the viewpoint of exhibiting excellent nuclease activity when formed as a zinc finger nuclease pair, it is preferable that they are linked in the following order from the N-terminus: zinc finger protein / optionally a linker / nuclease domain.

[0042] In the zinc finger nuclease of the present invention, the zinc finger protein, the nuclease domain, and, if necessary, the linker can be linked at the nucleic acid level and the amino acid level. That is, for example, a polynucleotide encoding the zinc finger nuclease of the present invention can be prepared by ligating each polynucleotide in the order of "polynucleotide encoding the zinc finger protein → polynucleotide encoding the linker → polynucleotide encoding the nuclease domain." The resulting polynucleotide can be inserted into an expression vector and expressed in an appropriate host cell to obtain the zinc finger nuclease of the present invention, in which the zinc finger protein, linker, and nuclease domain are linked at the amino acid level.

[0043] The expression vector can be appropriately selected from vectors commonly used in the art, including, for example, plasmid vectors, viral vectors, phage vectors, phagemid vectors, BAC vectors, YAC vectors, MAC vectors, and HAC vectors. Host cells into which the expression vector is introduced can be appropriately selected taking into consideration compatibility with the expression vector, including, for example, prokaryotic cells such as Escherichia coli, actinomycetes, and archaea, and eukaryotic cells such as yeast, sea urchin, silkworm, zebrafish, mouse, rat, frog, tobacco, Arabidopsis, and rice. The expression vector can also be introduced into cells whose target DNA is to be edited, as in the production method of the present invention described below, to directly express the zinc finger nuclease of the present invention in the cells. The term "polynucleotide" used in the preparation of the zinc finger protein and zinc finger nuclease described above encompasses both DNA and RNA (mRNA), and each polynucleotide may be codon-optimized for purposes such as increasing intracellular expression efficiency.

[0044] The zinc finger nucleases of the present invention can also be prepared by artificial synthesis based on amino acid sequence information. Furthermore, the zinc finger nucleases of the present invention may be tagged with epitope tags (e.g., flag tags, HA tags) for purification or detection, or with various localization signals (e.g., nuclear localization signals, mitochondrial localization signals, plastid localization signals), etc.

[0045] <Zinc finger nuclease pair> The zinc finger nuclease pair of the present invention is a first zinc finger nuclease pair (herein sometimes referred to as "first ZFNs") consisting of ZFN-I and ZFN-II, and a second zinc finger nuclease pair (herein sometimes referred to as "second ZFNs") consisting of ZFN-III and ZFN-IV.

[0046] In the first ZFNs, the combination of zinc finger proteins is a combination of ZF-I and ZF-II (first combination). The combination of nuclease domains is a combination of nuclease domain I and nuclease domain II. Such a combination may be any combination that forms a dimer and exhibits nuclease activity, and preferred embodiments thereof are as described above. By including the first combination as the combination of zinc finger proteins, the first ZFNs bind to the ZF recognition sequences (L, R) that flank the target sequence, respectively, and the nuclease domains form a dimer with each other. This nuclease activity allows the first ZFNs to specifically cleave the target sequence, i.e., the target sequence between the nucleotide sequence set forth in SEQ ID NO: 6 and the nucleotide sequence set forth in SEQ ID NO: 7, more preferably the sequence represented by 5'-GTCTCC-3' on the nucleotide sequence set forth in SEQ ID NO: 10 within the AAVS1 region, and its complementary sequence.

[0047] Furthermore, in the second ZFNs, the combination of zinc finger proteins is a combination of ZF-III-1 or ZF-III-2 with ZF-IV (second combination). The combination of nuclease domains is a combination of nuclease domain III and nuclease domain IV. Such combinations may be any combination that forms a dimer and exhibits nuclease activity, as described above, including preferred embodiments. By including the second combination of zinc finger proteins, the second ZFNs bind to the ZF recognition sequences (L, R) that flank the target sequence, respectively, and the nuclease domains form a dimer with each other. This nuclease activity allows the second ZFNs to specifically cleave the target sequence, i.e., the target sequence between the nucleotide sequence set forth in SEQ ID NO: 8 and the nucleotide sequence set forth in SEQ ID NO: 9, more preferably the sequence represented by 5'-CGCCCC-3' on the nucleotide sequence set forth in SEQ ID NO: 11 in the AAVS1 region, and its complementary sequence.

[0048] In the zinc finger nuclease pair of the present invention, each zinc finger nuclease may be independently in the form of a protein, a polynucleotide (DNA, RNA) encoding the protein, or a vector (expression vector) expressing the protein. Furthermore, the zinc finger nuclease pair of the present invention may be in the form of a composition containing a pair of zinc finger nucleases, or in the form of a combination containing the zinc finger nucleases separately without being mixed.

[0049] <Method for Editing Target DNA> The method for editing target DNA of the present invention (sometimes simply referred to herein as the "editing method") comprises the steps of: contacting target DNA with the zinc finger nuclease pair (first ZFNs and / or second ZFNs) of the present invention; and cleaving, with the first ZFNs, 5 to 7 bases between the nucleotide sequence set forth in SEQ ID NO: 6 and the nucleotide sequence set forth in SEQ ID NO: 7 and their complementary sequence as a target sequence; and / or cleaving, with the second ZFNs, 5 to 7 bases between the nucleotide sequence set forth in SEQ ID NO: 8 and the nucleotide sequence set forth in SEQ ID NO: 9 and their complementary sequence as a target sequence. In the editing method of the present invention, the zinc finger nuclease pair (first ZFNs, second ZFNs) and target DNA are as described above, including preferred embodiments thereof.

[0050] In the editing method of the present invention, the zinc finger nuclease pair is contacted with the target DNA, and the target sequence of the target DNA is cleaved by its nuclease activity. When the zinc finger nuclease pair of the present invention, i.e., a pair of zinc finger nucleases comprising a combination of zinc finger proteins of the present invention, is contacted with the target DNA, each zinc finger protein recognizes and binds to a corresponding ZF recognition sequence (L, R) on the target DNA, and each nuclease domain linked to the zinc finger protein is guided to the target DNA. As a result, two nuclease domains are guided to the target sequence sandwiched between the ZF recognition sequences to form a dimer, and the nuclease activity cleaves the double-stranded DNA of the target sequence.

[0051] One embodiment of the positional relationship between a zinc finger nuclease pair and a target DNA in the editing method of the present invention is shown, for example, in the conceptual diagram of Figure 1. In one embodiment of the target DNA editing method of the present invention, for example, when first ZFNs are used, ZF-I (in Figure 1, ZF-Left(L)) binds to the ZF recognition sequence (L), and ZF-II (in Figure 1, ZF-Right(R)) binds to the ZF recognition sequence (R) in target DNA, which consists of, from the 5' end, a complementary sequence of the ZF recognition sequence (L), a target sequence (Target sequences), and a ZF recognition sequence (R). As a result, the nuclease domains of ZFN-I and ZFN-II (in Figure 1, Nuc(L) and Nuc(R)) form a dimer with each other, and the target sequence is cleaved.

[0052] As a result of the cleavage of the target sequence, the self-repair mechanism of double-stranded DNA (non-homologous end joining repair) can efficiently cause substitution or deletion of bases in the target sequence. Furthermore, for example, when a base substitution occurs in the target sequence, for example, in a cell, due to a mismatch in double-stranded DNA, the base on the opposite strand of the strand where the substitution occurred can be repaired to pair with the substituted base, or replaced with another base during repair, or a deletion or insertion of one base or several dozen bases can occur, resulting in the introduction of various mutations. Furthermore, by further contacting the target DNA with a knock-in fragment that can be introduced into the target sequence or a template DNA for homologous recombination repair, it is also possible to introduce (knock-in) the desired base sequence at the cleavage site. Conventional known methods can be appropriately adopted for such knock-in technology.

[0053] Therefore, editing of target DNA according to the present invention includes deletion of one or more bases in the target sequence, substitution with one or more other bases, insertion of one or more bases, or a combination of these mutations. As described above, since the zinc finger nuclease pair of the present invention can specifically cleave a specific target sequence within the AAVS1 region, these edits can be made to the AAVS1 region according to the editing method of the present invention.

[0054] The editing method of the present invention may be performed intracellularly or in a cell-free system. The "intracellular" where the target DNA editing method of the present invention is performed may be a eukaryotic cell or a prokaryotic cell, preferably a eukaryotic cell. Examples of eukaryotic cells include animal cells (e.g., cells of mammals, fish, birds, reptiles, amphibians, insects), plant cells, algae cells, and yeast. Examples of prokaryotic cells include Escherichia coli, Salmonella, Bacillus subtilis, lactic acid bacteria, and extreme thermophiles.

[0055] "Animal cells" include, for example, cells constituting an individual animal, cells constituting organs or tissues extracted from an animal, and cultured cells derived from animal tissues. Specific examples include germ cells such as oocytes and sperm; germ cells of various stages of embryos (e.g., 1-cell embryos, 2-cell embryos, 4-cell embryos, 8-cell embryos, 16-cell embryos, morula embryos, etc.); stem cells such as induced pluripotent stem (iPS) cells and embryonic stem (ES) cells; and somatic cells such as fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, liver cells, pancreatic cells, brain cells, and kidney cells. Pre- and post-fertilization oocytes can be used as oocytes for the method of producing a non-human individual described below, but post-fertilization oocytes, i.e., fertilized eggs, are preferred. Particularly preferred are fertilized eggs at the pronuclear stage. Oocytes can be used by thawing cryopreserved oocytes.

[0056] "Plant cells" include, for example, cells that constitute an individual plant, cells that constitute organs or tissues separated from a plant, cultured cells derived from plant tissue, etc. Examples of plant organs and tissues include leaves, stems, shoot tips (growing points), roots, tubers, calluses, etc.

[0057] Furthermore, the "cell-free system" used in the editing method of the present invention refers to a system that does not contain living cells (eukaryotic cells or prokaryotic cells). The cell-free system according to the present invention is not particularly limited as long as it allows contact between the zinc finger nuclease pair and the target DNA, and examples thereof include a buffer solution, a cell lysate of the eukaryotic or prokaryotic cells, and a cell extract.

[0058] The method for contacting the zinc finger nuclease pair with the target DNA is not particularly limited. In cells, for example, the zinc finger nuclease pair can be introduced into or expressed in cells containing the target DNA, as in the production method of the present invention described below. In a cell-free system, for example, a solution of the target DNA and a solution of the zinc finger nuclease pair can be mixed. The solvent for these solutions is not particularly limited, but buffers such as phosphate buffer, Tris buffer, Good's buffer, and borate buffer are preferred.

[0059] <Method for producing cells in which target DNA has been edited> The method for producing cells in which target DNA has been edited of the present invention (sometimes simply referred to as the "production method" herein) comprises the steps of: introducing into a cell or expressing in the cell the zinc finger nuclease pair of the present invention (first ZFNs and / or second ZFNs) and contacting the target DNA; and cleaving, with the first ZFNs, 5 to 7 bases between the nucleotide sequence set forth in SEQ ID NO: 6 and the nucleotide sequence set forth in SEQ ID NO: 7 and their complementary sequence as a target sequence; and / or cleaving, with the second ZFNs, 5 to 7 bases between the nucleotide sequence set forth in SEQ ID NO: 8 and the nucleotide sequence set forth in SEQ ID NO: 9 and their complementary sequence as a target sequence. In the production method of the present invention, the zinc finger nuclease pair (first ZFNs, second ZFNs) and target DNA are as described above, including preferred embodiments thereof.

[0060] The cells into which the zinc finger nuclease pair is introduced or expressed include the cells mentioned when the above-mentioned editing method of the present invention is carried out intracellularly, and are preferably eukaryotic cells, preferably animal cells, and more preferably cells having DNA containing the AAVS1 region as genomic DNA. By targeting cells having DNA containing the AAVS1 region as genomic DNA, it is possible to produce cells in which the AAVS1 region has been edited.

[0061] In the production method of the present invention, the zinc finger nuclease pair is brought into contact with the target DNA in a cell by introducing each zinc finger nuclease constituting the zinc finger nuclease pair into the cell in the form of a protein, or in the form of a polynucleotide and / or in the form of an expression vector, thereby expressing the zinc finger nuclease in the cell. Thus, the zinc finger nuclease constituting the zinc finger nuclease pair may be introduced into the cell in the form of a protein, or in the form of RNA or DNA (polynucleotide) encoding the protein and expressed in the cell, or in the form of a vector expressing the protein (expression vector).

[0062] When each zinc finger nuclease constituting the zinc finger nuclease pair is introduced into a cell in the form of an expression vector and expressed within the cell, vectors expressing each zinc finger nuclease separately may be introduced into the cell, or a vector expressing a combination of the constituent zinc finger nucleases may be introduced into the cell.

[0063] Furthermore, when each zinc finger nuclease constituting the zinc finger nuclease pair is introduced into a cell in the form of an expression vector and expressed in the cell, the polynucleotides encoding each zinc finger nuclease may be independently codon-optimized as appropriate for the cell into which they are introduced. Furthermore, the expression vector preferably includes a promoter and / or other regulatory sequence operably linked to the polynucleotide to be expressed. Furthermore, the expression vector preferably is one that can stably express the encoded protein without being integrated into the host genome. Such expression vectors can be prepared according to conventionally known methods.

[0064] As a method for introducing each of the zinc finger nucleases, polynucleotides encoding the zinc finger nucleases, or vectors expressing the zinc finger nucleases into cells, known methods for introducing proteins, DNA, or RNA fragments into cells can be appropriately adopted depending on the type of cell. Examples of such methods include electroporation, microinjection, particle gun, calcium phosphate, polyethyleneimine (PEI), liposome (lipofection), DEAE-dextran, cationic lipid-mediated transfection, viruses (adenovirus, lentivirus, adeno-associated virus, baculovirus, etc.), Agrobacterium, lithium acetate, spheroplast, and heat shock methods (calcium chloride, rubidium chloride). Such methods are described in many standard laboratory manuals, such as Davis et al., Basic methods in molecular biology, New York: Elsevier, 1986.

[0065] When each zinc finger nuclease is introduced into or expressed within a cell, it comes into contact with the target DNA within the cell, and by editing the target DNA as described in the editing method of the present invention above, cleavage and editing occur in the target sequence, resulting in the production of a cell in which the target DNA has been edited.

[0066] The present invention also provides a method for producing a non-human individual containing cells in which the target DNA has been edited. This method includes a step of producing a non-human individual from cells obtained by the above-mentioned production method. Examples of the non-human individual include non-human animals and plants. Examples of the non-human animal include mammals (e.g., mice, rats, guinea pigs, hamsters, rabbits, monkeys, pigs, cows, goats, sheep), fish, birds, reptiles, amphibians, and insects. When producing a model animal, the mammal is preferably a rodent such as a mouse, rat, guinea pig, or hamster, with mice being particularly preferred. Examples of the plant include grains, oilseed crops, forage crops, fruits, and vegetables. Specific examples of crops include rice, corn, banana, peanut, sunflower, tomato, rapeseed, tobacco, wheat, barley, potato, soybean, cotton, and carnation.

[0067] Known methods can be used to create non-human individuals from cells in which the target DNA has been edited. When creating non-human individuals from cells in animals, germ cells or pluripotent stem cells are typically used. For example, the DNA editing system is microinjected into oocytes, and the resulting oocytes are implanted into the uterus of a pseudopregnant female non-human mammal, after which offspring can be obtained. It has long been known that somatic cells of plants possess totipotency. For example, a plant in which the desired DNA has been edited can be obtained by microinjecting the DNA editing system into plant cells and regenerating a plant from the resulting plant cells. Furthermore, from the resulting non-human individuals, offspring or clones in which the desired DNA has been edited can also be obtained.

[0068] Confirmation of the presence or absence of target DNA editing and determination of the genotype can be performed based on conventionally known techniques, such as PCR, sequencing, Southern blotting, etc.

[0069] <Kit> The kit of the present invention is a kit for use in the above-mentioned editing method of the present invention, the manufacturing method of the present invention, or the above-mentioned method for producing a non-human individual of the present invention, and comprises at least one zinc finger nuclease selected from the group consisting of ZFN-I, ZFN-II, ZFN-III, and ZFN-IV, a polynucleotide encoding the zinc finger nuclease, and an expression vector for the zinc finger nuclease.

[0070] Each zinc finger nuclease, including its preferred embodiments, is as described above. Each of these may independently be in the form of a protein, a polynucleotide encoding the protein, or a vector expressing the protein (expression vector), and the kit of the present invention also includes these forms.

[0071] In the kit of the present invention, the zinc finger nucleases are preferably used as the nuclease pair described above, and are therefore preferably a combination of ZFN-I and ZFN-II (first combination of ZFNs) and / or a combination of ZFN-III and ZFN-IV (second combination of ZFNs). In this case, the kit of the present invention may be in the form of a composition containing the combinations, or in the form of a combination containing each zinc finger nuclease individually.

[0072] The kit of the present invention may further include one or more additional reagents. Examples of such additional reagents include, but are not limited to, a dilution buffer, a reconstitution solution, a wash buffer, a nucleic acid transfer reagent, a protein transfer reagent, and a control reagent. The kit may also include instructions for carrying out the method of the present invention.

[0073] The components included in the kit of the present invention may be contained in separate containers or in the same container. Each component may be contained in a single-use amount in a container, or multiple doses may be contained in a single container. Each component may be contained in a container in a dry form, or in a form dissolved in an appropriate solvent (a solvent containing a buffer, stabilizer, preservative, antiseptic, etc.).

[0074] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0075] <Test Example 1> (1) Preparation of Zinc Finger Nucleases Zinc finger modules (Fingers 1 to 6) were amplified from ZF Module Plasmids (ZF58 to 106, Addgene) using primers (each set of Fw and Rw in the table) listed in Table 1 below.

[0076]

[0077] The amplified zinc finger modules were ligated using the Golden Gate Assembly method to prepare polynucleotides encoding the amino acid sequences of zinc finger proteins (ZF-I(L), ZF-II(R), ZF-III-1(L), and ZF-IV(R)) that bind to the ZF recognition sequences (underlined) in the target DNAs listed in Table 2 below. Polynucleotides encoding these amino acid sequences were added to each polynucleotide so that a nuclear localization signal (SV40 NLS (amino acid SEQ ID NO: 21) / c-myc NLS (amino acid SEQ ID NO: 22) / SV40 NLS) was added to the N-terminus of the amino acid. The target DNA sequences listed in Table 2 are sequences within intron 1 of the AAVS1 region, and the numbers in each target DNA name indicate the position of the base at the 5' end of the sense strand of the target DNA on chromosome 19 of the human genome.

[0078] The prepared polynucleotides were inserted into an ND1RRR expression plasmid (nucleotide sequence: 46) expressing the first nuclease domain 1 (ND1RRR, amino acid sequence: 13, ND1(L)) or an ND1DDD expression plasmid (nucleotide sequence: 47) expressing the second nuclease domain 1 (ND1DDD, amino acid sequence: 14, ND1(R)), respectively, to prepare an expression plasmid (L plasmid) containing a nucleotide sequence encoding the amino acid sequence of each zinc finger protein (L) / linker (amino acid sequence: 15) / ND1(L), and an expression plasmid (R plasmid) containing a nucleotide sequence encoding the amino acid sequence of each zinc finger protein (R) / linker (amino acid sequence: 15) / ND1(R). The sequences inserted into each plasmid were confirmed by Sanger sequencing. Table 3 below shows the structure and amino acid sequence of the zinc finger protein / linker / nuclease domain (zinc finger nuclease: Example) for each target DNA listed in the table, the name of the prepared plasmid, and a list of their combinations. The nuclease domain ND1(L) has the same meaning in Table 3, and the nuclease domain ND1(R) also has the same meaning in Table 3, with ND1(L) and ND1(R) forming a dimer to exert nuclease activity. In Table 3, for "ZF-III-2" and "490Lv2", see Test Example 2 below.

[0079]

[0080]

[0081] Similarly, polynucleotides encoding the amino acid sequences of zinc finger proteins that bind to the ZF recognition sequences were prepared for other target DNAs. These were used to prepare expression plasmids (L plasmids) containing nucleotide sequences encoding the amino acid sequences of each zinc finger protein (L) / linker / ND1(L) and expression plasmids (R plasmids) containing nucleotide sequences encoding the amino acid sequences of each zinc finger protein (R) / linker / ND1(R) (Comparative Example). Table 4 below shows some of the target DNAs (Target DNAs), their ZF recognition sequences (underlined), and the names of the corresponding plasmids prepared. The target DNA sequences listed in Table 4 are sequences within intron 1 of the AAVS1 region, and the numbers in each target DNA name indicate the position of the base at the 5' end of the sense strand of the target DNA on chromosome 19 of the human genome.

[0082]

[0083] (2) Cell Culture First, HEK293T cells (manufactured by Takara Bio) were maintained and cultured at 37°C in a medium (10% medium) containing 10% FBS (Hyclone Serum, manufactured by GE Healthcare) and 0.5% Penicillin Streptomycin (manufactured by Nacalai Tesq) in DMEM (manufactured by Nacalai Tesq). The maintained cells were then detached with 0.25% Trypsin-EDTA (manufactured by Nacalai Tesq) and collected at a concentration of 0.5 × 10 ５ The cells were seeded onto a 24-well plate at 100 cells / well.

[0084] (3) T7E1 Assay The day after seeding HEK293T cells onto a 24-well plate in (2) above, the cells were transfected with 150 ng / well of the L plasmid and 150 ng / well of the R plasmid prepared in (1) above using Lipofectamine 2000 (Thermofisher) in the combinations shown in Tables 3 and 4. 24 hours after transfection, the medium was replaced with the 10% medium described above, and the cells were further cultured at 37°C for 24 hours. 48 hours after transfection, the cultured cells were harvested, and genomic DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen). Using this as a template, PCR was carried out using the following primers: AAVS-1 T7E1 Fw (nucleotide sequence number: 43) and AAVS-1 T7E1 Rv (nucleotide sequence number: 44), and KOD One PCR Master Mix (manufactured by Toyobo).

[0085] 200 ng of the resulting PCR product, 10x NEB2 Buffer (NEB), and DDW (Deuterium-depleted water) were mixed to prepare a total of 20 μL of reaction mix, which was reacted under the following conditions: 95 ° C for 5 min, 95 → 85 ° C (-2 ° C / sec), 85 → 25 ° C (-0.1 ° C / sec), 25 → 4 ° C. 0.5 μL of T7 Endonuclease 1 (T7E1, NEB) was added to 20 μL of the reaction mix, and T7E1 treatment was performed at 37 ° C for 30 minutes. After T7E1 treatment, electrophoresis was performed on a 2% agarose gel (Nippongene).

[0086] The results of electrophoresis (gel appearance) are shown in Figure 2. From the results of electrophoresis, band intensity was quantified using ImageJ (NIH), and the percentage of base insertion or deletion between 6 bp of the target sequence calculated using the following formula: % gene modification = 100 × (1-(1-fraction cleaved) × 1 / 2) was taken as the genome editing efficiency (Indel%), which is also shown in Figure 2. Insertion or deletion of bases within the target sequence, that is, cleavage activity of the target sequence, was confirmed in only two combinations of L plasmid and R plasmid for the 10 target sequences shown in Figure 2: 418L + 418R and 490L + 490R.

[0087] Test Example 2 (1) Preparation of Zinc Finger Nuclease In the same manner as in Test Example 1(1), an expression plasmid (490Lv2 plasmid) was prepared so that the amino acid sequence of zinc finger protein (L) / linker / ND1(L) was the sequence set forth in SEQ ID NO: 19. The zinc finger protein ZF-III-2(L) of the zinc finger nuclease expressed by this plasmid contains a skip linker consisting of the amino acid sequence set forth in SEQ ID NO: 12 instead of "GE" at positions 81-82 of the amino acid sequence of the zinc finger protein ZF-III-1(L) prepared in Test Example 1(1). Therefore, the "T" shown in bold in Table 2 in the ZF recognition sequence (underlined) of the target DNA: 490L+R is skipped and recognized.

[0088] (2) T7E1 Assay The day after seeding HEK293T cells onto a 24-well plate in the same manner as in Test Example 1 (2), Lipofectamine 2000 (Thermofisher) was used to transfect 150 ng / well of the 490Lv2 plasmid prepared in (1) above with 150 ng / well of the 490R plasmid prepared in (1) above (the combinations are shown in Table 3 above). Also, 150 ng / well of the L plasmid and 150 ng / well of the R plasmid prepared in (1) above were transfected in the combinations shown in Table 3. 24 hours after transfection, the medium was replaced with the 10% medium and further cultured at 30 ° C. for 48 hours. The cultured cells were harvested 72 hours after transfection, and genomic DNA was extracted using a DNeasy Blood and Tissue Kit (Qiagen). Using this as a template, T7E1 treatment was performed and electrophoresis was performed in the same manner as in Test Example 1 (3).

[0089] The results of electrophoresis (gel appearance) are shown in Figure 3. From the results of electrophoresis, band intensity was quantified using ImageJ (NIH), and the percentage of base insertion or deletion between the 6 bp target sequence calculated by % gene modification = 100 × (1-(1-fraction cleaved) × 1 / 2) was taken as the genome editing efficiency (Indel%), which is also shown in Figure 3. As shown in Figure 3, when the combination of L plasmid and R plasmid is 418L + 418R, 490L + 490R, 490Lv2 + 490R, it showed excellent genome editing efficiencies of 22%, 16%, and 26%, respectively, and it was confirmed that it exhibited high site-specific DNA cleavage activity.

[0090] As described above, zinc finger nuclease pairs comprising a combination of zinc finger proteins of the present invention cleave specific target sequences within the AAVS1 region with high activity, thereby enabling editing of the target sequence with a high probability. Therefore, according to the present invention, it is possible to provide zinc finger proteins and combinations thereof that can be applied to zinc finger nuclease pairs to cleave and edit specific target sequences within the AAVS1 region with a high probability, zinc finger nucleases and zinc finger nuclease pairs comprising the same, methods for editing target DNA using these, methods for producing cells with edited target DNA, and kits for use in the editing and production methods. Therefore, the present invention is expected to be used as an excellent genome editing tool in a wide range of industrial fields, such as medicine, agriculture, and industry.

Claims

1. A zinc finger protein that is at least one selected from the group consisting of zinc finger protein I consisting of the amino acid sequence set forth in SEQ ID NO: 1, zinc finger protein II consisting of the amino acid sequence set forth in SEQ ID NO: 2, zinc finger protein III-1 consisting of the amino acid sequence set forth in SEQ ID NO: 3, zinc finger protein III-2 consisting of the amino acid sequence set forth in SEQ ID NO: 4, and zinc finger protein IV consisting of the amino acid sequence set forth in SEQ ID NO:

5.

2. A combination of zinc finger proteins for a zinc finger nuclease pair, the combination of zinc finger proteins being at least one selected from the group consisting of: a first combination consisting of zinc finger protein I consisting of the amino acid sequence set forth in SEQ ID NO: 1 and zinc finger protein II consisting of the amino acid sequence set forth in SEQ ID NO: 2; and a second combination consisting of zinc finger protein III-1 consisting of the amino acid sequence set forth in SEQ ID NO: 3 or zinc finger protein III-2 consisting of the amino acid sequence set forth in SEQ ID NO: 4 and zinc finger protein IV consisting of the amino acid sequence set forth in SEQ ID NO:

5.

3. A zinc finger nuclease which is at least one selected from the group consisting of zinc finger nuclease I comprising a zinc finger protein I consisting of the amino acid sequence set forth in SEQ ID NO: 1 and nuclease domain I; zinc finger nuclease II comprising a zinc finger protein II consisting of the amino acid sequence set forth in SEQ ID NO: 2 and nuclease domain II; zinc finger nuclease III comprising a zinc finger protein III-1 consisting of the amino acid sequence set forth in SEQ ID NO: 3 or a zinc finger protein III-2 consisting of the amino acid sequence set forth in SEQ ID NO: 4 and nuclease domain III; and zinc finger nuclease IV comprising a zinc finger protein IV consisting of the amino acid sequence set forth in SEQ ID NO: 5 and nuclease domain IV.

4. A zinc finger nuclease pair which is at least one selected from the group consisting of: a first zinc finger nuclease pair consisting of a zinc finger nuclease I comprising a zinc finger protein I and nuclease domain I having the amino acid sequence set forth in SEQ ID NO:1, and a zinc finger nuclease II comprising a zinc finger protein II and nuclease domain II having the amino acid sequence set forth in SEQ ID NO:2; and a second zinc finger nuclease pair consisting of a zinc finger nuclease III comprising a zinc finger protein III-1 having the amino acid sequence set forth in SEQ ID NO:3 or a zinc finger protein III-2 having the amino acid sequence set forth in SEQ ID NO:4, and nuclease domain III, and a zinc finger nuclease IV comprising a zinc finger protein IV and nuclease domain IV having the amino acid sequence set forth in SEQ ID NO:

5.

5. The zinc finger nuclease pair according to claim 4, wherein the pair of nuclease domain I and nuclease domain II, and the pair of nuclease domain III and nuclease domain IV are each pairs that form dimers with each other.

6. The zinc finger nuclease pair of claim 4, wherein the first zinc finger nuclease pair cleaves a target sequence of 5 to 7 bases between the nucleotide sequence set forth in SEQ ID NO: 6 and the nucleotide sequence set forth in SEQ ID NO: 7 and its complementary sequence.

7. The zinc finger nuclease pair of claim 4, wherein the second zinc finger nuclease pair cleaves a target sequence of 5 to 7 bases between the nucleotide sequence set forth in SEQ ID NO: 8 and the nucleotide sequence set forth in SEQ ID NO: 9 and its complementary sequence.

8. The zinc finger nuclease pair of claim 4, which targets the AAVS1 region.

9. A method for editing a target DNA, comprising the steps of contacting the zinc finger nuclease pair of claim 4 with the target DNA, and cleaving, with the first zinc finger nuclease pair, 5 to 7 bases between the nucleotide sequence set forth in SEQ ID NO:6 and the nucleotide sequence set forth in SEQ ID NO:7 and their complementary sequence as the target sequence, and / or cleaving, with the second zinc finger nuclease pair, 5 to 7 bases between the nucleotide sequence set forth in SEQ ID NO:8 and the nucleotide sequence set forth in SEQ ID NO:9 and their complementary sequence as the target sequence.

10. The method of claim 9, wherein the target DNA comprises the AAVS1 region and the method is for editing the AAVS1 region.

11. A method for producing a cell in which target DNA has been edited, comprising the steps of: introducing or expressing the zinc finger nuclease pair according to claim 4 into a cell and contacting it with the target DNA; and cleaving, with a first zinc finger nuclease pair, 5 to 7 bases between the nucleotide sequence set forth in SEQ ID NO:6 and the nucleotide sequence set forth in SEQ ID NO:7 and their complementary sequence as the target sequence; and / or cleaving, with a second zinc finger nuclease pair, 5 to 7 bases between the nucleotide sequence set forth in SEQ ID NO:8 and the nucleotide sequence set forth in SEQ ID NO:9 and their complementary sequence as the target sequence.

12. The method of claim 11, wherein the target DNA comprises the AAVS1 region and the method is for producing a cell in which the AAVS1 region has been edited.

13. A kit for use in the method of any one of claims 9 to 12, comprising at least one zinc finger nuclease selected from the group consisting of: zinc finger nuclease I comprising zinc finger protein I and nuclease domain I consisting of the amino acid sequence set forth in SEQ ID NO:1; zinc finger nuclease II comprising zinc finger protein II and nuclease domain II consisting of the amino acid sequence set forth in SEQ ID NO:2; zinc finger nuclease III comprising zinc finger protein III-1 consisting of the amino acid sequence set forth in SEQ ID NO:3 or zinc finger protein III-2 consisting of the amino acid sequence set forth in SEQ ID NO:4 and nuclease domain III; and zinc finger nuclease IV comprising zinc finger protein IV and nuclease domain IV consisting of the amino acid sequence set forth in SEQ ID NO:5; a polynucleotide encoding the zinc finger nuclease; and an expression vector for the zinc finger nuclease.

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