Mutants of the FokI nuclease domain

Abstract

It is found that four nuclease domain variants according to the present invention have nuclease activity superior to that of a wild type nuclease domain, and it is possible to improve genome editing efficiency in a combination of various nucleic acid-binding domains.

Description

[Technical Field]

[0001] The present invention relates to mutants of the FokI nuclease domain, artificial nucleic acid-cleaving enzymes containing the mutants, and uses thereof. [Background technology]

[0002] Genome editing requires a high probability of introducing DNA deletions, insertions, base substitutions, and other modifications into target regions of genes to be modified. The molecules used for genome editing (genome editing tools) consist of a nucleic acid-binding domain that binds to target sequences in the genome and a catalytic domain that cleaves double-stranded DNA near the target sequence. When constructing genome editing tools using DNA-binding proteins such as zinc finger proteins (ZFNs), transcription activator-like effector (TALE) proteins, or pentatricopeptide repeat (PPR) proteins, the catalytic domain (FokI-CD) of the Type IIS restriction enzyme FokI, which has the activity of cleaving double-stranded DNA, is typically used. For FokI to cleave double-stranded DNA, a dimer must be formed between two FokI-CD molecules (Non-Patent Document 1). Therefore, even in genome editing tools constructed by fusing FokI-CD, two molecules are used that target the sense and antisense strands near the target region to be modified.

[0003] In genome editing tools constructed by fusing FokI-CD, such as zinc finger nucleases, attempts have been made to improve the editing efficiency of the genome editing tool by modifying the FokI-CD. For example, an improved FokI-CD (Sharky mutation) with improved genome editing efficiency was isolated by introducing random mutations into ZFNs and using an artificial evolution system in Escherichia coli (Non-Patent Document 2). However, no difference in editing efficiency was observed between TALENs fused with FokI-CD containing the Sharky mutation and the wild type (Non-Patent Document 3). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Bitinaite J, et al., (1998) Proc Natl Acad Sci US A. 95:10570-10575. [Non-patent document 2] Guo J, et al., (2010) J Mol Biol. 400:96-107. Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors further evaluated the performance of the FokI-CD having the Sharky mutation described in Non-Patent Document 2 and found that even when fused with a PPR protein as a nucleic acid-binding domain, the FokI-CD did not exhibit improved genome editing efficiency compared to the wild-type. Therefore, the FokI-CD having the Sharky mutation has the problem that it cannot be used in combination with a wide range of nucleic acid-binding domains in genome editing systems.

[0006] The present invention has been made in light of these circumstances, and its purpose is to provide a nuclease domain mutant that can be used in combination with various nucleic acid binding domains in genome editing and that can improve genome editing efficiency. [Means for solving the problem]

[0007] In order to solve the above problems, the present inventors used an artificial evolution system of Escherichia coli to screen for highly active nuclease domain mutants. As a result, it was found that four nuclease domain mutants bound to TALE had superior nuclease activity compared to the wild-type nuclease domain, and that the use of these nuclease domain mutants improved genome editing efficiency. Of these nuclease domain mutants, the mutation at position 470, which two nuclease domain mutants shared, significantly contributed to the improvement of nuclease activity. Furthermore, the nuclease domain mutants also showed improved genome editing efficiency when fused with PPR. Based on the above, the present inventors discovered that the nuclease domain mutants they created have excellent nuclease activity and can be used for genome editing in combination with various nucleic acid binding domains, leading to the completion of the present invention.

[0008] The present invention relates to highly active nuclease domain mutants, artificial nucleic acid-cleaving enzymes containing the nuclease domain mutants, and uses thereof, and more specifically provides the following.

[0009] [1] A mutant of the nuclease domain of a FokI protein or a homologous protein thereof, which has a mutation according to any one of (a) to (d) below, and which has improved nuclease activity due to the mutation:

[0010] (a) Substitution of the amino acid at position 386 of the FokI protein or the corresponding site in a homologous protein with Met, substitution of the amino acid at position 399 of the FokI protein or the corresponding site in a homologous protein with Arg, substitution of the amino acid at position 421 of the FokI protein or the corresponding site in a homologous protein with Tyr, substitution of the amino acid at position 424 of the FokI protein or the corresponding site in a homologous protein with Phe, substitution of the amino acid at position 432 of the FokI protein or the corresponding site in a homologous protein with Leu, substitution of the amino acid at position 433 of the FokI protein or the corresponding site in a homologous protein with Leu, substitution of the amino acid at position 440 of the FokI protein or the corresponding site in a homologous protein with Leu, substitution of the amino acid at position 441 of the FokI protein or the corresponding site in a homologous protein with Leu, substitution of the amino acid at position 442 of the FokI protein or the corresponding site in a homologous protein with Leu, substitution of the amino acid at position 443 of the FokI protein with Leu, substitution of the amino acid at position 444 of the FokI protein with Leu, substitution of the amino acid at position 445 of the FokI protein with Leu, substitution of the amino acid at position 446 of the FokI protein with Leu, substitution of the amino acid at position 447 of the FokI protein with Leu, substitution of the amino acid at position 448 of the FokI protein with Leu, substitution of the amino acid at position 449 of the FokI protein with Leu, substitution of the amino acid at position 450 of the FokI protein with Leu, substitution of the amino acid at position 451 of the FokI protein with Leu, substitution of the amino acid at position 452 of the FokI protein with Leu, substitution of the amino acid at position 453 of the FokI protein with Leu, substitution of the amino acid at at least one mutation selected from the group consisting of substitution of the amino acid at position 33 or the corresponding position in a homologous protein with Val, substitution of the amino acid at position 448 in the FokI protein or the corresponding position in a homologous protein with Asn, substitution of the amino acid at position 466 in the FokI protein or the corresponding position in a homologous protein with Ile, substitution of the amino acid at position 484 in the FokI protein or the corresponding position in a homologous protein with Ala, and substitution of the amino acid at position 486 in the FokI protein or the corresponding position in a homologous protein with Leu (b) substitution of the amino acid at position 418 in the wild-type protein or at the corresponding position in a homologous protein with Pro, substitution of the amino acid at position 420 in the FokI protein or at the corresponding position in a homologous protein with Leu, substitution of the amino acid at position 452 in the FokI protein or at the corresponding position in a homologous protein with Val, substitution of the amino acid at position 473 in the FokI protein or at the corresponding position in a homologous protein with Arg, substitution of the amino acid at position 486 in the FokI protein or at the corresponding position in a homologous protein with His, FokI protein substitution of the amino acid at position 490 of the FokI protein or the corresponding site in a homologous protein with Val; substitution of the amino acid at position 498 of the FokI protein or the corresponding site in a homologous protein with Leu; substitution of the amino acid at position 542 of the FokI protein or the corresponding site in a homologous protein with Lys; substitution of the amino acid at position 559 of the FokI protein or the corresponding site in a homologous protein with Arg; and substitution of the amino acid at position 570 of the FokI protein or the corresponding site in a homologous protein with Ser. (c) Substitution of the amino acid at position 386 in the wild-type protein or at the corresponding position in a homologous protein with Met, substitution of the amino acid at position 395 in the FokI protein or at the corresponding position in a homologous protein with Phe, substitution of the amino acid at position 399 in the FokI protein or at the corresponding position in a homologous protein with Arg, substitution of the amino acid at position 421 in the FokI protein or at the corresponding position in a homologous protein with Tyr, substitution of the amino acid at position 424 in the FokI protein or at the corresponding position in a homologous protein with Phe, FokI protein substitution of the amino acid at position 432 of the FokI protein or the corresponding position in a homologous protein with Leu, substitution of the amino acid at position 433 of the FokI protein or the corresponding position in a homologous protein with Val, substitution of the amino acid at position 448 of the FokI protein or the corresponding position in a homologous protein with Asn, substitution of the amino acid at position 484 of the FokI protein or the corresponding position in a homologous protein with Ala, and substitution of the amino acid at position 486 of the FokI protein or the corresponding position in a homologous protein with Leu. (d) substitution of the amino acid at position 385 in the wild-type protein or at the corresponding position in a homologous protein with Pro, substitution of the amino acid at position 395 in the FokI protein or at the corresponding position in a homologous protein with Ala, substitution of the amino acid at position 427 in the FokI protein or at the corresponding position in a homologous protein with Met, substitution of the amino acid at position 452 in the FokI protein or at the corresponding position in a homologous protein with Val, substitution of the amino acid at position 460 in the FokI protein or at the corresponding position in a homologous protein with Phe, substitution of the amino acid at position 473 in the FokI protein or at the corresponding position in a homologous protein substitution of the amino acid at position 481 of the FokI protein or the corresponding position in a homologous protein with His; substitution of the amino acid at position 490 of the FokI protein or the corresponding position in a homologous protein with Gln; substitution of the amino acid at position 542 of the FokI protein or the corresponding position in a homologous protein with Thr; substitution of the amino acid at position 559 of the FokI protein or the corresponding position in a homologous protein with Arg; substitution of the amino acid at position 570 of the FokI protein or the corresponding position in a homologous protein with Ser. [2] A mutant of the nuclease domain of the FokI protein or its homologous protein, which has a mutation in which the amino acid at position 473 of the FokI protein or the corresponding position of the homologous protein is substituted with Arg, and the mutation improves nuclease activity.

[0011] [3] An artificial nucleic acid-cleaving enzyme comprising a nucleic acid-binding domain and a mutant of the nuclease domain according to [1] or [2].

[0012] [4] The artificial nucleic acid cleaving enzyme described in [3], wherein the nucleic acid binding domain is a TALE, a zinc finger, a PPR, or a CRISPR-Cas.

[0013] [5] A polynucleotide encoding a mutant of the nuclease domain according to [1] or [2] or an artificial nucleic acid-cleaving enzyme according to [3] or [4].

[0014] [6] A vector comprising the polynucleotide described in [5].

[0015] [7] A cell into which the polynucleotide according to [5] or the vector according to [6] has been introduced.

[0016] [8] A method for producing a genome-edited cell or non-human organism, comprising introducing the artificial nucleic acid cleaving enzyme described in [3], a polynucleotide encoding the artificial nucleic acid cleaving enzyme, or a vector containing the polynucleotide into a cell or non-human organism.

[0017] [9] A kit for editing the genome of a cell or organism, comprising the artificial nucleic acid cleaving enzyme described in [3], a polynucleotide encoding the artificial nucleic acid cleaving enzyme, or a vector containing the polynucleotide. [Effects of the Invention]

[0018] The nuclease domain mutants of the present invention can exhibit superior nuclease activity compared to wild-type nuclease domains. Artificial nucleic acid cleaving enzymes created by fusing the nuclease domains of the present invention with nucleic acid binding domains such as TALE and PPR enable efficient genome editing. [Brief explanation of the drawings]

[0019] [Figure 1] This figure shows the amino acid sequences of four types of FokI nuclease domain mutants. The amino acid sequence from positions 384 to 579, which corresponds to the FokI nuclease domain, is shown. The underlined parts indicate mutation sites that differ from those of wild-type FokI. [Figure 2]This figure shows the results of evaluating the nuclease activity of FokI nuclease domain mutants using survival rates in an artificial evolution system (B2P) of E. coli. The survival rate is calculated by dividing the number of colonies on a plate with arabinose by the number of colonies on a plate without arabinose. WT represents a wild-type FokI nuclease domain fused to a TALEN-A effector, and the numbers in the figure indicate the survival rate when using wild-type FokI nuclease. [Figure 3] This figure shows the results of evaluating the effect of the G473R mutation in the FokI nuclease domain on nuclease activity using survival rate as an indicator in an artificial evolution system (B2P) of E. coli. The survival rate is calculated by dividing the number of colonies on a plate with arabinose by the number of colonies on a plate without arabinose. WT represents a wild-type FokI nuclease domain fused to a TALEN-A effector. The numbers in the figure represent the survival rate when using wild-type FokI nuclease and when using M50 (M50(G473)) with a G473 reversion mutation introduced. [Figure 4] This figure shows the results of a target reporter assay using TALENs targeting the B2M gene or the GFP gene. The graph shows the fold increase in activity of the FokI nuclease domain mutants relative to the wild-type FokI nuclease domain. Values ​​are the results of three independent experiments. [Figure 5] This figure shows the results of a target reporter assay using PPR targeting the B2M gene or the GFP gene. The graph shows the fold increase in activity (nLUC / fLUC) of M50 relative to the wild-type FokI nuclease domain. Values ​​show the mean and standard error of three independent experiments. * indicates a significant difference (P<0.1), ** indicates a significant difference (P<0.05) relative to the reporter activity of the wild-type FokI effector. DETAILED DESCRIPTION OF THE INVENTION

[0020] <Nuclease domain mutant> The present invention provides mutants of the nuclease domain of the FokI protein or its homologous proteins.

[0021] The "FokI protein" of the present invention is a type IIS restriction enzyme naturally found in Flavobacterium okeanokoites. The FokI protein catalyzes cleavage of double-stranded DNA at a position 9 nucleotides away from the recognition site on one strand and at a position 13 nucleotides away from the recognition site on the other strand. The FokI protein has a nucleic acid binding domain at the N-terminus and a nuclease domain (DNA cleavage domain) at the C-terminus.

[0022] The amino acid sequence of a typical wild-type FokI protein is shown in SEQ ID NO: 1, and the amino acid sequence of its nuclease domain is shown in SEQ ID NO: 2. In SEQ ID NO: 1, the nuclease domain corresponds to positions 384 to 579. The nuclease domain mutant of the present invention has a specific mutation introduced into the nuclease domain of the FokI protein, and has improved nuclease activity compared to the wild-type nuclease domain.

[0023] One embodiment of the FokI nuclease domain mutant of the present invention has a mutation introduced into M47 in the Examples described below, and specifically, is a nuclease domain mutant having at least one mutation selected from the group consisting of a substitution of the amino acid at position 386 of the FokI protein with Met, a substitution of the amino acid at position 399 of the FokI protein with Arg, a substitution of the amino acid at position 421 of the FokI protein with Tyr, a substitution of the amino acid at position 424 of the FokI protein with Phe, a substitution of the amino acid at position 432 of the FokI protein with Leu, a substitution of the amino acid at position 433 of the FokI protein with Val, a substitution of the amino acid at position 448 of the FokI protein with Asn, a substitution of the amino acid at position 466 of the FokI protein with Ile, a substitution of the amino acid at position 484 of the FokI protein with Ala, and a substitution of the amino acid at position 486 of the FokI protein with Leu.

[0024] Another embodiment of the FokI nuclease domain mutant of the present invention is a nuclease domain mutant having a mutation introduced into M48 in the Examples described below, specifically, a nuclease domain mutant having at least one mutation selected from the group consisting of a substitution of the amino acid at position 418 in the wild-type protein with Pro, a substitution of the amino acid at position 420 in the FokI protein with Leu, a substitution of the amino acid at position 452 in the FokI protein with Val, a substitution of the amino acid at position 473 in the FokI protein with Arg, a substitution of the amino acid at position 486 in the FokI protein with His, a substitution of the amino acid at position 490 in the FokI protein with Val, a substitution of the amino acid at position 498 in the FokI protein with Leu, a substitution of the amino acid at position 542 in the FokI protein with Lys, a substitution of the amino acid at position 559 in the FokI protein with Arg, and a substitution of the amino acid at position 570 in the FokI protein with Ser.

[0025] Another embodiment of the FokI nuclease domain mutant of the present invention is a nuclease domain mutant having a mutation introduced into M49 in the Examples described below, specifically, a nuclease domain mutant having at least one mutation selected from the group consisting of a substitution of the amino acid at position 386 in the wild-type protein with Met, a substitution of the amino acid at position 395 in the FokI protein with Phe, a substitution of the amino acid at position 399 in the FokI protein with Arg, a substitution of the amino acid at position 421 in the FokI protein with Tyr, a substitution of the amino acid at position 424 in the FokI protein with Phe, a substitution of the amino acid at position 432 in the FokI protein with Leu, a substitution of the amino acid at position 433 in the FokI protein with Val, a substitution of the amino acid at position 448 in the FokI protein with Asn, a substitution of the amino acid at position 484 in the FokI protein with Ala, and a substitution of the amino acid at position 486 in the FokI protein with Leu.

[0026] Another embodiment of the FokI nuclease domain mutant of the present invention is a nuclease domain mutant having a mutation introduced into M50 in the Examples described below, specifically, a nuclease domain mutant having at least one mutation selected from the group consisting of a substitution of the amino acid at position 385 in the wild-type protein with Pro, a substitution of the amino acid at position 395 in the FokI protein with Ala, a substitution of the amino acid at position 427 in the FokI protein with Met, a substitution of the amino acid at position 452 in the FokI protein with Val, a substitution of the amino acid at position 460 in the FokI protein with Phe, a substitution of the amino acid at position 473 in the FokI protein with Arg, a substitution of the amino acid at position 481 in the FokI protein with His, a substitution of the amino acid at position 490 in the FokI protein with Gln, a substitution of the amino acid at position 542 in the FokI protein with Thr, a substitution of the amino acid at position 559 in the FokI protein with Arg, and a substitution of the amino acid at position 570 in the FokI protein with Ser.

[0027] An example of a preferred mutation in the FokI nuclease domain mutant of the present invention is a substitution of the amino acid at position 473 of the FokI protein with Arg.

[0028] Each mutant may have a combination of two or more mutations, a combination of three or more mutations, a combination of four or more mutations, or a combination of five or more (e.g., six or more, seven or more, eight or more, nine or more, ten or more) mutations.

[0029] Furthermore, the nuclease domain mutant of the present invention may be a mutant of the nuclease domain of a homologous protein of a naturally occurring FokI protein (hereinafter referred to as a "homologous protein nuclease domain mutant"). Here, "homologous protein" refers to a protein whose nuclease domain has at least 70% or more, preferably 80% or more, and more preferably 90% or more (e.g., 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) amino acid sequence identity with the nuclease domain of a naturally occurring FokI protein. In the homologous protein nuclease domain mutant, the amino acid at the site corresponding to the mutation site in the FokI nuclease domain mutant is substituted with the same amino acid as the mutation site in the FokI nuclease domain mutant. The homologous protein may be derived from an organism other than Flavobacterium okeanokoites.

[0030] "Amino acid sequence homology" can be determined using sequence analysis software (e.g., BLAST; http: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Furthermore, the amino acid at a "site corresponding to" a particular amino acid in the FokI protein can be identified as the amino acid that is aligned with the amino acid at the mutation site when aligned with the amino acid sequence of the FokI protein using the above-mentioned amino acid sequence analysis software (e.g., BLAST; http: / / blast.ncbi.nlm.nih.gov / Blast.cgi). When analyzing amino acid sequences using software, for example, default parameter settings can be used.

[0031] One preferred embodiment of the nuclease domain mutant of the present invention is a mutant having an amino acid substitution of Arg at position 473 of the FokI protein or the corresponding site in a homologous protein.

[0032] Furthermore, the nuclease domain mutants of the present invention (FokI nuclease domain mutants, homologous protein nuclease domain mutants) may further have one or more amino acid mutations (substitutions, deletions, additions, and / or insertions) introduced into the above mutation sites. Here, "multiple" is not particularly limited, but typically refers to 2 to 50, preferably 2 to 30, more preferably 2 to 20, and even more preferably 2 to 10 (e.g., 2 to 8, 2 to 4, or 2). Useful examples of mutations include heterodimer formation mutations (Doyon Y, et al. (2011) Nat Methods 8:74-79).

[0033] Methods for site-specific mutagenesis include known methods such as the Kunkel method (Kunkel, TA Proc Natl Acad Sci USA (1985), 82(2):488-492), the ODA method (Hashimoto-Gotoh et al., (1995) Gene 152:271-276), and the splicing-by-overlap-extension (SOE) PCR method (Ho, SN et al., (1989) Gene 77:51-59). Commercially available site-directed mutagenesis kits may also be used.

[0034] Nuclease domain mutants of the present invention (FokI nuclease domain mutants, homologous protein nuclease domain mutants) have improved nuclease activity compared to wild-type proteins due to the introduction of mutations. The improvement in nuclease activity is preferably 10% or more, more preferably 30% or more, and more preferably 50% or more (e.g., 70% or more, 100% or more, 150% or more, or 200% or more). The improvement in nuclease activity can be evaluated, for example, by the reporter assay described in Example 5 (Figures 4 and 5). Specifically, an effector plasmid expressing a fusion protein of a nucleic acid-binding domain such as TALEN or PPR with a nuclease domain mutant of the present invention and a reporter plasmid containing a recognition sequence for the nucleic acid-binding domain that expresses a reporter when the fusion protein recognizes the recognition sequence and causes DNA cleavage are introduced into cultured cells, and the reporter activity in the cultured cells is measured and compared to that when a fusion protein containing a wild-type nuclease domain is expressed. In this reporter assay system, for example, HEK293-T cells can be used as cultured cells, and in each plasmid, for example, the CMV promoter can be used as a promoter for expressing the fusion protein or reporter, and for example, the recognition sequence of the nucleic acid binding domain can be the recognition sequence on the human B2M gene or the GFP gene.

[0035] The nuclease domain mutants of the present invention may contain modified amino acids and / or unnatural amino acids. Modified amino acids include, but are not limited to, methylation, esterification, amidation, acetylation, alkylation, halogenation, etc. Modified amino acids and unnatural amino acids can be introduced by known methods.

[0036] <Artificial nucleic acid cleaving enzyme> The present invention provides an artificial nucleic acid cleaving enzyme comprising a nucleic acid-binding domain and the above-described nuclease domain mutant. The artificial nucleic acid cleaving enzyme of the present invention binds to a target sequence on a nucleic acid (a recognition sequence for the nucleic acid-binding domain) via the nucleic acid-binding domain, and cleaves the nucleic acid near the target sequence (a target cleavage site) via the nuclease domain. Therefore, the artificial nucleic acid cleaving enzyme of the present invention can function as a sequence-specific nucleic acid cleaving enzyme.

[0037] As used herein, "nucleic acid" encompasses both DNA and RNA. The nucleic acid cleaved by the artificial nucleic acid cleaving enzyme of the present invention is primarily DNA. DNA includes both double-stranded DNA and single-stranded DNA. There are no particular limitations on DNA, and examples include eukaryotic nuclear genomic DNA, mitochondrial DNA, plastid DNA, prokaryotic genomic DNA, phage DNA, and plasmid DNA. Preferably, the artificial nucleic acid cleaving enzyme of the present invention cleaves double-stranded DNA in the genome.

[0038] The target sequence of the artificial nuclease of the present invention is any sequence on a nucleic acid. When targeting genomic DNA, the target sequence may be set to any gene region or extragenic region. There are no particular limitations on the length of the target sequence, but it is, for example, 10 to 30 bases. When using a nucleic acid binding domain containing CRISPR / Cas, Cas must recognize the protospacer adjacent motif (PAM) sequence, so a sequence located near the PAM sequence is set as the target sequence.

[0039] When double-stranded DNA is cleaved using the artificial nucleic acid enzyme of the present invention, two target sequences are preferably set with a spacer sequence sandwiched between them. The length of the spacer sequence is not particularly limited, but is, for example, 1 to 20 bases. Those skilled in the art can appropriately set a target sequence of the desired length and base sequence. The two target sequences may be either palindromic or non-palindromic. When the two target sequences are non-palindromic, two artificial nucleases are used that target each sequence.

[0040] The nucleic acid-binding domain in the nucleic acid cleaving enzyme of the present invention may be any protein domain that specifically binds to any nucleic acid sequence (target sequence), and examples thereof include zinc fingers, TALEs, CRISPR / Cas (a complex of Cas protein and guide RNA), and pentatricopeptide repeats (PPRs). In the artificial nucleic acid cleaving enzyme of the present invention, the nuclease domain mutant and the nucleic acid-binding domain may be linked directly or via a linker. The length of the linker is not particularly limited and may be, for example, 1 to 20 amino acids.

[0041] A nucleic acid binding domain containing zinc fingers may preferably contain two or more zinc fingers, and may include, but is not limited to, a zinc finger array consisting of, for example, 3 to 9 zinc fingers. It is known that one zinc finger recognizes three consecutive bases, and for example, a zinc finger array consisting of 3 to 9 zinc fingers can recognize 9 to 27 bases. For examples of ZFNs, see International Publication No. 2003 / 087341.

[0042] A nucleic acid binding domain containing a TALE may preferably contain six or more repeat modules, and may contain, but is not limited to, a TALE consisting of, for example, 9 to 27 repeat modules. The repeat modules have a repeat structure with 34 amino acids per unit, and each module is known to recognize one base. For examples of TALENs, see the literature (WO 2012 / 104729, WO 2011 / 072246, WO 2015 / 019672).

[0043] A nucleic acid binding domain containing a PPR may preferably contain six or more repeat modules, and may contain, for example, a PPR consisting of 9 to 27 repeat modules, but is not limited thereto. The repeat modules have a repeat structure with 35 amino acids per unit, and each module is known to recognize one base. For examples of PPRs, see Nakamura et al. (2012) Plant Cell Physiol 53:1171-1179, International Publication WO 2014 / 175284.

[0044] The nucleic acid binding domain containing CRISPR-Cas recognizes the target sequence via the targeting RNA (crRNA) in the guide RNA that constitutes CRISPR-Cas. That is, the targeting RNA and the target sequence have complementary base sequences and can hybridize. Cas forms a complex with the guide RNA and is guided to the target sequence by the action of the guide RNA. In the present invention, a complex of guide RNA and Cas can be used as the nucleic acid binding domain, and in this case, the nuclease mutant of the present invention is usually fused to Cas. In the present invention, since nucleic acid cleavage is performed by the nuclease mutant, it is not necessarily necessary for the Cas in CRISPR / Cas to retain nuclease activity. Cas is preferably Cas with inactivated nuclease activity (e.g., dCas). It is also possible to modify PAM recognition by modifying the Cas protein (e.g., by introducing mutations) (Benjamin, P. et al., (2015) Nature 523:481-485; Hirano, S. et al., (2016) Molecular Cell 61:886-894; WO 2018 / 221685), thereby expanding the range of potential target sequences.

[0045] Examples of CRISPR / Cas used in the present invention include, but are not limited to, class 2 / II CRISPR / Cas such as CRISPR-Cas9, class 2 / V CRISPR / Cas such as CRISPR-Cpf1 (Cas12a), CRISPR-Cas12b, CRISPR-CasX (Cas12e), and CRISPR-Cas14, class 2 / VI CRISPR / Cas such as CRISPR-Cas13a, and class 1 / I CRISPR / Cas such as CRISPR-Cas3. For examples of CRISPR / Cas, see the literature (International Publication No. 2014 / 093712, International Publication No. 2013 / 176772, International Publication No. 2013 / 142578, International Publication No. 2016 / 205711, Strecker J, et al., Nature Communications 10:212(2019), Liu JJ, et al., Nature 566:218-223(2019), International Publication No. 2018 / 225858).

[0046] <Method for producing genome-edited cells or non-human organisms> The present invention provides a method for producing a genome-edited cell or non-human organism, comprising introducing the above-mentioned artificial nucleic acid cleaving enzyme, a polynucleotide encoding the artificial nucleic acid cleaving enzyme, or a vector containing the polynucleotide into a cell or non-human organism.

[0047] When the artificial nuclease of the present invention is introduced into cells (including cells in the body of non-human organisms), the nucleic acid-binding domain binds to a target sequence on the nucleic acid, and the nuclease domain mutant cleaves the nucleic acid at the target cleavage site. This cleavage is followed by repair by non-homologous end joining repair (NHEJ) or homology-directed repair (HR), resulting in genome editing. In repair by NHEJ, which is the main repair pathway, one or more mutations are inserted at the cleavage site, modifying the nucleic acid. Meanwhile, by using donor DNA, as described below, desired DNA within the donor DNA can be inserted into the target DNA region by homology-directed repair (HDR), which occurs in the region surrounding the target cleavage site.

[0048] The artificial nuclease of the present invention to be introduced into cells may be in the form of a protein, a polynucleotide, or a vector containing the polynucleotide. When CRISPR-Cas is used as the nucleic acid binding domain of the artificial nuclease, it may also be in the form of an RNA-protein complex. The polynucleotide may be DNA or RNA, and may be codon-optimized for high expression in cells.

[0049] When an expression vector is employed, it contains one or more regulatory elements operably linked to the DNA to be expressed. Here, "operably linked" means that the DNA is linked to the regulatory elements in a manner that allows it to be expressed. "Regulatory elements" include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements (e.g., transcription termination signals (e.g., polyadenylation signals, polyU sequences)). Depending on the purpose, regulatory elements may be those that direct constitutive expression of DNA in various host cells, or those that direct expression of DNA only in specific cells, tissues, or organs. Furthermore, they may be those that direct expression of DNA only at specific times, or those that direct artificially inducible expression of DNA. Examples of promoters include pol III promoters (e.g., U6 and H1 promoters), pol II promoters (e.g., retroviral Rous sarcoma virus (RSV) LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, phosphoglycerol kinase (PGK) promoter, and EF1α promoter), pol I promoters, or combinations thereof. Those skilled in the art will be able to select an appropriate expression vector depending on the type of cell to be introduced, etc.

[0050] In the method of producing genome-edited cells or non-human organisms of the present invention, donor DNA may be introduced into the cells in addition to the artificial nucleic acid cleaving enzyme described above. This allows the desired DNA to be inserted into the target DNA region by utilizing homology-directed repair (HDR) that occurs in the region surrounding the target cleavage site. The homology-directed repair process requires homology between the base sequence of the target DNA region and the base sequence of the donor DNA. The donor DNA is used as a template for repair of the target DNA region containing the target cleavage site, resulting in the transfer of genetic information from the donor DNA to the target DNA region. This allows the base sequence of the target DNA region to be changed (e.g., inserted, deleted, or substituted). Therefore, the donor DNA contains two base sequences (homology arms) that are highly identical to the base sequences in the target DNA region, with the desired DNA (DNA to be inserted into the target DNA region) positioned between them.

[0051] The homology arms need only be large enough to perform homologous recombination, and may vary depending on the form and length of the donor DNA. For example, in the case of double-stranded donor DNA, the length is 500 to 1,000 base pairs, and in the case of single-stranded donor DNA, the length is 30 to 1,000 bases. Furthermore, the homology arms do not need to be 100% identical to the base sequence in the target DNA region, as long as they are sufficiently identical to the base sequence in the target DNA region to perform homologous recombination. For example, the homology arms have an identity of 95% or more, preferably 97% or more, more preferably 99% or more, and even more preferably 99.9% or more.

[0052] Furthermore, the length of the desired DNA present between the homology arms is not particularly limited, and DNA of various sizes can be used. If the desired DNA contains a nucleotide sequence that needs to be subsequently removed, it is possible to add, for example, a recombinase recognition sequence (e.g., a loxP sequence or an FRT sequence) to both ends of the nucleotide sequence. The nucleotide sequence flanked by the recognition sequences can be removed by the action of a recombinase (e.g., a Cre recombinase or an FLP recombinase). Furthermore, for purposes such as confirming the success of DNA knock-in, a selectable marker sequence (e.g., a fluorescent protein or a drug resistance gene) can be incorporated into the desired DNA. Furthermore, a gene operably linked to one or more regulatory elements can also be used as the desired DNA.

[0053] The donor DNA may be linear or circular, and may be single-stranded or double-stranded.

[0054] The artificial nuclease of the present invention (in the various forms described above) can be introduced into cells by known methods such as electroporation, microinjection, the DEAE-dextran method, lipofection, nanoparticle-mediated transfection, and virus-mediated nucleic acid delivery. Direct administration into the body can be achieved by parenteral administration such as injection, or by oral administration. In this case, it is preferable to combine it with a delivery system that is directed toward the target site, if necessary. When donor DNA is used, the donor DNA can also be introduced into cells or the body by similar methods.

[0055] The cells into which the artificial cleavage enzyme of the present invention is introduced may be either prokaryotic cells (prokaryotic cells) or eukaryotic cells (eukaryotic cells), and are not particularly limited. Examples of prokaryotic cells include Escherichia coli, actinomycetes, and archaea. Examples of eukaryotic cells include animal cells, plant cells, algae cells, and fungal cells. Examples of animal cells include mammalian cells as well as cells of fish, birds, reptiles, amphibians, and insects.

[0056] Examples of animal cells include cells constituting individual animals, cells constituting organs and tissues extracted from animals, 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, 2-cell, 4-cell, 8-cell, 16-cell, and morula stages); 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 creating genome-edited animals, but post-fertilization oocytes, i.e., fertilized eggs, are preferred. Pronuclear stage fertilized eggs are particularly preferred. Oocytes can be thawed from cryopreserved oocytes. However, application to human germ cells and embryonic cells is excluded if ethical considerations are not acceptable.

[0057] The term "mammal" encompasses both humans and non-human mammals. Examples of non-human mammals include ungulates such as cattle, boars, pigs, sheep, and goats, perissodactyls such as horses, rodents such as mice, rats, guinea pigs, hamsters, and squirrels, lagomorphs such as rabbits, and carnivores such as dogs, cats, and ferrets. Non-human mammals may be livestock or companion animals (pets), or wild animals.

[0058] Examples of plant cells include cells of grains, oil crops, forage crops, fruits, and vegetables. Plant cells include, for example, cells constituting individual plants, cells constituting organs or tissues separated from plants, and cultured cells derived from plant tissue. Examples of plant organs and tissues include leaves, stems, shoot tips (growing points), roots, tubers, and calli. Examples of plants include rice, corn, banana, peanut, sunflower, tomato, rapeseed, tobacco, wheat, barley, potato, soybean, cotton, and carnation, as well as their propagation materials (e.g., seeds, tuberous roots, tubers, etc.).

[0059] In the method of producing genome-edited cells or non-human organisms of the present invention, two or more of the above-mentioned artificial nucleases having different nucleic acid binding domains may be introduced into cells. For example, if the region of the genome to be edited does not contain a palindromic sequence, two artificial nucleases targeting different sequences may be used. In this case, further mutations may be introduced into the nuclease domain mutants of the two artificial nucleases to promote heterodimer formation of the nuclease domain mutants. This increases the number of recognition sequences for the artificial nucleases and reduces the probability of off-target binding.

[0060] <Kit> The present invention provides a kit for editing the genome of a cell or organism, comprising the above-mentioned artificial nuclease, a polynucleotide encoding the artificial nuclease, and a vector containing the polynucleotide. The kit may further comprise one or more additional reagents, including, but not limited to, a dilution buffer, a reconstitution solution, a washing buffer, a nucleic acid introduction reagent, a protein introduction reagent, and a control reagent. Typically, the kit comes with an instruction manual. [Example]

[0061] Example 1. Principle of B2P screening The B2P screening system uses two plasmids: a reporter plasmid carrying the lethal gene ccdB gene, and an effector plasmid expressing TALEN-A (Chen Z, Zhao H. (2005) Nucleic Acids Res. 33:e154). The sequence recognized by TALEN-A (Table 1) is inserted into the sense and antisense strands downstream of the ccdB gene of the reporter plasmid (from the sense strand, the sequence is "target sequence - spacer sequence - complementary sequence of target sequence") (Table 2). In Table 2, capital letters indicate the sequence recognized by TALEN-A, and lowercase letters indicate the spacer sequence between the two sequences.

[0062] [Table 1]

[0063] [Table 2]

[0064] Expression of the ccdB gene is controlled by an arabinose-inducible promoter, and E. coli transformed with the reporter cannot survive when arabinose is added to the culture medium (this plasmid is referred to as the "TALEN-A reporter plasmid"). In the effector plasmid, TALEN-A is fused to a maltose-binding protein at its N-terminus, allowing the MBP-TALEN-A fusion protein to be induced with IPTG (this plasmid is referred to as the "TALEN-A effector plasmid"). When these two plasmids are transformed into E. coli (XL1-Blue), the transformants cannot survive in the presence of arabinose, but can survive in the presence of both arabinose and IPTG. This is because MBP-TALEN-A introduces a double-stranded DNA break in the target sequence of the TALEN-A reporter plasmid, preventing DNA replication of the cleaved plasmid DNA. Consequently, the reporter plasmid is lost from the transformants, eliminating the lethal effect of the ccdB gene. When a TALEN-A effector mutant library is introduced into E. coli carrying a TALEN-A reporter and screened in the presence of arabinose and IPTG, E. coli carrying more active TALEN-A effectors survive, while E. coli carrying less active TALEN-A effectors do not. The TALEN-A effector plasmid DNA is extracted from the surviving E. coli and transformed back into E. coli carrying the TALEN-A reporter, further enriching for E. coli carrying the most active TALEN-A effectors. Repeating this cycle allows the isolation of plasmids containing the most active TALEN-A effectors.

[0065] Example 2. Construction of a mutant library The TALEN-A effector mutant library was constructed as follows. A destination vector was constructed by PCR amplification using TALEN-A effector plasmid DNA as a template with PrimeSTAR® HS DNA Polymerase (TaKaRa). The FokI-CD region was removed and an Esp3I site was introduced. The nucleotide sequence of the FokI-CD region of the MBP-TALEN-A gene was amplified using the GeneMorph II Random Mutagenesis kit (Agilent Technologies), and an insert with random mutations introduced into the FokI-CD sequence was prepared. The destination vector was cleaved with Esp3I and the insert was ligated to construct a TALEN-A effector mutant library. Evaluation of the rate of random mutations revealed an average of four base substitutions in the FokI-CD region (595 bp).

[0066] Example 3. Screening of a mutant library In the first screening of Stage 1, 1 μg of plasmid DNA from the constructed mutant library was transformed into E. coli carrying the TALEN-A reporter by electroporation (Bio-Rad). After electroporation, the E. coli was transferred to 30 mL of SOB and cultured for recovery at 37°C for 1 hour. IPTG was added to a final concentration of 1 mM, and the cells were incubated at 30°C for 4 hours before plating on LB agar plates containing 100 mg / L chloramphenicol (Cm) and 0.2% arabinose. At this time, portions of the bacterial suspension were plated on LB+Cm agar plates with and without arabinose, and the survival rate was determined by comparing the colony counts. The total number of colonies in the first screening was 2 x 10 7 The number of colonies obtained on the medium with arabinose was 6 × 10 6 Therefore, the survival rate was 30%. The colonies on the arabinose-containing medium were suspended in SOB medium, and plasmid DNA was extracted from the bacterial solution using NucleoSpin (registered trademark) Plasmid (TaKaRa).

[0067] The second screening was carried out in the same manner as the first, using the extracted plasmid DNA. The total number of colonies was 6.8 × 10 8 The survival rate was 58% for the first 100 cells. In the third screening, the survival rate reached 100%. Because better TALEN-A effectors could not be selected under these conditions, in the fourth screening, the IPTG induction time was shortened from 4 hours to 2 hours, making the selection conditions more stringent. The survival rate in the fourth screening was 20%, but in the fifth screening under the same conditions, the survival rate reached 90%. In the sixth to eighth screenings, the IPTG induction time was increased to 1 hour, and the survival rates were 1.3%, 10%, and 6.5%, respectively. The survival rate did not improve with repeated screening, and the number of independent clones in the first screening, 6 × 10 6 The number of independent clones calculated from the survival rate of each of the eight screenings was 55 or less, so Stage 1 was terminated after the eighth screening.

[0068] Using the plasmid DNA obtained in the eighth screening in Stage 1 as a template, a FokI-CD insert with further random mutations was inserted into a destination vector to construct a mutation reintroduction library. This library was transformed into E. coli carrying a TALEN-A reporter to begin Stage 2 screening. Screening was performed by inducing the first round with 1 mM IPTG for 1 hour, the second and third rounds for 30 minutes, the fourth round for 15 minutes, and the fifth and sixth rounds for 15 minutes with 0.1 mM IPTG. The first round of screening was induced at 30°C, and all subsequent rounds were induced at 37°C. The number of independent clones in the first round was 2.8 x 10 5 The number of independent clones calculated from the survival rates of the six screenings was one.

[0069] Using the plasmid DNA obtained in the sixth screening in Stage 2 as a template, a FokI-CD insert with further random mutations was inserted into a destination vector to construct a mutation reintroduction library. This library was transformed into E. coli carrying a TALEN-A reporter to begin Stage 3 screening. Screening was performed by inducing the first round with 20 μM IPTG, the second and third rounds with 10 μM IPTG, the fourth round with 1 μM IPTG, and the fifth and sixth rounds without IPTG for 15 minutes. The first round of screening was induced at 30°C, and all subsequent rounds were induced at 37°C. The number of independent clones in the first round was 1.5 x 10 5 The number of independent clones calculated from the survival rates of the six screenings was one, and stage 3 screening was completed.

[0070] Using the plasmid DNA obtained in the sixth screening in Stage 3 as a template, a FokI-CD insert with further random mutations was inserted into the destination vector to construct a remutation library. This library was transformed into E. coli carrying a TALEN-A reporter to begin Stage 4 screening. All five screenings were performed under the same conditions (in the presence of 0.5 μM IPTG, induced at 37°C for 5 minutes). The number of independent clones in the first screening was 8.0 × 10 4 The number of independent clones calculated from the survival rates of the six screenings was six, and stage 4 screening was completed.

[0071] In stage 4, the plasmid DNA obtained in the fifth round was transformed into E. coli XL1Blue, and the plasmid DNA was extracted from the resulting colonies to confirm the base sequence of the FokI-CD region. As a result, four mutant FokI types (M47, M48, M49, and M50) were obtained. M47, M48, and M49 each contained 10 amino acid substitutions, and M50 contained 11 amino acid substitutions (Figure 1).

[0072] The amino acid substitutions found in the four isolated mutant FokIs were compared with those found in existing mutant FokIs. The Sharkey mutation, which exhibits high zinc finger nuclease activity, consists of the S418P and K441E substitutions (see Non-Patent Document 2). The S418P substitution is also present in M48, but the K441E substitution is not. Non-Patent Document 2 also examined the effects of the Q481H and N527D mutations, but their effects were smaller than those of the Sharkey mutation. The Q481H substitution is also present in M50. Furthermore, heterodimerization mutations, Q486E / I499L / N496D and E490K / I538K / H537R, which cause FokI to form dimers only between different molecules, have been reported (Doyon Y, et al., (2011) Nat Methods. 8: 74-79). Substitutions were introduced at Q486 in M47, M48, and M49, with Q486L substitutions in M47 and M49 and Q486H substitution in M48. M50 contains an E490Q substitution, which differs from the E490K substitution in Doyon et al. Similar heterodimer-forming mutations have been reported in the literature (Ramalingam S, et al., (2011) J Mol Biol. 405: 630-641; Szczepek M, et al., (2007) Nat Biotechnol. 25: 786-793), but there are no common amino acid substitutions other than those common to the four mutants isolated in Doyon et al. and this example. Although the Q481A substitution has been reported as a mutation that reduces off-target mutations (Miller JC, et al., (2019) Nat Biotechnol. 37: 945-952), this does not match as M47 and M49 had a Q486L substitution, while M48 had a Q486H substitution.

[0073] Example 4. Evaluation of mutant FokI using E. coli B2P (1) First, we examined the extent to which the survival rates of four mutant FokIs (M47, M48, M49, and M50) were improved compared to wild-type FokI using E. coli B2P. 10 ng of TALEN-A effector plasmid DNA harboring wild-type and four mutant FokIs (M47, M48, M49, and M50) was transformed into E. coli carrying a TALEN-A reporter by electroporation (Bio-Rad). After 1 mL of SOB was added and the cells were incubated at 37°C for 1 hour, TALEN-A was induced by adding 6 mL of SOB containing 0.1 mM IPTG and incubating at 37°C for 15 minutes. The transformants were plated on plates with and without arabinose and incubated overnight at 37°C, after which colonies were counted. The survival rate was calculated by dividing the number of colonies on the arabinose-supplemented plates by the number of colonies on the non-arabinose-supplemented plates.

[0074] As a result, the survival rate of E. coli expressing TALEN-A effectors with wild-type FokI was 0.0001%, while the survival rates of E. coli expressing FokI with M47, M48, M49, or M50 were 29%, 37%, 45%, and 28%, respectively (Figure 2). The activity of the mutant FokI-TALEN-A effectors was increased by 210,000-fold (M47, M50) to 330,000-fold (M49) compared to the wild-type.

[0075] (2) Of the four mutant FokIs (M47, M48, M49, and M50), M48 and M50 commonly contain a G473R mutation. The G473R mutation was identified in the first stage of screening (Stage 1) and is adjacent to the K469 residue, which is the active site of FokI nuclease. Therefore, the importance of the G473R mutation in the nuclease activity of M50 was examined using B2P in E. coli. First, a reversion mutation (M50(G473)) was introduced by PCR to restore the wild-type G473 residue in M50. Ten nanograms of TALEN-A effector plasmid DNA containing wild-type FokI, M50, and M50(G473) was transformed into E. coli carrying a TALEN-A reporter by electroporation (Bio-Rad). After 1 mL of SOB was added and the culture was recovered at 37°C for 1 hour, 6 mL of SOB containing 0.1 mM IPTG was added and incubated at 37°C for 15 minutes to induce TALEN-A. Transformants were plated on plates with and without arabinose and cultured overnight at 37°C, after which colonies were counted. The survival rate was calculated by dividing the number of colonies on the arabinose-supplemented plate by the number of colonies on the non-arabinose-supplemented plate.

[0076] As a result, the survival rate of E. coli expressing the TALEN-A effector with M50 was 24%, while the survival rate of E. coli expressing the TALEN-A effector with M50(G473) was less than 0.05% (Figure 3), demonstrating the importance of the G473R mutation in the nuclease activity of M50.

[0077] Example 5. Evaluation of mutant FokI by reporter assay in cultured animal cells (1) Next, to investigate whether mutant FokI improves genome editing efficiency, we evaluated the performance of an effector in which mutant FokI was linked to a TALE using an SSA (Single Strand Annealing) reporter, which is a commonly used method to evaluate genome editing efficiency.

[0078] To enable efficient expression of four mutant FokIs (M47, M48, M49, and M50) in mammalian cells, we synthesized genes optimized for human codons. The four human codon-optimized FokI-CD mutants were fused to the C-terminus of a TALEN targeting the GFP gene (GFP_TALEN) or a TALEN targeting the human B2M gene (B2M_TALEN), and then inserted into an expression vector with a CMV promoter using the Golden-Gate method to construct effector plasmids for mammalian cells. The sequences recognized by the TALENs used are listed in Table 1.

[0079] The SSA reporter containing the target sequence recognized by the above TALEN has the following structure: downstream of the CMV promoter, the 5' 1131 bp sequence of firefly luciferase (fLUC), the target sequence, and the 3' 1317 bp sequence of fLUC are linked in this order. There are 795 bp overlapping sequences on the 5' and 3' ends of fLUC, and when homologous recombination occurs between these overlapping sequences, normal fLUC is generated and expressed. The target sequence is shown in Table 2 above.

[0080] The SSA reporter assay was performed as follows. 1,500 human cultured HEK293-T cells were precultured for 1 day and then transfected with 2.0 ng of reporter plasmid DNA, 10 ng of effector plasmid DNA, and 0.5 ng of reference plasmid DNA using FuGENE® Transfection Reagent (Promega). After 2 days of incubation at 37°C in the presence of 5% CO2, the activities of fLUC and rLUC were measured using the Dual-Glo® Luciferase Assay System (Promega). Each sample was assayed three times in quadruplicate. The results showed that all four mutant FokIs exhibited higher activity than the wild-type FokI (Figure 4).

[0081] (2) We investigated whether the use of mutant FokI in PPR improves genome editing efficiency by using Fn( F irefly luciferase & NThis was investigated using the anoLUC reporter.

[0082] A pair of PPRs (B2M_PPR, GFP_PPR) targeting the human B2M gene or GFP gene was fused to the C-terminus of wild-type FokI or M50, which were optimized for human codons, and inserted into an expression vector with a CMV promoter to construct effector plasmids for animal cells. The sequences recognized by the PPRs used are shown in Table 3.

[0083] [Table 3]

[0084] The Fn reporter with the target sequence recognized by the PPR described above has the following structure. The Fn reporter plasmid was constructed by ligating firefly luciferase (fLUC), the target sequence, and NanoLuc (nLUC) downstream of a CMV promoter in this order. In the Fn reporter system, fLUC and the target sequence are connected in frame. Normally, translation occurs up to fLUC, but nLUC is not translated due to an upstream stop codon. Therefore, only fLUC activity is normally detected. On the other hand, if the target sequence is edited to introduce an indel, a frameshift occurs in the target sequence, resulting in expression of a reporter gene fused to fLUC and nLUC, allowing the activity of both fLUC and nLUC to be detected. The target sequences of the human B2M gene and GFP gene inserted into the Fn reporter system are shown in Table 4.

[0085] [Table 4]

[0086] The Fn reporter assay was performed as follows. 1,500 human cultured HEK293-T cells were pre-cultured for one day. 2.5 ng of Fn reporter plasmid DNA and 50 ng of effector plasmid DNA for each of PPR(L) and PPR(R) were transfected into the pre-cultured HEK293-T cells using FuGENE® Transfection Reagent (Promega). After overnight incubation at 37°C in the presence of 5% CO2, the activities of fLUC and nLUC were measured using the Nano-Glo® Luciferase Assay System (Promega). Each sample was assayed in quadruplicate, and activity was evaluated by dividing nLUC by fLUC.

[0087] As a result, when the B2M gene was targeted, M50 showed 2.2-fold higher activity than wild-type FokI, and when the GFP gene was targeted, M50 showed 1.5-fold higher activity than wild-type FokI (Fig. 5). These results demonstrate that M50 exhibits higher activity than wild-type FokI even when linked to PPR. [Industrial Applicability]

[0088] As described above, the novel nuclease domain mutant of the present invention has superior activity to the wild-type FokI nuclease domain when combined with various nucleic acid binding domains. The artificial nucleic acid cleaving enzyme of the present invention is useful as a genome editing tool not only for basic research but also for various industrial applications, including medicine, agriculture, and manufacturing. [Sequence List Free Text]

[0089] SEQ ID NO:3 <223> TALEN-A target sequence SEQ ID NO:4 <223> GFP_TALEN(L) target sequence SEQ ID NO:5 <223> GFP_TALEN(R) target sequence SEQ ID NO:6 <223> Target sequence of B2M_TALEN(L) SEQ ID NO:7 <223> B2M_TALEN(R) target sequence SEQ ID NO:8 <223> TALEN-A target site SEQ ID NO:9 <223> GFP_TALEN target site SEQ ID NO:10 <223> B2M_TALEN target site SEQ ID NO:11 <223> M47 mutant SEQ ID NO:12 <223> M48 mutant SEQ ID NO:13 <223> M49 mutant SEQ ID NO:14 <223> M50 mutant SEQ ID NO:15 <223> Target sequence of B2M_PPR(L) SEQ ID NO:16 <223> Target sequence of B2M_PPR(R) SEQ ID NO:17 <223> Target sequence of GFP_PPR(L) SEQ ID NO:18 <223> GFP_PPR(R) target sequence SEQ ID NO:19 <223> B2M_PPR target site SEQ ID NO:20 <223> GFP_PPR target site

Claims

1. A mutant of the nuclease domain of the FokI protein, which has a mutation described in any one of (a) to (d) below, and which has improved nuclease activity due to the mutation: (a) Substitution of the amino acid at position 386 of the FokI protein with Met, substitution of the amino acid at position 399 with Arg, substitution of the amino acid at position 421 with Tyr, substitution of the amino acid at position 424 with Phe, substitution of the amino acid at position 432 with Leu, substitution of the amino acid at position 433 with Val, substitution of the amino acid at position 448 with Asn, substitution of the amino acid at position 466 with Ile, substitution of the amino acid at position 484 with Ala, and substitution of the amino acid at position 486 with Leu (b) Substitution of the amino acid at position 418 with Pro, the amino acid at position 420 with Leu, the amino acid at position 452 with Val, the amino acid at position 473 with Arg, the amino acid at position 486 with His, the amino acid at position 490 with Val, the amino acid at position 498 with Leu, the amino acid at position 542 with Lys, the amino acid at position 559 with Arg, and the amino acid at position 570 with Ser of the FokI protein. (c) Substitution of the amino acid at position 386 with Met, the amino acid at position 395 with Phe, the amino acid at position 399 with Arg, the amino acid at position 421 with Tyr, the amino acid at position 424 with Phe, the amino acid at position 432 with Leu, the amino acid at position 433 with Val, the amino acid at position 448 with Asn, the amino acid at position 484 with Ala, and the amino acid at position 486 with Leu of the FokI protein. (d) Substitution of the amino acid at position 385 with Pro, the amino acid at position 395 with Ala, the amino acid at position 427 with Met, the amino acid at position 452 with Val, the amino acid at position 460 with Phe, the amino acid at position 473 with Arg, the amino acid at position 481 with His, the amino acid at position 490 with Gln, the amino acid at position 542 with Thr, the amino acid at position 559 with Arg, and the amino acid at position 570 with Ser of the FokI protein.

2. An artificial nucleic acid-cleaving enzyme comprising a nucleic acid-binding domain and a mutant of the nuclease domain of claim 1.

3. The artificial nucleic acid-cleaving enzyme according to claim 2, wherein the nucleic acid binding domain is a TALE, a zinc finger, a PPR, or a CRISPR-Cas.

4. A polynucleotide encoding the mutant of the nuclease domain of claim 1 or the artificial nucleic acid-cleaving enzyme of claim 2 or 3.

5. A vector comprising the polynucleotide of claim 4.

6. A cell into which the polynucleotide of claim 4 or the vector of claim 5 has been introduced.

7. A method for producing a genome-edited cell or non-human organism, the method comprising introducing the artificial nucleic acid cleaving enzyme described in claim 2, a polynucleotide encoding the artificial nucleic acid cleaving enzyme, or a vector containing the polynucleotide into a cell or non-human organism.

8. A kit for editing the genome of a cell or organism, comprising the artificial nucleic acid cleaving enzyme described in claim 2, a polynucleotide encoding the artificial nucleic acid cleaving enzyme, or a vector containing the polynucleotide.

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