Zinc finger protein, zinc finger nuclease, vector and genome editing method using zinc finger protein, zinc finger nuclease and vector

Abstract

Provided is a ZFN having three or more nuclear localization signals (NLSs) at the N-terminus as a technique that can be used to improve the efficiency of genome editing by zinc finger nuclease (ZFN), particularly in non-dividing cells.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to zinc finger proteins, zinc finger nucleases, vectors, and genome editing methods using them. More specifically, it relates to zinc finger proteins and the like having three or more nuclear localization signals attached to the N terminus. BACKGROUND

[0002] A zinc finger nuclease (ZFN) is composed of FokI-ND as a nucleic acid cleavage domain, Zinc-finger protein (ZFP) as a nucleic acid binding domain, and a linker connecting them. Patent Literature 1 discloses a ZFN containing nucleic acid cleavage domains called nuclease domain 1 (ND1) and nuclease domain 2 (ND2) instead of FokI-ND. ND1 is considered to be a nuclease domain derived from Bacillus SGD-V-76, and ND2 is considered to be a nuclease domain derived from Clostridium botulinum. ND1 and ND2 are considered to be superior to the conventional FokI-ND in terms of cleavage activity, specificity, selectivity of target sequence, and the like.

[0003] Regarding the present disclosure, Non-Patent Literature 1 reports that the efficiency of genome editing is improved by attaching multiple nuclear localization signals (NLS) to ZF-FokI-ND in cultured cells (dividing cells). In addition, Non-Patent Literature 2 reports that by attaching one NLS to each of the N terminus and the C terminus of a Cas nuclease, efficient implementation of the HITI (Homology-independent Targeted Integration) method in non-dividing cells can be achieved.

[0004] PRIOR ART DOCUMENTS

[0005] PATENT LITERATURE

[0006] Patent Literature 1: International Publication No. 2020 / 045281

[0007] NON-PATENT LITERATURE

[0008] Non-Patent Literature 1: “Improved Cell-Penetrating Zinc-Finger Nuclease Proteins for Precision Genome Engineering Molecular Therapy”, Jia Liu et al., Nucleic Acids, 2015, 4, e232

[0009] Non-patent literature 2: "In vivo genome editing via CRISPR / Cas9 mediated homology-independent targeted integration", Keiichiro Suzuki et al., Nature, 2016, 540, 144-149

[0010] Non-patent literature 3: "Standardized reagents and protocols for engineering zinc finger nucleases by modular assembly", Wright et al., Nature Protocols, 2006, Vol. 1, No. 3, p. 1637-52 SUMMARY

[0011] PROBLEMS TO BE SOLVED BY THE INVENTION

[0012] The main object of the present disclosure is to provide a technique that can be used to improve the efficiency of genome editing by ZFN, particularly in non-dividing cells.

[0013] MEANS FOR SOLVING THE PROBLEMS

[0014] In order to solve the above problems, the present disclosure provides the following [1] to

[13] .

[0015] [1] A zinc finger protein (ZFP) having three or more nuclear localization signals (NLSs) at the N terminus.

[0016] [2] The ZFP of [1], wherein the NLS is an SV40 T antigen NLS and / or a c-myc NLS.

[0017] [3] The ZFP of [2] having an SV40 T antigen NLS and a c-myc NLS arranged alternately.

[0018] [4] The ZFP of [3] having two SV40 T antigen NLSs and one c-myc NLS.

[0019] [5] A vector comprising a nucleic acid sequence encoding the ZFP of any one of [1] to [4].

[0020] [6] A zinc finger nuclease (ZFN) consisting of the ZFP of any one of [1] to [4] and a nucleic acid cleavage domain.

[0021] [7] The ZFN of [6], wherein the nucleic acid cleavage domain is ND1.

[0022] [8] The ZFN of [7], wherein the nucleic acid cleavage domain comprises:

[0023] the amino acid sequence of SEQ ID NO: 9 at positions 391 to 585,

[0024] the amino acid sequence of SEQ ID NO: 14, or

[0025] the amino acid sequence of SEQ ID NO: 15.

[0026] [9] A vector comprising a nucleic acid sequence encoding the ZFN of any one of [6] to [8].

[0027]

[10] A method for genome editing, comprising a step of introducing the vector of [9] into a cell.

[0028]

[11] The method for genome editing of

[10] , which is an in vitro method.

[0029]

[12] The method for genome editing of

[10] or

[11] , wherein the aforementioned cell is a non-dividing cell.

[0030]

[13] The method of

[12] , wherein the aforementioned non-dividing cell is a photoreceptor cell.

[0031] Effects of the Invention

[0032] According to the present disclosure, a technique useful for improving the efficiency of genome editing by ZFNs, particularly in non-dividing cells, is provided. BRIEF DESCRIPTION OF DRAWINGS

[0034] [ Figure 1 ] shows the sequence upstream of the start codon of exon 1 of the mouse Rho gene and the recognition sequence of the ZFPs (ZF-5057S, ZF5057AS) contained in the ZF-ND1 expression vector.

[0035] [ Figure 2 ] shows the constitution of the ZF-ND1 expression vector (pRho2k-ZF-ND1DDD, pRho2k-ZF-ND1RRR).

[0036] [ Figure 3 ] shows the constitution of the donor gene vector (mRho-ZF-HITI-donor).

[0037] [ Figure 4Results of genome editing of rod cells by applying HITI (homology-independent targeted integration) method using ZF-ND1 expression vectors into which 3 kinds of nuclear localization signal sequences (1xSV40NLS, 3xSV40NLS, 3xMultiNLS) were inserted are shown. The upper part is a fluorescence image of a retinal tissue (eye cup) recovered at P21 into which a fluorescent protein (AcGFP) gene was introduced into a rhodopsin gene locus of rod cells of a mouse retina immediately after birth (P0-2). The lower part is a fluorescence staining image of the retinal tissue by an anti-GFP antibody. Green indicates AcGFP, and red indicates fluorescence of mCherry, which is a positive control. DETAILED DESCRIPTION

[0038] 1. Zinc finger protein (ZFP)

[0039] The ZFP according to the present disclosure contains a nucleic acid binding domain characterized in that the N-terminal end of the nucleic acid binding domain has 3 or more nuclear localization signals (NLS). The ZFP according to the present disclosure exhibits improved genome editing efficiency by binding to an effector domain with 3 or more NLS at the N-terminal end. The improvement in genome editing efficiency is particularly achieved in non-dividing cells.

[0040] The nucleic acid binding domain of the ZFP according to the present disclosure contains a zinc finger array that recognizes a specific nucleic acid sequence. The zinc finger array is composed of 1 zinc finger (ZF) arranged multiple times that recognizes a 3-base sequence. The zinc finger array can contain 2 or more ZFs, for example, can be composed of 3 to 9, preferably 4 to 8, more preferably 5 to 7, typically 6 ZFs. The nucleic acid sequence recognized by the zinc finger array can be appropriately set in correspondence with the nucleic acid sequence of a target nucleic acid strand that is the object of genome editing and the nucleic acid sequence of an editing target site in the target nucleic acid strand. The relationship between the amino acid sequence of the ZF and the 3-base sequence recognized by the ZF is known, and the amino acid sequence of the ZF can be set in correspondence with the 3-base sequence as the recognition object, for example, the amino acid sequence described in Supplemental Table 1 of Non-Patent Literature 1. Since there are multiple amino acid sequences of ZFs that recognize the same 3-base sequence, these ZFs that recognize the same 3-base sequence can be used interchangeably.

[0041] The ZFP according to the present disclosure has 3 or more NLS, and preferably can be 3 to 5, in particular 3 or 5.

[0042] The ZFPs according to the present disclosure can have an NLS that is not particularly limited and can be, for example, an NLS known in the art, such as an SV40 T antigen NLS (PKKKRKV: SEQ ID NO: 6), a c-myc NLS (PAAKRVKLD: SEQ ID NO: 7), a Nucleoplasmin NLS (KRXXXXXXXXXXKKKLD: SEQ ID NO: 8, X represents any amino acid), and an NLS having an amino acid sequence derived from these sequences.

[0043] The NLS is preferably an SV40 T antigen NLS or a c-myc NLS, and more preferably an SV40 T antigen NLS and a c-myc NLS.

[0044] In the case where the NLS is an SV40 T antigen NLS and a c-myc NLS, they can be arranged alternately, and in particular, the three NLSs can be arranged in the order of "SV40 T antigen NLS, c-myc NLS, SV40 T antigen NLS".

[0045] Each NLS can be arranged directly, or can be arranged via a peptide linker. The amino acid sequence of the peptide linker is not particularly limited and can be, for example, AAA and GSG.

[0046] 2. Zinc finger nuclease (ZFN)

[0047] The effector domain to which the ZFP according to the present disclosure binds is not particularly limited and can be, for example, a nucleic acid cleavage domain. As other effector domains, there are cited cytosine base editors (UGI, APOBEC), adenine base editors (TadA), transcription activators (VP16) / repressors (SID), epigenome editors (DNMT3A, TET1), imaging proteins (dyes, fluorescent proteins), transposases (PiggyBac), recombinases (Flippase), and the like.

[0048] The effector domain can function alone (monomeric type), or can function via a multimer (divided type).

[0049] The ZFP and the effector domain can be connected directly, and in addition, can be connected via a linker. The linker is composed of, for example, two or more amino acid residues, and the length is not particularly limited and can be, for example, 2 to 20 amino acids long, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 14, 16, 17, 18, 19, or 20 amino acids long. In addition, the kind of the linker is not particularly limited and can be, for example, TGGS (SEQ ID NO: 13). The presence or absence of the linker and the length and kind of the linker are appropriately selected by those skilled in the art in consideration of the kind of the effector domain and the like.

[0050] The ZFPs according to the present disclosure are preferably combined with a nucleic acid cleavage domain to constitute a zinc finger nuclease (ZFN). The ZFN according to the present disclosure recognizes and binds to a specific nucleic acid sequence by the zinc finger array, and causes cleavage of DNA double strand (DNA double strand break; DSB) by the nucleic acid cleavage domain.

[0051] The nucleic acid cleavage domain is not particularly limited, and can be Fokl-ND, ND1 and ND2 described in Patent Literature 2, and variants thereof. The amino acid sequence of the full-length protein comprising ND1 (derived from Bacillus SGD-V-76) is shown in SEQ ID NO: 9, and the base sequence thereof is shown in SEQ ID NO: 10. The amino acid sequence of the full-length protein comprising ND2 (derived from Clostridium botulinum) is shown in SEQ ID NO: 11, and the base sequence thereof is shown in SEQ ID NO: 12. ND1 is typically a partial peptide corresponding to positions 391 to 585 of SEQ ID NO: 9, and ND2 is typically a partial peptide corresponding to positions 389 to 579 of SEQ ID NO: 11. The nucleic acid cleavage domain is preferably considered to be ND1.

[0052] The variants of ND1 and ND2 can be polypeptides comprising an amino acid sequence in which 1 to several amino acid residues in the amino acid sequence shown in positions 391 to 585 of SEQ ID NO: 9 or positions 389 to 579 of SEQ ID NO: 11 are substituted, deleted, inserted, or added, and having nuclease activity. Here, "1 to several" is, for example, 1 to 10, preferably 1 to 6, for example, 1, 2, 3, 4, or 5.

[0053] The variants of ND1 and ND2 can be polypeptides comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% sequence identity with respect to the amino acid sequence shown in positions 391 to 585 of SEQ ID NO: 9 or positions 389 to 579 of SEQ ID NO: 11, and having nuclease activity. Here, the "sequence identity" of the amino acid sequence is determined by comparing two sequences aligned in a state where the sequence identity is maximized. The method for obtaining the value (%) of the sequence identity is known to those skilled in the art. As the algorithm for obtaining the optimal alignment and sequence identity, any algorithm known to those skilled in the art (for example, BLAST algorithm, FASTA algorithm, etc.) can be used. The sequence identity of the amino acid sequence is determined, for example, using sequence analysis software such as BLASTP, FASTA, etc.

[0054] As variants of ND1 and ND2, specifically, variants in which the positions corresponding to positions 483, 487, and 496 in the amino acid sequence of Fokl contain aspartic acid (D483, D487, and D496) (DDD-type mutants), or variants in which the positions corresponding to positions 483, 487, and 537 in the amino acid sequence of Fokl contain arginine (R483, R487, and R537) (RRR-type mutants) are exemplified. The DDD-type mutant of ND1 (ND1 DDD) has an amino acid sequence in which the amino acids at positions 103 and 113 in the partial peptide corresponding to positions 391 to 585 of SEQ ID NO: 9 are substituted with aspartic acid (SEQ ID NO: 14). In addition, the RRR-type mutant of ND1 (ND1 RRR) has an amino acid sequence in which the amino acids at positions 100 and 154 in the partial peptide corresponding to positions 391 to 585 of SEQ ID NO: 9 are substituted with arginine (SEQ ID NO: 15).

[0055] The nucleic acid cleavage domain of the ZFNs to which the present disclosure relates can be a polypeptide containing a modified amino acid and / or a non-natural amino acid. As a modified amino acid, there is no limitation, and, for example, methylation, esterification, amidation, acetylation, alkylation, halogenation, and the like are exemplified. The modified amino acid and the non-natural amino acid can be introduced by a publicly known method.

[0056] The ZFPs and ZFNs to which the present disclosure relates can be produced in vitro or in vivo by a method known in the art. For example, a method in which a nucleic acid encoding a ZFN is artificially synthesized based on amino acid sequence information, inserted into a suitable expression vector, and then introduced into a suitable host cell, and the ZFN is expressed in the cell is exemplified.

[0057] 3. Vector

[0058] The present disclosure also provides a vector containing a nucleic acid sequence encoding the above-described ZFP or ZFN.

[0059] The vector can contain elements possessed by conventional gene expression vectors in addition to the nucleic acid sequence encoding the ZFP or ZFN. As such elements, for example, a promoter (CMV promoter, human EF1a (elongation factor alpha-1) promoter, and the like), a polyA addition sequence (SV40 pA, HGH pA, rabbit globin pA, and the like) are exemplified.

[0060] As the vector, there is no particular limitation, and a vector selected from those used in the field can be used. As specific examples of the vector, there are listed a phage vector, a plasmid vector, a viral vector, a retroviral vector, a chromosomal vector, an episomal vector, a viral-derived vector (bacterial plasmid, phage, yeast episome, etc.), a yeast chromosomal element, a virus (baculovirus, papovavirus, vaccinia virus, adenovirus, adeno-associated virus, avipox virus, pseudorabies virus, herpes virus, lentivirus, retrovirus, etc.), a vector from a combination thereof (cosmid, phagemid, etc.), and the like. Among the above, from the viewpoint of versatility, a plasmid vector is preferred. Furthermore, from the viewpoint of progress in clinical application, a viral vector is preferred, and an adeno-associated virus vector (AAV vector) is more preferred.

[0061] 4. Genome editing method

[0062] The present disclosure also provides a genome editing method, which comprises a step of introducing the above-described vector into a cell in vivo or in vitro, preferably in vitro.

[0063] The introduction of the vector into the cell can be performed by a method known in the art. As the introduction method, there is no limitation, and for example, there are listed electroporation, particle gun method, microinjection, lipofection, protein transduction, and the like.

[0064] According to the purpose of the genome editing, a donor DNA can be introduced into the cell together with the vector. The donor DNA can be, for example, a donor DNA encoding a normal gene. The donor DNA can be configured so that a normal gene can be introduced into the locus by a repair mechanism such as nonhomologous end joining (NHEJ) or homologous recombination (HR) at the cleavage site of the DNA double strand by the ZFN.

[0065] In the case of the genome editing without using the donor DNA, the cleavage site is mainly repaired by NHEJ. Since NHEJ is prone to errors, a deletion, an insertion, or a substitution of at least one nucleotide, or a combination thereof can be generated in the repair of the cleavage. Thus, the target locus is altered at the cleavage site.

[0066] The cell can be of human or non-human animal origin. The non-human animal includes an even-toed ungulate (cattle, wild boar, pig, sheep, goat, etc.), an odd-toed ungulate (horse, etc.), a rodent (mouse, rat, hamster, squirrel, etc.), a lagomorph (rabbit, etc.), a carnivore (dog, cat, ferret, etc.), and the like.

[0067] The cells can be either dividing cells or non-dividing cells, and non-dividing cells can be particularly preferably selected in the present disclosure. As non-dividing cells, for example, there are listed photoreceptor cells, lymphocytes, monocytes, neutrophils, eosinophils, basophils, endothelial cells, epithelial cells, hepatocytes, osteocytes, platelets, adipocytes, cardiomyocytes, neurons, retinal cells, smooth muscle cells, skeletal muscle cells, spermatocytes, oocytes, pancreatic beta cells, and the like.

[0068] Examples

[0069] [Example 1: Verification of the effect of NLS in the insertion of an exogenous gene into a non-dividing cell by the HITI method using ZF-ND1]

[0070] The HITI (homology-independent targeted integration) method is a technique for efficiently and in the correct orientation inserting an exogenous gene (donor gene) into the genome in a non-dividing cell. In the present example, the effectiveness of NLS in the HITI method using ZF-ND1 among genome editing factors was investigated.

[0071] The following plasmid vectors were prepared, and gene introduction into mouse rod cells (non-dividing cells) was performed by in vivo electroporation.

[0072] (1) ZF-ND1 expression vector

[0073] Since ND1 used in the genome DNA cleavage showed cleavage activity as a dimer, two ZFPs (a pair of ZFPs) corresponding to the sense strand and the antisense strand were designed for the sequence to be a cleavage target. Specifically, a pair of ZFPs that recognize sequences of 18 bases each located upstream of the start codon of exon 1 of the mouse Rho gene were designed. The sequence upstream of the start codon of exon 1 of the mouse Rho gene and the recognition sequences of the pair of ZFPs are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Figure 1 The sense strand ZFP (ZF-5057S) having a zinc finger array recognizing and binding the sequence CTACGAAGAGCCCGTGGG (SEQ ID NO: 1) of the sense strand was linked to the DDD-type mutant of ND1 (ND1DDD, SEQ ID NO: 14) as ZF-5057S-ND1DDD. The amino acid sequence of ZF-5057S-ND1DDD is shown in SEQ ID NO: 3. 16 The antisense strand ZFP (ZF-5057AS) having a zinc finger array recognizing and binding the sequence ACCAAGGCTGCTTGGCGA (SEQ ID NO: 2) of the antisense strand was linked to the RRR-type mutant of ND1 (ND1RRR, SEQ ID NO: 15) as ZF-5057AS-ND1RRR. The amino acid sequence of ZF-5057AS-ND1RRR is shown in SEQ ID NO: 4. 17The linker between ND1 and ZFPs uses a peptide linker (sequence Thr-Gly-Gly-Ser: SEQ ID NO: 13).

[0074] The nucleic acid sequences encoding ZF-5057AS-ND1RRR and ZF-5057S-ND1DDD were integrated into plasmid vectors, respectively, to prepare expression vectors pRho2k-ZF-ND1DDD and pRho2k-ZF-ND1RRR.

[0075] The promoter used was a Rho promoter of bovine origin (2174 bp), and the polyA addition sequence used was a rabbit β-globin polyA. The constitution of the expression vector is shown in Figure 2 In each of the expression vectors, a nuclear localization signal sequence of any one of the following three was inserted at the N-terminus of the ZF.

[0076] A) SV40 NLS 1 sequence (1xSV40 NLS): PKKKRKV (SEQ ID NO: 3)

[0077] B) SV40 NLS 3 sequence (3xSV40 NLS): PKKKRKV AAA PKKKRKV GSG PKKKRKV (SEQ ID NO: 4)

[0078] C) sequence arranged in the order of SV40 NLS, c-Myc NLS, SV40 NLS (3xMultiNLS): PKKKRKV AAA PAA KRVKLD GSG PKKKRKV (SEQ ID NO: 5)

[0079] (underlined amino acid sequence is NLS sequence, AAA and GSG indicate linker sequence)

[0080] (2) Donor gene vector

[0081] The donor gene is one in which a nucleic acid sequence encoding a fluorescent protein GFP is added to the cDNA of a mouse Rho gene. A construct in which a chimeric intron sequence, a mouse rhodopsin cDNA (Rho cDNA), a self-digestion peptide sequence (Furin + Spacer + P2A), AcGFP, and a 3'UTR sequence derived from a mouse Rho gene locus are sequentially linked was introduced into a pLeaklessIII plasmid, and further a sequence in which the recognition sequence of the ZF is inverted (ZF-ND1 target) was inserted at both ends of the construct. The resulting gene cassette was named mRho-ZF-HITI-donor (refer to Figure 3 ).

[0082] (3) mCherry expression vector

[0083] Plasmid expressing mCherry red fluorescent protein under control of CAG promoter was prepared.

[0084] The carrier solution adjusted to a concentration of 5-7 ug / μL was injected 0.3-0.4 μL into the subretinal space of mice immediately after birth (P0-2). The head was clamped with a pin electrode (Nepagene CUY650-7) with the retinal side as the anode side, and gene introduction was performed by applying an electric pulse. The mixing ratio of the carrier solution was as follows in percentage.

[0085] pRho2k-ZF-ND1DDD 15%

[0086] pRho2k-ZF-ND1RRR 15%

[0087] mRho-ZF-HITI-donor 60%

[0088] pCAG-mCherry 10%

[0089] Since the Rho promoter of pRho2k-ZF-ND1DDD and pRho2k-ZF-ND1RRR is activated after differentiation of the rod cells after P7-10, ZF-ND1DDD, ZF-ND1RRR starts to express in the rod cells after differentiation (i.e., after cell division stops). The eyeballs were recovered at P21 when the cell differentiation of the retina was substantially complete. The outer sclera portion was peeled off, and the remaining tissue (eye cup) was fixed with a 4% paraformaldehyde solution (Nacalai), and the fluorescence images of GFP and mCherry were photographed with a fluorescence stereomicroscope. In addition, tissue sections were prepared, and fluorescent immunostaining was performed with an anti-GFP antibody.

[0090] Fluorescence images of the eye cup in the case of pRho2k-ZF-ND1DDD and pRho2k-ZF-ND1RRR inserted with 3 kinds of nuclear localization signal sequences (1xSV40NLS, 3xSV40NLS, 3xMultiNLS) are shown in Figure 4 The upper part (green indicates AcGFP, and red indicates mCherry fluorescence).

[0091] In the construct (1xSV40NLS) into which 1 sequence of NLS was introduced, green fluorescence was hardly seen, to the same extent as the negative control group (no ZF) into which the ZF-ND1 carrier was not introduced. In the construct into which 3 sequences of NLS were introduced, an increase in green fluorescence was seen in the order of 3xSV40NLS, 3xMulti40NLS.

[0092] Fluorescent images by anti-GFP antibody staining of cases in which pRho2k-ZF-ND1 DDD and pRho2k-ZF-ND1 RRR into which 3 kinds of nuclear localization signal sequences (1xSV40NLS, 3xSV40NLS, 3xMultiNLS) were inserted are shown in Figure 4 Lower part.

[0093] Since AcGFP-positive cells were seen only in the outer retinal granular layer (ONL) where photoreceptor cells are located, mCherry-positive cells were also seen in the inner retinal granular layer (INL) where horizontal cells, bipolar cells, and amacrine cells are located, it was confirmed that the knock-in of mRho-ZF-HITI-donor cassette occurred photoreceptor cell-definitively. AcGFP-positive photoreceptor cells were seen in order of increase in 1xSV40NLS, 3xSV40NLS, 3xMulti40NLS.

[0094] According to the results shown above, by attaching multiple (3) NLS to ZFP, efficient genome editing in photoreceptor cells (non-dividing cells) is possible.

[0095] Free text of sequence listing

[0096] SEQ ID NO: 1: Recognition sequence of ZF-5057S

[0097] SEQ ID NO: 2: Recognition sequence of ZF-5057AS

[0098] SEQ ID NO: 3: Amino acid sequence of SV40 NLS 1 sequence (1xSV40NLS)

[0099] SEQ ID NO: 4: Amino acid sequence of SV40 NLS 3 sequence (3xSV40NLS)

[0100] SEQ ID NO: 5: Amino acid sequence of sequence arranged in order of SV40 NLS, c-Myc NLS, SV40 NLS (3xMultiNLS)

[0101] SEQ ID NO: 6: Amino acid sequence of SV40 T antigen NLS

[0102] SEQ ID NO: 7: Amino acid sequence of c-myc NLS

[0103] SEQ ID NO: 8: Amino acid sequence of nucleoplasmin NLS

[0104] SEQ ID NO: 9: Amino acid sequence of full-length protein containing ND1 (derived from Bacillus SGD-V-76)

[0105] SEQ ID NO: 10: Nucleotide sequence of full-length protein containing ND1 (derived from Bacillus SGD-V-76)

[0106] SEQ ID NO: 11: Amino acid sequence of full length protein comprising ND2 (Clostridium botulinum origin)

[0107] SEQ ID NO: 12: Nucleotide sequence of amino acid sequence of full length protein comprising ND2 (Clostridium botulinum origin)

[0108] SEQ ID NO: 13: Amino acid sequence of peptide linker

[0109] SEQ ID NO: 14: Amino acid sequence of ND1DDD

[0110] SEQ ID NO: 15: Amino acid sequence of ND1RRR

[0111] SEQ ID NO: 16: Amino acid sequence of ZF-5057S-ND1DDD

[0112] SEQ ID NO: 17: Amino acid sequence of ZF-5057AS-ND1RRR

Claims

1. Zinc finger protein (ZFP) has more than three nuclear localization signals (NLS) at its N-terminus.

2. The ZFP according to claim 1, wherein, NLS is SV40 T antigen NLS and / or c-myc NLS.

3. The ZFP of claim 2, having alternating SV40 T antigen NLS and c-myc NLS.

4. The ZFP of claim 3, having two SV40 T antigen NLS and one c-myc NLS.

5. A vector comprising a nucleic acid sequence encoding the ZFP of any one of claims 1-4.

6. A zinc finger nuclease (ZFN) comprising the ZFP as described in any one of claims 1-4 and a nucleic acid cleavage domain.

7. The ZFN according to claim 6, wherein, The nucleic acid cleavage domain is ND1.

8. A vector comprising a nucleic acid sequence encoding the ZFN of claim 6.

9. A genome editing method comprising the step of introducing the vector of claim 8 into a cell.

10. The method of claim 9, wherein, The cells in question are non-dividing cells.

11. The method of claim 10, wherein, The non-dividing cells are photoreceptor cells.

Images