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

Abstract

As a technique that can be used to improve the efficiency of genome editing with zinc finger nucleases (ZFNs), particularly in non-dividing cells, the present invention provides a ZFN having three or more nuclear localization signals (NLSs) at the N-terminus thereof.

Description

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

[0001] The present disclosure relates to a zinc finger protein, a zinc finger nuclease, a vector, and a genome editing method using them, and more particularly to a zinc finger protein having three or more nuclear localization signals added to its N-terminus.

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

[0003] In relation to the present disclosure, Non-Patent Document 1 reports that genome editing efficiency is improved in cultured cells (dividing cells) by adding multiple nuclear localization signals (NLS) to ZF-FokI-ND. Non-Patent Document 2 also reports that adding one NLS each to the N-terminus and C-terminus of Cas nuclease enables efficient implementation of the HITI (homology-independent targeted integration) method in non-dividing cells.

[0004] International Publication No. 2020 / 045281

[0005] "Improved Cell-Penetrating Zinc-Finger Nuclease Proteins for Precision Genome Engineering Molecular Therapy", Jia Liu et al., Nucleic Acids,2015, 4, e232"In vivo genome editing via CRISPR / Cas9 mediated homology-independent targeted integration", Keiichiro Suzuki et al., Nature, 2016, 540, 144-149“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

[0006] The primary objective of the present disclosure is to provide techniques that can be used to improve the efficiency of ZFN-mediated genome editing, particularly in non-dividing cells.

[0007] To solve the above problems, the present disclosure provides the following [1] to

[13] . [1] A zinc finger protein (ZFP) having three or more nuclear localization signals (NLSs) at its N-terminus. [2] The ZFP of [1], wherein the NLS is an SV40 T antigen NLS and / or a c-myc NLS. [3] The ZFP of [2], which has an SV40 T antigen NLS and a c-myc NLS arranged alternately. [4] The ZFP of [3], which has two SV40 T antigen NLSs and one c-myc NLS.

[0008] [5] A vector comprising a nucleic acid sequence encoding any one of the ZFPs described in [1] to [4].

[0009] [6] A zinc finger nuclease (ZFN) comprising any of the ZFPs of [1] to [4] and a nucleic acid cleavage domain. [7] The ZFN of [6], wherein the nucleic acid cleavage domain is ND1. [8] The ZFN of [7], wherein the nucleic acid cleavage domain comprises the amino acid sequence of positions 391 to 585 of SEQ ID NO: 9, the amino acid sequence of SEQ ID NO: 14, or the amino acid sequence of SEQ ID NO: 15.

[0010] [9] A vector comprising a nucleic acid sequence encoding any one of ZFNs [6] to [8].

[0011]

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

[11] The genome editing method of

[10] , which is an in vitro method.

[12] The genome editing method of

[10] or

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

[13] The method of

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

[0012] The present disclosure provides techniques that can be used to improve the efficiency of ZFN-mediated genome editing, particularly in non-dividing cells.

[0013] The upstream sequence of the exon 1 start codon of the mouse Rho gene and the recognition sequences of ZFPs (ZF-5057S, ZF5057AS) contained in ZF-ND1 expressed by the ZF-ND1 expression vector are shown. The structures of the ZF-ND1 expression vectors (pRho2k-ZF-ND1DDD, pRho2k-ZF-ND1RRR) are shown. The structure of the donor gene vector (mRho-ZF-HITI-Donor) is also shown. The results of genome editing of rod photoreceptors by the Homology-Independent Targeted Integration (HITI) method using a ZF-ND1 expression vector containing three nuclear localization signal sequences (1xSV40NLS, 3xSV40NLS, 3xMultiNLS) are shown. The top panel shows a fluorescent image of retinal tissue (eyecup) harvested on P21 after a fluorescent protein (AcGFP) gene was introduced into the rhodopsin locus of rod photoreceptors in mouse retina immediately after birth (P0-2). The bottom panel shows fluorescent staining of retinal tissue with an anti-GFP antibody. Green indicates AcGFP fluorescence, and red indicates mCherry fluorescence as a positive control.

[0014] 1. Zinc Finger Proteins (ZFPs) The ZFPs of the present disclosure comprise a nucleic acid binding domain and are characterized by having three or more nuclear localization signals (NLSs) at the N-terminus of the nucleic acid binding domain. By having three or more NLSs at the N-terminus, the ZFPs of the present disclosure can exhibit improved genome editing efficiency when combined with an effector domain. The improved genome editing efficiency can be achieved particularly in non-dividing cells.

[0015] The nucleic acid binding domain of the ZFP disclosed herein comprises a zinc finger array that recognizes a specific nucleic acid sequence. The zinc finger array comprises multiple zinc fingers (ZFs), each of which recognizes a three-base sequence. The zinc finger array may contain two or more ZFs, for example, 3-9, preferably 4-8, more preferably 5-7, and typically 6 ZFs. The nucleic acid sequence recognized by the zinc finger array can be appropriately selected depending on the nucleic acid sequence of the target nucleic acid strand to be subjected to genome editing and the nucleic acid sequence of the editing target site in the target nucleic acid strand. The relationship between the amino acid sequence of a ZF and the three-base sequence recognized by the ZF is publicly known. For example, the amino acid sequence listed in Supplemental Table 1 of Non-Patent Document 1 can be used for the ZF amino acid sequence depending on the target three-base sequence. Since there are multiple amino acid sequences of ZFs that recognize the same three-base sequence, these ZFs that recognize the same three-base sequence can be used interchangeably.

[0016] The number of NLSs possessed by the ZFPs of the present disclosure may be three or more, preferably 3-5, and particularly 3 or 5. The NLS possessed by the ZFPs of the present disclosure may be any conventionally known NLS, including, without limitation, the SV40 T antigen NLS (PKKKRKV: SEQ ID NO: 6), the c-myc NLS (PAAKRVKLD: SEQ ID NO: 7), the nucleoplasmin NLS (KRXXXXXXXXXXKKKLD: SEQ ID NO: 8, where X represents any amino acid), and NLSs having amino acid sequences derived from these sequences. The NLS is preferably the SV40 T antigen NLS or the c-myc NLS, and more preferably the SV40 T antigen NLS and the c-myc NLS. When the NLS is the SV40 T antigen NLS and the c-myc NLS, they may be arranged alternately, and particularly, the three NLSs may be arranged in the order of "SV40 T antigen NLS, c-myc NLS, SV40 T antigen NLS."

[0017] The NLSs may be directly connected or may be connected via a peptide linker, the amino acid sequence of which may be, for example, AAA and GSG.

[0018] 2. Zinc Finger Nuclease (ZFN) The effector domain to which the ZFP of the present disclosure binds is not particularly limited, and may be, for example, a nucleic acid cleavage domain. Other effector domains include cytosine base editors (UGI, APOBEC), adenine base editors (TadA), transcriptional activators (VP16) / repressors (SID), epigenomic editors (DNMT3A, TET1), imaging proteins (Dye, fluorescent proteins), transposases (PiggyBac), and recombinases (Flippase). The effector domain may function alone (monomer type) or as a multimer (split type).

[0019] The ZFP and effector domain may be linked directly or via a linker. The linker may consist of, for example, two or more amino acid residues. The length is not particularly limited, but may be, for example, 2 to 20 amino acids, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, 19, or 20 amino acids. The type of linker is also not particularly limited, but examples include TGGS (SEQ ID NO: 13). The presence or absence of a linker and the length and type of the linker can be appropriately selected by those skilled in the art, taking into account the type of effector domain, etc.

[0020] The ZFPs of the present disclosure are preferably combined with a nucleic acid cleavage domain to form zinc finger nucleases (ZFNs), which recognize and bind to specific nucleic acid sequences via a zinc finger array, resulting in DNA double-strand breaks (DSBs) via the nucleic acid cleavage domain.

[0021] The nucleic acid cleavage domain is not particularly limited and may be FokI-ND, ND1 and ND2 described in Patent Document 2, or modified versions thereof. The amino acid sequence of a full-length protein containing ND1 (derived from Bacillus SGD-V-76) is shown in SEQ ID NO: 9, and its nucleotide sequence is shown in SEQ ID NO: 10. The amino acid sequence of a full-length protein containing ND2 (derived from Clostridium botulinum) is shown in SEQ ID NO: 11, and its nucleotide sequence 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 ND1.

[0022] The ND1 and ND2 variants may be polypeptides having nuclease activity, comprising an amino acid sequence in which one to several amino acid residues have been substituted, deleted, inserted or added in the amino acid sequence shown at positions 391 to 585 of SEQ ID NO: 9 or positions 389 to 579 of SEQ ID NO: 11. Here, "one to several" means, for example, 1 to 10, preferably 1 to 6, e.g., 1, 2, 3, 4 or 5.

[0023] The ND1 and ND2 variants may be polypeptides having nuclease activity and comprising 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% sequence identity to the amino acid sequence shown at positions 391 to 585 of SEQ ID NO:9 or positions 389 to 579 of SEQ ID NO:11. Here, the "sequence identity" of an amino acid sequence is determined by comparing two sequences aligned to maximize sequence identity. Methods for determining the numerical value (%) of sequence identity are known to those skilled in the art. Any algorithm known to those skilled in the art (e.g., the BLAST algorithm, the FASTA algorithm, etc.) may be used to obtain optimal alignment and sequence identity. The sequence identity of an amino acid sequence can be determined using sequence analysis software such as BLASTP or FASTA.

[0024] Specific examples of ND1 and ND2 variants include variants containing aspartic acid (D483, D487, and D496) at positions corresponding to positions 483, 487, and 496 in the amino acid sequence of FokI (DDD variants), and variants containing arginine (R483, R487, and R537) at positions corresponding to positions 483, 487, and 537 in the amino acid sequence of FokI (RRR variants). The ND1 DDD variant (ND1DDD) has an amino acid sequence corresponding to positions 391 to 585 of SEQ ID NO: 9, in which the amino acids at positions 103 and 113 are substituted with aspartic acid (SEQ ID NO: 14). The ND1 RRR variant (ND1RRR) has an amino acid sequence corresponding to positions 391 to 585 of SEQ ID NO: 9, in which the amino acids at positions 100 and 154 are substituted with arginine (SEQ ID NO: 15).

[0025] The nucleic acid cleavage domain of a ZFN according to the present disclosure may be a polypeptide containing modified amino acids and / or unnatural amino acids. Modified amino acids include, but are not limited to, methylation, esterification, amidation, acetylation, alkylation, halogenation, etc. The modified amino acids and unnatural amino acids can be introduced by known methods.

[0026] The ZFPs and ZFNs of the present disclosure can be produced in vitro or in vivo by methods known in the art, such as artificial synthesis based on amino acid sequence information, or artificial synthesis of nucleic acids encoding ZFNs, insertion into an appropriate expression vector, and subsequent introduction into an appropriate host cell to express the ZFN in the cell.

[0027] 3. Vectors The present disclosure also provides vectors containing a nucleic acid sequence encoding the above-described ZFP or ZFN. In addition to the nucleic acid sequence encoding the ZFP or ZFN, the vector may also contain elements found in conventional gene expression vectors. Such elements include, for example, a promoter (such as a CMV promoter or a human EF1a (Elongation Factor alpha-1) promoter) and a polyA addition sequence (such as SV40 pA, HGH pA, or rabbit globin pA).

[0028] The vector is not particularly limited and may be selected from vectors used in the relevant field. Specific examples of vectors include phage vectors, plasmid vectors, viral vectors, retroviral vectors, chromosomal vectors, episomal vectors, virus-derived vectors (bacterial plasmids, bacteriophages, yeast episomes, etc.), yeast chromosomal elements, viruses (baculoviruses, papovaviruses, vaccinia viruses, adenoviruses, adeno-associated viruses, avian poxviruses, pseudorabies viruses, herpes viruses, lentiviruses, retroviruses, etc.), and vectors derived from combinations thereof (cosmids, phagemids, etc.). Among the above, plasmid vectors are preferred from the viewpoint of their versatility. Furthermore, from the viewpoint of their advanced clinical applications, viral vectors are preferred, and adeno-associated viral vectors (AAV vectors) are more preferred.

[0029] 4. Genome Editing Method The present disclosure also provides a genome editing method comprising the step of introducing the above-described vector into a cell in vivo or in vitro, preferably in vitro.

[0030] Vectors can be introduced into cells by conventionally known techniques, including, but not limited to, electroporation, particle gun, microinjection, lipofection, and protein transduction.

[0031] Depending on the purpose of genome editing, donor DNA may be introduced into cells together with the vector. The donor DNA may be, for example, donor DNA encoding a normal gene. The donor DNA may be configured to be able to introduce a normal gene into a gene locus using repair mechanisms such as nonhomologous end joining (NHEJ) or homologous recombination (HR) at the DNA double-strand break site induced by ZFN.

[0032] In genome editing without donor DNA, the breakage site is mainly repaired by NHEJ.Because NHEJ is error-prone, at least one nucleotide deletion, insertion, or substitution, or their combination, may occur during the repair of the breakage.In this way, the target gene locus is modified at the breakage site.

[0033] The cells may be derived from humans or non-human animals, including ungulates (such as cattle, boars, pigs, sheep, and goats), perissodactyls (such as horses), rodents (such as mice, rats, hamsters, and squirrels), lagomorphs (such as rabbits), and carnivores (such as dogs, cats, and ferrets).

[0034] The cells may be dividing or non-dividing cells, but non-dividing cells may be particularly preferably selected in the present disclosure, such as photoreceptors, lymphocytes, monocytes, neutrophils, eosinophils, basophils, endothelial cells, epithelial cells, hepatocytes, bone cells, platelets, adipocytes, cardiac myocytes, neurons, retinal cells, smooth muscle cells, skeletal muscle cells, spermatocytes, oocytes, and pancreatic β cells.

[0035] Example 1: Verification of the effectiveness of NLS in foreign gene insertion into non-dividing cells by HITI method using ZF-ND1. HITI (homology-independent targeted integration) is a technology for inserting a foreign gene (donor gene) into the genome of non-dividing cells with high efficiency and precise orientation. In this example, the effectiveness of NLS in HITI method using ZF-ND1 as a genome editing element was examined.

[0036] The following plasmid vectors were constructed and transduced into mouse rod photoreceptors (non-dividing cells) by in vivo electroporation.

[0037] (1) ZF-ND1 Expression Vector. ND1, used for genomic DNA cleavage, exhibits cleavage activity as a dimer. Therefore, two ZFPs (a pair of ZFPs) were designed, one recognizing the sense strand and the other the antisense strand of the target sequence. Specifically, a pair of ZFPs was designed, each recognizing an 18-base sequence located upstream of the start codon of exon 1 of the mouse Rho gene. The sequence upstream of the start codon of exon 1 of the mouse Rho gene and the recognition sequence of the ZFP pair are shown in Figure 1. A DDD mutant of ND1 (ND1DDD, SEQ ID NO: 14) was ligated to a sense strand ZFP (ZF-5057 S) with a zinc finger array that recognizes and binds to the sense strand sequence CTACGAAGAGCCCGTGGG (SEQ ID NO: 1), resulting in ZF-5057S-ND1DDD. The amino acid sequence of ZF-5057S-ND1DDD is shown in SEQ ID NO: 16. An antisense strand ZFP (ZF-5057AS) with a zinc finger array that recognizes and binds to the antisense strand sequence ACCAAGGCT GCTTGGCGA (SEQ ID NO: 2) was linked to an RRR-type mutant of ND1 (ND1RRR, SEQ ID NO: 15) to create ZF-5057AS-ND1RRR. The amino acid sequence of ZF-5057AS-ND1RRR is shown in SEQ ID NO: 17. A peptide linker (sequence Thr-Gly-Gly-Ser: SEQ ID NO: 13) was used to link ND1 and the ZFP.

[0038] The nucleic acid sequences encoding ZF-5057AS-ND1RRR and ZF-5057S-ND1DDD were inserted into plasmid vectors to construct the expression vectors pRho2k-ZF-ND1DDD and pRho2k-ZF-ND1RRR. The bovine Rho promoter (2174 bp) was used, and the rabbit beta-globin polyA was used as the polyA addition sequence. The structure of the expression vectors is shown in Figure 2. Each expression vector contained one of the following three nuclear localization signal sequences inserted at the N-terminus of ZF. A) SV40 NLS 1 sequence (1xSV40NLS): PKKKRKV (SEQ ID NO: 3) B) SV40 NLS 3 sequence (3xSV40NLS): PKKKRKV AAA PKKKRKV GSG PKKKRKV (SEQ ID NO: 4) C) Sequence in which SV40 NLS, c-Myc NLS, and SV40 NLS are arranged in this order (3xMultiNLS): PKKKRKV AAA PAA KRVKLD GSG PKKKRKV (SEQ ID NO: 5) (The underlined amino acid sequence is the NLS sequence, and AAA and GSG represent linker sequences.)

[0039] (2) Donor Gene Vector. The donor gene was constructed by adding a nucleic acid sequence encoding the fluorescent protein GFP to the cDNA of the mouse Rho gene. A chimeric intron sequence, mouse rhodopsin cDNA (Rho cDNA), an autolytic peptide sequence (Furin+Spacer+P2A), AcGFP, and the 3'UTR sequence derived from the mouse Rho locus were sequentially ligated into the pLeaklessIII plasmid. Furthermore, a sequence with the ZF recognition sequence reversed (ZF-ND1 target) was inserted at both ends of this construct. The resulting gene cassette was designated mRho-ZF-HITI-Donor (see Figure 3).

[0040] (3) mCherry Expression Vector A plasmid expressing mCherry red fluorescent protein under the control of the CAG promoter was constructed.

[0041] 0.3-0.4 μL of vector solution adjusted to a concentration of 5-7 μg / μL was injected subretinally into mice immediately after birth (P0-2). The head was clamped with tweezers (Nepagene CUY650-7) with the retina side as the anode, and an electric pulse was applied to perform gene transfer. The vector solution was mixed in the following percentage ratios: pRho2k-ZF-ND1DDD 15%, pRho2k-ZF-ND1RRR 15%, mRho-ZF-HITI-Donor 60%, pCAG-mCherry 10%.

[0042] The Rho promoters in pRho2k-ZF-ND1DDD and pRho2k-ZF-ND1RRR are activated after rod photoreceptor differentiation (P7-10 onward), and ZF-ND1DDD and ZF-ND1RRR are expressed in differentiated rod photoreceptors (i.e., after cell division ceases). Eyeballs were harvested at P21, when retinal cell differentiation is largely complete. The outer sclera was peeled off and removed, and the remaining tissue (eyecup) was fixed in 4% paraformaldehyde solution (Nacalai). Fluorescent images of GFP and mCherry were captured under a fluorescent stereomicroscope. Tissue sections were also prepared and immunostained with anti-GFP antibodies.

[0043] The upper panel of Figure 4 shows the fluorescence images of the eyecups of pRho2k-ZF-ND1DDD and pRho2k-ZF-ND1RRR, which contained three nuclear localization signal sequences (1xSV40NLS, 3xSV40NLS, and 3xMultiNLS). Green fluorescence was barely observed in the construct containing a single NLS sequence (1xSV40NLS), and was comparable to that of the negative control group (No ZF) without the ZF-ND1 vector. In the constructs containing three NLS sequences, green fluorescence increased in the order 3xSV40NLS and 3xMulti40NLS.

[0044] The bottom panel of Figure 4 shows the anti-GFP immunofluorescence staining images of pRho2k-ZF-ND1DDD and pRho2k-ZF-ND1RRR, which contain three nuclear localization signal sequences (1xSV40NLS, 3xSV40NLS, and 3xMultiNLS). AcGFP-positive cells were observed only in the outer nuclear layer (ONL), where photoreceptors are located, while mCherry-positive cells were also observed in the inner nuclear layer (INL), where horizontal cells, bipolar cells, and amacrine cells are located. This confirms that knock-in of the mRho-ZF-HITI-Donor cassette is restricted to photoreceptors. The number of AcGFP-positive photoreceptors increased in the following order: 1xSV40NLS, 3xSV40NLS, and 3xMulti40NLS.

[0045] These results demonstrate that efficient genome editing in photoreceptor cells (non-dividing cells) is possible by adding multiple (3) NLSs to ZFP.

[0046] SEQ ID NO: 1: Recognition sequence of ZF-5057S SEQ ID NO: 2: Recognition sequence of ZF-5057AS SEQ ID NO: 3: Amino acid sequence of SV40 NLS 1 sequence (1xSV40NLS) SEQ ID NO: 4: Amino acid sequence of SV40 NLS 3 sequence (3xSV40NLS) SEQ ID NO: 5: Amino acid sequence of sequence in which SV40 NLS, c-Myc NLS, SV40 NLS are arranged in this order (3xMultiNLS) SEQ ID NO: 6: Amino acid sequence of SV40 T antigen NLS SEQ ID NO: 7: Amino acid sequence of c-myc NLS SEQ ID NO: 8: Amino acid sequence of Nucleoplasmin NLS SEQ ID NO: 9: Amino acid sequence of full-length protein containing ND1 (derived from Bacillus SGD-V-76) SEQ ID NO: 10: Nucleotide sequence of full-length protein containing ND1 (derived from Bacillus SGD-V-76) SEQ ID NO: 11: Amino acid sequence of full-length protein containing ND2 (derived from Clostridium botulinum) SEQ ID NO: 12: Nucleotide sequence of the amino acid sequence of the full-length protein (derived from Clostridium botulinum) containing ND2 SEQ ID NO: 13: Amino acid sequence of the peptide linker SEQ ID NO: 14: Amino acid sequence of ND1DDD SEQ ID NO: 15: Amino acid sequence of ND1RRR SEQ ID NO: 16: Amino acid sequence of ZF-5057S-ND1DDD SEQ ID NO: 17: Amino acid sequence of ZF-5057AS-ND1RRR

Claims

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

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

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

4. The ZFP of claim 3, which has two SV40 T antigen NLSs 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 a ZFP according to any one of claims 1 to 4 and a nucleic acid cleavage domain.

7. The ZFN of 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 described in claim 8 into a cell.

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

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

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