WO2022097663A1 - ＦｏｋＩヌクレアーゼドメインの変異体 - Google Patents

ＦｏｋＩヌクレアーゼドメインの変異体 Download PDF

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WO2022097663A1
WO2022097663A1 PCT/JP2021/040503 JP2021040503W WO2022097663A1 WO 2022097663 A1 WO2022097663 A1 WO 2022097663A1 JP 2021040503 W JP2021040503 W JP 2021040503W WO 2022097663 A1 WO2022097663 A1 WO 2022097663A1
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protein
foki
amino acid
substitution
corresponding site
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French (fr)
Japanese (ja)
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祐介 八木
賢 太田
崇裕 中村
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Editforce Inc
Kyushu University
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Editforce Inc
Kyushu University
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Priority to JP2022560800A priority Critical patent/JP7752342B2/ja
Priority to EP21889216.4A priority patent/EP4242237A4/en
Priority to CN202180074640.5A priority patent/CN116406383A/zh
Priority to US18/035,430 priority patent/US12637667B2/en
Publication of WO2022097663A1 publication Critical patent/WO2022097663A1/ja
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • the present invention relates to variants of the FokI nuclease domain, artificial nucleic acid cleavage enzymes containing the variants, and their utilization.
  • the molecule used for genome editing consists of a nucleic acid binding domain that binds to a target sequence on the genome and a catalytic domain that cleaves double-stranded DNA near the target sequence.
  • a genome-binding tool using a DNA-binding protein such as a zinc finger protein (ZFN), a transcription activator-like effector (TALE) protein, or a pentricopeptide repeat (PPR) protein, it is usually a type IIS type limiting enzyme.
  • ZFN zinc finger protein
  • TALE transcription activator-like effector
  • PPR pentricopeptide repeat
  • a catalytic domain (FokI-CD) having the activity of cleaving the double-stranded DNA of FokI is used.
  • FokI cleaves double-stranded DNA, it is necessary to form a dimer between two molecules of FokI-CD (Non-Patent Document 1). Therefore, even in the genome editing tool constructed by fusing FokI-CD, two molecules whose target sequences are the sense strand and the antisense strand around the target site to be modified are used.
  • Non-Patent Document 2 an improved FokI-CD (Sharky mutation) in which a random mutation is introduced into ZFN and the genome editing efficiency is improved by using an artificial evolution system of Escherichia coli has been isolated.
  • Non-Patent Document 3 In TALEN fused with FokI-CD containing the Shaky mutation, no difference in editing efficiency was observed with the wild type (Non-Patent Document 3).
  • the present inventors further evaluated the performance of FokI-CD having the Shaky mutation described in Non-Patent Document 2, and found that even when the PPR protein was fused as a nucleic acid binding domain, the FokI-CD was found. It was found that no improvement in genome editing efficiency was observed compared to the wild type. Therefore, FokI-CD having a Shaky mutation has a problem that it cannot be used in combination with a wide range of nucleic acid binding domains in a genome editing system.
  • the present invention has been made in view of such a situation, and an object thereof is to be able to be used in combination with various nucleic acid binding domains in genome editing and to improve the efficiency of genome editing.
  • the purpose is to provide possible nuclease domain variants.
  • nuclease domain mutants bound to TALE are wild-type. It has been found that it has excellent nuclease activity as compared with the nuclease domain, and that the efficiency of genome editing is improved by using the nuclease domain mutant.
  • the mutation at position 470 which is commonly retained by the two nuclease domain variants, contributed significantly to the improvement of nuclease activity.
  • the nuclease domain mutant was found to have improved genome editing efficiency even when fused with PPR. From the above, the present inventors have found that the prepared nuclease domain variant has 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. rice field.
  • the present invention provides, in more detail, the following regarding highly active nuclease domain mutants, artificial nucleic acid cleavage enzymes containing the nuclease domain mutants, and their utilization.
  • a mutant of the nuclease domain of the FokI protein or its homologous protein which has the mutation described in any of the following (a) to (d) and whose nuclease activity is improved by the mutation. ..
  • At least one mutation selected from the group consisting of substitutions with Leu (b) Substitution of the amino acid at position 418 or the corresponding site of the homologous protein in the wild-type protein with Pro, the corresponding position 420 or homologous protein of the FokI protein.
  • Substitution of amino acid at site to Leu, substitution of amino acid at position 542 or homologous protein with Lys of FokI protein, substitution of amino acid at position 559 or homologous protein of FokI protein with Arg, FokI At least one mutation selected from the group consisting of substitution of the amino acid at position 570 or the corresponding site of the homologous protein with Ser of the protein (c) Me of the amino acid at position 386 or the corresponding site of the homologous protein in the wild-type protein
  • substitution with t substitution of the amino acid at position 395 or the corresponding site of the FokI protein with Phe, substitution of the amino acid at position 399 of the FokI protein or the corresponding site of the homologous protein with Arg, position 421 of the FokI protein.
  • substitution with Leu substitution of amino acid at position 433 of FokI protein or corresponding site of homologous protein with Val, substitution of amino acid at position 448 of FokI protein or corresponding site of homologous protein with Asn, position 484 of FokI protein
  • An artificial nucleic acid-cleaving enzyme containing a [3] nucleic acid-binding domain and a variant of the nuclease domain according to [1] or [2].
  • nucleic acid-binding domain is TALE, zinc finger, PPR, or CRISPR-Cas.
  • [5] A polynucleotide encoding a variant of the nuclease domain according to [1] or [2] or the artificial nucleic acid cleaving enzyme according to [3] or [4].
  • [8] A method for producing a cell or a non-human organism whose genome has been edited, wherein the artificial nucleic acid-cleaving enzyme according to [3], a polynucleotide encoding the artificial nucleic acid-cleaving enzyme, or a vector containing the polynucleotide is used. Methods involving introduction into cells or non-human organisms.
  • a kit for editing the genome of a cell or an organism which comprises the artificial nucleic acid-cleaving enzyme according to [3], a polynucleotide encoding the artificial nucleic acid-cleaving enzyme, or a vector containing the polynucleotide. ..
  • the nuclease domain mutant according to the present invention can exhibit excellent nuclease activity as compared with the wild-type nuclease domain.
  • An artificial nucleic acid-cleaving enzyme produced by fusing the nuclease domain of the present invention with a nucleic acid-binding domain such as TALE or PPR enables efficient genome editing.
  • FIG. 1 It is a figure which shows the amino acid sequence of 4 kinds of FokI nuclease domain mutants. The amino acid sequences from positions 384 to 579 corresponding to the FokI nuclease domain are shown. The underlined part shows the mutation site different from the wild type FokI. It is a figure which shows the result of having evaluated the nuclease activity of the FokI nuclease domain mutant type using the survival rate in the artificial evolution system (B2P) of Escherichia coli as an index. Survival rate is the value obtained by dividing the number of colonies on the plate to which arabinose is added by the number of colonies growing on the plate to which arabinose is not added.
  • survival rate is the value obtained by dividing the number of colonies on the plate to which arabinose is added by the number of colonies growing on the plate to which arabinose is not added.
  • WT is a fusion of a wild-type FokI nuclease domain with a TALEN-A effector, and the numerical values in the figure indicate the survival rate when the wild-type FokI nuclease is used. It is a figure which shows the result of having evaluated the influence which the G473R mutation has on the nuclease activity in the FokI nuclease domain using the survival rate in the artificial evolution system (B2P) of Escherichia coli as an index. Survival rate is the value obtained by dividing the number of colonies on the plate to which arabinose is added by the number of colonies growing on the plate to which arabinose is not added.
  • WT is a fusion of the wild-type FokI nuclease domain with the TALEN-A effector, and the numerical values in the figure are the case where the wild-type FokI nuclease was used and the M50 (M50 (G473)) into which the G473 reversion mutation was introduced. The survival rate of the case is shown. It is a figure which shows the result of the target reporter assay using TALEN which targets the B2M gene or GFP gene. The graph shows the rate of increase (fold) in the activity of the FokI nuclease domain variant relative to the wild-type FokI nuclease domain. The values are the result of 3 independent experiments.
  • nuclease domain mutant The present invention provides variants of the nuclease domain of FokI proteins or homologous proteins thereof.
  • the "FokI protein” in the present invention is an IIS type restriction enzyme found in Flavobacterium okeanocoites in nature.
  • the FokI protein has the effect of catalyzing cleavage in double-stranded DNA at a position 9 nucleotides away from the recognition site on one strand and 13 nucleotides away from the recognition site on the other strand.
  • the FokI protein has a nucleic acid binding domain on the N-terminal side and a nuclease domain (DNA cleavage domain) on the C-terminal side.
  • 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.
  • positions 384 to 579 correspond to the nuclease domain.
  • the nuclease domain variant of the present invention is one in which a specific mutation is introduced into the nuclease domain of the FokI protein, and the nuclease activity is improved as compared with the wild-type nuclease domain.
  • FokI nuclease domain variant of the present invention has a mutation introduced into M47 of Examples described below, specifically, substitution of the FokI protein with the amino acid at position 386 to Met, FokI. Substitution of amino acid at position 399 with Arg, substitution of amino acid at position 421 of FokI protein with Tyr, substitution of amino acid at position 424 with Phe, substitution of amino acid at position 432 with Leu , FokI protein at position 433 replaced with Val, FokI protein at position 448 replaced with Asn, FokI protein at position 466 replaced with Ile, FokI protein at position 484 amino acid Ala
  • a nuclease domain variant having at least one mutation selected from the group consisting of substitution with and substitution of the amino acid at position 486 of the FokI protein with Leu.
  • FokI nuclease domain variant of the invention has a mutation introduced into M48 of Examples described below, specifically the amino acid at position 418 in a wild protein to Pro. Substitution, substitution of FokI protein at position 420 with Leu, substitution of FokI protein with amino acid at position 452 with Val, substitution of FokI protein with amino acid at position 473 with Arg, substitution of FokI protein with amino acid at position 486, His Substitution of FokI protein with Val of amino acid at position 490, substitution of FokI protein with amino acid at position 498 with Leu, substitution of FokI protein with amino acid at position 542 with Lys, amino acid at position 559 of FokI protein. Is a nuclease domain variant having at least one mutation selected from the group consisting of substitution of Arg and substitution of the FokI protein at position 570 with Ser.
  • FokI nuclease domain variant of the invention has a mutation introduced into M49 of Examples described below, specifically the amino acid at position 386 in wild-type proteins to Met. Substitution, substitution of FokI protein with amino acid at position 395 to Phe, substitution of FokI protein with amino acid at position 399 with Arg, substitution of FokI protein with amino acid at position 421 with Tyr, substitution of amino acid at position 421 of FokI protein with Phe. Substitution of FokI protein with amino acid at position 432 to Leu, substitution of amino acid at position 433 with Val, substitution of amino acid at position 448 of FokI protein with Asn, amino acid at position 484 of FokI protein. Is a nuclease domain variant having at least one mutation selected from the group consisting of substitution of Ala and substitution of the amino acid at position 486 of FokI protein with Leu.
  • FokI nuclease domain variant of the invention has a mutation introduced into M50 of the Examples described below, specifically the amino acid at position 385 in a wild protein to Pro. Substitution, FokI protein 395th amino acid substitution with Ala, FokI protein 427th amino acid substitution with Met, FokI protein 452th amino acid substitution with Val, FokI protein 460th amino acid Phe Substitution of FokI protein with Arg at position 473 of FokI protein, substitution of amino acid at position 481 with His of FokI protein, substitution of amino acid at position 490 with Gln of FokI protein, amino acid at position 542 of FokI protein A nuclease domain variant having at least one mutation selected from the group consisting of substitution of the FokI protein with Arg at position 559 of the FokI protein and substitution of the amino acid at position 570 of FokI protein with Ser. ..
  • An example of a preferred mutation in the FokI nuclease domain variant of the present invention is the substitution of the amino acid at position 473 of the FokI protein with Arg.
  • Each variant may have a combination of 2 or more mutations, a combination of 3 or more mutations, or a combination of 4 or more mutations, 5 or more. It may have a combination of mutations (eg, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more).
  • nuclease domain variant of the present invention may be a variant of the nuclease domain of a homologous protein of a natural FokI protein (hereinafter referred to as "homologous protein nuclease domain variant").
  • homologous protein means that the homology of the amino acid sequence is at least 70% or more, preferably 80% or more, more preferably 90% or more when the nuclease domain is compared with the nuclease domain of the natural FokI protein. (For example, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) means a protein having.
  • homologous protein nuclease domain variant the amino acid at the site corresponding to the mutation site in the FokI nuclease domain variant described above is replaced with the same amino acid as the mutation site in the FokI nuclease domain variant.
  • the homologous protein may be derived from an organism other than Flavobacterium okeanocoites.
  • amino acid sequence homology can be specified using sequence analysis software (for example, BLAST; http: //blast.ncbi.nlm.nih.gov/Blast.cgi).
  • amino acid of the "corresponding site" corresponding to a specific amino acid of the FokI protein uses the above-mentioned amino acid sequence analysis software (for example, BLAST; http: //blast.ncbi.nlm.nih.gov/Blast.cgi).
  • amino acid sequence analysis software for example, BLAST; http: //blast.ncbi.nlm.nih.gov/Blast.cgi.
  • nuclease domain variant of the invention is a variant having a substitution of the amino acid at position 473 of the FokI protein or the corresponding site of the homologous protein with Arg.
  • nuclease domain mutant of the present invention is further mutated with one or more amino acids (substitution, deletion, addition, and / /) in addition to the above-mentioned mutation site. Or insertion) may be introduced.
  • the term "plurality” is not particularly limited, but is usually 2 to 50, preferably 2 to 30, more preferably 2 to 20, and even more preferably 2 to 10 (for example, 2 to 8). 2-4 pieces, 2 pieces). Examples of useful mutations include heterodimeric formation mutations (Doyon Y, et al. (2011) Nat Methods 8: 74-79).
  • Examples of the method for introducing mutations in a region-specific manner include the Kunkel method (Kunkel, TA Proc Natl Acad Sci USA (1985), 82 (2): 488-492), and the ODA method (Hashimoto-Gotoh et.). al., (1995) Gene 152: 271-276), SOE (splicing-by-overlap-extension) -PCR method (Ho, S.N. et al., (1989) Gene 77: 51-59), etc.
  • a known method can be used.
  • Commercially available site-specific mutagenesis kits may be used.
  • the nuclease domain mutant of the present invention has improved nuclease activity as compared with wild-type protein due to the introduction of the mutation.
  • the improvement in nuclease activity is preferably 10% or more, more preferably 30% or more, more preferably 50% or more (for example, 70% or more, 100% or more, 150% or more, 200% or more).
  • the improvement in nuclease activity can be assessed, for example, by the reporter assay described in Example 5 (FIGS. 4 and 5).
  • an effector plasmid expressing a fusion protein of a nucleic acid binding domain such as TALEN or PPR and a nuclease domain variant of the present invention, and a recognition sequence of the nucleic acid binding domain, and the fusion protein recognizes the recognition sequence.
  • a reporter plasmid that expresses a reporter when DNA cleavage occurs is introduced into cultured cells, and the reporter activity in the cultured cells is measured and evaluated by comparing with the case where a fusion protein containing a wild-type nuclease domain is expressed. be able to.
  • HEK293-T cells can be used as the cultured cells, and in each plasmid, for example, the CMV promoter can be used as the promoter for expressing the fusion protein or the reporter.
  • the recognition sequence of the nucleic acid binding domain for example, a recognition sequence on the human B2M gene or the GFP gene can be used.
  • the nuclease domain variant of the present invention may contain modified amino acids and / or unnatural amino acids.
  • modified amino acid include, but are not limited to, methylation, esterification, amidation, acetylation, alkylation, halogenation and the like.
  • Modified amino acids and unnatural amino acids can be introduced by known methods.
  • the present invention provides an artificial nucleic acid cleavage enzyme containing a nucleic acid binding domain and the above-mentioned nuclease domain variant.
  • the artificial nucleic acid cleavage enzyme of the present invention binds to a target sequence on a nucleic acid (recognition sequence of a nucleic acid binding domain) via a nucleic acid binding domain, and cleaves the nucleic acid in the vicinity of the target sequence (target cleavage site) by the nuclease domain. .. Therefore, the artificial nucleic acid cleavage enzyme of the present invention can function as a sequence-specific nucleic acid cleavage enzyme.
  • nucleic acid includes both DNA and RNA.
  • the nucleic acid cleaved by the artificial nucleic acid cleaving enzyme of the present invention is mainly DNA.
  • DNA includes both double-stranded DNA and single-stranded DNA.
  • the DNA is not particularly limited, and examples thereof include eukaryotic nuclear genomic DNA, mitochondrial DNA, plastide DNA, prokaryotic genomic DNA, phage DNA, and plasmid DNA.
  • the artificial nucleic acid cleavage enzyme of the present invention cleaves double-stranded DNA on the genome.
  • the target sequence of the artificial nucleic acid cleavage enzyme of the present invention is an arbitrary sequence on the nucleic acid.
  • the target sequence may be set to any gene region or extragene region.
  • the length of the target sequence is not particularly limited, but is, for example, 10 to 30 bases.
  • a sequence located in the vicinity of the PAM sequence is set as the target sequence.
  • two target sequences sandwiching a spacer sequence are set.
  • 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 consisting of a desired length and a base sequence.
  • the two target sequences may be palindromic sequences or non-palindromic sequences with each other. If the two target sequences are non-palindromic sequences, two artificial nucleic acid cleavage enzymes targeting each sequence are used.
  • the nucleic acid binding domain in the nucleic acid cleavage enzyme of the present invention may be any protein domain that specifically binds to an arbitrary nucleic acid sequence (target sequence), for example, zinc finger, TALE, CRISPR / Cas (Cas protein and guide RNA). Complex), Pentricopeptide repeat (PPR) and the like.
  • target sequence for example, zinc finger, TALE, CRISPR / Cas (Cas protein and guide RNA). Complex), Pentricopeptide repeat (PPR) and the like.
  • the nuclease domain mutant and the nucleic acid binding domain may be directly linked or may be linked via a linker.
  • the chain length of the linker is not particularly limited, and is, for example, 1 to 20 amino acids.
  • the nucleic acid binding domain containing the zinc finger may preferably include, but is not limited to, a zinc finger array consisting of 3 to 9 zinc fingers. good.
  • a zinc finger array consisting of 3 to 9 zinc fingers can recognize 9 to 27 bases. See literature (International Publication 2003/087341) for examples of ZFNs.
  • the nucleic acid binding domain containing TALE may preferably contain 6 or more repeat modules, and may include, but is not limited to,, for example, a TALE consisting of 9 to 27 repeat modules. It is known that a repeat module has a repeat structure having 34 amino acids as one unit, and one module recognizes one base.
  • TALEN refer to the literature (International Publication No. 2012/104729, International Publication No. 2011/072246, International Publication No. 2015/019672).
  • the nucleic acid binding domain containing PPR may preferably contain 6 or more repeat modules, and may include, but is not limited to,, for example, a PPR consisting of 9 to 27 repeat modules. It is known that a repeat module has a repeat structure having 35 amino acids as one unit, and one module recognizes one base.
  • PPR refer to the literature (Nakamura et al., (2012) Plant Cell Physiol 53: 1171-1179, International Publication No. 2014/175284).
  • the nucleic acid binding domain containing CRISPR-Cas recognizes the target sequence by the targeted RNA (crRNA) in the guide RNA constituting CRISPR-Cas. That is, the targeted RNA and the target sequence have base sequences complementary to each other and can hybridize. Since Cas forms a complex with the guide RNA, it is guided to the target sequence by the action of the guide RNA.
  • the complex of guide RNA and Cas can be utilized as a nucleic acid binding domain, in which case the nuclease variant of the present invention is usually fused to Cas.
  • Cas in CRISPR / Cas does not necessarily retain the nuclease activity.
  • Cas is preferably Cas (eg, dCas) with nuclease activity inactivated. It is also possible to modify PAM recognition by modifying the Cas protein (eg, introduction of mutations) (Benjamin, P. et al., (2015) Nature 523: 481-485, Hirano, S. et al. ., (2016) Molecular Cell 61: 886-894, International Publication No. 2018/221685), thereby expanding the candidates for the target sequence.
  • CRISPR / Cas used in the present invention include class 2 / II type CRISPR / Cas such as CRISPR-Cas9, CRISPR-Cpf1 (Cas12a), CRISPR-Cas12b, CRISPR-CasX (Cas12e), and CRISPR-Cas14.
  • Examples include, but are not limited to, class 2 / V type CRISPR / Cas, class 2 / VI type CRISPR / Cas such as CRISPR-Cas13a, and class 1 / I type CRISPR / Cas such as CRISPR-Cas3. ..
  • CRISPR / Cas For examples of CRISPR / Cas, refer to the literature (International Publication 2014/093712, International Publication 2013/176772, International Publication 2013/142578, International Publication 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).
  • the present invention comprises introducing the above-mentioned artificial nucleic acid-cleaving enzyme, a polynucleotide encoding the artificial nucleic acid-cleaving enzyme, and a vector containing the polynucleotide into a cell or a non-human organism, the cell or non-genome-edited cell.
  • a method for producing a human organism is provided.
  • a nucleic acid-binding domain binds to a target sequence on the nucleic acid, and a nuclease domain variant cleaves the nucleic acid at the target cleavage site. do.
  • repair is performed, such as by non-homologous end linkage repair (NHEJ) or homologous recombination repair (HR), and the genome is edited.
  • NHEJ non-homologous end linkage repair
  • HR homologous recombination repair
  • one or more mutations are inserted into the cleavage site to modify the nucleic acid.
  • the desired DNA in the donor DNA can be inserted into the target DNA region by homologous recombination repair (HDR) occurring in the region around the target cleavage site.
  • HDR homologous recombination repair
  • the artificial nucleic acid-cleaving enzyme of the present invention introduced into a cell may be in the form of a protein, a polynucleotide, or a vector containing the polynucleotide.
  • CRISPR-Cas is used as the nucleic acid binding domain of the artificial nucleic acid-cleaving enzyme, the form of a complex of RNA and protein can be further adopted.
  • the polynucleotide may be DNA or RNA, and may be codon-optimized for high expression in cells.
  • regulatory elements When adopting the form of an expression vector, it contains one or more regulatory elements that are operably bound to the DNA to be expressed.
  • operably bound means that the DNA is operably bound to the regulatory element.
  • regulatory elements include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements such as transcription termination signals (eg, polyadenylation signals, polyU sequences).
  • Regulatory elements depending on the purpose, for example, those that direct the constitutive expression of DNA in various host cells, but those that direct the expression of DNA only in specific cells, tissues, or organs. May be. Further, it may be directed to the expression of DNA only at a specific time, or may be directed to the expression of artificially inducible DNA.
  • promoters examples include the polIII promoter (eg, U6 and H1 promoters), the polII promoter (eg, retrovirus Laus sarcoma virus (RSV) LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, etc.
  • the ⁇ -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1 ⁇ promoter), the polI promoter, or a combination thereof can be mentioned.
  • a person skilled in the art can select an appropriate expression vector according to the type of cells to be introduced and the like.
  • donor DNA may be introduced into the cell in addition to the above-mentioned artificial nucleic acid-cleaving enzyme.
  • This allows the desired DNA to be inserted into the target DNA region by utilizing homologous recombination repair (HDR) that occurs in the region surrounding the target cleavage site.
  • HDR homologous recombination repair
  • the process of homologous recombinant repair requires homology between the base sequence of the target DNA region and the base sequence of the donor DNA, and the donor DNA is used for template repair of the target DNA region including the target cleavage site to target from the donor DNA. It results in the transfer of genetic information to the DNA region.
  • the donor DNA is two base sequences (homologous arms) having high identity with the base sequence in the target DNA region and the desired DNA arranged between them (DNA for insertion into the target DNA region). including.
  • the homology arm may be large enough to perform homologous recombination and may vary depending on the morphology and strand length of the donor DNA, but in the case of double-stranded donor DNA, for example, 500 to It is 1000 base pairs, and in the case of single-stranded donor DNA, it is, for example, 30 to 1000 bases. Further, the homology arm does not have to be 100% identical as long as it has the same identity with the base sequence in the target DNA region to the extent that homologous recombination can be performed. For example, it has an identity of 95% or more, preferably 97% or more, more preferably 99% or more, still more preferably 99.9% or more.
  • the length of the desired DNA existing between the homology arms is not particularly limited, and various sizes can be used.
  • a recognition sequence of a recombinant enzyme for example, loxP sequence or FRT sequence
  • the base sequence sandwiched between the recognition sequences can be removed by allowing a recombinant enzyme (for example, Cre recombinase or FLP recombinant enzyme) to act on the sequence.
  • a selectable marker sequence for example, a fluorescent protein, a drug resistance gene, etc.
  • a gene operably linked to one or more regulatory elements can be used.
  • the donor DNA may be linear DNA or circular DNA. Further, it may be single-stranded DNA or double-stranded DNA.
  • the introduction of the artificial nucleic acid-cleaving enzyme (various forms described above) of the present invention into cells includes, for example, electroporation, microinjection, DEAE-dextran method, lipofection method, nanoparticle-mediated transfection method, virus-mediated nucleic acid delivery method. It can be carried out by a known method such as.
  • the direct administration is performed in vivo, it can be performed by parenteral administration such as injection or oral administration. In this case, it is preferable to combine it with a delivery system having directivity to the target site, if necessary.
  • the donor DNA can also be introduced into cells or living organisms in the same manner.
  • the cell into which the artificial cleaving enzyme of the present invention is introduced may be either a prokaryotic cell (prokaryotic cell) or a eukaryotic cell (eukaryotic cell), and is not particularly limited.
  • prokaryotic cells include Escherichia coli, actinomycetes, archaea and the like.
  • eukaryotic cells include animal cells, plant cells, algae cells, and fungal cells. Examples of animal cells include mammalian cells, fish, birds, reptiles, amphibians, and insect cells.
  • Animal cells include, for example, cells constituting an individual animal, cells constituting an organ / tissue excised from an animal, cultured cells derived from an animal tissue, and the like.
  • germ cells such as egg mother cells and sperm; embryo cells of embryos at each stage (for example, 1-cell stage embryo, 2-cell stage embryo, 4-cell stage embryo, 8-cell stage embryo, 16-cell stage). Embryos, mulberry stage embryos, etc.); Stem cells such as induced pluripotent stem (iPS) cells and embryonic stem (ES) cells; fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, hepatocytes, pancreatic cells , Brain cells, somatic cells such as kidney cells and the like.
  • iPS induced pluripotent stem
  • ES embryonic stem
  • pre-fertilized and post-fertilized oocytes can be used, but a post-fertilized oocyte, that is, a fertilized egg is preferable.
  • the fertilized egg is from a pronuclear stage embryo. Oocytes can be thawed and used after being cryopreserved. However, if it is not acceptable from an ethical point of view, its application to human germ cells and embryonic cells is excluded.
  • Mammals are a concept that includes humans and non-human mammals.
  • non-human mammals include artiodactyls such as cows, wild boars, pigs, sheep and goats, odd-toed ungtiles such as horses, rodents such as mice, rats, guinea pigs, hamsters and squirrels, and rabbits such as rabbits. , Dogs, cats, squirrels and other meats.
  • the non-human mammal may be a domestic animal or a companion animal (pet) or a wild animal.
  • plant cells include cells of cereals, oil crops, feed crops, fruits, and vegetables.
  • Plant cells include, for example, cells constituting an individual plant, cells constituting an organ or tissue separated from a plant, cultured cells derived from a plant tissue, and the like.
  • plant organs and tissues include leaves, stems, shoot apex (growth point), roots, tubers, callus and the like.
  • plants include rice, corn, bananas, peanuts, sunflowers, tomatoes, oilseed rape, tobacco, wheat, barley, potatoes, soybeans, cotton, carnations, etc., and their breeding materials (eg, seeds, roots, tubers, etc.) ) Is also included.
  • two or more kinds of the above-mentioned artificial nucleic acid cleavage enzymes having different nucleic acid binding domains may be introduced into the cell.
  • two kinds of artificial nucleic acid cleavage enzymes that target different sequences may be used.
  • further mutations may be further introduced into the nuclease domain variants of the two artificial nucleic acid cleavage enzymes to promote heterodimer formation of the nuclease domain variants. This can increase the recognition sequence of the artificial nucleic acid-cleaving enzyme and reduce the probability of off-targeting.
  • the present invention provides a kit for editing the genome of a cell or organism, which comprises the above-mentioned artificial nucleic acid-cleaving enzyme, a polynucleotide encoding the artificial nucleic acid-cleaving enzyme, and a vector containing the polynucleotide.
  • the kit may further include one or more additional reagents, such as dilution buffer, reconstruction solution, wash buffer, nucleic acid transfer reagent, protein transfer reagent, control reagent. However, it is not limited to these.
  • the kit usually comes with an instruction manual.
  • Example 1 Principle of B2P screening
  • two plasmids a reporter plasmid having the lethal gene ccdb gene and an effector plasmid expressing TALEN-A
  • the sequence recognized by TALEN-A (Table 1) is inserted downstream of the ccdB gene of the reporter plasmid on the sense strand side and the antisense side, respectively (when viewed from the sense strand side, "target sequence-spacer sequence-target”. It becomes the complementary sequence of the sequence "(Table 2).
  • uppercase letters indicate the sequence recognized by TALEN-A
  • lowercase letters indicate the spacer sequence between the two sequences.
  • TALEN-A reporter plasmid The expression of the ccdb gene can be controlled by an arabinose-inducible promoter, and Escherichia coli transformed with a reporter cannot survive when arabinose is added to the medium (this plasmid is referred to as "TALEN-A reporter plasmid").
  • TALEN-A has a maltose-binding protein fused to the N-terminal side, and the MBP-TALEN-A fusion protein can be induced by IPTG (this plasmid is referred to as "TALEN-A effector plasmid").
  • E. coli having a TALEN-A reporter and screened in the presence of arabinose and IPTG Escherichia coli having a more active TALEN-A effector can survive but is active. E. coli with a weak TALEN-A effector cannot survive.
  • the plasmid DNA of the TALEN-A effector is extracted from the surviving E. coli and transformed into E. coli having the TALEN-A reporter again, the E. coli having the most active TALEN-A effector is further concentrated. By repeating such a cycle, a plasmid containing the most active TALEN-A effector can be isolated.
  • Example 2 Construction of mutant library
  • the mutant library of TALEN-A effector was constructed as follows. A destination vector was constructed by PCR amplification using TALEN-A effector plasmid DNA as a template and PCR amplification using PrimeSTAR® HS DNA Polymerase (TaKaRa), removing the FokI-CD moiety and introducing the Esp3I site. ..
  • the nucleotide sequence of the FokI-CD portion of the MBP-TALEN-A gene was amplified using the GeneMorph II Random Mutagenesis kit (Agilent Technologies) to prepare an insert in which a random mutation was introduced into the Nucleotide sequence of FokI-CD.
  • a mutant library of TALEN-A effectors was constructed by ligating the destination vector cleaved with Esp3I and the insert. When the introduction rate of the random mutation was evaluated, an average of 4 base substitutions were introduced in the base sequence (595 bp) of the FokI-CD portion.
  • Example 3 In the first screening of the mutant library screening stage 1, 1 ⁇ g of the constructed mutant library plasmid DNA was transformed into Escherichia coli having a TALEN-A reporter by electroporation (Bio-Rad). After electroporation, E. coli was transferred to 30 mL of SOB and recultured at 37 ° C. for 1 hour. IPTG was added to a final concentration of 1 mM and incubated at 30 ° C. for 4 hours before sowing on LB agar plates containing 100 mg / L chloramphenicol (Cm) and 0.2% arabinose.
  • Cm chloramphenicol
  • the second screening was performed in the same manner as the first screening using the extracted plasmid DNA. At this time, the total number of colonies was 6.8 ⁇ 108 , and the survival rate was 58%. In the third screening, the survival rate was 100%. Under these conditions, better TALEN-A effectors could not be selected, so in the fourth screening, the IPTG induction time was shortened from 4 hours to 2 hours to make the selection conditions more stringent. The survival rate of the 4th time was 20%, but when the 5th screening was performed under the same conditions, the survival rate reached 90%. In the 6th to 8th times, when the IPTG induction time was set to 1 hour, the survival rates were 1.3%, 10%, and 6.5%, respectively.
  • the survival rate did not improve even if the number of times was repeated, and the number of independent clones calculated from the survival rate of 6 ⁇ 10 6 which was the first independent clone and the survival rate of 8 screenings was 55 or less, so stage 1 was 8 It was completed in the second screening.
  • a FokI-CD insert further introduced with a random mutation was inserted into a destination vector to construct a mutation reintroduction library.
  • This library was transformed into E. coli with a TALEN-A reporter to initiate stage 2 screening.
  • the first screening was performed with 1 mM IPTG for 1 hour, the second to third times with 30 minutes, the fourth time with 15 minutes, and the fifth to sixth times with 0.1 mM IPTG for 15 minutes.
  • the first induction of the screening was performed at 30 ° C., and the second and subsequent inductions were performed at 37 ° C.
  • the number of independent clones calculated from the survival rate of 2.8 ⁇ 10 5 which was the first independent clone and the survival rate of 6 screenings became 1.
  • a FokI-CD insert further introduced with a random mutation was inserted into a destination vector to construct a mutation reintroduction library.
  • This library was transformed into E. coli with a TALEN-A reporter to initiate stage 3 screening.
  • the first screening was performed with 20 ⁇ M IPTG, the second to third with 10 ⁇ M IPTG, the fourth with 1 ⁇ M IPTG, and the fifth to sixth without IPTG for 15 minutes.
  • the first induction of the screening was performed at 30 ° C., and the second and subsequent inductions were performed at 37 ° C. Since the number of independent clones calculated from the survival rate of 1.5 ⁇ 10 5 in the first screening and the survival rate in the 6 screenings became 1, the stage 3 screening was completed.
  • a FokI-CD insert further introduced with a random mutation was inserted into a destination vector to construct a mutation reintroduction library.
  • This library was transformed into E. coli with a TALEN-A reporter to initiate stage 4 screening. All five screenings were performed under the same conditions (induced at 37 ° C. for 5 minutes in the presence of 0.5 ⁇ M IPTG). Since the number of independent clones calculated from the survival rate of 8.0 ⁇ 10 4 which is the first independent clone and the survival rate of 6 screenings became 6, the stage 4 screening was completed.
  • stage 4 the plasmid DNA obtained by transforming the plasmid DNA obtained in the 5th time into an Escherichia coli XL1Blue strain was extracted from the colony obtained, and the nucleotide sequence of the FokI-CD portion was confirmed.
  • M47, M48, M49, M50 four types of mutant FokI (M47, M48, and M49 each contained 10 amino acid substitutions, and M50 contained 11 amino acid substitutions (FIG. 1).
  • the amino acid substitutions found in the four isolated mutant FokIs were compared with the amino acid substitutions contained in the existing mutant FokIs.
  • the Sharkey mutation which is highly active with zinc finger nucleases, is composed of S418P substitutions and K441E substitutions (Non-Patent Document 2 above).
  • the S418P substitution is also present in M48, but the K441E substitution is not.
  • Non-Patent Document 2 also verifies the effects of the Q481H and N527D mutations, but the effects are smaller than those of the Sharkey mutation.
  • the Q481H substitution is also present in M50.
  • Example 4 Evaluation of mutant FokI by E. coli B2P (1) First, how much the survival rate of four types of mutant FokI (M47, M48, M49, M50) is improved compared to wild-type FokI by E. coli B2P. confirmed. TALEN-A effector plasmid DNA 10 ng with wild-type and four variant FokI (M47, M48, M49, M50) was transformed into E. coli with TALEN-A reporter by electroporation (Bio-Rad). After 1 mL SOB was added and the cells were recovered and cultured at 37 ° C. for 1 hour, 6 mL SOB containing 0.1 mM IPTG was added and incubated at 37 ° C.
  • the transformants were sown on plates with and without arabinose and cultured at 37 ° C. overnight, and then the number of colonies was counted.
  • the survival rate was the value obtained by dividing the number of colonies on the plate to which arabinose was added by the number of colonies on the plate to which arabinose was not added.
  • the survival rate of Escherichia coli expressing the TALEN-A effector having wild-type FokI is 0.0001%, whereas the survival rate of Escherichia coli using M47, M48, M49, or M50 as FokI.
  • the mutant FokI-TALEN-A effector was 210,000 times (M47, M50) to 330,000 times (M49) more active than the wild type.
  • M48 and M50 commonly contain the G473R mutation.
  • the G473R mutation is a mutation already identified in the first stage of screening (stage 1) and is adjacent to the K469 residue, which is the active center of the FokI nuclease. Therefore, the importance of the G473R mutation in the activity of the nuclease of M50 was examined by B2P of Escherichia coli.
  • a TALEN-A effector plasmid was constructed by introducing a reversion mutation (M50 (G473)) that returns the R473 residue of M50 to the wild-type G473 residue by the PCR method.
  • Example 5 Evaluation of mutant FokI by reporter assay in cultured animal cells (1) Next, in order to investigate whether mutant FokI improves genome editing efficiency, SSA used as a conventional method for evaluating genome editing efficiency. The performance of the effector in which the mutant FokI was ligated to TALE was evaluated using a (Single Strand Annealing) reporter.
  • Mutant FokI-CD optimized for four human codons is fused to the C-terminal side of TALEN (GFP_TALEN) targeting the GFP gene or TALEN (B2M_TALEN) targeting the human B2M gene and CMV.
  • GFP_TALEN C-terminal side of TALEN
  • B2M_TALEN TALEN
  • An effector plasmid for animal cells was constructed by inserting into an expression vector having a promoter using the Golden-Gate method. The sequences recognized by TALEN used are shown in Table 1 above.
  • the SSA reporter having the target sequence recognized by the above TALEN has the following configuration. Downstream of the CMV promoter, the sequence of 1131bp on the 5'side of firefly luciferase (fLUC), the target sequence, and the sequence of 1317bp on the 3'side of fLUC were ligated in this order. There are 795 bp overlapping sequences on the 5'and 3'sides of the fLUC, and when homologous recombination occurs between these overlapping sequences, a normal fLUC is generated and expressed.
  • the target sequences are shown in Table 2 above.
  • the SSA reporter assay was performed as follows. 1500 human cultured cells HEK293-T cells were pre-cultured for 1 day to obtain 2.0 ng of reporter plasmid DNA, 10 ng of effector plasmid DNA, and 0.5 ng of reference plasmid DNA. ) was used for transfection. After culturing at 37 ° C. for 2 days in the presence of 5% CO 2 , the activity of fLUC and rLUC was measured using Dual-Glo® Luciferase Assay System (Promega). The assay was performed 3 times in 4 series for each sample. As a result, all four types of mutant FokI showed higher activity than the wild type (Fig. 4).
  • a pair of PPRs (B2M_PPR, GFP_PPR) targeting the human B2M gene or GFP gene are fused to the C-terminal side with wild-type FokI or M50 optimized for human codons to form an expression vector with a CMV promoter. It was inserted to construct an effector plasmid for animal cells.
  • the sequences recognized by the PPR used are shown in Table 3.
  • the Fn reporter having the target sequence recognized by the above PPR has the following configuration.
  • a firefly luciferase (fLUC), a target sequence, and NanoLuc (nLUC) were ligated downstream of the CMV promoter in this order to construct an Fn reporter plasmid.
  • fLUC firefly luciferase
  • nLUC NanoLuc
  • the fLUC and the target sequence are linked in-frame, and normally, up to fLUC is translated, but nLUC is not translated because there is a stop codon upstream. Therefore, usually only the activity of fLUC is detected.
  • the Fn reporter assay was performed as follows. 1500 human cultured cells HEK293-T cells were pre-cultured for 1 day. 2.5 ng of Fn reporter plasmid DNA and 50 ng of effector plasmid DNA for each of PPR (L) and PPR (R) into HEK2933-T cells precultured with FuGENE® Transfection Reagent (Promega). Transfected. After culturing at 37 ° C. overnight in the presence of 5% CO 2 , the activity of fLUC and nLUC was measured using Nano-Glo® Luciferase Assay System (Promega). Assays were performed on each sample in quadruples and activity was assessed by dividing nLUC by fLUC.
  • M50 showed 2.2 times the activity of wild-type FokI, and when targeting the GFP gene, M50 showed 1.5 times the activity of wild-type FokI. (Fig. 5). From the above results, it was found that M50 showed higher performance than wild-type FokI even when linked to PPR.
  • the novel nuclease domain variant of the present invention has better activity than the wild-type FokI nuclease domain in combination with various nucleic acid binding domains.
  • the artificial nucleic acid cleavage enzyme of the present invention is useful as a genome editing tool not only in basic research but also in various industrial applications including medical care, agriculture, and industry.

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JP7125727B1 (ja) 2021-09-07 2022-08-25 国立大学法人千葉大学 核酸配列改変用組成物および核酸配列の標的部位を改変する方法
JP2023038817A (ja) * 2021-09-07 2023-03-17 国立大学法人千葉大学 核酸配列改変用組成物および核酸配列の標的部位を改変する方法

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