WO2022244544A1 - Composition utilisée dans la production d'une lignée parentale d'une plante ou d'un animal modifié par un épigénome et son utilisation - Google Patents

Composition utilisée dans la production d'une lignée parentale d'une plante ou d'un animal modifié par un épigénome et son utilisation Download PDF

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WO2022244544A1
WO2022244544A1 PCT/JP2022/017327 JP2022017327W WO2022244544A1 WO 2022244544 A1 WO2022244544 A1 WO 2022244544A1 JP 2022017327 W JP2022017327 W JP 2022017327W WO 2022244544 A1 WO2022244544 A1 WO 2022244544A1
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nucleic acid
epigenome
acid sequence
animal
recognition module
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Japanese (ja)
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出穂 畑田
拓郎 堀居
純代 森田
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国立大学法人群馬大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C—CHEMISTRY; METALLURGY
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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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)

Definitions

  • the present invention relates to a composition for use in the production of epigenome-modified animal and plant parent strains and uses thereof.
  • Non-Patent Document 1 Gene expression in animals and plants is epigenetically modified.
  • genomic DNA methylation and demethylation histone acetylation, deacetylation, phosphorylation, dephosphorylation, ubiquitination, and sumoylation; , has been shown to regulate gene expression differently (Non-Patent Document 1).
  • mice with modified epigenetic modification of genomic DNA that is, mice with modified epigenome
  • the demethylase TET (ten-eleven translocation) 1 TET1
  • TET1 ten-eleven translocation 1
  • the nucleic acid encoding dCas9 and TET1 was introduced into genomic DNA, and the expression of dCas9 and TET1 was induced systemically, and the epigenome of the target region was modified.
  • the resulting epigenome-modified mice had poor growth, died before sexual maturity, and the like, making maintenance by breeding difficult.
  • the object of the present invention is to provide a composition for producing parent line animals and plants that enable the production and maintenance of epigenome-modified animals and plants.
  • the composition of the present invention is a composition for use in producing epigenome-modified animals and plants or producing parent line animals and plants for maintenance, the composition comprises a nucleic acid;
  • the nucleic acid is a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence of a target region that modifies the epigenome in genomic DNA; an epigenome modifying enzyme capable of forming a complex with the nucleic acid sequence recognition module and modifying the epigenome; comprising a nucleic acid encoding
  • the nucleic acid is operably linked to a gametogenesis-specific promoter that is activated in gametogenesis so that the sequence recognition module and the epigenome modifying enzyme are induced to be expressed in the gametogenesis. configured, In the gametogenesis, the nucleic acid sequence recognition module whose expression is induced and the epigenome modifying enzyme form a complex to modify the epigenome of the target region in the genomic DNA of the forming gamete.
  • the production method of the present invention (hereinafter also referred to as the "first production method") is a method for producing parent lines of animals and plants with modified epigenomes, The step of introducing the composition of the present invention into the animal or plant of interest.
  • the parental line of animals and plants of the present invention is a parental line of animals and plants used for the production or maintenance of epigenome-modified animals and plants,
  • the animals and plants contain exogenous nucleic acids
  • the nucleic acid is a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence of a target region that modifies the epigenome in genomic DNA; an epigenome modifying enzyme capable of forming a complex with the nucleic acid sequence recognition module and modifying the epigenome; comprising a nucleic acid encoding
  • the nucleic acid is operably linked to a gametogenesis-specific promoter that is activated in gametogenesis so that the sequence recognition module and the epigenome modifying enzyme are induced to be expressed in the gametogenesis. configured, In the gametogenesis, the nucleic acid sequence recognition module whose expression is induced and the epigenome modifying enzyme form a complex to modify the epigenome of the target region in the genomic DNA of the forming
  • the gamete of the present invention is a gamete used in the production of parent line animals and plants used in the production or maintenance of epigenome-modified animals and plants, It is isolated from plants and animals of the parent line of the present invention.
  • the production method of the present invention (hereinafter also referred to as "second production method") is a method for producing animals and plants in which the epigenome of the target region is modified, crossing the first parent and the second parent and obtaining an epigenome-altered individual from the resulting progeny individual; Said first parent and/or said second parent are plants and animals of the parent strain of the present invention.
  • the animals and plants of the present invention are animals and plants in which the epigenome of the target region is modified,
  • the animals and plants are
  • the target region comprises a target region in which the epigenome derived from the gamete of the present invention is modified, It does not contain said exogenous nucleic acid.
  • the animals and plants of the present invention are animals and plants in which the epigenome of the target region is modified,
  • the animals and plants contain exogenous nucleic acids
  • the nucleic acid is a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence of a target region that modifies the epigenome in genomic DNA; an epigenome modifying enzyme capable of forming a complex with the nucleic acid sequence recognition module and modifying the epigenome; comprising a nucleic acid encoding
  • the nucleic acid is operably linked to a gametogenesis-specific promoter that is activated in gametogenesis so that the sequence recognition module and the epigenome modifying enzyme are induced to be expressed in the gametogenesis.
  • the animal or plant is derived from a gamete in which the epigenome of the target region is modified by forming a complex between the nucleic acid sequence recognition module whose expression is induced in the gametogenesis and the epigenome modifying enzyme as the target region. Contains the target region.
  • composition of the present invention it is possible to produce parent line animals and plants that are capable of producing and maintaining epigenome-modified animals and plants.
  • FIG. 1 is a schematic diagram showing an example of epigenomic alterations in parental lines produced using the composition of the present invention.
  • FIG. 2 is a schematic diagram showing other examples of epigenomic alterations in parental lines generated using the compositions of the present invention.
  • 3 is a schematic diagram showing the structure of the epigenome editing vector in Example 1.
  • FIG. 4 is a graph showing the methylation rate in sperm derived from subline mice in Example 1.
  • FIG. 5 is a graph showing body weights of F1 progeny individuals derived from the male parent line in Example 1.
  • FIG. 6 is a graph showing body weights of F1 progeny individuals derived from male or female parental lines in Example 1.
  • FIG. 7 is a graph showing the methylation rate of the target region of F1 progeny individuals derived from the male or female parent line in Example 1.
  • FIG. 8 is a graph showing the methylation rate and body weight with and without insertion of an epigenome editing vector in Example 1.
  • epigenome refers to a state of chemical modification of DNA and/or histone proteins in the genome without alteration of the nucleic acid sequence (base sequence). Said epigenome can also be referred to as epigenetic modification or genomic modification, for example.
  • epigenome modification or "epigenome modification” means changing the chemical modification of DNA and/or histone proteins in the genome.
  • the change in modification is preferably a change that does not involve a change in the nucleic acid sequence (base sequence). Examples of the modification include addition or removal of modification, change in modification type, increase or decrease in modification frequency, and the like.
  • the above-mentioned “epigenome modification” or “epigenome modification” can also be referred to as, for example, “epigenetic modification modification” or "genome modification modification”.
  • epidermatitis modifying enzyme means a protein that alters the chemical modification of DNA and/or histone proteins in the genome.
  • genomic DNA means genomic DNA within the cell nucleus of eukaryotic cells.
  • nucleic acid means a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and/or modified nucleotides.
  • the nucleic acid may be a single-stranded nucleic acid or a double-stranded nucleic acid. Said nucleic acid can also be referred to, for example, as a “nucleic acid molecule”.
  • hybridize means annealing with complementary polynucleotides resulting from complementarity of nucleotides, specifically complementarity of bases at the nucleotides, i.e., two polynucleotides undergo hydrogen bonding. It means that it can non-covalently pair via.
  • complementarity means that a polynucleotide and another polynucleotide can form a nucleotide pair, that is, a base pair.
  • protein or “peptide” means a polymer composed of unmodified amino acids (natural amino acids), modified amino acids, and/or artificial amino acids.
  • polypeptide means a polymer composed of unmodified amino acids (natural amino acids), modified amino acids, and/or artificial amino acids. Said polypeptide is a peptide having a length of 10 amino acids or more.
  • domain means a three-dimensionally or functionally integrated region in a protein, polypeptide, and/or peptide.
  • antibody means a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes.
  • Immunoglobulin genes include, for example, genes encoding constant regions such as ⁇ , ⁇ , ⁇ (including ⁇ 1 and ⁇ 2), ⁇ (including ⁇ 1, ⁇ 2, ⁇ 3 and ⁇ 4), ⁇ , ⁇ and ⁇ ; , D regions, J regions, and genes that can encode a myriad of immunoglobulin variable regions.
  • Said antibody for example, comprises a heavy chain and a light chain. Said light chains comprise kappa and lambda, constituting kappa and lambda chains, respectively.
  • the heavy chains can be gamma, mu, alpha, delta, or epsilon and constitute the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively.
  • the antibody may be a typical immunoglobulin (antibody) structural unit composed of a tetramer. In this case, the antibody is composed of two identical pairs of polypeptide chains, each pair composed of one light chain (about 25 kDa) and one heavy chain (about 50-70 kDa). The N-terminus of each chain also defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition.
  • the antibody may be a full-length immunoglobulin or an antigen-binding fragment thereof.
  • an "antigen-binding fragment” is a polypeptide comprising a portion of an antibody, more specifically a polypeptide comprising the variable region.
  • the antigen-binding fragments can be produced, for example, by digestion of the full-length immunoglobulin with various peptidases.
  • the antigen-binding fragment include F(ab') 2 , Fab', Fab, Fv (variable fragment of antibody), disulfide-bonded Fv, single-chain antibody (scFv), and polymers thereof.
  • gamete means a germ cell, a cell that can generate a new individual by mating or fertilization. Said gametes can also be referred to, for example, as “gametes” or “gametes”.
  • the gametes are, for example, heterogametes, and specific examples thereof include spermatozoa and eggs in animals and pollen and embryo sacs in plants.
  • imprinted gene means a gene whose gene expression is regulated by genomic imprinting.
  • regulatory region means a region that controls gene expression in genomic DNA.
  • the control regions of the imprinted genes are generally controlled by methylation. Therefore, the control region of the imprinting gene is also called DMR (differentially methylated region) or ICR (imprinting control region).
  • promoter or “promoter region” is a region that exists upstream of a DNA region encoding a gene or polynucleotide and contains a nucleic acid sequence (base sequence) to which a transcription factor binds. It means a region that regulates the amount of transcription of the polynucleotide.
  • the "promoter” or “promoter region” can also be referred to as, for example, a “transcriptional regulatory region.”
  • an "expression vector” or “vector” is a recombinant plasmid, virus, or virus-like particle (VLP) containing a nucleic acid that is delivered to a host cell in vitro or in vivo . means.
  • animal and plant means taxonomic animals and plants.
  • the animals and plants include any animals and plants that form gametes.
  • Said animal means, for example, human and non-human animals.
  • non-human animals include mammals such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, and sea lions.
  • Examples of the plant include angiosperms.
  • exogenous means introduced into cells from outside the cells that constitute animals and plants.
  • isolated means the state of being identified and separated, and/or the state of being recovered from components in their natural state. Said “isolation” can be carried out, for example, by obtaining at least one purification step.
  • target region means a region in genomic DNA that aims to induce a desired effect.
  • Said desired effect is, for example, modification of said epigenome.
  • targeting means binding or accumulating in a target region to induce a desired effect.
  • Said desired effect is, for example, modification of said epigenome.
  • the present invention provides a nucleic acid or a composition comprising said nucleic acid for use in the production of parental line animals and plants that can be used to produce and/or maintain epigenome-modified animals and plants.
  • the present invention provides a nucleic acid or composition for use in the production of epigenome-modified animals and plants or in the production of parent line animals and plants for maintenance, wherein the composition comprises a nucleic acid, wherein the nucleic acid is epigenome-modified in genomic DNA. and an epigenome modifying enzyme capable of forming a complex with the nucleic acid sequence recognition module and capable of modifying the epigenome.
  • nucleic acid is configured such that the sequence recognition module and the epigenome modifying enzyme are induced to be expressed in the gametogenesis, and in the gametogenesis, the nucleic acid sequence recognition module whose expression is induced and the epigenome A modifying enzyme forms a complex and modifies the epigenome of a target region in the genomic DNA of the forming gamete.
  • parent line animals and plants that can be used for the production and/or maintenance of epigenome-modified animals and plants can be produced as follows.
  • the animal or plant is a mouse
  • the gamete in which epigenome modification occurs is a male parental gamete, that is, a sperm
  • the nucleic acid is introduced into one chromosome (genomic DNA) in the mouse genomic DNA.
  • the nucleic acid is a transgene will be described with reference to FIG.
  • the present invention is not limited in any way by the following description. First, the male parent line will be explained.
  • the male parent line has a chromosome (TG1) containing the nucleic acid and a chromosome (WT1) not containing the nucleic acid.
  • sperm are formed from primordial germ cells. The spermatogenesis induces the expression of the sequence recognition module and the epigenome modifying enzyme in the nucleic acid so that they form a complex and are targeted to the target region in the genomic DNA. Then, since the epigenome of the target region in the complex is modified, the epigenome is modified, the chromosome (TG2) containing the nucleic acid or the epigenome is modified, and a sperm containing the chromosome (WT2) not containing the nucleic acid is formed. be done.
  • an epigenome-modified mouse By fertilizing the epigenome-modified sperm with a non-epigenome-modified egg, an epigenome-modified mouse can be produced in which one chromosome is an epigenome-modified chromosome and the other is a non-epigenome-modified chromosome.
  • an epigenome-modified sperm and an epigenome-modified egg By combining an epigenome-modified sperm and an epigenome-modified egg, it is possible to obtain an epigenome-modified mouse in which both chromosomes are epigenome-modified chromosomes.
  • the female parent strain has a chromosome (TG1) containing the nucleic acid and a chromosome (WT1) not containing the nucleic acid.
  • TG1 chromosome
  • WT1 chromosome
  • ovary of the female parent line ova are formed from primordial germ cells.
  • said oogenesis the expression of said sequence recognition module and said epigenome modifying enzyme, which are induced in spermatogenesis in said nucleic acid, are not induced.
  • the target regions of the chromosome containing said nucleic acid (TG1) and the chromosome without said nucleic acid (WT1) are not epigenome modified, the chromosome containing said nucleic acid (TG1) or epigenome modified, An ovum is formed that contains a chromosome (WT1) that does not contain said nucleic acid.
  • a mouse containing a chromosome (TG1) containing the nucleic acid and a chromosome (WT1) not containing the nucleic acid, and two nucleic acids A chromosome (WT1) that does not contain The former mouse has the same genomic DNA as the male parent strain and the female parent strain, and therefore can be used as a mouse for strain maintenance. Therefore, according to the composition of the present invention, it is possible to generate parental lines of animals and plants that can be used for the production and/or maintenance of epigenome-modified animals and plants. Also, as shown in FIGS.
  • the epigenome of the target region is modified by using the composition of the present invention, the chromosome (WT2) that does not contain the nucleic acid, and the non-modified epigenome, the Epigenomic mice can be generated that contain a chromosome (WT1) that does not contain nucleic acid.
  • the epigenome-modified mouse is the same as a mouse containing two sets of chromosomes that do not contain the nucleic acid except that the epigenome has not been modified, except that the epigenome of the target region has been modified.
  • An epigenome-modified mouse can be generated that is excluded and only the effects of the epigenome modification can be studied.
  • the gamete in which the epigenome modification occurs is a sperm is explained as an example, but as shown in FIG.
  • parental strains of animals and plants can be generated that can be used for the production and/or maintenance of epigenome-modified animals and plants.
  • mouse animals are used as the animals and plants, the present invention can also be applied to plants that form gametes (eg, pollen and embryo sacs).
  • the target region is an arbitrary region in genomic DNA and can be set appropriately according to the purpose.
  • Examples of the target region include control regions or promoter regions of imprinted genes.
  • the imprinting gene for example, the gene described in IMPRINTED GENE DATABASES (https://www.geneimprint.com/site/home) can be referred to.
  • the atypical imprinted genes include, for example, Igf2 gene, Gab1 (GRB2-associated-binding protein 1) gene, Gm32885 gene, Jade1/Phf17 (Plant Homeo-domain- 17) gene, Platr20 gene, Sfmbt2 (Scm-like with four MBT domains protein 2) gene, Slc38a4 gene, Smoc1 gene, Xist gene and the like.
  • the imprinted gene is the Igf2 gene
  • the regulatory region is, for example, H19-DMR.
  • the length of the target region is not particularly limited, and can be set appropriately according to the purpose of epigenome modification, for example.
  • the nucleic acid sequence recognition module is a molecule that specifically binds to the nucleic acid sequence of the target region that modifies the epigenome in genomic DNA.
  • the nucleic acid sequence recognition module can form a complex with the epigenome modifying enzyme. Therefore, the nucleic acid sequence recognition module can recruit (accumulate) the epigenome modifying enzyme to the target region by binding to the nucleic acid sequence of the target region. Thereby, the epigenome modifying enzyme modifies the epigenetic modification of the genome of the target region to modify the epigenome.
  • nucleic acid sequence recognition module can recruit the epigenome-modifying enzyme to a target region, by combining with the epigenome-modifying enzyme according to the type of modification to be performed on the epigenome, the desired of the epigenome can be performed.
  • the nucleic acid sequence recognition module is, for example, a protein that recognizes the nucleic acid sequence of DNA, or a complex of protein and nucleic acid.
  • the nucleic acid sequence recognition module includes, for example, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas system, Zinc finger motif, transcription activator-like (TAL) effector, PRR (Pentatricopeptide repeat) motif, restriction enzyme, transcription Factors, RNA polymerase, DNA polymerase, and other proteins that specifically bind to DNA or their DNA-binding domains, preferably CRISPR-Cas system, Zinc finger motif, TAL effector, or PRR motif, or their DNA-binding is a domain.
  • the nucleic acid sequence recognition module preferably does not cut both strands of the double-stranded DNA in order to suppress the occurrence of changes in the nucleic acid sequence of the genome, and the nuclease activity for the double-stranded DNA is inactivated. It is more preferable to be
  • the CRISPR-Cas system is composed of a Cas protein with nuclease activity and a guide strand (guide RNA) that forms a complex with the Cas protein and hybridizes with the target nucleic acid sequence.
  • the Cas protein is not particularly limited, and examples thereof include types IV Cas proteins.
  • the Cas proteins include, for example, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12, Cas14, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2 , Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf2 , Csf3, Csf4, and the like.
  • the origin of the Cas protein is not particularly limited.
  • the Cas9 protein is, for example, Cas9 (SaCas9) derived from Staphylococcus aureus ( Staphylococcus aureus ), Cas9 derived from Streptococcus pyogenes ( Streptococcus pyogenes ) (SpCas9), Streptococcus thermophilus ( Streptococcus thermophilus )-derived Cas9 (StCas9) and the like.
  • the Cas protein preferably has inactivated nuclease activity (DNA cleaving ability) in at least one of the DNA cleavage domains (cleavage sites) of the Cas protein.
  • the Cas9 protein has HNH and RuvC domains as DNA cleavage domains. Therefore, in the Cas9 protein, at least one of the HNH domain and the RuvC domain is preferably inactivated, more preferably both are inactivated. Inactivation of the nuclease activity can be performed, for example, by introducing an amino acid substitution into the nucleic acid sequence encoding the DNA cleavage domain in the DNA encoding the Cas protein.
  • the HNH domain of the SpCas9 for example, the 836th asparagine (Asn) and / or 840th histidine residue (His) to alanine residue (Ala) to replace
  • the ability to cleave the complementary strand with the guide RNA can be inactivated.
  • the RuvC domain of the SpCas9 can be inactivated by replacing the 10th aspartic acid residue (Asp) with an alanine residue (Ala), i.e., on the opposite side of the complementary strand with the guide RNA
  • the cleavability of the strand (reverse complementary strand) can be inactivated.
  • the SpCas9 protein for example, the 840th histidine residue (His) is substituted with an alanine residue (Ala), the 10th aspartic acid residue (Asp) is substituted with an alanine residue (Ala) , the nuclease activity is inactivated (dCas9 protein).
  • the SpCas9 protein for example, the 836th asparagine (Asn) is substituted with an alanine residue (Ala), and the 10th aspartic acid residue (Asp) is substituted with an alanine residue (Ala) , the nuclease activity is inactivated (dCas9 protein).
  • the guide RNA comprises a polynucleotide having a nucleic acid sequence complementary to a target nucleic acid sequence existing in the target region or in the vicinity of the target region, and is hybridizable with the target nucleic acid sequence by the polynucleotide.
  • the guide RNA is, for example, an RNA molecule containing a nucleic acid sequence that specifically binds to the nucleic acid sequence of the target region, that is, the nucleic acid sequence of the target region.
  • a RNA molecule comprising a complementary polynucleotide.
  • the guide RNA can be appropriately set according to the type of the Cas protein.
  • the guide RNA may contain only crRNA (CRISPR RNA), or may contain crRNA (CRISPR RNA) and tracrRNA (trans-activating CRISPR RNA).
  • CRISPR RNA crRNA
  • CRISPR RNA crRNA
  • tracrRNA trans-activating CRISPR RNA
  • the guide RNA may consist of two RNAs or a single RNA (sgRNA).
  • the guide RNA is composed of crRNA and tracrRNA, and crRNA and tracrRNA hybridize with complementary polynucleotides to form a complex and function as guide RNA.
  • the sgRNA is crRNA and tracrRNA, or these are linked via a linker.
  • the crRNA preferably contains, for example, a polynucleotide complementary to the target nucleic acid sequence at the 5' end.
  • the crRNA can recruit (accumulate) the Cas protein in the nucleic acid sequence of the target region, thereby allowing the nucleic acid sequence of the target region to , can recruit (accumulate) the epigenome-modifying enzyme.
  • the length of the complementary polynucleotide is, for example, 15 to 25 nucleotides, 18 to 22 nucleotides.
  • the length of the guide RNA can be appropriately set, for example, according to the length of the target nucleic acid sequence.
  • the nucleic acid sequences targeted by the guide RNA may be one type or two or more types.
  • the number of types of guide RNA can be set, for example, according to the length of the target region.
  • the nucleic acid of the present invention is specific to a nucleic acid sequence at a different site in the target region based on the length of the modifiable region of the epigenome modifying enzyme.
  • the epigenome can be modified throughout the target region.
  • the vicinity of the target region can be set, for example, according to the length of the modifiable region of the epigenome-modifying enzyme. Alternatively, it is within 1 kbp, preferably within 1 kbp.
  • the zinc finger motif is formed by linking a plurality of different C 2 H 2 (Cys 2 His 2 ) type zinc finger units.
  • One zinc finger unit recognizes a nucleic acid sequence of about 3 bases.
  • the plurality can be appropriately set according to the nucleic acid sequence of the target region, but generally it is 3 to 6.
  • the zinc finger motif can recognize, for example, a nucleic acid sequence of about 9-18 bases.
  • the Zinc finger motif for example, modular assembly method (Nat Biotechnol (2002) 20: 135-141), OPEN method (Mol Cell (2008) 31: 294-301), CoDA method (Nat Methods (2011) 8: 67 -69), Escherichia coli one-hybrid method (Nat Biotechnol (2008) 26:695-701).
  • the zinc finger motif can be produced, for example, by the method described in WO03/087341.
  • the TAL effector has a repeating structure of modules with about 34 amino acids as a unit, and the 12th and 13th amino acid residues (repeat variable diresidues: RVD) of one module determine Specificity is determined.
  • Each module is highly independent, and by arranging the modules consecutively, it is possible to create a TAL effector specific to the target nucleic acid sequence.
  • the TAL effector for the target nucleic acid sequence is, for example, the REAL method (Curr Protoc Mol Biol (2012) Chapter 12: Unit 12.15), the FLASH method (Nat Biotechnol (2012) 30: 460-465), the Golden Gate method (Nucleic Acids Res (2011) 39: e82) and other open resources can be used for design.
  • the TAL effector can be produced, for example, by the method described in WO2011/072246.
  • the PPR motif is configured to recognize a specific nucleic acid sequence by continuously arranging PPR motifs consisting of 35 amino acids and recognizing one nucleic acid (base).
  • the PPR motif recognizes a target nucleic acid (base) at the 1st, 4th, and 2nd (ii(-2)th) amino acids from the C-terminus of each motif.
  • base a target nucleic acid
  • ii(-2)th a target nucleic acid sequence
  • PPR motif can be produced, for example, by the method described in WO2014/175284.
  • the DNA binding domain of the restriction enzyme When using the DNA binding domain of the restriction enzyme, the transcription factor, the RNA polymerase, or the DNA polymerase, the DNA binding domain of these proteins is known and the DNA binding domain is used as the nucleic acid sequence recognition module. can.
  • the nucleic acid sequence recognition module may be, for example, one type, or two or more types.
  • the nucleic acid of the present invention encodes multiple types of nucleic acid sequence recognition modules that specifically bind to nucleic acid sequences at different sites in the target region, thereby recognizing the epigenome of the entire target region. can be modified.
  • the modification target of the epigenome modifying enzyme may be chemical modification of genomic DNA or chemical modification of histone protein.
  • Examples of the epigenome-modifying enzyme include methylase, demethylase, acetylase, deacetylase, phosphorylation enzyme, dephosphorylation enzyme, ubiquitinase, and sumoylation enzyme.
  • the epigenome modification enzyme includes, for example, a methylation enzyme or a demethylation enzyme.
  • the methyltransferase methylates, for example, cytosine nucleotides at CpG sites in the genomic DNA.
  • the methyltransferase includes, for example, DNMT (DNA Methyltransferase) 3A, DNMT3B, and DNMT3L.
  • DNMT3A or DNMT3B is used as the epigenome-modifying enzyme, these methyltransferases are preferably used in combination with DNMT3L.
  • the methyltransferase methylates, for example, cytosine nucleotides at CpG, CpHpG, or CpHpH sites (H is a nucleotide other than guanine) in the genomic DNA.
  • examples of the methyltransferase include DRMa (DRM-type cytosine DNA-methyltransferase), DRM2, CMT (chromomethylase) 2, and the like.
  • the demethylase demethylates, for example, the methyl group of the cytosine nucleotide at the CpG site in the genomic DNA.
  • the demethylase includes, for example, TET (ten-eleven translocation) 1, TET2, and TET3.
  • the demethylase demethylates, for example, a methyl group of a cytosine nucleotide at a CpG, CpHpG, or CpHpH site (H is a nucleotide other than guanine) in the genomic DNA.
  • examples of the demethylase include DME (DEMETER), ROS (SILENCING) 1, and the like.
  • the epigenome modifying enzyme includes, for example, methylase, demethylase, acetylase, deacetylase, kinase, dephosphorylation, and ubiquitination. Enzymes, SUMOylation enzymes, and the like can be mentioned.
  • the methyltransferase methylates, for example, lysine residues and/or arginine residues in the histone proteins.
  • Examples of the methyltransferase include EZH2 (enhancer of zeste homolog 2) classified as PRC2, G9, SUV39H1, and the like.
  • the demethylase demethylates, for example, methyl groups of lysine and/or arginine residues in the histone protein.
  • Examples of the demethylase include LSD1 (Lysine-specific demethylase 1), KDM4D (Lysine-specific demethylase 4D), KDM6B (Lysine Demethylase 6B) and the like.
  • the acetylating enzyme acetylates, for example, lysine residues in the histone protein.
  • the acetylating enzyme includes, for example, Gcn5, P300/CBP (CREB-binding protein), and the like.
  • the deacetylase deacetylates, for example, the acetyl group of a lysine residue in the histone protein. Examples of the deacetylase include histone deacetylase (HDAC), SIRT (Sirtuin) 1, SIRT2 and the like.
  • the kinase phosphorylates, for example, serine and/or threonine residues in the histone protein.
  • examples of the kinase include Haspin (GSG2), Aurora B, ChK1 (Checkpoint kinase 1) and the like.
  • the dephosphorylation enzyme dephosphorylates, for example, the phosphate groups of serine and/or threonine residues in the histone protein.
  • examples of the phosphatase include PP1 ⁇ (Protein phosphatase 1 ⁇ ), PP4C (Protein Phosphatase 4 Catalytic Subunit), DUSP1 (Dual specificity protein phosphatase 1) and the like.
  • the ubiquitinase for example, ubiquitinates lysine residues in the histone protein.
  • the ubiquitination enzymes include, for example, Ring1, RNF8, UBC13, UHRF1 and the like.
  • the sumoylation enzyme for example, sumoylates a lysine residue in the histone protein.
  • the SUMOylation enzyme includes, for example, UBC9.
  • the origin of the epigenome-modifying enzyme may be the same as that of the animal or plant using the composition of the present invention, ie, the same species, or different, ie, heterologous.
  • the epigenome-modifying enzyme may be all or part of the enzyme protein.
  • the epigenome-modifying enzyme should retain the enzymatic activity of the enzyme protein.
  • the nucleic acid sequence recognition module and the epigenome modifying enzyme are capable of forming a complex.
  • the formation of the complex may be formation of a complex by linkage via a covalent bond, or formation of a complex utilizing intermolecular interaction.
  • the nucleic acid sequence recognition module and the epigenome modifying enzyme are directly or indirectly ligated, i.e., the fusion protein and By doing so, a complex may be formed.
  • the nucleic acid sequence recognition module may form a complex with one epigenome modifying enzyme, or may form a complex with multiple epigenome modifying enzymes.
  • the epigenome-modifying enzyme may form a complex with one nucleic acid sequence recognition module, or may form a complex with a plurality of nucleic acid sequence recognition modules.
  • the resulting complex can perform epigenome modification more efficiently than a complex formed with one epigenome modifying enzyme. and the epigenome at locations more distant from the target nucleic acid sequence within the target region can also be modified.
  • the number of epigenome-modifying enzymes forming a complex with one nucleic acid sequence recognition module is one or more, preferably two or more (plurality), 3-10, 3-7, or 3-5.
  • the epigenome modifying enzymes may be of one type or multiple types.
  • the complex can modify, for example, a plurality of types of epigenomes in the target region.
  • the N-terminal or C-terminal amino acid of the protein in the nucleic acid sequence recognition module is the C-terminal or N-terminal amino acid in the epigenome modifying enzyme. forms a covalent bond with
  • the order of the nucleic acid sequence recognition module and the epigenome modifying enzyme is not particularly limited, and can be any order.
  • the nucleic acid sequence recognition module and the epigenome modifying enzyme may be arranged in this order from the N-terminal side.
  • the epigenome modifying enzyme and the nucleic acid sequence recognition module may be arranged in this order.
  • the order of the nucleic acid sequence recognition module and the plurality of epigenome modifying enzymes is not particularly limited, but the epigenome modifying enzymes are preferably arranged consecutively.
  • the nucleic acid is a nucleic acid encoding a fusion protein in which the amino acid sequences of the nucleic acid sequence recognition module and the epigenome modifying enzyme are integrated. .
  • the amino acid at the N-terminus or C-terminus of the protein in the nucleic acid sequence recognition module is linked via a linker to the C-terminus or C-terminus of the epigenome modifying enzyme. It binds to the N-terminal amino acid.
  • the nucleic acid sequence recognition module is indirectly linked to a plurality of epigenome modifying enzymes, only the nucleic acid sequence recognition module and the epigenome modifying enzyme are linked via the linker, and the epigenome modifying enzyme may be directly linked, or both between the nucleic acid sequence recognition module and the epigenome modifying enzyme and between the epigenome modifying enzymes may be linked via the linker.
  • the order of the nucleic acid sequence recognition module and the plurality of epigenome modifying enzymes is not particularly limited, but the epigenome modifying enzymes are preferably arranged consecutively.
  • the nucleic acid is a nucleic acid encoding a fusion protein in which the amino acid sequences of the nucleic acid sequence recognition module and the epigenome modifying enzyme are integrated. .
  • any sequence can be selected for the linker as long as it does not interfere with the functions of the nucleic acid sequence recognition module and the epigenome modifying enzyme.
  • the linker include a repeating sequence of glycine and serine.
  • the length of the linker is, for example, 5-100 amino acids, 5-50 amino acids, 10-50 amino acids, 15-50 amino acids, 15-40 amino acids, 17-30 amino acids, or 22 amino acids.
  • the linker may be, for example, GSGSG (SEQ ID NO: 1), GSGGS (SEQ ID NO: 2), SGSGS (SEQ ID NO: 3), or GGGGS (SEQ ID NO: 4), or 2-3 repeat sequences thereof; ); GSGSGGSGSGGSGSGGSGSGGSGGSGSGGSGSGGGSGSGGSGSG (SEQ ID NO: 6);
  • the nucleic acid sequence recognition module and the epigenome modifying enzyme are composed of a tag domain and a tag domain capable of binding to the tag domain.
  • a binding partner may be used to form a complex through interaction of the tag domain with the binding partner.
  • the nucleic acid sequence recognition module may be linked to the tag domain
  • the epigenome modifying enzyme may be linked to the binding partner
  • the nucleic acid sequence recognition module may be linked to the binding partner
  • the epigenome modifying enzyme is linked to the binding partner.
  • nucleic acid sequence recognition module and the epigenome modifying enzyme form a complex through interaction between the tag domain and the binding partner
  • the nucleic acid sequence recognition module and the tag domain are, for example, directly or indirectly linked, ie forming a fusion protein.
  • the epigenome-modifying enzyme and the binding partner are, for example, directly or indirectly linked, ie, form a fusion protein.
  • the nucleic acid sequence recognition module may be linked with one tag domain, or may be linked with a plurality of tag domains.
  • the tag domain may be linked to one nucleic acid sequence recognition module, or may be linked to a plurality of nucleic acid sequence recognition modules.
  • the resulting complex is more efficient in epigenome modification compared to complexes formed with nucleic acid sequence recognition modules having one tag domain. It is well-performed and can also modify the epigenome at locations more distant from the target nucleic acid sequence within the target region.
  • the number of tag domains linked to one nucleic acid sequence recognition module is 1 or more, preferably 2 or more (plurality), 3-10, 3-7, or 3-5.
  • the epigenome-modifying enzyme may be ligated with one binding partner, or may be ligated with a plurality of binding partners.
  • the binding partner may be linked to one epigenome-modifying enzyme, or may be linked to a plurality of epigenome-modifying enzymes.
  • the resulting complex is more efficient in performing epigenome modification compared to a complex comprising binding partners with one epigenome modifying enzyme;
  • the epigenome at more distant locations from the target nucleic acid sequence within the target region can also be modified.
  • the number of epigenome-modifying enzymes linked to one binding partner is 1 or more, preferably 2 or more (plurality), 3-10, 3-7, or 3-5.
  • the nucleic acid sequence recognition module and the epigenome modifying enzyme are configured such that one nucleic acid sequence recognition module and a plurality of epigenome modifying enzymes form a complex.
  • Epigenome modification can be performed more efficiently and the epigenome can be modified more distantly from the target nucleic acid sequence within the target region than when the enzyme forms a complex.
  • the nucleic acid sequence recognition module may, for example, be linked with multiple tag domains and one binding partner may be linked with multiple epigenome modifying enzymes.
  • the N-terminal or C-terminal amino acid of the protein in the nucleic acid sequence recognition module is shared with the C-terminal or N-terminal amino acid in the tag domain. forming a bond.
  • the order of the nucleic acid sequence recognition module and the tag domain is not particularly limited, and can be any order.
  • the nucleic acid sequence recognition module and the tag domain may be arranged in this order from the N-terminal side.
  • said tag domain and said nucleic acid sequence recognition module may be arranged in this order.
  • the order of the nucleic acid sequence recognition module and the plurality of tag domains is not particularly limited, but the tag domains are preferably arranged consecutively.
  • the nucleic acid is a first nucleic acid encoding a fusion protein in which the amino acid sequences of the nucleic acid sequence recognition module and the tag domain are integrated.
  • the amino acid at the N-terminus or C-terminus of the protein in the nucleic acid sequence recognition module is linked via a linker to the C-terminus or N-terminus of the tag domain. is bound to the amino acid of
  • the nucleic acid sequence recognition module is indirectly linked to a plurality of tag domains, only the nucleic acid sequence recognition module and the tag domain are linked via the linker, and the tag domains are linked directly.
  • both the nucleic acid sequence recognition module and the tag domain and between the tag domains may be linked via the linker.
  • the order of the nucleic acid sequence recognition module and the plurality of tag domains is not particularly limited, but the tag domains are preferably arranged consecutively.
  • the nucleic acid is a first nucleic acid encoding a fusion protein in which the amino acid sequences of the nucleic acid sequence recognition module and the tag domain are integrated.
  • the linker the description of the linker between the nucleic acid sequence recognition module and the epigenome modifying enzyme can be used.
  • the linker the description of the linker between the nucleic acid sequence recognition module and the epigenome modifying enzyme can be used.
  • the GCN4 peptide epitope described below is used as the tag domain and the GCN4 peptide epitope antibody is used as the binding partner
  • the length of the linker is preferably 17 to 30 amino acids, or 22 amino acids.
  • the N-terminal or C-terminal amino acid of the protein in the epigenome-modifying enzyme forms a covalent bond with the C-terminal or N-terminal amino acid in the binding partner.
  • the order of the epigenome-modifying enzyme and the binding partner is not particularly limited and can be any order.
  • the epigenome-modifying enzyme and the binding partner may be arranged in this order from the N-terminal side, A binding partner and said epigenome modifying enzyme may be arranged in this order.
  • the order of the epigenome-modifying enzyme and the plurality of binding partners is not particularly limited, but the binding partners are preferably arranged consecutively.
  • the nucleic acid constitutes a second nucleic acid encoding a fusion protein in which the amino acid sequences of the epigenome-modifying enzyme and the binding partner are integrated. .
  • the N-terminal or C-terminal amino acid of the protein in the epigenome-modifying enzyme is linked via a linker to the C-terminal or N-terminal amino acid in the binding partner. is connected with
  • the binding partner is indirectly linked to a plurality of epigenome-modifying enzymes, only the epigenome-modifying enzyme and the binding partner are linked via the linker, and the binding partners are directly linked Alternatively, both the epigenome-modifying enzyme and the binding partner and the epigenome-modifying enzyme may be connected via the linker.
  • the order of the binding partner and the plurality of epigenome-modifying enzymes is not particularly limited, but the epigenome-modifying enzymes are preferably arranged consecutively.
  • the nucleic acid constitutes a second nucleic acid encoding a fusion protein in which the amino acid sequences of the epigenome-modifying enzyme and the binding partner are integrated.
  • the linker the description of the linker between the nucleic acid sequence recognition module and the epigenome modifying enzyme can be used.
  • the GCN4 peptide epitope described below is used as the tag domain and the GCN4 peptide epitope antibody is used as the binding partner
  • the length of the linker is preferably 17 to 30 amino acids, or 22 amino acids.
  • the tag domain and the binding partner are specifically Any combination that binds can be used.
  • the combination of the tag domain and the binding partner include a combination of a peptide epitope and an antibody or aptamer that recognizes it, a combination of a split protein small fragment and a large fragment having self-assembly ability, and the like.
  • the tag domain is composed of a peptide
  • the tag domain can also be called a peptide tag.
  • Combinations of the peptide epitope and an antibody that recognizes it include, for example, GCN (General Control Non-derepressible) 4 peptide epitope and anti-GCN4 peptide epitope antibody; His tag and anti-His tag antibody; EE hexapeptide and anti-EE hexapeptide; Peptide antibody; c-Myc tag and anti-c-Myc tag antibody; HA tag and anti-HA tag antibody; S tag and anti-S tag antibody; FLAG tag and anti-FLAG tag antibody; ; vol.24 (5): pp.419-428 (2011)).
  • the GCN4 peptide may be an epitope contained in GCN4, and a specific example thereof is the amino acid sequence represented by ELLSKNYHLENEVARLKK (SEQ ID NO: 7).
  • a split protein that has the ability to self-assemble is a protein that can form the same structure as the original protein by reorganizing the two protein fragments when a protein is split into two.
  • a short peptide (small fragment) obtained by dividing the original protein into two may be used as the tag domain (peptide tag) and a long peptide (large fragment) may be used as the binding partner, or vice versa.
  • the split protein having the ability to self-assemble include GFP (Green Fluorescent Protein).
  • GFP Green Fluorescent Protein
  • the tag domain and the binding partner for example, a combination of a peptide and a protein domain that binds to the peptide may be used.
  • the combination of the peptide and the protein domain can be obtained, for example, from databases (PepBDB: http://huanglab.phys.hust.edu.cn/pepbdb/, PiSITE: https://pisite.sb.ecei.tohoku.ac. jp/cgi-bin/top.cgi, STRING: https://string-db.org/).
  • the PDZ Alpha-Syntrophin PDZ protein interaction domain can bind to GVKESLV (SEQ ID NO: 8).
  • GVKESLV can be used as the tag domain and the PDZ domain as the binding partner.
  • the binding strength of the pair of the peptide and the protein domain is such that another inert domain is linked to the linker. It may be strengthened by connecting via and improving with evolutionary engineering. Epigenome modification can be controlled more efficiently by using the pair obtained by the above improvement as the tag domain and the binding partner (Proc. Natl. Acad. Sci. USA, 2008, vol. 105 no. 18, 6578- 6583).
  • the nucleic acid sequence recognition module is a CRISPR-Cas system
  • the nucleic acid sequence recognition module and the epigenome modifying enzyme form a complex via the tag domain and the binding partner
  • the nucleic acid sequence recognition module Preferably, the Cas protein is linked to said tag domain.
  • Sequence information of the proteins, fusion proteins, or nucleic acids (eg, DNA or RNA) encoding them described herein can be obtained from Protein Data Bank, UniPort, GenBank, or the like.
  • the nucleic acid sequence of RNA can also be obtained from the corresponding DNA nucleic acid sequence by using sequence conversion software or the like as appropriate.
  • the DNA encoding the nucleic acid sequence recognition module and the DNA encoding the epigenome modifying enzyme described herein may be obtained by cloning from mRNA by molecular biological methods, or based on the sequence information, It may be obtained by chemically synthesizing DNA. In obtaining the DNA, codon optimization may be performed according to the animal or plant (host) into which the nucleic acid is to be introduced.
  • the nucleic acid can be expected to, for example, increase the protein expression level in the host.
  • Data on the frequency of codon usage in the host to be used can be obtained, for example, from the genetic code usage frequency database (http://www.kazusa.or.jp/codon/index. html), or refer to the literature describing the codon usage in each host.
  • the nucleic acids encoding the nucleic acid sequence recognition module and the epigenome modifying enzyme are configured such that the nucleic acid sequence recognition module and the epigenome modifying enzyme are induced to be expressed in the gametogenesis.
  • the nucleic acid sequence recognition module and the epigenome modifying enzyme form a complex, bind to the nucleic acid sequence of the target region in the genomic DNA of the forming gamete, and form the epigenome of the target region. can be modified.
  • the nucleic acid is preferably constructed so that the expression-inducing timing of the nucleic acid sequence recognition module and the expression-inducing timing of the epigenome-modifying enzyme overlap in the gametogenesis.
  • the nucleic acid may be configured such that both the nucleic acid sequence recognition module and the epigenome-modifying enzyme are expressed at overlapping times during gametogenesis. Further, the nucleic acid is configured such that one of the nucleic acid sequence recognition module and the epigenome modifying enzyme is expressed during the gametogenesis and at a stage other than the gametogenesis, and the other is expressed during the gametogenesis. It may be configured as In addition, the nucleic acid encoding the nucleic acid sequence recognition module and the epigenome modifying enzyme is constantly expressed, and suppresses the expression of the nucleic acid sequence recognition module and/or the epigenome modifying enzyme at a time other than the gametogenesis. It may be configured to induce expression in gametogenesis by inducing the expression of a molecule (eg, siRNA, miRNA, shRNA, etc.).
  • a molecule eg, siRNA, miRNA, shRNA, etc.
  • the timing of the expression induction in the gametogenesis can be, for example, any timing until the precursor cells of the gametes differentiate into gametes.
  • the time of expression induction in the gametogenesis is, for example, the time from primordial germ cells to spermatogonia and spermatocytes to differentiate into sperm.
  • the time of expression induction in the gametogenesis is, for example, the time from primordial germ cells to oogonia and oocyte differentiation to ova.
  • the time of expression induction in gametogenesis is, for example, the time from pollen oocytes to differentiation into pollen.
  • the time of expression induction in the gametogenesis is, for example, the time from the embryo sac oocyte to the embryo sac.
  • the expression timing of the nucleic acid sequence recognition module and the epigenome modifying enzyme can be regulated, for example, by functionally linking a timing-specific promoter to the nucleic acids encoding them.
  • a timing-specific promoter to the nucleic acids encoding them.
  • the control of the expression time activates a gametogenesis-specific promoter that is activated in the gametogenesis.
  • the gametogenesis-specific promoters include spermatogenesis-specific promoters, oogenesis-specific promoters, pollen formation-specific promoters, germ sac formation-specific promoters, and the like.
  • the gametogenesis-specific promoter includes spermatogenesis-specific promoters, and specific examples thereof include STRA8 promoter, Pgk2 promoter, Dazl promoter, Gsg2 promoter and the like. Each promoter means the promoter sequence of each gene, and as a specific example, the STRA8 promoter is the promoter of the STRA8 gene.
  • the gametogenesis-specific promoter includes oogenesis-specific promoters, and specific examples thereof include Gdf-9 promoter, Zp3 promoter, Msx2 promoter and the like.
  • ⁇ Sperm-specific promoter Stra8 Patricia I Sadate-Ngatchou et.al., “Cre recombinase activity specific to postnatal, premeiotic male germ cells in transgenic mice”, Genesis, 2008 Dec;46(12):738-42
  • Pgk2 Tatsuo Kido et.al., "The testicular fatty acid binding protein PERF15 regulates the fate of germ cells in PERF15 transgenic mice", Dev Growth Differ, 2005 Jan;47(1):15-24.
  • Dazl Cory R Nicholas et.al., “Characterization of a Dazl-GFP germ cell-specific reporter”, Genesis, 2009 Feb;47(2):74-84 GSG2(haspin): Keizo Tokuhiro et al., “The 193-base pair Gsg2 (haspin) promoter region regulates germ cell-specific expression bidirectionally and synchronously”, Biol Reprod, 2007 Mar;76(3):407-14 ⁇ Egg-specific promoter Gdf-9: Zi-Jian Lan et.al., "Differential oocyte-specific expression of Cre recombinase activity in GDF-9-iCre, Zp3cre, and Msx2Cre transgenic mice", Biol Reprod, 2004 Nov;71(5):1469 -74 Zp3: M Lewandoski et.al., "Zp3-cre, a transgenic mouse line for the activation or inactivation of loxP-flanked target genes specifically in the female germ line
  • the expression stage can be regulated using, for example, a promoter whose expression is constantly induced.
  • a promoter whose expression is constantly induced.
  • the promoter include CAG promoter, SR ⁇ promoter, SV50 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) promoter, HSV-TK. (herpes simplex virus thymidine kinase) promoter and the like.
  • the nucleic acid is preferably inserted into an expression vector.
  • the nucleic acid encoding the nucleic acid sequence recognition module and the nucleic acid encoding the epigenome modifying enzyme are respectively linked to the expression vector so that the nucleic acid sequence recognition module and the epigenome modifying enzyme can be expressed.
  • the expression vector can be prepared, for example, by inserting a nucleic acid encoding the nucleic acid sequence recognition module and/or a nucleic acid encoding the epigenome modifying enzyme into a backbone vector (hereinafter also referred to as a "basic vector"). .
  • the nucleic acid encoding the nucleic acid sequence recognition module and the nucleic acid encoding the epigenome modifying enzyme may be linked to the same expression vector or may be linked to different vectors.
  • the expression vector is the first expression vector.
  • a second expression vector wherein the first expression vector is operably linked to the first nucleic acid so that the nucleic acid sequence recognition module can be expressed, and the second expression vector comprises:
  • the second nucleic acid is operably linked so that the epigenome modifying enzyme can be expressed.
  • the nucleic acid sequence recognition module is composed of a plurality of elements, for example, when using the CRISPR-Cas system, part or all of each element may be linked to different expression vectors.
  • the basic vector can be appropriately selected according to the animal or plant that uses the composition of the present invention, that is, the host.
  • the expression vector include non-viral vectors such as plasmid vectors and viral vectors.
  • Plasmid vectors for animals include, for example, pCDM8, pMT2PC, pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo and the like.
  • Examples of plasmid vectors for plants include vectors containing T-DNA, and specific examples thereof include pGEM-T and the like.
  • the viral vectors include viral vectors such as retroviruses, vaccinia viruses, and adenoviruses.
  • the expression vector preferably has regulatory sequences that regulate the expression of the nucleic acid sequence recognition module and the epigenome modifying enzyme.
  • the regulatory sequences include, for example, promoters, terminators, enhancers, polyadenylation signal sequences, replication origin sequences (ori) and the like. Arrangement of the regulatory sequence in the expression vector is not particularly limited as long as it is arranged so that the expression of the nucleic acid sequence recognition module and/or the epigenome modifying enzyme can be functionally regulated. can be placed
  • the regulatory sequence for example, a sequence provided in advance in the basic vector may be used, the regulatory sequence may be further inserted into the basic vector, or the regulatory sequence provided in the basic vector may be may be replaced by the regulatory sequences of
  • the expression vector may, for example, further have a coding sequence for a selectable marker.
  • selectable marker include drug resistance markers, fluorescent protein markers, enzyme markers, cell surface receptor markers and the like.
  • Insertion of nucleic acid (DNA), insertion of the regulatory sequence, and/or insertion of the coding sequence of the selectable marker into the expression vector may be performed by, for example, a method using restriction enzymes and ligase, A commercially available kit or the like may be used.
  • composition of the present invention contains multiple expression vectors
  • the composition of the present invention can also be called, for example, a nucleic acid kit or an expression vector kit.
  • the kit may further include an instruction manual and the like, for example.
  • the present invention provides methods for producing parental line animals and plants that can be used to produce and/or maintain epigenome-modified animals and plants.
  • the production method of the present invention is a method of producing a parent line of an animal or plant with modified epigenome, and includes a step of introducing the composition of the present invention into a target animal or plant (introduction step). According to the first production method of the present invention, it is possible to produce parent line animals and plants that can be used for the production and/or maintenance of epigenome-modified animals and plants.
  • the introduction step by introducing the composition of the present invention, that is, the nucleic acid or an expression vector containing the same, into the target animal or plant, for example, the nucleic acid in the composition of the present invention, that is, the nucleic acid sequence recognition module and a step of producing a parent line animal or plant containing a nucleic acid encoding the epigenome-modifying enzyme.
  • the target animals and plants are, for example, plant cells that form callus and the like and can develop into plant individuals; animal cells that can develop into animal individuals such as fertilized eggs and animal embryos (embryos, blastocysts); etc.
  • the animals and plants exclude, for example, humans.
  • the introduction method in the introduction step can be implemented, for example, by a known method for producing transformants or genetically modified animals and plants.
  • the target animals and plants when introducing into cells such as plant cells such as plant callus; animal cells such as ES cells, fertilized eggs, and animal embryos (embryos, blastocysts); , for example, introduction methods using gene guns such as particle guns, calcium phosphate methods, polyethylene glycol methods, lipofection methods using liposomes, electroporation methods, ultrasonic nucleic acid introduction methods, DEAE-dextran methods, direct methods using micro glass tubes, etc.
  • gene guns such as particle guns, calcium phosphate methods, polyethylene glycol methods, lipofection methods using liposomes, electroporation methods, ultrasonic nucleic acid introduction methods, DEAE-dextran methods, direct methods using micro glass tubes, etc.
  • Examples thereof include an injection method, a microinjection method, a hydrodynamic method, a cationic liposome method, a method using an introduction aid, and a method via Agrobacterium.
  • Examples of the liposome include lipofectamine and cationic liposome, and examples of the introduction aid include atelocollagen, nanoparticles, and polymers.
  • the composition of the present invention may be introduced into the plant using the Agrobacterium method.
  • the introduction step is preferably carried out by an introduction method in which the nucleic acid in the composition of the present invention is integrated into the genomic DNA of the target animal or plant.
  • the nucleic acid of the composition of the present invention is introduced (inserted) into the genomic DNA of the target animal or plant by introducing the composition of the present invention into the target animal or plant. into (insert) a nucleic acid encoding the nucleic acid sequence recognition module and a nucleic acid encoding the epigenome modifying enzyme.
  • the introduction method for integration into the genomic DNA a method combining the aforementioned introduction method into cells with a method using homologous recombination or a method using genome editing technology can be used.
  • the introduction method may be performed by introducing the composition of the present invention into germ cells.
  • the introduction method for example, a method of introducing exogenous DNA into germ cells of mammals can be used (eg, Gordon, et al., PNAS, 77: 7380-84 (1980); Gordon and Ruddle , Science, 214:1244-46 (1981); Palmiter and Brinster, Cell, 41:343-45 (1985); Brinster, et al., PNAS, 82:4438-42 (1985), etc.).
  • a method of introducing exogenous DNA into germ cells of mammals can be used (eg, Gordon, et al., PNAS, 77: 7380-84 (1980); Gordon and Ruddle , Science, 214:1244-46 (1981); Palmiter and Brinster, Cell, 41:343-45 (1985); Brinster, et al., PNAS, 82:4438-42 (1985), etc.).
  • RNA encoding Cas protein such as Cas9 RNA or Cas protein such as Cas9 protein and guide RNA are introduced into germ cells together with foreign DNA.
  • Cas protein such as Cas9 RNA
  • Cas protein such as Cas9 protein and guide RNA
  • the first production method of the present invention includes, after the introducing step, the plant cell or the animal It is preferable to include the step of generating plant or animal individuals from cells.
  • the method for generating the plant individual can be carried out, for example, by a known method of forming shoots from callus and regenerating the plant individual.
  • the animal individual in the method for generating the animal individual, for example, the animal individual can be obtained as a litter by transplanting the animal cell into a pseudopregnant mother to develop and give birth.
  • the first production method of the present invention may include, after the introducing step, the step of selecting animals and plants in which the nucleic acid encoding the nucleic acid sequence recognition module and the nucleic acid encoding the epigenome modifying enzyme have been integrated into their genomic DNA. .
  • the selection is performed, for example, by decoding the genomic DNA of the animal or plant after the introduction step, examining whether the nucleic acid encoding the nucleic acid sequence recognition module and the nucleic acid encoding the epigenome modifying enzyme is included, and selecting.
  • the genomic DNA of animals and plants after the introduction step may be detected and selected using primers and/or probes for the nucleic acid encoding the nucleic acid sequence recognition module and the nucleic acid encoding the epigenome modifying enzyme. and may be selected using selectable markers.
  • the present invention provides parental strains of animals and plants that can be used for the production and/or maintenance of epigenome-modified animals and plants.
  • the parental line of animals and plants of the present invention is a parental line of animals and plants for production or maintenance of epigenome-modified animals and plants, wherein the animals and plants contain exogenous nucleic acids, and the nucleic acids modify the epigenome in genomic DNA.
  • a nucleic acid encoding a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence of a target region, and an epigenome-modifying enzyme capable of forming a complex with the nucleic acid sequence recognition module and capable of modifying the epigenome
  • the nucleic acid is configured such that the sequence recognition module and the epigenome modifying enzyme are induced to be expressed in the gametogenesis, and the nucleic acid sequence recognition module and the epigenome modifying enzyme whose expression is induced in the gametogenesis form a complex to modify the epigenome of target regions in the genomic DNA of forming gametes.
  • the parent line used for producing the animal or plant in which the epigenome of the target region is modified and the epigenome of the target region are used according to the type of gamete whose epigenome is modified. can produce a parental line used for maintenance as a parental line of animals and plants that have been modified.
  • Said exogenous nucleic acid is, for example, derived from the nucleic acid of said composition of the invention.
  • the exogenous nucleic acid is preferably inserted into the genomic DNA of the parent strain because it allows the parent strain to be stably maintained.
  • the gamete whose epigenome is modified is a sperm
  • modification of the epigenome of the target region occurs in spermatogenesis of the male parent line. Therefore, when the gamete whose epigenome is modified is a sperm, the parent line used for producing the epigenome-modified animal or plant is a male parent line, and the line used for maintaining the parent line as an animal or plant is Female parent line.
  • the epigenome of the target region is modified in cells at the stage of differentiation from primordial germ cells to sperm, but differentiation from the primordial germ cell to sperm constituting the male parent line
  • the epigenome of the target region is not modified in cells other than the staged cells.
  • the cells that make up the female parental line are not epigenome-modified in the target region.
  • the exogenous nucleic acid is preferably operably linked to a spermatogenesis-specific promoter.
  • the parent line used for producing the animal or plant with the epigenome modified is a female parent line, and the line used for maintaining the parent line as an animal or plant is It is the male parent line.
  • the female parent line for example, in cells at the stage of differentiation from primordial germ cells to eggs, the epigenome of the target region is modified.
  • the epigenome of the target region is not modified in cells other than cells at the stage of differentiation.
  • the cells that make up the male parental line are not epigenome modified in the target region.
  • the exogenous nucleic acid is preferably operably linked to a spermatogenesis-specific promoter.
  • the parent line of animals and plants includes a chromosome into which the exogenous nucleic acid is integrated and the exogenous nucleic acid into which the exogenous nucleic acid is integrated. It is preferred to have a wild-type chromosome with no
  • the present invention provides gametes that can be used to produce epigenome-modified animals and plants and/or to produce parental lines for maintenance.
  • Gametes of the present invention are gametes isolated from plants and animals of the parent line of the present invention. According to the gamete of the present invention, it is possible to produce animals and plants in which the epigenome of the target region is modified, and animals and plants of the parent line for production or maintenance of animals and plants in which the epigenome is modified.
  • the gametes can be isolated, for example, from plants and animals of the parent line.
  • the isolation method can be appropriately set according to, for example, the type of gamete.
  • Said gametes may, for example, be stored after isolation from said parent line of animals or plants.
  • the method of preservation can be appropriately selected according to the type of gamete, and examples thereof include preservation under liquid nitrogen.
  • the gamete preferably has a modified epigenome in the target region.
  • the nucleic acid sequence recognition module whose expression is induced and the epigenome modifying enzyme form a complex in the gamete formation, and the epigenome of the target region in the genomic DNA of the gamete being formed. can be isolated from the modified parental line of plants and animals.
  • the gamete When the gamete is used to produce an animal or plant in which the epigenome of the target region is modified, the gamete may or may not contain the exogenous nucleic acid, but preferably does not contain it. In addition, when the gametes are used for the production of the epigenome-modified animals and plants or the production of the maintenance parent line animals and plants, the gametes contain the exogenous nucleic acid.
  • the present invention provides a method for producing an animal or plant in which the epigenome of the target region is modified.
  • the production method of the present invention is a method for producing an animal or plant in which the epigenome of the target region is modified, wherein the first parent and the second parent are crossed, and the obtained progeny individual is an individual whose epigenome is modified. (crossbreeding step), wherein the first parent and/or the second parent are plants and animals of the parent line of the present invention.
  • animals and plants in which the epigenome of the target region is modified can be produced.
  • one of the first parent and the second parent may be the animal or plant of the parent line of the present invention, or both may be the animal or plant of the parent line of the present invention.
  • the one parent an animal or plant of the parent line of the present invention
  • in the second production method of the present invention in the genomic DNA, the epigenome of the target region in one chromosome of the set of chromosomes is modified. plants or animals in which the epigenome of the target region of one chromosome (eg, the Y chromosome) has been modified.
  • the epigenome of the target region in both chromosomes of one set of chromosomes is modified in the genomic DNA. animals and plants can be obtained.
  • gametes of the first parent and/or the second parent may be used instead of the animal or plant individual.
  • the crossbreeding of the first parent and the second parent can be carried out by a known method.
  • the second production method of the present invention individuals that do not contain the exogenous nucleic acid may be selected from the progeny individuals.
  • the second production method of the present invention is similar to animals and plants in which the epigenome of the target region is not modified except that the epigenome of the target region is modified, and which does not have the exogenous nucleic acid. individual can be obtained.
  • the exogenous nucleic acid can be detected, for example, by detecting the nucleic acid encoding the nucleic acid sequence recognition module and/or the nucleic acid encoding the epigenome modifying enzyme.
  • the invention provides plants and animals in which the epigenome of the target region is modified.
  • the first animal or plant of the present invention is an animal or plant in which the epigenome of the target region is modified, and the animal or plant contains, as the target region, the target region in which the epigenome derived from the gamete of the present invention is modified, It does not contain said exogenous nucleic acid.
  • the first animals and plants of the present invention are animals and plants in which the epigenome of the target region is not modified except that the epigenome of the target region is modified and which does not have the exogenous nucleic acid (wild-type animals and plants). They are similar individuals. Therefore, according to the first animal and plant of the present invention, for example, by comparing with the wild-type animal and plant, it can be suitably used for functional analysis of the epigenome in the target region.
  • the first animal or plant of the present invention is such that the epigenome of the target region in the cells constituting the first animal or plant is the epigenome of a wild-type animal or plant. different.
  • the second animal or plant of the present invention is an animal or plant in which the epigenome of the target region is modified, the animal or plant contains an exogenous nucleic acid, the nucleic acid is the nucleic acid sequence of the target region in which the epigenome is modified in the genomic DNA.
  • the sequence recognition module and the epigenome modifying enzyme are configured to be induced to be expressed in the gametogenesis by being functionally linked to a gametogenesis-specific promoter that is activated in the animal and plant
  • the target region includes a target region derived from a gamete in which the epigenome of the target region is modified by forming a complex between the nucleic acid sequence recognition module whose expression is induced in the gametogenesis and the epigenome modifying enzyme.
  • the second animal or plant of the present invention is an individual similar to the animal or plant having the exogenous nucleic acid, in which the epigenome of the target region is not modified except that the epigenome of the target region is modified. Therefore, according to the second animal or plant of the present invention, for example, it can be suitably used for the functional analysis of the epigenome in the target region by comparing with the animal or plant of the parent line used for the maintenance.
  • Example 1 Using the composition of the present invention, it is possible to produce a parent line of animals and plants that can be used for the production and maintenance of animals and plants with modified epigenomes, and to obtain offspring lines in which the target region of the epigenome is modified from the parent line. confirmed.
  • Target region Igf2 gene is an imprinting gene, and in chromosomes derived from male parents, the DMR region (H19-DMR) of the Igf2 gene is methylated, whereas in chromosomes derived from female parents is unmethylated in the DMR region (H19-DMR) of the Igf2 gene.
  • systemic demethylation of H19-DMR causes abnormal methylation of the DMR region of the mouse Igf2 gene, that is, H19-DMR in male parent-derived chromosomes. It has been separately confirmed that demethylation of the DMR renders the mice stunted and unable to reproduce, symptoms of Silver-Russell syndrome. Therefore, the DMR region of the Igf2 gene was selected as a model for the target region.
  • Example 2 Vector construction The expression vector used in Example 1 was constructed from the all-in-one epigenome editing vector (pPlatTET-gRNA2-H19DMRx9) of Reference 1 below.
  • the epigenome editing vector of Reference Document 1 5 copies of the GCN4 peptide (ELLSKNYHLENEVARLKK (SEQ ID NO: 9)) are linked between each peptide via a 22 amino acid linker (GSGSGGGSGSGSGGGSGSGGSGSG: SEQ ID NO: 10) to form a tag domain (SunTag). and the tag domain is linked to dCas9 to form a fusion protein.
  • an anti-GCN4 single-chain antibody (scFv) is linked to the catalytic site (TET1CD) of the TET1 protein via sfGFP (superfolder green fluorescent protein) to form a fusion protein.
  • scFv anti-GCN4 single-chain antibody
  • TET1CD catalytic site of the TET1 protein
  • sfGFP superfolder green fluorescent protein
  • Example 3(B) the nucleic acid encoding these fusion proteins and the nucleic acid encoding sgRNA are arranged consecutively downstream of the CAG promoter. Therefore, in Example 1, the CAG promoter was replaced with the promoter of the Stra8 gene, which is specifically expressed during spermatogenesis, and used as the epigenome editing vector (pStrPlATTET-gRNA2-H19DMRx9) of the Examples.
  • the epigenome-editing vectors of the above Examples were used after being linearized with a restriction enzyme (ApaLI) prior to microinjection, which will be described later.
  • the recovered zygotes were treated with M2 medium (manufactured by Sigma-Aldrich) containing 0.1% hyaluronidase (manufactured by Sigma-Aldrich) for several minutes, and then the obtained zygotes were washed with the M2 medium.
  • the washed fertilized eggs were transferred into drops of M16 medium containing penicillin and streptomycin (manufactured by Sigma-Aldrich) at 37°C.
  • the linearized epigenome editing vector of the example was introduced into the fertilized egg. Specifically, by microinjection, the linearized epigenome editing vector (35 ng/ ⁇ l) of Example 1 (2) was injected into the pronucleus of a fertilized egg in the M16 medium of Example 1 (3). injected into. The injected embryos were cultured in air at 37° C., 5% CO 2 using M16 medium. The next day, the embryos that had developed to the 2-cell stage were transplanted into the oviduct ampulla of pseudopregnant female ICR mice (purchased from CLEA Japan). The number of embryos transferred was 20 to 25 per oviduct.
  • genomic DNA was extracted from the tip of the tail of the mouse using a genomic DNA extraction kit (DirectPCR Lysis Reagent, Mouse Tail, manufactured by Viagenbiotech). The obtained genomic DNA was subjected to PCR using the primer sets shown in Table 2 below, and it was examined whether an amplified fragment derived from the epigenome editing vector of the above example could be obtained. As a result, it was confirmed that the epigenome editing vector of the above example was inserted into the genomic DNA in 9 of the 41 individuals.
  • a genomic DNA extraction kit DirectPCR Lysis Reagent, Mouse Tail, manufactured by Viagenbiotech
  • mice in which the insertion of the epigenome editing vector of the above example was confirmed By backcrossing the mice in which the insertion of the epigenome editing vector of the above example was confirmed to B6 mice, three fertile recombinant mouse strains (441-2, 445-7, 445-14) and their sublines was established. Newborn mice of each subline were weighed. In addition, mouse-derived sperm from the subline was collected and frozen at -80°C until DNA extraction.
  • the frozen sperm pellet was treated with an RSB solution containing sodium dodecyl sulfate (final concentration 2%), 2-mercaptoethanol (final concentration 2%) and proteinase K (final concentration 1 mg/ml). was suspended using The composition of the RSB solution was 10 mmol/l NaCl, 10 mmol/l Tris (pH 7.5), and 25 mmol/l EDTA. After the suspension, the suspension was incubated at 56° C. overnight (about 8 hours), extracted with phenol/chloroform, and precipitated with ethanol to isolate DNA.
  • the isolated DNA was treated using a bisulfite treatment kit (Epitect Plus DNA Bisulfite Kit, manufactured by QIAGEN). Then, the treated DNA was amplified by PCR using the primer set shown in Table 3 below. Demethylation rate of CpG sites was determined by combined bisulfite restriction analysis (COBRA). Specifically, the amplified fragments obtained by PCR were cleaved with the restriction enzymes shown in Table 3 below. The sites (CpG sites) recognized by each restriction enzyme are shown in Table 3 below. Separation and quantification of the PCR-amplified fragments were performed using a capillary microchip electrophoresis device (MCE-202 MultiNA, manufactured by Shimadzu Corporation). The methylation rate was calculated by the following formula (1).
  • FIG. 4 is a graph showing the methylation rate in mouse-derived sperm of the subline.
  • the horizontal axis indicates the mouse strain, and the vertical axis indicates the methylation rate.
  • sperm-specific epigenome-altered mice had significantly reduced methylation in sperm compared to wild-type mice. Therefore, it was confirmed that introduction of the composition of the present invention enables gamete-specific modification of the epigenome of the target region.
  • mice of the above sublines were used as male parent strains and crossed with wild-type B6 mice to obtain F1 progeny individuals.
  • the female mouse of the subline was used as a female parent strain, and crossed with a wild-type B6 mouse to obtain an F1 progeny individual.
  • the epigenome editing vector was inserted into the genomic DNA.
  • genomic DNA was extracted using the F1 progeny newborn mouse and a DNA extraction kit (AllPrep DNA/RNA Mini Kit, manufactured by QIAGEN).
  • the methylation rate was measured in the same manner as in Example 1 (5) above, except that the genomic DNA was used instead of the isolated DNA. These results are shown in FIGS. 5-8.
  • Fig. 5 is a graph showing the body weight of F1 progeny individuals derived from the male parent line.
  • the horizontal axis indicates the mouse strain, and the vertical axis indicates the body weight.
  • progeny individuals of sperm-specifically modified epigenome mice lost weight compared to wild-type mice. That is, it was presumed that the modification of the epigenome causes developmental delay, similar to Silver-Russell syndrome.
  • FIG. 6 is a graph showing the body weight of the F1 progeny individuals derived from the male parent line or the female parent line.
  • the horizontal axis indicates the mouse strain
  • the vertical axis indicates the body weight.
  • F1 progeny individuals from the female parental line exhibited similar body weights to wild-type mice.
  • F1 progeny derived from male parental strains had significantly decreased body weight compared to F1 progeny derived from wild-type mice and female parental strains. That is, in the female parent line, the spermatogenesis-specific promoter does not operate, the epigenome is not modified, and weight loss does not occur, whereas in the male parent line, the spermatogenesis-specific promoter operates. However, it was presumed that the epigenome was altered and developmental delay occurred as in Silver-Russell syndrome.
  • FIG. 7 is a graph showing the methylation rate of the target region of F1 progeny individuals derived from the male or female parent line.
  • (A) shows the results for the CpG site (m2)
  • (B) shows the results for the CpG site (m3).
  • the horizontal axis indicates the mouse strain
  • the vertical axis indicates the methylation rate.
  • F1 progeny individuals derived from the female parental line showed a methylation rate comparable to that of wild-type mice.
  • the F1 progeny derived from the male parent strain had a significantly decreased methylation rate compared to the F1 progeny derived from the wild-type mouse and the female parent strain. That is, in the female parent line, the spermatogenesis-specific promoter does not operate, the epigenome is not modified, and methylation does not change, whereas in the male parent line, the spermatogenesis-specific promoter operates. However, it was confirmed that the epigenome of the target region was modified by being targeted after demethylating enzyme was induced.
  • FIG. 8 is a graph showing the methylation rate and body weight with and without epigenome editing vector insertion.
  • (A) shows the results of the methylation rate of the CpG site (m3)
  • (B) shows the results of the body weight of the newborn.
  • the horizontal axis indicates the presence or absence of insertion of the mouse strain or epigenome editing vector into the genomic DNA
  • the vertical axis indicates the methylation rate.
  • the horizontal axis indicates the presence or absence of insertion of the mouse strain or epigenome editing vector into the genomic DNA
  • the vertical axis indicates body weight. As shown in FIG.
  • the F1 progeny strain of the male parent strain has a lower methylation rate than the wild-type mouse. rice field.
  • the F1 progeny strain of the male parent strain has decreased body weight compared to the wild-type mouse. rice field. From these results, if epigenome modification occurs during gametogenesis, then even if there is no epigenome editing vector, that is, the sequence recognition module dCas9 and sgRNA, and the epigenome modification enzyme TET1CD It was found that epigenome modifications were maintained even without induction.
  • the epigenome editing vector can be passaged by backcrossing to B6 mice, it can be said that the mouse in which the epigenome editing vector has been introduced into the genomic DNA can also be used as a parental strain for strain maintenance. .
  • composition of the present invention can be used to produce a parent line of animals and plants that can be used for the production and maintenance of epigenome-modified animals and plants, and a offspring line in which the target region of the epigenome is modified from the parent line. was found to be obtained.
  • the nucleic acid is a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence of a target region that modifies the epigenome in genomic DNA; an epigenome modifying enzyme capable of forming a complex with the nucleic acid sequence recognition module and modifying the epigenome; comprising a nucleic acid encoding
  • the nucleic acid is operably linked to a gametogenesis-specific promoter that is activated in gametogenesis so that the sequence recognition module and the epigenome modifying enzyme are induced to be expressed in the gametogenesis.
  • the nucleic acid comprises a first nucleic acid and a second nucleic acid;
  • the first nucleic acid is a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence of a target region that modifies the epigenome in genomic DNA; a tag domain linked to the nucleic acid sequence recognition module; comprising a nucleic acid encoding
  • the second nucleic acid is an epigenome-modifying enzyme capable of modifying the epigenome; a tag domain binding partner linked to the epigenome modifying enzyme and capable of binding to the tag domain; comprising a nucleic acid encoding
  • the first nucleic acid and/or the second nucleic acid are operably linked to a gametogenesis-specific promoter that is activated in gametogenesis
  • the nucleic acid sequence recognition module is the CRISPR-Cas system;
  • the CRISPR-Cas system is a guide strand comprising a nucleic acid sequence that specifically binds to a nucleic acid sequence in the target region; a Cas protein; including 5.
  • the nucleic acid sequence recognition module is the CRISPR-Cas system;
  • the CRISPR-Cas system is a guide strand comprising a nucleic acid sequence that specifically binds to a nucleic acid sequence in the target region; a Cas protein; 5.
  • (Appendix 8) 8.
  • a composition according to any one of Appendices 1 to 7, wherein said target region is the control region and/or the promoter region of an imprinted gene. (Appendix 9) 9.
  • Appendix 10 Appendices 1 to 9, wherein the epigenome-modifying enzyme is a methylase, demethylase, acetylase, deacetylase, phosphorylation enzyme, dephosphorylation enzyme, ubiquitinase, and/or sumoylation enzyme
  • composition according to any one of Appendices 1 to 10 wherein the epigenome-modifying enzyme is TET (ten-eleven translocation) 1, TET2, TET3, DNMT (DNA Methyltransferase) 1, DNMT3A, and/or DNMT3B.
  • TET ten-eleven translocation
  • DNMT DNA Methyltransferase
  • Appendix 12 12.
  • Appendix 13 13.
  • (Appendix 14) 13 The composition according to any one of paragraphs 1 to 12, wherein said gametogenesis-specific promoter is Gdf-9 promoter, Zp3 promoter and/or Msx2 promoter.
  • (Appendix 15) comprising an expression vector; 15.
  • (Appendix 16) comprising a first expression vector and a second expression vector; The first expression vector is functionally linked to the nucleic acid so that the nucleic acid sequence recognition module can be expressed, 16.
  • ⁇ Production method of parent line> (Appendix 17) A method for producing an epigenome-modified animal or plant parent line, comprising: 17. A production method, comprising the step of introducing the composition according to any one of Appendixes 1 to 16 into a target animal or plant. (Appendix 18) 18. The production method according to Appendix 17, wherein the animal or plant is a non-human animal.
  • the nucleic acid is a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence of a target region that modifies the epigenome in genomic DNA; an epigenome-modifying enzyme capable of forming a complex with the nucleic acid sequence recognition module and capable of modifying the epigenome; comprising a nucleic acid encoding
  • the nucleic acid is operably linked to a gametogenesis-specific promoter that is activated in gametogenesis so that the sequence recognition module and the epigenome modifying enzyme are induced to be expressed in the gametogenesis.
  • the nucleic acid sequence recognition module whose expression is induced and the epigenome modifying enzyme form a complex to modify the epigenome of the target region in the genomic DNA of the gamete being formed.
  • the nucleic acid comprises a first nucleic acid and a second nucleic acid;
  • the first nucleic acid is a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence of a target region that modifies the epigenome in genomic DNA; a tag domain linked to the nucleic acid sequence recognition module; comprising a nucleic acid encoding
  • the second nucleic acid is an epigenome-modifying enzyme capable of modifying the epigenome; a tag domain binding partner linked to the epigenome modifying enzyme and capable of binding to the tag domain; comprising a nucleic acid encoding
  • the first nucleic acid and/or the second nucleic acid are operably linked to a gametogenesis-specific promoter that is activated in gametogenesis so that the nucleic acid sequence recognition module and the epigenome modifying enzyme are , configured to be induced to be expressed in gametogenesis, In the gametogenesis, the nucleic acid sequence recognition module whose expression is induced and the epigenome
  • the nucleic acid sequence recognition module is the CRISPR-Cas system;
  • the CRISPR-Cas system is a guide strand comprising a nucleic acid sequence that specifically binds to a nucleic acid sequence in the target region; a Cas protein; including 23.
  • the nucleic acid sequence recognition module is the CRISPR-Cas system;
  • the CRISPR-Cas system is a guide strand comprising a nucleic acid sequence that specifically binds to a nucleic acid sequence in the target region; a Cas protein; 24.
  • (Appendix 27) 27 The animal or plant according to any one of Appendices 19 to 26, wherein the epigenome-modifying enzyme is a base-modifying enzyme.
  • Appendix 28 Appendices 19 to 27, wherein the epigenome-modifying enzyme is a methylase, demethylase, acetylase, deacetylase, phosphorylation enzyme, dephosphorylation enzyme, ubiquitinase, and/or sumoylation enzyme Animals and plants according to any one of (Appendix 29) 29.
  • the epigenome-modifying enzyme is TET (ten-eleven translocation) 1, TET2, TET3, DNMT (DNA Methyltransferase) 1, DNMT3A, and/or DNMT3B.
  • the gametogenesis-specific promoter is a spermatogenesis-specific promoter or an oogenesis-specific promoter.
  • Appendix 33 comprising an expression vector; 33.
  • Appendix 34 comprising a first expression vector and a second expression vector;
  • the first expression vector is functionally linked to the nucleic acid so that the nucleic acid sequence recognition module can be expressed, 34.
  • Appendix 35 35.
  • Appendix 36 36.
  • the gametogenesis-specific promoter is a spermatogenesis-specific promoter;
  • the parent line used for the production is a male parent line, 37.
  • the animal or plant according to any one of Appendices 19 to 36, wherein the parental line used for maintenance is a female parental line.
  • the gametogenesis-specific promoter is an oogenesis-specific promoter,
  • the parent line used for the production is a female parent line, 37.
  • the animal or plant according to any one of Appendices 19 to 36, wherein the parental line used for maintenance is a male parental line.
  • Gametes used for the production of epigenome-modified animals and plants or the production of parent line animals and plants for maintenance, 40.
  • Appendix 41 41.
  • Appendix 42 42.
  • Appendix 44 A method for producing an animal or plant in which the epigenome of the target region is modified, crossing the first parent and the second parent and obtaining an epigenome-altered individual from the resulting progeny individual; The production method, wherein the first parent and/or the second parent are plants and animals of the parent line according to any one of Appendices 19 to 39. (Appendix 45) 45.
  • the production method according to Appendix 44 comprising the step of selecting individuals that do not contain the nucleic acid from the progeny individuals.
  • Appendix 46 An animal or plant in which the epigenome of the target region is modified, The animals and plants are The target region comprises a target region in which the gamete-derived epigenome according to any one of Appendices 40 to 42 is modified, does not contain the exogenous nucleic acid; flora and fauna.
  • Appendix 47 The animal or plant according to appendix 46, which is obtained by the production method according to appendix 44 or 45.
  • Appendix 48 48.
  • the animal or plant according to Appendix 46 or 47 wherein the animal or plant is a non-human animal.
  • the nucleic acid is a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence of a target region that modifies the epigenome in genomic DNA; an epigenome-modifying enzyme capable of forming a complex with the nucleic acid sequence recognition module and capable of modifying the epigenome; comprising a nucleic acid encoding
  • the nucleic acid is operably linked to a gametogenesis-specific promoter that is activated in gametogenesis so that the sequence recognition module and the epigenome modifying enzyme are induced to be expressed in the gametogenesis.
  • the animal or plant is derived from a gamete in which the epigenome of the target region is modified by forming a complex between the nucleic acid sequence recognition module whose expression is induced in the gametogenesis and the epigenome modifying enzyme as the target region. Animals and plants, including target areas.
  • the first nucleic acid is a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence in a target region to modify the epigenome in the genome; a tag domain linked to the nucleic acid sequence recognition module; comprising a nucleic acid encoding
  • the second nucleic acid is an epigenome-modifying enzyme capable of modifying the epigenome; a tag domain binding partner linked to the epigenome modifying enzyme and capable of binding to the tag domain; comprising a nucleic acid encoding said first nucleic acid and/or said second nucleic acid is operably linked to a gamete-specific promoter that is activated in gametogenesis;
  • the animal or plant is derived from a gamete in which the epigenome of the target region is modified by forming a complex between the nucleic acid sequence recognition module whose expression is induced in the gametogenesis and the epigenome modifying enzyme as the target region.
  • composition of the present invention it is possible to produce parent line animals and plants that are capable of producing and maintaining epigenome-modified animals and plants. Therefore, according to the present invention, it can be suitably used for analysis of diseases caused by changes in the epigenome. Therefore, the present invention is extremely useful in, for example, the fields of life science and medicine.

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Abstract

L'invention concerne une composition pour la production d'un animal ou d'une plante de lignée parentale permettant de produire et de maintenir un animal ou une plante génétiquement modifié.　Une composition selon la présente invention est utilisée dans la production d'un animal ou d'une plante de lignée parentale utilisé dans la production ou le maintien d'un animal ou d'une plante modifié par un épigénome : la composition contenant des acides nucléiques; les acides nucléiques contenant des acides nucléiques codant un module de reconnaissance de séquence d'acides nucléiques qui se lie spécifiquement à une séquence d'acides nucléiques dans une région cible, dans laquelle une modification d'épigénome est réalisée dans un ADN génomique, et une enzyme de modification d'épigénome pouvant modifier un épigénome; le module de reconnaissance de séquence et l'enzyme de modification d'épigénome pouvant former un complexe; les acides nucléiques étant conçus de telle sorte que, à la suite de la liaison fonctionnelle avec un promoteur spécifique de la gamétogenèse activé dans une gamétogenèse, les expressions du module de reconnaissance de séquence et de l'enzyme de modification d'épigénome sont induites dans la gamétogenèse; et, dans la gamétogenèse, le module de reconnaissance de séquence d'acide nucléique et l'enzyme de modification d'épigénome, dont les expressions ont été induites, forment un complexe et modifient un épigénome dans la région cible dans l'ADN génomique d'un gamète dans le processus de la gamétogenèse.
PCT/JP2022/017327 2021-05-20 2022-04-08 Composition utilisée dans la production d'une lignée parentale d'une plante ou d'un animal modifié par un épigénome et son utilisation WO2022244544A1 (fr)

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Citations (4)

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Publication number Priority date Publication date Assignee Title
JP2009532053A (ja) * 2006-04-07 2009-09-10 セルセントリック・リミテッド インヴィヴォでの核酸配列のエピジェネティック修飾用組成物および方法
WO2017090724A1 (fr) * 2015-11-25 2017-06-01 国立大学法人 群馬大学 Kit d'édition de méthylation d'adn et procédé d'édition de méthylation d'adn
JP2020132584A (ja) * 2019-02-21 2020-08-31 株式会社豊田中央研究所 エピゲノム状態を制御する融合タンパク質及びその利用
WO2021167101A1 (fr) * 2020-02-21 2021-08-26 国立大学法人群馬大学 Procédé pour induire une expression synergique d'un gène spécifique à l'aide de déméthylase et facteur associé à la transcription ou facteur associé à la chromatine

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Publication number Priority date Publication date Assignee Title
JP2009532053A (ja) * 2006-04-07 2009-09-10 セルセントリック・リミテッド インヴィヴォでの核酸配列のエピジェネティック修飾用組成物および方法
WO2017090724A1 (fr) * 2015-11-25 2017-06-01 国立大学法人 群馬大学 Kit d'édition de méthylation d'adn et procédé d'édition de méthylation d'adn
JP2020132584A (ja) * 2019-02-21 2020-08-31 株式会社豊田中央研究所 エピゲノム状態を制御する融合タンパク質及びその利用
WO2021167101A1 (fr) * 2020-02-21 2021-08-26 国立大学法人群馬大学 Procédé pour induire une expression synergique d'un gène spécifique à l'aide de déméthylase et facteur associé à la transcription ou facteur associé à la chromatine

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Title
PRATIKSHA I THAKORE, JOSHUA B BLACK, ISAAC B HILTON, CHARLES A GERSBACH: "Editing the epigenome: technologies for programmable transcription and epigenetic modulation", NATURE METHODS, NATURE PUBLISHING GROUP US, NEW YORK, vol. 13, no. 2, 1 February 2016 (2016-02-01), New York, pages 127 - 137, XP055623879, ISSN: 1548-7091, DOI: 10.1038/nmeth.3733 *

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