Compositions for use in producing parental lines of epigenome-modified animals and plants and uses thereof

Abstract

Provided is a composition for producing a parental-line animal / plant capable of producing and maintaining an epigenome-modified animal / plant.　A composition according to the present invention is employed in producing a parental-line animal / plant employed in producing or maintaining an epigenome-modified animal / plant, wherein: the composition contains nucleic acids; the nucleic acids contain nucleic acids coding for a nucleic-acid-sequence recognition module that specifically binds to a nucleic acid sequence in a target region, in which an epigenome modification is performed in a genomic DNA, and an epigenome modifying enzyme that is capable of modifying an epigenome; the sequence recognition module and the epigenome modifying enzyme can form a complex; the nucleic acids are configured so that, as a result of being functionally joined with a gametogenesis-specific promoter activated in a gametogenesis, the expressions of the sequence recognition module and the epigenome modifying enzyme are induced in the gametogenesis; and, in the gametogenesis, the nucleic-acid-sequence recognition module and the epigenome modifying enzyme, the expressions of which have been induced, form a complex and modify an epigenome in the target region in the genomic DNA of a gamete in the process of the gametogenesis.

Description

[Technical Field]

[0001] The present invention relates to compositions for use in producing parental lines of epigenome-modified animals and plants, and uses thereof. [Background technology]

[0002] Gene expression in animals and plants is epigenetically modified. Examples of such epigenetic modifications include methylation and demethylation of genomic DNA, and acetylation, deacetylation, phosphorylation, dephosphorylation, ubiquitination, and sumoylation of histones. It has been revealed that each modification exerts different controls on gene expression (Non-Patent Document 1).

[0003] In recent years, causative genes for various diseases have been identified by using genetic engineering techniques to generate genetically engineered mice. On the other hand, it has also been suggested that changes in epigenetic modifications may be the cause of the diseases. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Allis, CD et al., “Epigenetics”, Cold Spring Harbor Lab. Press, (2015) Summary of the Invention [Problem to be solved by the invention]

[0005] To investigate the role of epigenetic modification in various diseases, the present inventors used the following method to generate mice with modified epigenetic modifications of genomic DNA, i.e., mice with modified epigenomes. Specifically, using dCas9, in which the nuclease activity of the Cas9 protein used in the CRISPR / Cas9 method was inactivated, the demethylase TET (ten-eleven translocation) 1 (TET1) was targeted to a target region, and the epigenome of the target region was modified. However, as with general genetically modified mice, when nucleic acids encoding dCas9 and TET1 were introduced into genomic DNA and systemic expression of dCas9 and TET1 was induced, mice with modified epigenomes in the target region were obtained, but depending on the target region, problems arose in that the resulting epigenetic modified mice exhibited poor growth, died before sexual maturity, and other problems made them difficult to maintain through breeding.

[0006] Therefore, an object of the present invention is to provide a composition for producing parental lines of animals and plants that can be used to produce and maintain animals and plants with modified epigenomes. [Means for solving the problem]

[0007] In order to achieve the above object, the composition of the present invention is a composition for use in producing an epigenome-modified animal or plant or a parent line for maintaining the epigenome, comprising: the composition comprises a nucleic acid; The nucleic acid is a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence in a target region of genomic DNA to be modified in the epigenome; an epigenome modification enzyme capable of forming a complex with the nucleic acid sequence recognition module and modifying the epigenome; and a nucleic acid encoding the nucleic acid is operably linked to a gametogenesis-specific promoter that is activated during gametogenesis, thereby configuring the sequence recognition module and the epigenome modification enzyme to be expressed and induced during gametogenesis; During gamete formation, the nucleic acid sequence recognition module whose expression is induced and the epigenome modification enzyme form a complex and modify the epigenome of the target region in the genomic DNA of the gamete being formed.

[0008] The production method of the present invention (hereinafter also referred to as "first production method") is a method for producing parent lines of epigenome-modified animals and plants, comprising the steps of: The method includes the step of introducing the composition of the present invention into a target animal or plant.

[0009] The parental strain of animals and plants of the present invention (hereinafter also referred to as "parental strain") is a parental strain of animals and plants used for producing or maintaining epigenome-modified animals and plants, The animal or plant contains an exogenous nucleic acid, The nucleic acid is a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence in a target region of genomic DNA to be modified in the epigenome; an epigenome modification enzyme capable of forming a complex with the nucleic acid sequence recognition module and modifying the epigenome; and a nucleic acid encoding the nucleic acid is operably linked to a gametogenesis-specific promoter that is activated during gametogenesis, thereby configuring the sequence recognition module and the epigenome modification enzyme to be expressed and induced during gametogenesis; During gamete formation, the nucleic acid sequence recognition module whose expression is induced and the epigenome modification enzyme form a complex and modify the epigenome of the target region in the genomic DNA of the gamete being formed.

[0010] The gametes of the present invention are gametes used to produce parental lineage animals or plants used in producing or maintaining epigenome-modified animals or plants, It has been isolated from the parental line of the present invention.

[0011] The production method of the present invention (hereinafter also referred to as "second production method") is a method for producing an animal or plant in which the epigenome of a target region is modified, comprising the steps of: The method includes a step of crossbreeding the first parent with the second parent and obtaining an individual having a modified epigenome from the resulting progeny individual, The first parent and / or the second parent are plants or animals of the parental lineage of the present invention.

[0012] The animal or plant of the present invention (hereinafter also referred to as "first animal or plant") is an animal or plant in which the epigenome of a target region has been modified, The animals and plants are The target region includes a target region in which the epigenome derived from the gamete of the present invention has been modified, It does not contain the exogenous nucleic acid.

[0013] The animals and plants of the present invention (hereinafter also referred to as "second animals and plants") are animals and plants in which the epigenome of a target region has been modified, The animal or plant contains an exogenous nucleic acid, The nucleic acid is a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence in a target region of genomic DNA to be modified in the epigenome; an epigenome modification enzyme capable of forming a complex with the nucleic acid sequence recognition module and modifying the epigenome; and a nucleic acid encoding the nucleic acid is operably linked to a gametogenesis-specific promoter that is activated during gametogenesis, thereby configuring the sequence recognition module and the epigenome modification enzyme to be expressed and induced during gametogenesis; The animal or plant includes, as the target region, 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 during gametogenesis and the epigenome modification enzyme. [Effects of the Invention]

[0014] The composition of the present invention enables the production of parental lines of animals and plants that can be used to produce and maintain animals and plants with modified epigenomes. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram showing an example of epigenetic changes in a parent line produced using the composition of the present invention. [Figure 2] FIG. 2 is a schematic diagram showing another example of epigenetic changes in parental lines produced using the composition of the present invention. [Figure 3] Figure 3 is a schematic diagram showing the structure of the epigenome editing vector in Example 1. [Figure 4] FIG. 4 is a graph showing the methylation rate in sperm derived from subline mice in Example 1. [Figure 5] FIG. 5 is a graph showing the body weights of F1 progeny individuals derived from the male parent line in Example 1. [Figure 6] FIG. 6 is a graph showing the body weights of F1 progeny individuals derived from male parent lines or female parent lines in Example 1. [Figure 7] FIG. 7 is a graph showing the methylation rates of target regions in F1 progeny individuals derived from male parent lines or female parent lines in Example 1. [Figure 8] Figure 8 is a graph showing the methylation rate and body weight in the presence or absence of insertion of the epigenome editing vector in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0016] <Definition> As used herein, the term "epigenome" refers to the state of chemical modifications of DNA and / or histone proteins in the genome without changes in the nucleic acid sequence (base sequence). The epigenome can also be referred to as, for example, epigenetic modification or genomic modification.

[0017] As used herein, "epigenome modification" or "epigenome modification" refers to 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 adding or removing a modification, changing the type of modification, and increasing or decreasing the frequency of modification. The "epigenome modification" or "epigenome modification" can also be referred to as, for example, "modification of epigenetic modification" or "modification of genome modification."

[0018] As used herein, "epigenome-modifying enzyme" refers to a protein that alters chemical modifications of DNA and / or histone proteins in the genome.

[0019] As used herein, "genomic DNA" refers to the genomic DNA within the cell nucleus of a eukaryotic cell.

[0020] As used herein, "nucleic acid" refers to 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. The nucleic acid may also be referred to as, for example, a "nucleic acid molecule."

[0021] As used herein, "hybridize" refers to annealing with a complementary polynucleotide that occurs due to nucleotide complementarity, specifically base complementarity in the nucleotides, i.e., two polynucleotides can pair non-covalently via hydrogen bonds.

[0022] As used herein, "complementary" or "complementary" means the ability to form nucleotide pairs, i.e., base pairs, between one polynucleotide and another polynucleotide.

[0023] As used herein, "protein" or "peptide" refers to a polymer composed of unmodified (naturally occurring), modified, and / or artificial amino acids.

[0024] As used herein, "polypeptide" refers to a polymer composed of unmodified (naturally occurring), modified, and / or artificial amino acids. The polypeptide is a peptide having a length of 10 amino acids or more.

[0025] As used herein, the term "domain" refers to a structurally or functionally organized region in a protein, polypeptide, and / or peptide.

[0026] As used herein, the term "antibody" refers to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Immunoglobulin genes include genes encoding constant regions such as κ, λ, α (including α1 and α2), γ (including γ1, γ2, γ3, and γ4), δ, ε, and μ, as well as genes capable of encoding numerous immunoglobulin variable regions such as V regions, D regions, and J regions. The antibody comprises, for example, a heavy chain and a light chain. The light chain includes κ and λ, constituting the κ chain and the λ chain, respectively. The heavy chain includes γ, μ, α, δ, or ε, constituting 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 a pair of two identical polypeptide chains, each pair consisting of one light chain (approximately 25 kDa) and one heavy chain (approximately 50-70 kDa). The N-terminus of each chain defines a variable region consisting of approximately 100-110 or more amino acids that is primarily involved in antigen recognition. The antibody may be a full-length immunoglobulin or an antigen-binding fragment thereof.

[0027] As used herein, an "antigen-binding fragment" refers to a polypeptide comprising a portion of an antibody, more specifically, a polypeptide comprising the variable region. The antigen-binding fragment can be produced, for example, by digesting the full-length immunoglobulin with various peptidases. Examples of the antigen-binding fragment include F(ab')2, Fab', Fab, Fv (variable fragment of antibody), disulfide-linked Fv, single-chain antibody (scFv), and polymers thereof.

[0028] As used herein, "gamete" refers to a reproductive cell that can generate a new individual through fusion or fertilization. The gamete can also be referred to as, for example, a "gamete" or a "gametome." The gamete can be, for example, a heterogamete, and specific examples include sperm and eggs in animals, and pollen and embryo sacs in plants.

[0029] As used herein, the term "imprinted gene" refers to a gene whose expression is controlled by genomic imprinting.

[0030] As used herein, the term "control region" refers to a region in genomic DNA that controls gene expression. The control region of an imprinted gene is generally regulated by methylation. For this reason, the control region of an imprinted gene is also called a differentially methylated region (DMR) or an imprinting control region (ICR).

[0031] As used herein, a "promoter" or "promoter region" refers to a region that is located upstream of a DNA region encoding a gene or a polynucleotide, contains a nucleic acid sequence (base sequence) to which a transcription factor binds, and regulates the amount of transcription of the gene or polynucleotide. The "promoter" or "promoter region" can also be referred to as, for example, a "transcriptional regulatory region."

[0032] As used herein, an "expression vector" or "vector" refers to a in In vitro or in vivo In the context of the present invention, the term refers to a recombinant plasmid, virus, or virus-like particle (VLP) that contains a nucleic acid to be delivered to a host cell.

[0033] As used herein, "animals and plants" refers to taxonomic animals and plants. The animals and plants include any animals and plants that form gametes. The animals refer to, for example, humans and non-human animals. The non-human animals include, for example, mammals such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, and sea lions. The plants include, for example, angiosperms.

[0034] As used herein, "exogenous" means introduced into a cell from outside the cells that constitute the plant or animal.

[0035] As used herein, "isolated" means identified and separated and / or recovered from components in their natural state. The "isolation" can be achieved, for example, by obtaining at least one purification step.

[0036] As used herein, the term "target region" refers to a region in genomic DNA intended to induce a desired effect, such as modification of the epigenome.

[0037] As used herein, "targeting" means binding to or accumulating in a target region and inducing a desired effect, such as modification of the epigenome.

[0038] The present invention will be described below using examples, but the present invention is not limited to the following examples and can be practiced with any modifications. Furthermore, each description in the present invention can be mutually incorporated unless otherwise specified. In this specification, when the expression "to" is used, it is used to mean including the numerical or physical values ​​before and after it. In addition, in this specification, the expression "A and / or B" includes "A only," "B only," and "both A and B."

[0039] <Nucleic acids or compositions used to produce parental lineage animals and plants> In one aspect, the present invention provides a nucleic acid or a composition comprising the nucleic acid for use in producing a parent line of animals or plants that can be used in producing and / or maintaining animals or plants with modified epigenomes. The present invention provides a nucleic acid or composition for use in producing a parent line of animals or plants for producing or maintaining animals or plants with modified epigenomes, the composition comprising a nucleic acid, the nucleic acid encoding a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence in a target region in genomic DNA for epigenome modification, and an epigenome modification enzyme that is capable of forming a complex with the nucleic acid sequence recognition module and modifying the epigenome, the nucleic acid being configured such that the sequence recognition module and the epigenome modification enzyme are induced to be expressed during gametogenesis, and during gametogenesis, the induced nucleic acid sequence recognition module and the epigenome modification enzyme form a complex and modify the epigenome of the target region in the genomic DNA of the gamete being formed.

[0040] The composition of the present invention can be used to create parental strains of animals and plants that can be used to produce and / or maintain epigenome-modified animals and plants, as described below. As a specific example, a case will be described with reference to FIG. 1 in which the animal or plant is a mouse, the gamete in which the epigenome modification occurs is a gamete of a male parent strain, i.e., sperm, and the nucleic acid is introduced into one chromosome (genomic DNA) of the mouse's genomic DNA, i.e., the nucleic acid is a transgene. The following description does not limit the present invention. First, the male parent strain will be described. The male parent strain has a chromosome (TG1) that contains the nucleic acid and a chromosome (WT1) that does not contain the nucleic acid. Sperm are formed from primordial germ cells in the testes of the male parent strain. During spermatogenesis, expression of the sequence recognition module and the epigenome modification enzyme in the nucleic acid is induced, forming a complex that is targeted to the target region in the genomic DNA. Then, because the epigenome of the target region in the complex is modified, sperm are formed that contain either a chromosome (TG2) containing the epigenome and the nucleic acid, or a chromosome (WT2) containing the epigenome and not containing the nucleic acid. Fertilizing the epigenome-modified sperm with an epigenome-unmodified egg produces an epigenome-modified mouse in which one chromosome is epigenome-modified and the other is not. Furthermore, by combining epigenome-modified sperm with an epigenome-modified egg, it is also possible to obtain an epigenome-modified mouse in which both chromosomes are epigenome-modified.

[0041] Next, the female parent strain will be described. The female parent strain has a chromosome (TG1) containing the nucleic acid and a chromosome (WT1) not containing the nucleic acid. In the ovaries of the female parent strain, ova are formed from primordial germ cells. During oogenesis, the expression of the sequence recognition module and the epigenome modification enzyme in the nucleic acid, which are induced during spermatogenesis, is not induced. Therefore, the target regions of the chromosome (TG1) containing the nucleic acid and the chromosome (WT1) not containing the nucleic acid are not epigenome-modified, and ova are formed containing the chromosome (TG1) containing the nucleic acid or the chromosome (WT1) not containing the nucleic acid. Then, by fertilizing the non-epigenome-modified ova with non-epigenome-modified sperm, mice containing the chromosome (TG1) containing the nucleic acid and the chromosome (WT1) not containing the nucleic acid, and two chromosomes (WT1) not containing the nucleic acid can be produced. The former mice have genomic DNA similar to that of the male parent strain and the female parent strain, and can therefore be used to maintain the lineage. Therefore, the compositions of the present invention can be used to produce parent strains of animals and plants that can be used to produce and / or maintain epigenome-modified animals and plants. Furthermore, as shown in Figures 1 and 2, the compositions of the present invention can be used to produce epigenetic mice in which the epigenome of the target region has been modified and which contain a chromosome (WT2) that does not contain the nucleic acid and a chromosome (WT1) that has not been epigenome-modified and does not contain the nucleic acid. These epigenome-modified mice are identical to mice that contain two sets of chromosomes that have not been epigenome-modified and do not contain the nucleic acid, except for the modification of the epigenome of the target region. Therefore, the effects of exogenous nucleic acids can be eliminated, and epigenome-modified mice can be produced that allow for the investigation of the effects of the epigenome modification alone. While the above description uses sperm as an example of gametes in which epigenome modification occurs, as shown in Figure 2, parent strains of animals and plants that can be used to produce and / or maintain epigenome-modified animals and plants can also be produced when the gametes in which epigenome modification occurs are gametes of a female parent strain, i.e., eggs. Furthermore, although mice were used as the animals and plants, the present invention can be similarly applied to plants that form gametes (for example, pollen and embryo sacs).

[0042] The target region is any region in genomic DNA and can be appropriately set depending on the purpose. Examples of the target region include the regulatory region or promoter region of an imprinted gene. For example, the genes listed in the IMPRINTED GENE DATABASES (https: / / www.geneimprint.com / site / home) can be referenced as examples of the imprinted gene. Specific examples of the atypical imprinted gene when the animal or plant is a mouse include the 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, and Xist gene. When the imprinted gene is the Igf2 gene, examples of the regulatory region include the H19-DMR.

[0043] The length of the target region is not particularly limited and can be set appropriately depending on, for example, the purpose of epigenome modification.

[0044] The nucleic acid sequence recognition module is a molecule that specifically binds to the nucleic acid sequence of a target region in genomic DNA where the epigenome is to be modified. The nucleic acid sequence recognition module can form a complex with the epigenome modification enzyme. Therefore, by binding to the nucleic acid sequence of the target region, the nucleic acid sequence recognition module can recruit (accumulate) the epigenome modification enzyme to the target region. As a result, the epigenome modification enzyme modifies the epigenetic modification of the genome in the target region, thereby modifying the epigenome. Furthermore, because the nucleic acid sequence recognition module can recruit the epigenome modification enzyme to the target region, by combining it with the epigenome modification enzyme appropriate for the type of modification to be performed on the epigenome, desired epigenome modification can be performed on the target region.

[0045] Examples of the nucleic acid sequence recognition module include a protein that recognizes a nucleic acid sequence in DNA, or a complex of a protein and a nucleic acid. Specific examples of the nucleic acid sequence recognition module include proteins that specifically bind to DNA, such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas system, zinc finger motifs, transcription activator-like (TAL) effectors, PRR (Pentatricopeptide repeat) motifs, restriction enzymes, transcription factors, RNA polymerases, and DNA polymerases, or DNA-binding domains thereof. The CRISPR-Cas system, zinc finger motifs, TAL effectors, or PRR motifs, or DNA-binding domains thereof, are preferred. To suppress changes in the nucleic acid sequence of a genome, the nucleic acid sequence recognition module preferably does not cleave both strands of double-stranded DNA, and more preferably has inactivated nuclease activity against double-stranded DNA.

[0046] 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 a target nucleic acid sequence. The Cas protein is not particularly limited, and examples include types I to V of Cas proteins. Specific examples of the Cas protein include 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, Csf1, Csf2, Csf3, and Csf4.

[0047] The origin of the Cas protein is not particularly limited. When Cas9 is used as the Cas protein, the Cas9 protein can be derived from, for example, Staphylococcus aureus ( Staphylococcus aureus ) derived Cas9 (SaCas9), Streptococcus pyogenes ( Streptococcus pyogenes ) derived Cas9 (SpCas9), Streptococcus thermophilus ( Streptococcus thermophilus Examples include Cas9 (StCas9) derived from

[0048] The nuclease activity (DNA cleavage ability) of at least one of the Cas protein's DNA cleavage domains (cleavage sites) is preferably inactivated. Specifically, the Cas9 protein has an HNH domain and a RuvC domain as DNA cleavage domains. Therefore, it is preferable that at least one of the HNH domain and the RuvC domain of the Cas9 protein is inactivated, and more preferably both. The nuclease activity can be inactivated, 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. Specifically, when the Cas9 protein is SpCas9, the HNH domain of SpCas9 can be inactivated by substituting the asparagine (Asn) at position 836 and / or the histidine (His) at position 840 with an alanine (Ala), thereby inactivating the ability to cleave the strand complementary to the guide RNA. The RuvC domain of SpCas9 can be inactivated by, for example, substituting the aspartic acid residue (Asp) at position 10 with an alanine residue (Ala), i.e., inactivating the ability to cleave the strand opposite the strand complementary to the guide RNA (reverse complementary strand). The nuclease activity of the SpCas9 protein can be inactivated by, for example, substituting the histidine residue (His) at position 840 with an alanine residue (Ala) and substituting the aspartic acid residue (Asp) at position 10 with an alanine residue (Ala) (dCas9 protein). The nuclease activity of the SpCas9 protein can be inactivated by, for example, substituting the asparagine (Asn) at position 836 with an alanine residue (Ala) and substituting the aspartic acid residue (Asp) at position 10 with an alanine residue (Ala) (dCas9 protein).

[0049] The guide RNA comprises a polynucleotide having a nucleic acid sequence complementary to a target nucleic acid sequence present in the target region or near the target region, and is RNA that can hybridize with the target nucleic acid sequence via the polynucleotide. When the CRISPR-Cas system is used as the nucleic acid sequence recognition module, the guide RNA is, for example, an RNA molecule comprising a nucleic acid sequence that specifically binds to the nucleic acid sequence of the target region, i.e., an RNA molecule comprising a polynucleotide complementary to the nucleic acid sequence of the target region.

[0050] The guide RNA can be appropriately designed depending on 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). When the Cas protein is Cas9 protein, the guide RNA may be composed of two RNAs or a single RNA (sgRNA). In the former case, the guide RNA is composed of crRNA and tracrRNA, and the crRNA and tracrRNA hybridize with complementary polynucleotides to form a complex and function as a guide RNA. In the latter case, the sgRNA is composed of crRNA and tracrRNA, or these are linked via a linker.

[0051] The crRNA preferably contains, for example, at its 5' end, a polynucleotide complementary to the target nucleic acid sequence. By using the target nucleic acid sequence as the nucleic acid sequence in the target region, the crRNA can recruit (accumulate) the Cas protein to the nucleic acid sequence in the target region, thereby recruiting (accumulating) the epigenome modification enzyme to the nucleic acid sequence in the target region. The length of the complementary polynucleotide is, for example, 15 to 25 bases or 18 to 22 bases.

[0052] The length of the guide RNA can be set appropriately depending on, for example, the length of the target nucleic acid sequence.

[0053] The nucleic acid sequence 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, depending on the length of the target region. As a specific example, when the target region is relatively long, the nucleic acid of the present invention can encode multiple types of guide RNAs that specifically bind to nucleic acid sequences at different sites in the target region based on the length of the region modifiable by the epigenome modification enzyme, thereby modifying the epigenome of the entire target region. The vicinity of the target region can be set, for example, depending on the length of the region modifiable by the epigenome modification enzyme. As a specific example, the vicinity of the target region can be set within 3 kbp, 2 kbp, or 1 kbp, preferably within 1 kbp, of the terminal nucleic acid residue of the target region.

[0054] The zinc finger motif is formed by linking multiple zinc finger units of different C2H2 (Cys2His2) types. One zinc finger unit recognizes a nucleic acid sequence of approximately 3 bases. The number of zinc finger units can be appropriately determined depending on the nucleic acid sequence of the target region, but is generally 3 to 6. In this case, the zinc finger motif can recognize, for example, a nucleic acid sequence of approximately 9 to 18 bases. The zinc finger motif can be prepared by known methods such as the modular assembly method (Nat Biotechnol (2002) 20: 135-141), the OPEN method (Mol Cell (2008) 31: 294-301), the CoDA method (Nat Methods (2011) 8: 67-69), or the E. coli one-hybrid method (Nat Biotechnol (2008) 26: 695-701). The zinc finger motif can be prepared, for example, by the method described in WO 03 / 087341.

[0055] TAL effectors have a repeating structure of modules consisting of approximately 34 amino acids. The 12th and 13th amino acid residues (repeat variable diresidues: RVD) of each module determine binding stability and nucleic acid (base) specificity. Each module is highly independent, and by arranging modules consecutively, it is possible to create TAL effectors specific to a target nucleic acid sequence. TAL effectors specific to the target nucleic acid sequence can be designed using open resources such as the REAL method (Curr Protoc Mol Biol (2012) Chapter 12: Unit 12.15), the FLASH method (Nat Biotechnol (2012) 30: 460-465), and the Golden Gate method (Nucleic Acids Res (2011) 39: e82). TAL effectors can be produced, for example, by the method described in WO 2011 / 072246.

[0056] The PPR motif is composed of 35 amino acids and is configured to recognize a specific nucleic acid sequence by arranging a series of PPR motifs that recognize a single nucleic acid (base). The PPR motifs recognize the target nucleic acid (base) at the first, fourth, and second amino acids from the C-terminus (ii(-2)) of each motif. Furthermore, the structure of each motif is independent, and there is no interference from PPR motifs on the C- or N-terminal side. Therefore, by arranging PPR motifs consecutively, a PPR protein specific to a target nucleic acid sequence can be produced. The PPR motif can be produced, for example, by the method described in International Publication No. 2014 / 175284.

[0057] When the DNA-binding domain of the restriction enzyme, transcription factor, RNA polymerase, or DNA polymerase is used, the DNA-binding domains of these proteins are known, and the DNA-binding domain can be used as the nucleic acid sequence recognition module.

[0058] The nucleic acid sequence recognition module may be, for example, one type, or two or more types. When the target region is long, the nucleic acid of the present invention can modify the epigenome of the entire target region by encoding multiple types of nucleic acid sequence recognition modules that specifically bind to nucleic acid sequences at different sites in the target region.

[0059] The target of modification by the epigenome-modifying enzyme may be chemical modification of genomic DNA or histone protein. Examples of the epigenome-modifying enzyme include methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, ubiquitinases, and sumoylases.

[0060] When the target of modification is a chemical modification of genomic DNA, examples of the epigenome modification enzyme include a methylation enzyme or a demethylation enzyme. When the animal or plant is an animal, the methylation enzyme methylates, for example, a cytosine nucleotide at a CpG site in the genomic DNA. In this case, examples of the methylation enzyme include DNMT (DNA methyltransferase) 3A, DNMT3B, and DNMT3L. When DNMT3A or DNMT3B is used as the epigenome modification enzyme, these methylation enzymes are preferably used in combination with DNMT3L. When the animal or plant is a plant, the methylation enzyme methylates, for example, a cytosine nucleotide at a CpG, CpHpG, or CpHpH site (where H is a nucleotide other than guanine) in the genomic DNA. In this case, examples of the methylation enzyme include DRMa (DRM-type cytosine DNA-methyltransferase), DRM2, CMT (chromomethylase) 2, etc.

[0061] When the animal or plant is an animal, the demethylase demethylates, for example, the methyl group of a cytosine nucleotide at a CpG site in the genomic DNA. In this case, examples of the demethylase include TET (ten-eleven translocation) 1, TET2, and TET3. When the animal or plant is a plant, the demethylase demethylates, for example, the methyl group of a cytosine nucleotide at a CpG, CpHpG, or CpHpH site (where H is a nucleotide other than guanine) in the genomic DNA. In this case, examples of the demethylase include DME (DEMETER), ROS (SILENCING) 1, etc.

[0062] When the target of modification is a chemical modification of a histone protein, examples of the epigenome-modifying enzyme include methylases, demethylases, acetylases, deacetylases, kinases, dephosphorylating enzymes, ubiquitinases, and sumoylases.

[0063] The methylation enzyme, for example, methylates lysine residues and / or arginine residues in the histone protein. Examples of the methylation enzyme include EZH2 (enhancer of zeste homolog 2), which is classified as PRC2, G9, SUV39H1, and the like. The demethylation enzyme, for example, demethylates the methyl group of a lysine residue and / or an arginine residue in the histone protein. Examples of the demethylation enzyme include lysine-specific demethylase 1 (LSD1), lysine-specific demethylase 4D (KDM4D), lysine demethylase 6B (KDM6B), and the like. The acetylation enzyme, for example, acetylates a lysine residue in the histone protein. In this case, examples of the acetylation enzyme include Gcn5 and P300 / CBP (CREB-binding protein). The deacetylation enzyme, for example, deacetylates the acetyl group of a lysine residue in the histone protein. Examples of the deacetylase include histone deacetylase (HDAC), SIRT (Sirtuin) 1, SIRT2, etc. The kinase phosphorylates, for example, serine and / or threonine residues in the histone protein. In this case, examples of the kinase include Haspin (GSG2), Aurora B, ChK1 (Checkpoint kinase 1), etc. The dephosphatase dephosphorylates, for example, the phosphate group of serine and / or threonine residues in the histone protein. Examples of the dephosphatase include PP1γ (Protein phosphatase 1γ), PP4C (Protein Phosphatase 4 Catalytic Subunit), DUSP1 (Dual specificity protein phosphatase 1), etc. The ubiquitinase ubiquitinates, for example, lysine residues in the histone protein. In this case, examples of the ubiquitination enzyme include Ring1, RNF8, UBC13, and UHRF1.The sumoylating enzyme sumoylates a lysine residue in the histone protein, for example. In this case, examples of the sumoylating enzyme include UBC9.

[0064] The origin of the epigenome-modifying enzyme may be the same as that of the animal or plant in which the composition of the present invention is used, i.e., the same species, or may be different, i.e., heterologous.

[0065] The epigenome-modifying enzyme may be the whole or a part of an enzyme protein. When the epigenome-modifying enzyme is a part of an enzyme protein, the epigenome-modifying enzyme may retain the enzymatic activity of the enzyme protein. For example, an enzyme catalytic site (catalytic site) may be used.

[0066] The nucleic acid sequence recognition module and the epigenome modification enzyme can form a complex. The formation of the complex may be by linkage via a covalent bond or by utilizing intermolecular interaction.

[0067] When the formation of the complex is by linkage via a covalent bond, the nucleic acid sequence recognition module and the epigenome modification enzyme may be linked directly or indirectly, i.e., formed into a fusion protein, to form the complex.

[0068] The nucleic acid sequence recognition module may form a complex with one epigenome modification enzyme or multiple epigenome modification enzymes. The epigenome modification enzyme may form a complex with one nucleic acid sequence recognition module or multiple nucleic acid sequence recognition modules. When one nucleic acid sequence recognition module forms a complex with multiple epigenome modification enzymes, the resulting complex can more efficiently modify the epigenome and modify the epigenome at a position farther from the target nucleic acid sequence in the target region than a complex formed with a single epigenome modification enzyme. The number of epigenome modification enzymes forming a complex with one nucleic acid sequence recognition module is one or more, preferably two or more (multiple), 3 to 10, 3 to 7, or 3 to 5. When the nucleic acid sequence recognition module forms a complex with multiple epigenome modification enzymes, the epigenome modification enzymes may be one type or multiple types. By using multiple types of epigenome modification enzymes, the complex can, for example, perform multiple types of epigenome modifications in the target region.

[0069] When the nucleic acid sequence recognition module and the epigenome modification enzyme are directly linked, the N- or C-terminal amino acid of the protein in the nucleic acid sequence recognition module forms a covalent bond with the C- or N-terminal amino acid in the epigenome modification enzyme. The order of the nucleic acid sequence recognition module and the epigenome modification enzyme is not particularly limited and can be any order. For example, from the N-terminus side, the nucleic acid sequence recognition module and the epigenome modification enzyme may be arranged in this order, or the epigenome modification 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 multiple epigenome modification enzymes is not particularly limited, but it is preferable that the epigenome modification enzymes are arranged consecutively. When the nucleic acid sequence recognition module and the epigenome modification enzyme are directly linked, 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 modification enzyme are integrated.

[0070] When the nucleic acid sequence recognition module and the epigenome modification enzyme are indirectly linked, the N- or C-terminal amino acid of the protein in the nucleic acid sequence recognition module is bound to the C- or N-terminal amino acid of the epigenome modification enzyme via a linker. When the nucleic acid sequence recognition module is indirectly linked to multiple epigenome modification enzymes, only the nucleic acid sequence recognition module and the epigenome modification enzyme are linked via the linker, and the epigenome modification enzymes may be linked directly to each other, or both the nucleic acid sequence recognition module and the epigenome modification enzyme and the epigenome modification enzymes may be linked via the linker. The order of the nucleic acid sequence recognition module and the multiple epigenome modification enzymes is not particularly limited, but it is preferable that the epigenome modification enzymes are arranged consecutively. When the nucleic acid sequence recognition module and the epigenome modification enzyme are indirectly linked, 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 modification enzymes are integrated.

[0071] The linker can have any sequence as long as it does not interfere with the functions of the nucleic acid sequence recognition module and the epigenome modification enzyme. Examples of the linker include a repeat sequence of glycine and serine. The linker may be, for example, 5 to 100 amino acids, 5 to 50 amino acids, 10 to 50 amino acids, 15 to 50 amino acids, 15 to 40 amino acids, 17 to 30 amino acids, or 22 amino acids long. By increasing the length of the linker, the resulting complex can modify, for example, an epigenome at a position farther from the target nucleic acid sequence within the target region. Examples of the linker include GSGSG (SEQ ID NO: 1), GSGGS (SEQ ID NO: 2), SGSGS (SEQ ID NO: 3), or GGGGS (SEQ ID NO: 4), or a sequence consisting of two or three repeats thereof; GSGSGGSGSGSGGSGSGGSGSG (SEQ ID NO: 5); GSGSGGSGSGGSGGSGGSGSGGSGSGGSGSGGSGSG (SEQ ID NO: 6); and the like.

[0072] When the formation of the complex is a complex formed using an intermolecular interaction, the nucleic acid sequence recognition module and the epigenome modification enzyme may use a tag domain and a binding partner of the tag domain that can bind to the tag domain, and form a complex through the interaction between the tag domain and the binding partner. In this case, the nucleic acid sequence recognition module may be linked to the tag domain, and the epigenome modification enzyme may be linked to the binding partner, or the nucleic acid sequence recognition module may be linked to the binding partner, and the epigenome modification enzyme may be linked to the tag domain. In the following explanation, an example will be given in which the nucleic acid sequence recognition module is linked to the tag domain, and the epigenome modification enzyme is linked to the binding partner. However, when the nucleic acid sequence recognition module is linked to the binding partner, and the epigenome modification enzyme is linked to the tag domain, the "tag domain" and the "binding partner" can be read interchangeably and the explanation therefor can be used.

[0073] When the nucleic acid sequence recognition module and the epigenome modification enzyme form a complex through the 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, i.e., form a fusion protein. Also, the epigenome modification enzyme and the binding partner are, for example, directly or indirectly linked, i.e., form a fusion protein.

[0074] The nucleic acid sequence recognition module may be linked to one tag domain or multiple tag domains. Furthermore, the tag domain may be linked to one nucleic acid sequence recognition module or multiple nucleic acid sequence recognition modules. When one nucleic acid sequence recognition module is linked to multiple tag domains, the resulting complex can more efficiently modify the epigenome and can also modify epigenomes located farther from the target nucleic acid sequence within the target region than a complex formed with a nucleic acid sequence recognition module having one tag domain. The number of tag domains linked to one nucleic acid sequence recognition module is one or more, preferably two or more (multiple), 3 to 10, 3 to 7, or 3 to 5.

[0075] The epigenome modification enzyme may be linked to one binding partner or to multiple binding partners. Furthermore, the binding partner may be linked to one epigenome modification enzyme or to multiple epigenome modification enzymes. When one binding partner is linked to multiple epigenome modification enzymes, the resulting complex can more efficiently modify the epigenome and can also modify epigenomes at locations farther from the target nucleic acid sequence within the target region than a complex containing a binding partner with one epigenome modification enzyme. The number of epigenome modification enzymes linked to one binding partner is one or more, preferably two or more (multiple), 3 to 10, 3 to 7, or 3 to 5.

[0076] The nucleic acid sequence recognition module and the epigenome modification enzyme are configured to form a complex between one nucleic acid sequence recognition module and multiple epigenome modification enzymes, which allows for more efficient epigenome modification and allows for modification of epigenomes at positions farther from the target nucleic acid sequence within the target region than when one nucleic acid sequence recognition module and one epigenome modification enzyme form a complex. Therefore, in the present invention, the nucleic acid sequence recognition module may be linked to, for example, multiple tag domains, and one binding partner may be linked to multiple epigenome modification enzymes.

[0077] When the nucleic acid sequence recognition module and the tag domain are directly linked, the N- or C-terminal amino acid of the protein in the nucleic acid sequence recognition module forms a covalent bond with the C- or N-terminal amino acid in the tag domain. The order of the nucleic acid sequence recognition module and the tag domain is not particularly limited and can be any order. For example, from the N-terminus, the nucleic acid sequence recognition module and the tag domain may be arranged in this order, or the tag domain and the nucleic acid sequence recognition module may be arranged in this order. The order of the nucleic acid sequence recognition module and the multiple tag domains is not particularly limited, but it is preferable that the tag domains are arranged consecutively. When the nucleic acid sequence recognition module and the tag domain are directly linked, the nucleic acid constitutes 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.

[0078] When the nucleic acid sequence recognition module and the tag domain are indirectly linked, the N- or C-terminal amino acid of the protein in the nucleic acid sequence recognition module is bound to the C- or N-terminal amino acid of the tag domain via a linker. When the nucleic acid sequence recognition module is indirectly linked to multiple tag domains, only the nucleic acid sequence recognition module and the tag domain may be linked via the linker, and the tag domains may be directly linked, or the nucleic acid sequence recognition module and the tag domain and the tag domains may both be linked via the linker. The order of the nucleic acid sequence recognition module and the multiple tag domains is not particularly limited, but it is preferable that the tag domains are arranged contiguously. When the nucleic acid sequence recognition module and the tag domain are indirectly linked, the nucleic acid constitutes 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 can be similar to the description of the linker between the nucleic acid sequence recognition module and the epigenome modification enzyme. The linker may be the same as that described for the linker between the nucleic acid sequence recognition module and the epigenome modification enzyme. When 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.

[0079] When the epigenome modification enzyme and the binding partner are directly linked, the N- or C-terminal amino acid of the epigenome modification enzyme forms a covalent bond with the C- or N-terminal amino acid of the binding partner. The order of the epigenome modification enzyme and the binding partner is not particularly limited and can be any order. For example, from the N-terminus, the epigenome modification enzyme and the binding partner may be arranged in this order, or the binding partner and the epigenome modification enzyme may be arranged in this order. The order of the epigenome modification enzyme and the multiple binding partners is not particularly limited, but it is preferable that the binding partners are arranged consecutively. When the epigenome modification enzyme and the binding partner are directly linked, the nucleic acid constitutes a second nucleic acid encoding a fusion protein in which the amino acid sequences of the epigenome modification enzyme and the binding partner are integrated.

[0080] When the epigenome modification enzyme and the binding partner are indirectly linked, the N- or C-terminal amino acid of the epigenome modification enzyme protein is linked to the N- or C-terminal amino acid of the binding partner via a linker. When the binding partner is indirectly linked to multiple epigenome modification enzymes, only the epigenome modification enzyme and the binding partner may be linked via the linker, and the binding partners may be linked directly, or both the epigenome modification enzyme and the binding partner and the epigenome modification enzymes may be linked via the linker. The order of the binding partner and the multiple epigenome modification enzymes is not particularly limited, but it is preferable that the epigenome modification enzymes are arranged consecutively. When the epigenome modification enzyme and the binding partner are indirectly linked, the nucleic acid constitutes a second nucleic acid encoding a fusion protein in which the amino acid sequences of the epigenome modification enzyme and the binding partner are integrated. The linker can be described in the same manner as for the linker between the nucleic acid sequence recognition module and the epigenome modification enzyme. When 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.

[0081] When the nucleic acid sequence recognition module and the epigenome modification enzyme form a complex through the interaction between the tag domain and the binding partner, any combination of the tag domain and the binding partner can be used as long as they specifically bind to each other. Examples of combinations of the tag domain and the binding partner include a combination of a peptide epitope and an antibody or aptamer that recognizes it, and a combination of a small fragment and a large fragment of a split protein with self-assembly ability. When the tag domain is composed of a peptide, it can also be called a peptide tag.

[0082] Examples of combinations of the peptide epitope and the antibody that recognizes it include a GCN4 (General Control Non-derepressible) peptide epitope and an anti-GCN4 peptide epitope antibody; a His tag and an anti-His tag antibody; an EE hexapeptide and an anti-EE hexapeptide antibody; a c-Myc tag and an anti-c-Myc tag antibody; an HA tag and an anti-HA tag antibody; an S tag and an anti-S tag antibody; a FLAG tag and an anti-FLAG tag antibody; etc. (Protein Engineering, Design & Selection; vol. 24 (5): pp. 419-428 (2011)). The GCN4 peptide may be any epitope contained in GCN4, and a specific example is the amino acid sequence represented by ELLSKNYHLENEVARLKK (SEQ ID NO: 7).

[0083] A self-assembling split protein is a protein that, when a protein is bisected, can reassemble into the same structure as the original protein. In this case, a short peptide (small fragment) obtained by bisecting the original protein can be used as a tag domain (peptide tag) and a long peptide (large fragment) can be used as the binding partner, or the reverse combination can be used (Current Opinion in Chemical Biology (2011) 15: pp. 789-797, WO 2005 / 074436). An example of the self-assembling split protein is GFP (Green Fluorescent Protein), where a small fragment of GFP can be used as the tag domain and a large fragment of GFP can be used as the binding partner.

[0084] The tag domain and the binding partner may be, for example, a combination of a peptide and a protein domain that binds to the peptide. Combinations of peptides and protein domains can be found in databases such as PepBDB: http: / / huanglab.phys.hust.edu.cn / pepbdb / , PiSITE: https: / / pisite.sb.ecei.tohoku.ac.jp / cgi-bin / top.cgi, and STRING: https: / / string-db.org / . As a specific example, the PDZ Alpha-Syntrophin PDZ protein interaction domain can bind to GVKESLV (SEQ ID NO: 8). Therefore, GVKESLV can be used as the tag domain, and a PDZ domain can be used as the binding partner.

[0085] When a combination of the peptide and a protein domain that binds to the peptide is used as the tag domain and the binding partner, the binding strength of the pair of the peptide and the protein domain may be strengthened by linking another inactive domain via a linker and improving the pair through evolutionary engineering. Using the pair obtained by the improvement as the tag domain and the binding partner allows for more efficient control of epigenome modification (Proc. Natl. Acad. Sci. USA, 2008, vol. 105 no. 18, 6578-6583).

[0086] When the nucleic acid sequence recognition module is a CRISPR-Cas system and the nucleic acid sequence recognition module and the epigenome modification enzyme form a complex via the tag domain and the binding partner, it is preferable that the Cas protein in the nucleic acid sequence recognition module is linked to the tag domain.

[0087] Sequence information for the proteins, fusion proteins, or nucleic acids (e.g., DNA or RNA) encoding them described herein is available from Protein Data Bank, UniPort, GenBank, or the like. Furthermore, RNA nucleic acid sequences can also be obtained from the corresponding DNA nucleic acid sequences using appropriate sequence conversion software. DNA encoding the nucleic acid sequence recognition module and DNA encoding the epigenome modification enzyme described herein may be obtained by cloning from mRNA using molecular biology methods, or by chemically synthesizing DNA based on the sequence information. Furthermore, when obtaining the DNA, codon optimization may be performed according to the animal or plant (host) into which the nucleic acid will be introduced. This can be expected to increase the amount of protein expressed in the host. Data on codon usage in the host to be used may be obtained, for example, from the genetic code usage database published on the website of the Kazusa DNA Research Institute (http: / / www.kazusa.or.jp / codon / index.html), or references listing codon usage in each host may be referenced.

[0088] The nucleic acid encoding the nucleic acid sequence recognition module and the epigenome modification enzyme is configured so that the nucleic acid sequence recognition module and the epigenome modification enzyme are expressed and induced during gametogenesis. As described above, the nucleic acid sequence recognition module and the epigenome modification enzyme form a complex and bind to a nucleic acid sequence in a target region in the genomic DNA of a gamete being formed, thereby modifying the epigenome of the target region. Therefore, the nucleic acid is preferably configured so that the induction period of expression of the nucleic acid sequence recognition module and the induction period of expression of the epigenome modification enzyme overlap during gametogenesis. Specifically, the nucleic acid may be configured so that both the nucleic acid sequence recognition module and the epigenome modification enzyme are expressed during gametogenesis such that their expression periods overlap during gametogenesis. Furthermore, the nucleic acid may be configured so that one of the nucleic acid sequence recognition module and the epigenome modification enzyme is expressed during gametogenesis or at a time other than gametogenesis, and the other is expressed during gametogenesis. Furthermore, the nucleic acids encoding the nucleic acid sequence recognition module and the epigenome modification enzyme may be configured to be constitutively expressed, and their expression may be induced during gametogenesis by inducing the expression of a molecule (e.g., siRNA, miRNA, shRNA, etc.) that suppresses the expression of the nucleic acid sequence recognition module and / or the epigenome modification enzyme at a time other than gametogenesis.

[0089] The time of expression induction in gametogenesis can be, for example, any time until the precursor cells of the gametes differentiate into gametes. Specifically, when the gametes are sperm, the time of expression induction in gametogenesis is, for example, the time from primordial germ cells to spermatogonia and spermatocytes until they differentiate into sperm. When the gametes are eggs, the time of expression induction in gametogenesis is, for example, the time from primordial germ cells to oogonia and oocytes until they differentiate into eggs. When the gametes are pollen, the time of expression induction in gametogenesis is, for example, the time from pollen oocytes to pollen. When the gametes are embryo sacs, the time of expression induction in gametogenesis is, for example, the time from embryo sac oocytes to embryo sacs.

[0090] The expression timing of the nucleic acid sequence recognition module and the epigenome modification enzyme can be controlled, for example, by functionally linking a timing-specific promoter to the nucleic acid encoding them. Specifically, when inducing expression of the nucleic acid sequence recognition module and the epigenome modification enzyme during gametogenesis, the expression timing can be controlled using a gametogenesis-specific promoter that is activated during gametogenesis. Examples of the gametogenesis-specific promoter include a spermatogenesis-specific promoter, an oogenesis-specific promoter, a pollen formation-specific promoter, and an embryo sac formation-specific promoter. Examples of the gametogenesis-specific promoter include a spermatogenesis-specific promoter, specific examples of which include the STRA8 promoter, Pgk2 promoter, Dazl promoter, and Gsg2 promoter. Each promoter refers to the promoter sequence of the respective gene, and a specific example of the STRA8 promoter is the promoter of the STRA8 gene. The gametogenesis-specific promoter includes an oogenesis-specific promoter, and specific examples thereof include the Gdf-9 promoter, the Zp3 promoter, and the Msx2 promoter.

[0091] 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 ·Oocyte-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”, Curr Biol, 1997 Feb 1;7(2):148-51 Msx2: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

[0092] When expression is induced during gametogenesis or at a stage other than gametogenesis, the expression timing can be controlled using, for example, a promoter that constitutively induces expression, etc. Specific examples of the promoter include a CAG promoter, an SRα promoter, an SV50 promoter, an LTR promoter, a CMV (cytomegalovirus) promoter, an RSV (Rous sarcoma virus) promoter, a MoMuLV (Moloney murine leukemia virus) promoter, and an HSV-TK (herpes simplex virus thymidine kinase) promoter.

[0093] In the present invention, the nucleic acid is preferably inserted into an expression vector. In this case, the nucleic acid encoding the nucleic acid sequence recognition module and the nucleic acid encoding the epigenome modification enzyme are linked to the expression vector so that the nucleic acid sequence recognition module and the epigenome modification enzyme can be expressed, respectively. The expression vector can be prepared, for example, by inserting the nucleic acid encoding the nucleic acid sequence recognition module and / or the nucleic acid encoding the epigenome modification enzyme into a backbone vector (hereinafter also referred to as a "basic vector").

[0094] The nucleic acid encoding the nucleic acid sequence recognition module and the nucleic acid encoding the epigenome modification enzyme may be linked to the same expression vector or to different vectors. When the nucleic acid encoding the nucleic acid sequence recognition module (first nucleic acid) and the nucleic acid encoding the epigenome modification enzyme (second nucleic acid) are linked to different vectors, the expression vectors include a first expression vector and a second expression vector, and the first expression vector is functionally linked to the first nucleic acid so as to enable expression of the nucleic acid sequence recognition module, and the second expression vector is functionally linked to the second nucleic acid so as to enable expression of the epigenome modification enzyme. Furthermore, when the nucleic acid sequence recognition module is composed of multiple elements, for example, when a CRISPR-Cas system is used, some or all of the elements may be linked to different expression vectors.

[0095] The basic vector can be appropriately selected depending on the animal or plant in which the composition of the present invention is used, i.e., the host. Examples of the expression vector include non-viral vectors such as plasmid vectors, or viral vectors. Examples of the plasmid vectors for animals include pCDM8, pMT2PC, pA1-11, pXT1, pRc / CMV, pRc / RSV, and pcDNAI / Neo. Examples of the plasmid vectors for plants include vectors containing T-DNA, specifically pGEM-T. Examples of the viral vectors include retroviruses, vaccinia viruses, and adenoviruses.

[0096] The expression vector preferably has a regulatory sequence that regulates the expression of the nucleic acid sequence recognition module and the epigenome modification enzyme. Examples of the regulatory sequence include a promoter, a terminator, an enhancer, a polyadenylation signal sequence, and an origin of replication (ori). The location of the regulatory sequence in the expression vector is not particularly limited, as long as it is located so as to functionally regulate the expression of the nucleic acid sequence recognition module and / or the epigenome modification enzyme, and can be located based on known methods. For example, the regulatory sequence may utilize a sequence already contained in the basic vector, or the regulatory sequence may be further inserted into the basic vector, or the regulatory sequence contained in the basic vector may be replaced with another regulatory sequence.

[0097] The expression vector may further comprise, for example, a coding sequence for a selection marker, such as a drug resistance marker, a fluorescent protein marker, an enzyme marker, or a cell surface receptor marker.

[0098] Insertion of nucleic acid (DNA), insertion of the regulatory sequence, and / or insertion of the coding sequence of the selection marker into the expression vector may be carried out, for example, by a method using restriction enzymes and ligase, or by using a commercially available kit, etc.

[0099] When the composition of the present invention contains multiple expression vectors, it can also be referred to as, for example, a nucleic acid kit or an expression vector kit. In this case, the kit may further include, for example, an instruction manual.

[0100] <Method for producing parental lines for producing or maintaining epigenome-modified animals and plants> In another aspect, the present invention provides a method for producing a parent line of an animal or plant that can be used to produce and / or maintain an epigenome-modified animal or plant. The production method of the present invention is a method for producing a parent line of an epigenome-modified animal or plant, 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 a parent line of an animal or plant that can be used to produce and / or maintain an epigenome-modified animal or plant.

[0101] The introduction step is a step of introducing the composition of the present invention, i.e., the nucleic acid or an expression vector containing the same, into the target animal or plant, thereby producing, for example, a parent lineage animal or plant containing the nucleic acids in the composition of the present invention, i.e., the nucleic acid encoding the nucleic acid sequence recognition module and the nucleic acid encoding the epigenome modification enzyme.

[0102] Examples of the target animals and plants include plant cells that form callus or the like and can develop into individual plants, and animal cells that can develop into individual animals such as fertilized eggs and animal embryos (embryos, blastocysts), etc. The animals and plants exclude, for example, humans.

[0103] The introduction method in the introduction step can be carried out, for example, by known methods for producing transformants or genetically modified animals and plants. Specific examples of the introduction method for the target animal or plant include plant cells such as plant callus; animal cells such as ES cells, fertilized eggs, and animal embryos (embryos and blastocysts); and the like. Examples of the introduction method include gene gun-based introduction using a particle gun or other gene gun, calcium phosphate method, polyethylene glycol method, lipofection using liposomes, electroporation, ultrasonic nucleic acid transfer, DEAE-dextran method, direct injection using glass microtubes, microinjection, hydrodynamic method, cationic liposome method, methods using introduction adjuvants, and Agrobacterium-mediated methods. Examples of the liposome include lipofectamine and cationic liposomes, and examples of the introduction adjuvants include atelocollagen, nanoparticles, and polymers. When the target animal or plant is a plant cell, the composition of the present invention may be introduced into the plant using the Agrobacterium method.

[0104] 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. In this case, the introduction step involves introducing the composition of the present invention into the target animal or plant, thereby introducing (inserting) the nucleic acid of the composition of the present invention into the genomic DNA of the target animal or plant; more specifically, introducing (inserting) the nucleic acid encoding the nucleic acid sequence recognition module and the nucleic acid encoding the epigenome modification enzyme. As an introduction method for integration into the genomic DNA, a method combining the above-mentioned introduction method into cells with a method using homologous recombination or a method using genome editing technology can be used.

[0105] For the method of producing a genetically modified animal using homologous recombination, see, for example, Hogan, et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986. The introduction method may also be carried out by introducing the composition of the present invention into germ cells. In this case, the introduction method can be, for example, a method of introducing foreign DNA into germ cells of a mammalian animal (e.g., 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)).

[0106] In a method for producing recombinant plants or animals using genome editing, RNA encoding a Cas protein, such as Cas9 RNA, or a Cas protein, such as Cas9 protein, and a guide RNA can be introduced into germ cells together with foreign DNA (e.g., Yang et al., Cell, 154:1370-9 (2013); Quadros et al., Genome Biol, 18:92 (2018)).

[0107] When the target plant or animal is a plant cell capable of developing into a plant individual or an animal cell capable of developing into an animal individual, the first production method of the present invention preferably includes, after the introduction step, a step of generating a plant individual or an animal individual from the plant cell or the animal cell. The method for generating a plant individual can be carried out, for example, by a known method of forming a shoot from a callus and regenerating a plant individual. Furthermore, the method for generating an animal individual can be carried out, for example, by transplanting the animal cell into a pseudopregnant mother and allowing it to develop and give birth, thereby obtaining an animal individual as a litter.

[0108] The first production method of the present invention may include a step of selecting, after the introduction step, animals or plants in which the nucleic acid encoding the nucleic acid sequence recognition module and the nucleic acid encoding the epigenome modification enzyme have been integrated into their genomic DNA. The selection may be performed, for example, by decoding the genomic DNA of the animals or plants after the introduction step to determine whether the nucleic acid encoding the nucleic acid sequence recognition module and the nucleic acid encoding the epigenome modification enzyme are contained, or by detecting the genomic DNA of the animals or plants after the introduction step using primers and / or probes for the nucleic acid encoding the nucleic acid sequence recognition module and the nucleic acid encoding the epigenome modification enzyme, or by using a selection marker.

[0109] <Animals and plants of parental lines for producing or maintaining epigenome-modified animals and plants> In another aspect, the present invention provides parental lines of animals and plants that can be used to produce and / or maintain epigenome-modified animals and plants. The parental lines of animals and plants of the present invention are parental lines for producing or maintaining epigenome-modified animals and plants, and the animals and plants contain exogenous nucleic acids, which include nucleic acids encoding a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence in a target region in genomic DNA for epigenome modification, and an epigenome modification enzyme that can form a complex with the nucleic acid sequence recognition module and modify the epigenome. The nucleic acids are configured so that the sequence recognition module and the epigenome modification enzyme are induced to express during gametogenesis, and during gametogenesis, the induced nucleic acid sequence recognition module and the epigenome modification enzyme form a complex to modify the epigenome of the target region in the genomic DNA of the gamete being formed. The parental lines of animals and plants of the present invention enable the production of epigenome-modified animals and plants and the maintenance of epigenome-modified animals and plants.

[0110] As described above, in the parental line of the animal or plant of the present invention, depending on the type of gamete in which the epigenome is modified, it is possible to produce a parental line used to produce an animal or plant in which the epigenome of the target region has been modified, and a parental line used to maintain the parental line of an animal or plant in which the epigenome of the target region has been modified. The exogenous nucleic acid is derived, for example, from the nucleic acid of the composition of the present invention. The exogenous nucleic acid is preferably inserted into the genomic DNA of the parental line, as this allows the parental line to be stably maintained.

[0111] When the gamete whose epigenome is to be modified is sperm, modification of the epigenome in the target region occurs during spermatogenesis in the male parent line. Therefore, when the gamete whose epigenome is to be modified is sperm, the parent line used to produce an animal or plant with the modified epigenome is a male parent line, and the line used to maintain the parent line as an animal or plant is a female parent line. Furthermore, in the male parent line, the epigenome in the target region is modified in cells at the differentiation stage from primordial germ cells to sperm, but the epigenome in the target region is not modified in cells other than the cells at the differentiation stage from primordial germ cells to sperm that constitute the male parent line. On the other hand, in the female parent line, the epigenome in the target region is not modified in cells that constitute the female parent line. When the gamete whose epigenome is to be modified is sperm, it is preferable that the exogenous nucleic acid is operably linked to a spermatogenesis-specific promoter.

[0112] When the gamete whose epigenome is to be modified is an egg, modification of the epigenome in the target region occurs during egg formation in the female parent line. Therefore, when the gamete whose epigenome is to be modified is an egg, the parent line used to produce an animal or plant whose epigenome is modified is a female parent line, and the line used to maintain the parent line as an animal or plant is a male parent line. In the female parent line, for example, the epigenome in the target region is modified in cells at the differentiation stage from primordial germ cells to eggs, but the epigenome in the target region is not modified in cells other than the cells at the differentiation stage from primordial germ cells to eggs that constitute the female parent line. On the other hand, in the male parent line, the epigenome in the target region is not modified in cells that constitute the male parent line. When the gamete whose epigenome is to be modified is a sperm, it is preferable that the exogenous nucleic acid is operably linked to a spermatogenesis-specific promoter.

[0113] When the exogenous nucleic acid is integrated into genomic DNA in the parent line of animals or plants, it is preferable that the parent line of animals or plants has a chromosome into which the exogenous nucleic acid has been integrated and a wild-type chromosome into which the exogenous nucleic acid has not been integrated.

[0114] <Animals and plants of parental lines for producing or maintaining epigenome-modified animals and plants> In another aspect, the present invention provides gametes that can be used to produce and / or maintain parent lines for epigenome-modified animals and plants. The gametes of the present invention are gametes isolated from the parent lines of animals and plants of the present invention. Using the gametes of the present invention, animals and plants in which the epigenome of the target region has been modified, and parent lines for producing or maintaining animals and plants with modified epigenomes, can be produced.

[0115] The gametes can be isolated from, for example, the parent lineage of animals or plants. The isolation method can be appropriately selected depending on, for example, the type of gamete. The gametes may be preserved after isolation from, for example, the parent lineage of animals or plants. The preservation method can be appropriately selected depending on the type of gamete, and examples thereof include preservation under liquid nitrogen.

[0116] The gametes preferably have an epigenome modified in the target region. In this case, the gametes can be isolated from a parental lineage animal or plant in which the epigenome of the target region in the genomic DNA of the forming gamete has been modified by forming a complex between the induced expression of the nucleic acid sequence recognition module and the epigenome modification enzyme during gamete formation.

[0117] When the gametes are used to produce an animal or plant having a modified epigenome in the target region, the gametes may or may not contain the exogenous nucleic acid, but preferably do not. Furthermore, when the gametes are used to produce an animal or plant having a modified epigenome or to produce a parent line of an animal or plant for maintenance, the gametes contain the exogenous nucleic acid.

[0118] <Method for producing epigenome-modified animals and plants> In another aspect, the present invention provides a method for producing an animal or plant in which the epigenome of a target region has been modified. The production method of the present invention is a method for producing an animal or plant in which the epigenome of a target region has been modified, and includes a step (hybridization step) of crossbreeding a first parent with a second parent and obtaining an individual with a modified epigenome from the resulting progeny, wherein the first parent and / or the second parent is an animal or plant of the parent lineage of the present invention. According to the second production method of the present invention, an animal or plant in which the epigenome of the target region has been modified can be produced.

[0119] In the hybridization step, one of the first parent and the second parent may be an animal or plant of the parent line of the present invention, or both may be an animal or plant of the parent line of the present invention. By using an animal or plant of the parent line of the present invention as one parent, the second production method of the present invention can obtain an animal or plant in which the epigenome of a target region in one chromosome of a set of chromosomes is modified, or an animal or plant in which the epigenome of a target region in one chromosome (e.g., the Y chromosome) is modified in the genomic DNA. On the other hand, by using an animal or plant of the parent line of the present invention as both parents, the second production method of the present invention can obtain an animal or plant in which the epigenome of a target region in both chromosomes of a set of chromosomes is modified in the genomic DNA.

[0120] In the crossbreeding step, gametes of the first parent and / or the second parent may be used as the first parent and / or the second parent instead of the individual animal or plant.

[0121] In the hybridization step, hybridization between the first parent and the second parent can be carried out by known methods.

[0122] The second production method of the present invention may select individuals that do not contain the exogenous nucleic acid from the progeny individuals. This allows the second production method of the present invention to obtain individuals that are similar to animals or plants in which the epigenome of the target region has not been modified and that do not contain the exogenous nucleic acid, except for the modification of the epigenome of the target region. The exogenous nucleic acid can be detected, for example, by detecting a nucleic acid encoding the nucleic acid sequence recognition module and / or a nucleic acid encoding the epigenome modification enzyme.

[0123] <Animals and plants with modified epigenomes> In another aspect, the present invention provides animals and plants in which the epigenome of a target region has been modified. The first animal or plant of the present invention is an animal or plant in which the epigenome of a target region has been modified, and the animal or plant contains, as the target region, a target region in which the epigenome derived from the gamete of the present invention has been modified, and does not contain the exogenous nucleic acid. The first animal or plant of the present invention is an individual in which the epigenome of the target region has not been modified, and is the same as an animal or plant that does not contain the exogenous nucleic acid (wild-type animal or plant), except for the modification of the epigenome of the target region. Therefore, the first animal or plant of the present invention can be suitably used for functional analysis of the epigenome in the target region, for example, by comparing it with the wild-type animal or plant.

[0124] When the target region is a regulatory region and / or promoter region of an imprinted gene, the epigenome of the target region in the cells constituting the first animal or plant of the present invention is different from the epigenome of a wild-type animal or plant.

[0125] A second animal or plant of the present invention is an animal or plant whose epigenome in a target region has been modified, the animal or plant comprising an exogenous nucleic acid, the nucleic acid comprising a nucleic acid encoding a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence in a target region in genomic DNA for epigenome modification, and an epigenome modification enzyme capable of forming a complex with the nucleic acid sequence recognition module and capable of epigenome modification, the nucleic acid being configured to be operably linked to a gametogenesis-specific promoter that is activated during gametogenesis, such that the sequence recognition module and the epigenome modification enzyme are expressed and induced during gametogenesis, and the animal or plant comprises, as the target region, a target region derived from a gamete in which the epigenome in the target region has been modified, wherein the nucleic acid sequence recognition module and the epigenome modification enzyme, whose expression has been induced during gametogenesis, form a complex. The second animal or plant of the present invention is an individual whose epigenome in the target region has not been modified, except for the modification of the epigenome in the target region, and is the same as an animal or plant having the exogenous nucleic acid. Therefore, the second animal or plant of the present invention can be suitably used for functional analysis of the epigenome in the target region, for example, by comparing it with the parent line of animals or plants used in the maintenance. [Example]

[0126] The present invention will be described in detail below using examples, but the present invention is not limited to the embodiments described in the examples. Unless otherwise specified, commercially available reagents, kits, etc. were used according to their protocols.

[0127] [Example 1] It has been confirmed that the composition of the present invention can be used to produce parental lines of animals and plants that can be used to produce and maintain epigenome-modified animals and plants, and that offspring lines with modified epigenomes in the target region can be obtained from the parental lines.

[0128] (1)Target area The Igf2 gene is an imprinted gene, and the DMR region (H19-DMR) of the Igf2 gene is methylated in chromosomes derived from male parents, whereas the DMR region (H19-DMR) of the Igf2 gene is unmethylated in chromosomes derived from female parents. Furthermore, the present inventors have separately confirmed that when abnormal methylation of the DMR region of the Igf2 gene in mice is caused by systemic demethylation of the H19-DMR, i.e., when the H19-DMR is demethylated in chromosomes derived from male parents, the mice exhibit growth retardation, a symptom of Silver-Russell syndrome, and become unable to reproduce. Therefore, the DMR region of the Igf2 gene was selected as a model for the target region.

[0129] (2) Vector construction The expression vector used in Example 1 was constructed from the all-in-one epigenome editing vector (pPlatTET-gRNA2-H19DMRx9) described in Reference 1 below. In the epigenome editing vector described in Reference 1 below, five copies of the GCN4 peptide (ELLSKNYHLENEVARLKK (SEQ ID NO: 9)) are linked via a 22-amino acid linker (GSGSGGSGSGSGGSGSGGSGSG: SEQ ID NO: 10) between each peptide to form a tag domain (SunTag), which is then linked to dCas9 to form a fusion protein. Furthermore, the epigenome editing vector described in Reference 1 also contains an anti-GCN4 single-chain antibody (scFv) linked to the catalytic domain of the TET1 protein (TET1CD) via sfGFP (superfold green fluorescent protein) to form a fusion protein. Furthermore, the epigenome editing vector of Reference 1 is equipped with sgRNAs capable of hybridizing to nine nucleic acid sequences (target nucleic acid sequences) in H19-DMR, as shown in Figure 3(A) and Table 1 below. In the epigenome editing vector, as shown in Figure 3(B), the nucleic acids encoding these fusion proteins and the 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) in the example. The epigenome editing vector in the example was linearized with a restriction enzyme (ApaLI) before microinjection, as described below. Reference 1: Horii T et.al., “Successful generation of epigenetic disease model mice by targeted demethylation of the epigenome.” Genome Biol., 2020 Apr 1;21(1):77.

[0130] [Table 1]

[0131] (3) Embryo preparation B6D2F1 female mice (8-10 weeks old, purchased from CLEA Japan) were administered 7.5 units of an ovulation inducer (SEROTROPIN, ASKA Pharmaceuticals) and then 7.5 units of human chorionic gonadotropin (hCG; GONATROPIN, ASKA Pharmaceuticals) 48 hours later to induce superovulation. After hCG administration, the mice were mated with B6D2F1 male mice. 21 hours after mating, zygotes were collected from the oviducts of the female mice. The collected zygotes were treated for several minutes with M2 medium (Sigma-Aldrich) containing 0.1% hyaluronidase (Sigma-Aldrich), and the resulting fertilized eggs were then washed with the M2 medium. The washed fertilized eggs were transferred to a drop of M16 medium (Sigma-Aldrich) containing penicillin and streptomycin at 37°C.

[0132] (4) Microinjection 24 to 72 hours after the hCG treatment, the linearized epigenome editing vector of the Example was introduced into the fertilized eggs. Specifically, the linearized epigenome editing vector of the Example (35 ng / μl) of Example 1(2) was microinjected into the pronuclei of the fertilized eggs in the M16 medium of Example 1(3). The injected embryos were cultured in M16 medium at 37°C and 5% CO2 in air. The following day, embryos that had developed to the two-cell stage were transplanted into the ampulla of the oviduct of pseudopregnant female ICR mice (purchased from CLEA Japan). 20 to 25 embryos were transplanted per oviduct. The insertion of the epigenome editing vector of the Example into mice derived from the ICR mice was confirmed by examining their genomic DNA. Specifically, genomic DNA was extracted from the tip of the mouse's tail using a genomic DNA extraction kit (Direct PCR Lysis Reagent, Mouse Tail, Viagenbiotech). PCR was performed on the obtained genomic DNA using the primer set in Table 2 below to examine 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 had been inserted into the genomic DNA of 9 out of 41 individuals. By backcrossing mice in which the insertion of the epigenome editing vector of the above example was confirmed with B6 mice, three fertile recombinant mouse strains (441-2, 445-7, 445-14) and their sublines were established. The weight of newborn mice from each subline was measured. Sperm from the subline mice was also collected and frozen and stored at -80°C until DNA extraction.

[0133] [Table 2]

[0134] (5) Methylation analysis The frozen sperm pellet was suspended in RSB solution containing sodium dodecyl sulfate (final concentration 2%), 2-mercaptoethanol (final concentration 2%), and proteinase K (final concentration 1 mg / ml). RSB solution consisted of 10 mmol / L NaCl, 10 mmol / L Tris (pH 7.5), and 25 mmol / L EDTA. After suspension, the suspension was incubated overnight (approximately 8 hours) at 56°C, followed by phenol / chloroform extraction and ethanol precipitation to isolate DNA.

[0135] The isolated DNA was treated with a bisulfite treatment kit (Epitect Plus DNA Bisulfite Kit, manufactured by QIAGEN). The treated DNA was then amplified by PCR using the primer set listed in Table 3 below. The demethylation rate of CpG sites was determined by combined bisulfite restriction analysis (COBRA). Specifically, the PCR-amplified fragments were cleaved with the restriction enzymes listed in Table 3 below. Table 3 below shows the sites (CpG sites) recognized by each restriction enzyme. The PCR-amplified fragments were separated and quantified using a capillary microchip electrophoresis system (MCE-202 MultiNA, manufactured by Shimadzu Corporation). The methylation rate was calculated using the following formula (1). The control was performed in the same manner, except that wild-type B6 mice were used. The results are shown in Figure 4. M = Dd / Dt × 100(%) (1) M: Methylation rate Dd: Amount of DNA cleaved by restriction enzyme (mV·μm) Dt: total DNA amount (mV·μm)

[0136] [Table 3]

[0137] Figure 4 is a graph showing the methylation rate in sperm derived from mice of the subline. In Figure 4, the horizontal axis represents the mouse strain, and the vertical axis represents the methylation rate. As shown in Figure 4, in mice whose epigenomes were modified in a sperm-specific manner, methylation in sperm was significantly reduced compared to wild-type mice. This confirmed that the introduction of the composition of the present invention enables gamete-specific modification of the epigenome in the target region.

[0138] (6) Analysis of epigenome-modified mice Male mice from the subline were used as male parent strains and mated with wild-type B6 mice to obtain F1 progeny. Female mice from the subline were used as female parent strains and mated with wild-type B6 mice to obtain F1 progeny. PCR was performed using the primers listed in Table 2 to confirm that the epigenome editing vector had been inserted into the genomic DNA of each of the F1 progeny.

[0139] Next, the weights of the newborn F1 progeny individuals were measured, and then genomic DNA was extracted from the newborn F1 progeny mice using a DNA extraction kit (AllPrep DNA / RNA Mini Kit, manufactured by QIAGEN).

[0140] 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. The results are shown in Figures 5 to 8.

[0141] Figure 5 is a graph showing the body weights of F1 progeny individuals derived from the sire line. In Figure 5, the horizontal axis represents the mouse lineage, and the vertical axis represents body weight. As shown in Figure 5, the progeny individuals of the sperm-specific epigenome-modified mice had reduced body weights compared to wild-type mice. In other words, it was presumed that the epigenome modification caused growth retardation, similar to Silver-Russell syndrome.

[0142] Next, Figure 6 is a graph showing the body weight of F1 progeny derived from the sire or dam strain. In Figure 6, the horizontal axis represents the mouse strain, and the vertical axis represents body weight. As shown in Figure 6, the F1 progeny derived from the dam strain exhibited body weights similar to those of wild-type mice. In contrast, the F1 progeny derived from the sire strain exhibited significantly reduced body weight compared to wild-type mice and F1 progeny derived from the dam strain. That is, in the dam strain, the spermatogenesis-specific promoter was not activated, the epigenome was not modified, and no weight loss occurred, whereas in the sire strain, the spermatogenesis-specific promoter was activated, the epigenome was modified, and growth retardation similar to that in Silver-Russell syndrome was presumed to occur.

[0143] Figure 7 is a graph showing the methylation rates of the target region in F1 progeny derived from the sire or dam strain. In Figure 7, (A) shows the results for CpG site (m2), and (B) shows the results for CpG site (m3). In Figures 7(A) and (B), the horizontal axis indicates the mouse strain, and the vertical axis indicates the methylation rate. As shown in Figures 7(A) and (B), the F1 progeny derived from the dam strain exhibited a methylation rate comparable to that of wild-type mice. In contrast, the F1 progeny derived from the sire strain exhibited a significantly reduced methylation rate compared to wild-type mice and the F1 progeny derived from the dam strain. In other words, in the dam strain, the spermatogenesis-specific promoter was not activated, resulting in no epigenome modification and no change in methylation. In contrast, in the sire strain, the spermatogenesis-specific promoter was activated, and demethylase was induced and targeted, thereby confirming that the epigenome of the target region was modified.

[0144] Figure 8 is a graph showing the methylation rate and body weight in mice with and without the insertion of an epigenome editing vector. In Figure 8, (A) shows the methylation rate at the CpG site (m3), and (B) shows the body weight of newborns. In Figure 8(A), the horizontal axis indicates the mouse strain or the presence or absence of insertion of an epigenome editing vector into the genomic DNA, and the vertical axis indicates the methylation rate. In Figure 8(B), the horizontal axis indicates the mouse strain or the presence or absence of insertion of an epigenome editing vector into the genomic DNA, and the vertical axis indicates body weight. As shown in Figure 8(A), regardless of the presence or absence of insertion of an epigenome editing vector into the genomic DNA, the methylation rate was reduced in the F1 progeny of the sire strain compared to wild-type mice. Furthermore, as shown in Figure 8(B), regardless of the presence or absence of insertion of an epigenome editing vector into the genomic DNA, the body weight was reduced in the F1 progeny of the sire strain compared to wild-type mice. These results indicate that if epigenome modification occurs during gamete formation, the epigenome modification is maintained even in the absence of the epigenome editing vector, i.e., even if the sequence recognition modules dCas9 and sgRNA and the epigenome modification enzyme TET1CD are not induced.

[0145] Furthermore, as mentioned above, since the epigenome editing vector can be propagated by backcrossing into B6 mice, mice into whose genomic DNA the epigenome editing vector has been introduced can also be used as parent strains for maintaining the lineage.

[0146] From the above, it was found that the composition of the present invention can be used to produce parent strains of animals and plants that can be used to produce and maintain animals and plants with modified epigenomes, and that offspring strains in which the epigenome of the target region has been modified can be obtained from the parent strains.

[0147] Although the present invention has been described above with reference to the embodiments and examples, the present invention is not limited to the above-described embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

[0148] This application claims priority based on Japanese Patent Application No. 2021-85099, filed on May 20, 2021, the disclosure of which is incorporated herein in its entirety.

[0149] <Additional Notes> Some or all of the above-described embodiments and examples can be described as, but are not limited to, the following supplementary notes. <Nucleic acid> (Appendix 1) A composition for use in producing an epigenome-modified animal or plant or a parent line for maintaining the animal or plant, comprising: the composition comprises a nucleic acid; The nucleic acid is a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence in a target region of genomic DNA to be modified in the epigenome; an epigenome modification enzyme capable of forming a complex with the nucleic acid sequence recognition module and modifying the epigenome; and a nucleic acid encoding the nucleic acid is operably linked to a gametogenesis-specific promoter that is activated during gametogenesis, thereby configuring the sequence recognition module and the epigenome modification enzyme to be expressed and induced during gametogenesis; A composition in which, during gamete formation, the induced expression of the nucleic acid sequence recognition module and the epigenome modification enzyme form a complex and modify the epigenome of the target region in the genomic DNA of the gamete being formed. (Appendix 2) 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 in a target region of genomic DNA to be modified in the epigenome; a tag domain linked to the nucleic acid sequence recognition module; and a nucleic acid encoding The second nucleic acid is an epigenome modification enzyme capable of modifying the epigenome; A tag domain binding partner linked to the epigenome modification enzyme and capable of binding to the tag domain; and a nucleic acid encoding the first nucleic acid and / or the second nucleic acid is operably linked to a gametogenesis-specific promoter that is activated during gametogenesis, thereby causing the nucleic acid sequence recognition module and the epigenome modification enzyme to be induced to be expressed during gametogenesis; The composition described in Appendix 1, wherein during gamete formation, the induced nucleic acid sequence recognition module and the epigenome modification enzyme form a complex and modify the epigenome of the target region in the genomic DNA of the gamete being formed. (Appendix 3) 3. The composition of claim 2, comprising a plurality of the tag domains. (Appendix 4) the tag domain is a peptide epitope; 4. The composition of claim 2 or 3, wherein the binding partner is an antibody against the peptide epitope. (Appendix 5) the nucleic acid sequence recognition module is a CRISPR-Cas system; The CRISPR-Cas system a guide strand comprising a nucleic acid sequence that specifically binds to a nucleic acid sequence in the target region; Cas protein and Including, 5. The composition of any of Appendices 2 to 4, wherein the Cas protein is linked to the tag domain. (Appendix 6) the nucleic acid sequence recognition module is a CRISPR-Cas system; The CRISPR-Cas system a guide strand comprising a nucleic acid sequence that specifically binds to a nucleic acid sequence in the target region; Cas protein and 5. The composition of any of Appendices 1 to 4, comprising: (Appendix 7) 7. The composition of any one of claims 1 to 6, wherein the nucleic acid sequence recognition module does not cleave both strands of the double stranded genomic DNA. (Appendix 8) 8. The composition of any of claims 1 to 7, wherein the target region is a regulatory and / or promoter region of an imprinted gene. (Appendix 9) The composition of any of Appendices 1 to 8, wherein the epigenome-modifying enzyme is a base-modifying enzyme. (Appendix 10) The composition of any of Appendices 1 to 9, wherein the epigenome-modifying enzyme is a methylase, demethylase, acetylase, deacetylase, kinase, phosphatase, ubiquitinase, and / or sumoylase. (Appendix 11) The composition of any 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. (Appendix 12) 12. The composition of any of claims 1 to 11, wherein the gametogenesis-specific promoter is a spermatogenesis-specific promoter or an oogenesis-specific promoter. (Appendix 13) 13. The composition of any of claims 1 to 12, wherein the gametogenesis-specific promoter is a STRA8 promoter, a Pgk2 promoter, a Dazl promoter, and / or a Gsg2 promoter. (Appendix 14) 13. The composition of any of claims 1 to 12, wherein the gametogenesis-specific promoter is a Gdf-9 promoter, a Zp3 promoter, and / or an Msx2 promoter. (Appendix 15) an expression vector, A composition described in any of Appendix 1 to 14, wherein the expression vector operably links the nucleic acid to the nucleic acid sequence recognition module so that the epigenome modification enzyme can be expressed. (Appendix 16) a first expression vector and a second expression vector, The first expression vector has the nucleic acid operably linked thereto so that the nucleic acid sequence recognition module can be expressed; A composition described in any of Appendices 1 to 15, wherein the second expression vector is operably linked to the nucleic acid so as to enable expression of the epigenome modification enzyme. <Production method of parent line> (Appendix 17) A method for producing parental lines of epigenome-modified animals and plants, comprising: A method of production comprising the step of introducing into a target animal or plant a composition described in any one of Appendices 1 to 16. (Appendix 18) The method of claim 17, wherein the plant or animal is a non-human animal. <Parent lineage animals and plants> (Appendix 19) A parental line of an animal or plant for producing or maintaining an epigenome-modified animal or plant, The animal or plant contains an exogenous nucleic acid, The nucleic acid is a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence in a target region of genomic DNA to be modified in the epigenome; an epigenome modification enzyme capable of forming a complex with the nucleic acid sequence recognition module and modifying the epigenome; and a nucleic acid encoding the nucleic acid is operably linked to a gametogenesis-specific promoter that is activated during gametogenesis, thereby configuring the sequence recognition module and the epigenome modification enzyme to be expressed and induced during gametogenesis; In the formation of gametes, the nucleic acid sequence recognition module whose expression is induced and the epigenome modification enzyme form a complex and modify the epigenome of the target region in the genomic DNA of the gamete being formed, in animals and plants. (Appendix 20) 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 in a target region of genomic DNA to be modified in the epigenome; a tag domain linked to the nucleic acid sequence recognition module; and a nucleic acid encoding The second nucleic acid is an epigenome modification enzyme capable of modifying the epigenome; A tag domain binding partner linked to the epigenome modification enzyme and capable of binding to the tag domain; and a nucleic acid encoding the first nucleic acid and / or the second nucleic acid is operably linked to a gametogenesis-specific promoter that is activated during gametogenesis, thereby causing the nucleic acid sequence recognition module and the epigenome modification enzyme to be induced to be expressed during gametogenesis; The animal or plant described in Appendix 19, wherein during gamete formation, the induced nucleic acid sequence recognition module and the epigenome modification enzyme form a complex and modify the epigenome of the target region in the genomic DNA of the gamete being formed. (Appendix 21) 21. The plant or animal described in Appendix 20, comprising a plurality of the tag domains. (Appendix 22) the tag domain is a peptide epitope; 22. The animal or plant according to claim 20 or 21, wherein the binding partner is an antibody against the peptide epitope. (Appendix 23) the nucleic acid sequence recognition module is a CRISPR-Cas system; The CRISPR-Cas system a guide strand comprising a nucleic acid sequence that specifically binds to a nucleic acid sequence in the target region; Cas protein and Including, 23. The plant or animal of any one of appendices 19 to 22, wherein the Cas protein is linked to the tag domain. (Appendix 24) the nucleic acid sequence recognition module is a CRISPR-Cas system; The CRISPR-Cas system a guide strand comprising a nucleic acid sequence that specifically binds to a nucleic acid sequence in the target region; Cas protein and 24. The plant or animal described in any one of appendices 19 to 23, including: (Appendix 25) 25. The animal or plant described in any one of appendices 19 to 24, wherein the nucleic acid sequence recognition module does not cleave both strands of the double strand of the genomic DNA. (Appendix 26) 26. The animal or plant according to any one of appendices 19 to 25, wherein the target region is a regulatory region and / or promoter region of an imprinted gene. (Appendix 27) 27. The animal or plant described in any one of Appendices 19 to 26, wherein the epigenome modification enzyme is a base-modifying enzyme. (Appendix 28) 28. The animal or plant described in any one of appendices 19 to 27, wherein the epigenome-modifying enzyme is a methylase, demethylase, acetylase, deacetylase, kinase, phosphatase, ubiquitinase, and / or sumoylase. (Appendix 29) The animal or plant described in any of Appendices 19 to 28, wherein the epigenome modification enzyme is TET (ten-eleven translocation) 1, TET2, TET3, DNMT (DNA methyltransferase) 1, DNMT3A, and / or DNMT3B. (Appendix 30) 30. The plant or animal described in any one of appendix 19 to 29, wherein the gametogenesis-specific promoter is a spermatogenesis-specific promoter or an oogenesis-specific promoter. (Appendix 31) 31. The animal or plant according to any one of appendices 19 to 30, wherein the gametogenesis-specific promoter is a STRA8 promoter, a Pgk2 promoter, a Dazl promoter, and / or a Gsg2 promoter. (Appendix 32) 31. The animal or plant according to any one of appendices 19 to 30, wherein the gametogenesis-specific promoter is a Gdf-9 promoter, a Zp3 promoter, and / or an Msx2 promoter. (Appendix 33) an expression vector, An animal or plant described in any of Appendices 19 to 32, wherein the expression vector functionally links the nucleic acid to the nucleic acid sequence recognition module so that the epigenome modification enzyme can be expressed. (Appendix 34) a first expression vector and a second expression vector, The first expression vector has the nucleic acid operably linked thereto so that the nucleic acid sequence recognition module can be expressed; An animal or plant described in any of Appendices 19 to 33, wherein the nucleic acid is operably linked to the second expression vector so that the epigenome modification enzyme can be expressed. (Appendix 35) 35. The plant or animal described in any one of appendices 19 to 34, wherein the animal is a non-human animal. (Appendix 36) 36. The plant or animal described in any one of appendices 19 to 35, wherein the nucleic acid is inserted into genomic DNA. (Appendix 37) the gametogenesis-specific promoter is a spermatogenesis-specific promoter, The parent line used in the production is a male parent line, 37. The animal or plant according to any one of appendices 19 to 36, wherein the parent line used for the maintenance is a female parent line. (Appendix 38) the gametogenesis-specific promoter is an oogenesis-specific promoter; The parent line used in the production is a female parent line, 37. The animal or plant according to any one of appendices 19 to 36, wherein the parent line used for the maintenance is a male parent line. (Appendix 39) 39. The animal or plant according to any one of Appendices 19 to 38, wherein the parent line has no epigenome modification of the target region in cells other than the gametes. <gametes> (Appendix 40) A gamete used to produce an epigenome-modified animal or plant or a parent line for maintaining the animal or plant, 39. A gamete isolated from an animal or plant of a parental lineage according to any one of appendices 19 to 39. (Appendix 41) 41. The gamete of claim 40, wherein the gamete is a sperm and / or an egg. (Appendix 42) A gamete described in Appendix 40 or 41, in which the nucleic acid sequence recognition module induced to express during gametogenesis and the epigenome modification enzyme form a complex, and the epigenome of the target region is modified. (Appendix 43) 43. A gamete according to any one of claims 40 to 42, comprising the exogenous nucleic acid. <Method for producing epigenome-modified animals and plants> (Appendix 44) A method for producing an animal or plant in which the epigenome of a target region is modified, comprising: The method includes a step of crossbreeding the first parent with the second parent and obtaining an individual having a modified epigenome from the resulting progeny individual, 40. The method of claim 19, wherein the first parent and / or the second parent is an animal or plant of a parental lineage as described in any one of appendices 19 to 39. (Appendix 45) 45. The production method according to claim 44, comprising the step of selecting individuals that do not contain the nucleic acid from the progeny individuals. <Animals and plants with modified epigenomes> (Appendix 46) An animal or plant in which the epigenome of a target region has been modified, The animals and plants are The target region comprises a target region in which the epigenome derived from a gamete according to any one of Supplementary Notes 40 to 42 has been modified; does not contain the exogenous nucleic acid, Animals and plants. (Appendix 47) 47. The plant or animal according to claim 46, obtained by the production method according to claim 44 or 45. (Appendix 48) 48. The animal or plant described in Appendix 46 or 47, wherein the animal or plant is a non-human animal. <Animals and plants with modified epigenomes> (Appendix 49) An animal or plant in which the epigenome of a target region has been modified, The animal or plant contains an exogenous nucleic acid, The nucleic acid is a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence in a target region of genomic DNA to be modified in the epigenome; an epigenome modification enzyme capable of forming a complex with the nucleic acid sequence recognition module and modifying the epigenome; and a nucleic acid encoding the nucleic acid is operably linked to a gametogenesis-specific promoter that is activated during gametogenesis, thereby configuring the sequence recognition module and the epigenome modification enzyme to be expressed and induced during gametogenesis; The animal or plant includes, as the target region, a target region derived from a gamete in which the nucleic acid sequence recognition module whose expression is induced during gametogenesis and the epigenome modification enzyme form a complex, and the epigenome of the target region is modified. (Appendix 50) 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 in a target region in a genome for modifying the epigenome; a tag domain linked to the nucleic acid sequence recognition module; and a nucleic acid encoding The second nucleic acid is an epigenome modification enzyme capable of modifying the epigenome; A tag domain binding partner linked to the epigenome modification enzyme and capable of binding to the tag domain; and a nucleic acid encoding the first nucleic acid and / or the second nucleic acid is operably linked to a gamete-specific promoter that is active during gametogenesis; The animal or plant is described in Appendix 49, wherein the target region is derived from a gamete in which the nucleic acid sequence recognition module induced to express during gametogenesis and the epigenome modification enzyme form a complex, and the epigenome of the target region is modified. (Appendix 51) 51. The animal or plant described in Appendix 49 or 50, wherein the animal or plant is a non-human animal. [Industrial Applicability]

[0150] As described above, the composition of the present invention enables the production of parent lineage animals and plants that can be used to produce and maintain epigenome-modified animals and plants. Therefore, the present invention can be suitably used for analyzing diseases caused by epigenome changes. Therefore, the present invention is extremely useful, for example, in the fields of life science and medicine.

Claims

1. A composition for use in producing an epigenome-modified animal or plant or a parent line for maintaining the animal or plant, comprising: the composition comprises a nucleic acid; The nucleic acid is a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence in a target region of genomic DNA for modifying the epigenome; an epigenome modification enzyme capable of forming a complex with the nucleic acid sequence recognition module and modifying the epigenome; and a nucleic acid encoding the nucleic acid is operably linked to a gametogenesis-specific promoter that is activated during gametogenesis, thereby causing the nucleic acid sequence recognition module and the epigenome modification enzyme to be expressed during gametogenesis; A composition in which, during gamete formation, the induced expression of the nucleic acid sequence recognition module and the epigenome modification enzyme form a complex and modify the epigenome of the target region in the genomic DNA of the gamete being formed.

2. the nucleic acid comprises a first nucleic acid and a second nucleic acid; The first nucleic acid comprises: a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence in a target region of genomic DNA for modifying the epigenome; a tag domain linked to the nucleic acid sequence recognition module; and a nucleic acid encoding The second nucleic acid comprises: an epigenome modification enzyme capable of modifying the epigenome; A tag domain binding partner linked to the epigenome modification enzyme and capable of binding to the tag domain; and a nucleic acid encoding the first nucleic acid and / or the second nucleic acid is operably linked to a gametogenesis-specific promoter that is activated during gametogenesis, thereby causing the nucleic acid sequence recognition module and the epigenome modification enzyme to be induced to be expressed during gametogenesis; The composition described in claim 1, wherein during gametogenesis, the induced expression of the nucleic acid sequence recognition module and the epigenome modification enzyme form a complex and modify the epigenome of the target region in the genomic DNA of the gamete being formed.

3. The composition of claim 2, comprising a plurality of said tag domains.

4. the tag domain is a peptide epitope; The composition of claim 2 , wherein the binding partner is an antibody against the peptide epitope.

5. The nucleic acid sequence recognition module is a CRISPR-Cas system; The CRISPR-Cas system a guide strand comprising a nucleic acid sequence that specifically binds to a nucleic acid sequence in the target region; a Cas protein; and Including, 3. The composition of claim 2, wherein the Cas protein is linked to the tag domain.

6. The composition of claim 1 , wherein the nucleic acid sequence recognition module does not cleave both strands of the double-stranded genomic DNA.

7. The composition of claim 1 , wherein the target region is a regulatory and / or promoter region of an imprinted gene.

8. The composition of claim 1 , wherein the epigenome-modifying enzyme is a base-modifying enzyme.

9. The composition of claim 1, wherein the epigenome-modifying enzyme is a methylase, demethylase, acetylase, deacetylase, kinase, phosphatase, ubiquitinase, and / or sumoylase.

10. The composition of claim 1, wherein the epigenome-modifying enzyme is TET (ten-eleven translocation) 1, TET2, TET3, DNMT (DNA methyltransferase) 1, DNMT3A, and / or DNMT3B.

11. The composition of claim 1 , wherein the gametogenesis-specific promoter is a spermatogenesis-specific promoter or an oogenesis-specific promoter.

12. The composition of claim 1 , wherein the gametogenesis-specific promoter is a STRA8 promoter, a Pgk2 promoter, a Dazl promoter, and / or a Gsg2 promoter.

13. The composition of claim 1, wherein the gametogenesis-specific promoter is a Gdf-9 promoter, a Zp3 promoter, and / or an Msx2 promoter.

14. an expression vector, The composition of claim 1, wherein the expression vector operably links the nucleic acid to the nucleic acid sequence recognition module so that the epigenome modification enzyme can be expressed.

15. a first expression vector and a second expression vector, The first expression vector has the nucleic acid operably linked thereto so that the nucleic acid sequence recognition module can be expressed; The composition of claim 1, wherein the second expression vector is operably linked to the nucleic acid so as to enable expression of the epigenome modification enzyme.

16. A method for producing parental lines of epigenome-modified animals and plants (excluding humans), comprising: A method of production comprising the step of introducing into a target animal or plant (excluding humans) a composition according to any one of claims 1 to 15.

17. The method of claim 16, wherein the plant or animal is a non-human animal.

18. An animal or plant (excluding humans) whose epigenome has been modified, or an animal or plant (excluding humans) whose parent lineage is used for maintenance, The animal or plant contains an exogenous nucleic acid, The nucleic acid is a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence in a target region of genomic DNA for modifying the epigenome; an epigenome modification enzyme capable of forming a complex with the nucleic acid sequence recognition module and modifying the epigenome; and a nucleic acid encoding the nucleic acid is operably linked to a gametogenesis-specific promoter that is activated during gametogenesis, thereby causing the nucleic acid sequence recognition module and the epigenome modification enzyme to be expressed during gametogenesis; During gamete formation, the induced expression of the nucleic acid sequence recognition module and the epigenome modification enzyme form a complex and modify the epigenome of the target region in the genomic DNA of the gamete being formed, in animals and plants (excluding humans).

19. the nucleic acid comprises a first nucleic acid and a second nucleic acid; The first nucleic acid comprises: a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence in a target region of genomic DNA for modifying the epigenome; a tag domain linked to the nucleic acid sequence recognition module; and a nucleic acid encoding The second nucleic acid comprises: an epigenome modification enzyme capable of modifying the epigenome; A tag domain binding partner linked to the epigenome modification enzyme and capable of binding to the tag domain; and a nucleic acid encoding the first nucleic acid and / or the second nucleic acid is operably linked to a gametogenesis-specific promoter that is activated during gametogenesis, thereby causing the nucleic acid sequence recognition module and the epigenome modification enzyme to be induced to be expressed during gametogenesis; The animal or plant described in claim 18, wherein during gamete formation, the induced expression of the nucleic acid sequence recognition module and the epigenome modification enzyme form a complex and modify the epigenome of the target region in the genomic DNA of the gamete being formed.

20. The plant or animal described in claim 19, comprising a plurality of said tag domains.

21. the tag domain is a peptide epitope; 20. The plant or animal of claim 19, wherein the binding partner is an antibody against the peptide epitope.

22. The nucleic acid sequence recognition module is a CRISPR-Cas system; The CRISPR-Cas system a guide strand comprising a nucleic acid sequence that specifically binds to a nucleic acid sequence in the target region; a Cas protein; and Including, 20. The plant or animal of claim 19, wherein the Cas protein is linked to the tag domain.

23. The plant or animal described in claim 18, wherein the nucleic acid sequence recognition module does not cleave both strands of the double strand of the genomic DNA.

24. The animal or plant of claim 18, wherein the target region is a regulatory region and / or promoter region of an imprinted gene.

25. The animal or plant described in claim 18, wherein the epigenome-modifying enzyme is a base-modifying enzyme.

26. The animal or plant described in claim 18, wherein the epigenome-modifying enzyme is a methylase, demethylase, acetylase, deacetylase, kinase, dephosphorylase, ubiquitinase, and / or sumoylase.

27. The animal or plant described in claim 18, wherein the epigenome modification enzyme is TET (ten-eleven translocation) 1, TET2, TET3, DNMT (DNA methyltransferase) 1, DNMT3A, and / or DNMT3B.

28. The plant or animal of claim 18, wherein the gametogenesis-specific promoter is a spermatogenesis-specific promoter or an oogenesis-specific promoter.

29. The plant or animal of claim 18, wherein the gametogenesis-specific promoter is a STRA8 promoter, a Pgk2 promoter, a Dazl promoter, and / or a Gsg2 promoter.

30. The animal or plant according to claim 18, wherein the gametogenesis-specific promoter is a Gdf-9 promoter, a Zp3 promoter, and / or an Msx2 promoter.

31. an expression vector, The animal or plant described in claim 18, wherein the expression vector functionally links the nucleic acid to the nucleic acid sequence recognition module so that the epigenome modification enzyme can be expressed.

32. a first expression vector and a second expression vector, The first expression vector has the nucleic acid operably linked thereto so that the nucleic acid sequence recognition module can be expressed; The animal or plant described in claim 18, wherein the nucleic acid is operably linked to the second expression vector so that the epigenome modification enzyme can be expressed.

33. The plant or animal of claim 18, wherein the plant or animal is a non-human animal.

34. The plant or animal of claim 18, wherein the nucleic acid is inserted into genomic DNA.

35. the gametogenesis-specific promoter is a spermatogenesis-specific promoter, The parent line used in the production is a male parent line, 19. The plant or animal of claim 18, wherein the parent line used for the maintenance is a female parent line.

36. the gametogenesis-specific promoter is an oogenesis-specific promoter; The parent line used in the production is a female parent line, The plant or animal according to claim 18, wherein the parent line used for the maintenance is a male parent line.

37. The animal or plant of claim 18, wherein the parent line has no modification of the epigenome of the target region in cells other than the gametes.

38. A gamete used to produce an epigenome-modified animal or plant or a parent line for maintaining the animal or plant, 38. A gamete isolated from an animal or plant of the parental lineage of any one of claims 18 to 37.

39. 39. The gamete of claim 38, wherein the gamete is a sperm and / or an egg.

40. The gamete described in claim 38, wherein the nucleic acid sequence recognition module and the epigenome modification enzyme, whose expression is induced during gametogenesis, form a complex, and the epigenome of the target region is modified.

41. 39. The gamete of claim 38, comprising said exogenous nucleic acid.

42. A method for producing an animal or plant in which the epigenome of a target region is modified, comprising: The method comprises a step of crossbreeding the first parent with the second parent and obtaining an individual having a modified epigenome from the resulting progeny individual, 38. A method of production wherein the first parent and / or the second parent is an animal or plant of the parental lineage described in any one of claims 18 to 37.

43. The production method according to claim 42, further comprising the step of selecting individuals that do not contain the nucleic acid from the progeny individuals.

44. An animal or plant (excluding humans) in which the epigenome of a target region has been modified, The animals and plants are The target region comprises a target region in which the epigenome derived from the gamete according to claim 38 has been modified; does not contain the exogenous nucleic acid, Animals and plants (excluding humans).

45. 45. The plant or animal according to claim 44, obtained by the method according to claim 42.

46. 45. The plant or animal of claim 44, wherein the plant or animal is a non-human animal.

47. An animal or plant (excluding humans) in which the epigenome of a target region has been modified, The animal or plant contains an exogenous nucleic acid, The nucleic acid is a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence in a target region of genomic DNA for modifying the epigenome; an epigenome modification enzyme capable of forming a complex with the nucleic acid sequence recognition module and modifying the epigenome; and a nucleic acid encoding the nucleic acid is operably linked to a gametogenesis-specific promoter that is activated during gametogenesis, thereby causing the nucleic acid sequence recognition module and the epigenome modification enzyme to be expressed during gametogenesis; The animals and plants (excluding humans) include, as the target region, a target region derived from a gamete in which the nucleic acid sequence recognition module whose expression is induced during gametogenesis and the epigenome modification enzyme form a complex, and the epigenome of the target region is modified.

48. a first nucleic acid and a second nucleic acid, The first nucleic acid comprises: a nucleic acid sequence recognition module that specifically binds to a nucleic acid sequence in a target region in a genome for modifying the epigenome; a tag domain linked to the nucleic acid sequence recognition module; and a nucleic acid encoding The second nucleic acid comprises: an epigenome modification enzyme capable of modifying the epigenome; A tag domain binding partner linked to the epigenome modification enzyme and capable of binding to the tag domain; and a nucleic acid encoding the first nucleic acid and / or the second nucleic acid is operably linked to a gametogenesis-specific promoter that is active in gametogenesis; The animal or plant described in claim 47, wherein the target region is derived from a gamete in which the nucleic acid sequence recognition module induced to express during gametogenesis and the epigenome modification enzyme form a complex, and the epigenome of the target region is modified.

49. 49. The plant or animal of claim 47 or 48, wherein the plant or animal is a non-human animal.

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