JP7823919B2 - Compositions for use in producing parental lines of epigenome-modified animals and plants and uses thereof - Google Patents

Description

The present invention relates to compositions and uses for producing parental lines of animals and plants with modified epigenomes.

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

In recent years, causative genes for various diseases have been identified by using genetic engineering techniques to generate genetically modified mice. Meanwhile, it has also been suggested that changes in epigenetic modifications may be the cause of the disease.

Allis, C.D. et al., “Epigenetics”, Cold Spring Harbor Lab. Press, (2015)

To investigate the role of epigenetic modifications in various diseases, the inventors used the following method to generate mice with epigenetic modifications of genomic DNA, i.e., mice with modified epigenomes. Specifically, using dCas9, a form of the Cas9 protein used in the CRISPR/Cas9 method in which the nuclease activity was inactivated, the demethylase TET (ten-eleven translocation) 1 (TET1) was targeted to a target region, thereby modifying the epigenome of the target region. However, as with conventional 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. However, depending on the target region, problems arose in that the resulting epigenome-modified mice exhibited poor growth and died before sexual maturity, making them difficult to maintain through breeding.

Therefore, the present invention aims to provide a composition for producing parent lineage animals and plants that can be used to produce and maintain animals and plants with modified epigenomes.

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 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 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.

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.

The parent strain of animals and plants of the present invention (hereinafter also referred to as "parent strain") is a parent 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 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 configuring the sequence recognition module and the epigenome modification enzyme to be expressed and induced 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.

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.

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 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,
The first parent and/or the second parent are plants or animals of the parental lineage of the present invention.

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.

The animal or plant of the present invention (hereinafter also referred to as "second animal or plant") is 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 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 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.

The composition of the present invention makes it possible to produce parental lines of animals and plants that can be used to produce and maintain animals and plants with modified epigenomes.

FIG. 1 is a schematic diagram showing an example of epigenetic changes in a parent line produced using the composition of the present invention. 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 is a schematic diagram showing the structure of the epigenome editing vector in Example 1. FIG. 4 is a graph showing the methylation rate in sperm derived from subline mice in Example 1. FIG. 5 is a graph showing the body weights of F1 progeny individuals derived from the male parent line in Example 1. 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. 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 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.

<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.

As used herein, "epigenome modification" or "epigenome modification" refers to changing the chemical modifications of DNA and/or histone proteins in the genome. The change in modification preferably 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, or increasing or decreasing the frequency of a modification. The "epigenome modification" or "epigenome modification" can also be referred to as, for example, "modification of epigenetic modifications" or "modification of genome modifications."

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

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

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."

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

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

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

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

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

As used herein, "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, D, and J regions. The antibody comprises, for example, a heavy chain and a light chain. The light chain comprises κ and λ, constituting the κ chain and λ chain, respectively. The heavy chain comprises 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 two identical pairs of polypeptide chains, each pair consisting of one light chain (about 25 kDa) and one heavy chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The antibody may be a full-length immunoglobulin or an antigen-binding fragment thereof.

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.

As used herein, "gamete" refers to a reproductive cell that can generate a new individual through union or fertilization. The gamete can also be referred to as, for example, a "gamete" or a "gametome." Specific examples of the gamete include heterogametes, such as sperm and eggs in animals, and pollen and embryo sacs in plants.

As used herein, "imprinted gene" refers to a gene whose expression is controlled by genomic imprinting.

As used herein, "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).

As used herein, the term "promoter" or "promoter region" refers to a region that is located upstream of a DNA region encoding a gene or 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."

As used herein, "expression vector" or "vector" refers to a recombinant plasmid, virus, or virus-like particle (VLP) containing a nucleic acid to be delivered to a host cell, either in vitro or in vivo .

As used herein, "animals and plants" refers to taxonomic animals and plants. The term "animals and plants" includes any animals and plants that form gametes. Examples of animals include humans and non-human animals. Examples of non-human animals include mammals such as mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, dolphins, and sea lions. Examples of plants include angiosperms.

As used herein, "exogenous" means introduced into a cell from outside the cells that make up an animal or plant.

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 at least one purification step.

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

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

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, all descriptions in this invention are mutually applicable unless otherwise specified. In this specification, the expression "~" is used to include the numerical or physical values before and after it. In this specification, the expression "A and/or B" includes "A only," "B only," and "both A and B."

<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.

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 follows. As a specific example, a case will be described using Figure 1 in which the animal or plant is a mouse, the gamete in which the epigenome modification occurs is a gamete of the 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. Note that the present invention is not limited in any way by the following explanation. 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. In the testes of the male parent strain, sperm are formed from primordial germ cells. 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 containing a chromosome (TG2) in which the epigenome is modified and contains the nucleic acid, or a chromosome (WT2) in which the epigenome is modified and does not contain the nucleic acid, is formed. Fertilizing the epigenome-modified sperm with an unmodified egg produces an epigenome-modified mouse in which one chromosome is epigenome-modified and the other is unmodified. 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.

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, eggs are formed from primordial germ cells. During egg formation, 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 eggs are formed containing the chromosome (TG1) containing the nucleic acid or the chromosome (WT1) not containing the nucleic acid that is not epigenome-modified. Then, by fertilizing the non-epigenome-modified egg 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 mouse has genomic DNA similar to that of the male and female parent strains, and therefore can be used to maintain the lineage. Therefore, the composition of the present invention can 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 composition of the present invention can be used to produce epigenome 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. The epigenome-modified mouse is identical to a mouse whose epigenome has not been modified and contains two sets of chromosomes that do not contain the nucleic acid, except for the modification of the epigenome of the target region. Therefore, the influence of exogenous nucleic acids can be eliminated, and epigenome-modified mice can be produced that allow for the examination of the influence of the epigenome modification alone. Although the above description has been given taking the example of a case where the gamete in which the epigenome modification occurs is a sperm, as shown in Figure 2, even when the gamete in which the epigenome modification occurs is a gamete of a female parent lineage, i.e., an egg, it is possible to similarly produce a parent lineage animal or plant that can be used to produce and/or maintain an epigenome-modified animal or plant. Furthermore, although mice were used as the animal or plant, the same can be applied to plants that form gametes (e.g., pollen and embryo sacs).

The target region can be 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 imprinted genes. Specific examples of atypical imprinted genes 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, the regulatory region can be, for example, H19-DMR.

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

The nucleic acid sequence recognition module is a molecule that specifically binds to the nucleic acid sequence of the 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 modifications 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, the desired epigenome modification can be performed on the target region.

The nucleic acid sequence recognition module may be, for example, 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 motif, transcription activator-like (TAL) effector, PRR (Pentatricopeptide repeat) motif, restriction enzyme, transcription factor, RNA polymerase, and DNA polymerase, or DNA-binding domains thereof. Preferably, the nucleic acid sequence recognition module is a CRISPR-Cas system, zinc finger motif, TAL effector, or PRR motif, or a DNA-binding domain thereof. To suppress changes in the nucleic acid sequence of the genome, for example, 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.

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 Cas proteins of types I to V. 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, Examples include 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.

The origin of the Cas protein is not particularly limited. When Cas9 is used as the Cas protein, examples of the Cas9 protein include Cas9 (SaCas9) derived from Staphylococcus aureus , Cas9 (SpCas9) derived from Streptococcus pyogenes , and Cas9 (StCas9) derived from Streptococcus thermophilus .

Preferably, the nuclease activity (DNA cleavage ability) of at least one of the Cas protein's DNA cleavage domains (cleavage sites) is 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 domains are inactivated. 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, for example, substituting the asparagine (Asn) at position 836 and/or the histidine residue (His) at position 840 with an alanine residue (Ala), thereby inactivating the ability to cleave the strand complementary to the guide RNA. Furthermore, the RuvC domain of the SpCas9 can be inactivated, for example, by substituting the 10th aspartic acid residue (Asp) with an alanine residue (Ala), i.e., the ability to cleave the strand opposite the complementary strand to the guide RNA (reverse complementary strand) can be inactivated. The SpCas9 protein, for example, has its nuclease activity inactivated by substituting the 840th histidine residue (His) with an alanine residue (Ala) and the 10th aspartic acid residue (Asp) with an alanine residue (Ala) (dCas9 protein). The SpCas9 protein also has its nuclease activity inactivated by substituting the 836th asparagine (Asn) with an alanine residue (Ala) and the 10th aspartic acid residue (Asp) with an alanine residue (Ala) (dCas9 protein).

The guide RNA comprises a polynucleotide having a nucleic acid sequence complementary to a target nucleic acid sequence present in 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 in the target region, i.e., an RNA molecule comprising a polynucleotide complementary to the nucleic acid sequence in the target region.

The guide RNA can be appropriately designed depending on the type of 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 that functions as the guide RNA. In the latter case, the sgRNA is composed of crRNA and tracrRNA, or these are linked via a linker.

The crRNA preferably includes, for example, at its 5' end, a polynucleotide complementary to the target nucleic acid sequence. By using the target nucleic acid sequence as a 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 long or 18 to 22 bases long.

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

The guide RNA may target one or more nucleic acid sequences. The number of types of guide RNAs can be set, for example, depending on the length of the target region. Specifically, 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. Specifically, 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.

The zinc finger motif is formed by linking multiple zinc finger units of different C ₂ H ₂ (Cys ₂ His ₂ ) types. One zinc finger unit recognizes a nucleic acid sequence of about 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 about 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 Escherichia coli one-hybrid method (Nat Biotechnol (2008) 26: 695-701). The zinc finger motif can be prepared by the method described in WO 03/087341, for example.

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 target nucleic acid sequences. TAL effectors specific to target nucleic acid sequences 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 International Publication No. 2011/072246.

PPR motifs are composed of 35 amino acids and are configured to recognize a specific nucleic acid sequence by arranging them consecutively, each recognizing 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 motifs can be produced, for example, by the method described in International Publication No. WO 2014/175284.

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

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 encode multiple types of nucleic acid sequence recognition modules that specifically bind to nucleic acid sequences at different sites in the target region, thereby modifying the epigenome of the entire target region.

The target of modification by the epigenome-modifying enzyme may be the chemical modification of genomic DNA or the chemical modification of histone proteins. Examples of the epigenome-modifying enzyme include methylases, demethylases, acetylases, deacetylases, kinases, dephosphorylating enzymes, ubiquitinases, and sumoylases.

When the modification target is a chemical modification of genomic DNA, the epigenome modification enzyme can be, for example, a methylation enzyme or a demethylation enzyme. When the animal or plant is an animal, the methylation enzyme can methylate, for example, cytosine nucleotides at CpG sites in the genomic DNA. In this case, the methylation enzyme can be, for example, DNMT (DNA methyltransferase) 3A, DNMT3B, or DNMT3L. When DNMT3A or DNMT3B is used as the epigenome modification enzyme, it is preferable to use these methylation enzymes in combination with DNMT3L. When the animal or plant is a plant, the methylation enzyme can methylate, for example, cytosine nucleotides at CpG, CpHpG, or CpHpH sites (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, and CMT (chromomethylase) 2.

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.

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, dephosphorylation enzymes, ubiquitination enzymes, and sumoylation enzymes.

The methylation enzyme, for example, methylates lysine and/or arginine residues in the histone protein. Examples of the methylation enzyme include EZH2 (enhancer of zeste homolog 2), G9, SUV39H1, and the like, which are classified as PRC2. The demethylation enzyme, for example, demethylates the methyl groups of lysine and/or arginine residues in the histone protein. Examples of the demethylation enzyme include LSD1 (lysine-specific demethylase 1), KDM4D (lysine-specific demethylase 4D), and KDM6B (lysine demethylase 6B). The acetylation enzyme, for example, acetylates lysine residues in the histone protein. In this case, examples of the acetylation enzyme include Gcn5 and P300/CBP (CREB-binding protein). The deacetylase, 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, for example, phosphorylates a serine residue and/or a threonine residue in the histone protein. In this case, examples of the kinase include Haspin (GSG2), Aurora B, ChK1 (Checkpoint kinase 1), etc. The dephosphatase, for example, dephosphorylates a phosphate group of a serine residue and/or a threonine residue 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 ubiquitinating enzyme, for example, ubiquitinates a lysine residue in the histone protein. In this case, examples of the ubiquitinating enzyme include Ring1, RNF8, UBC13, and UHRF1. The sumoylating enzyme, for example, sumoylates a lysine residue in the histone protein. In this case, examples of the sumoylating enzyme include UBC9.

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

The epigenome-modifying enzyme may be the entire enzyme protein or a portion thereof. When the epigenome-modifying enzyme is a portion of an enzyme protein, the epigenome-modifying enzyme needs only to retain the enzymatic activity of the enzyme protein; for example, an enzyme catalytic site (catalytic site) can be used.

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 may be by formation of a complex utilizing intermolecular interactions.

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.

The nucleic acid sequence recognition module may form a complex with one epigenome modification enzyme or multiple epigenome modification enzymes. Furthermore, 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 epigenomes located farther from the target nucleic acid sequence within 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. Furthermore, when the nucleic acid sequence recognition module forms a complex with multiple epigenome modification enzymes, the epigenome modification enzymes may be of one type or multiple types. By using multiple types of epigenome modification enzymes, the complex can, for example, modify multiple types of epigenomes in the target region.

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, 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.

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 may be 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.

Any sequence can be selected for the linker as long as it does not interfere with the function 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 length of the linker is, 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. By increasing the length of the linker relatively, the resulting complex can also modify the 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; GSGSGGSGSGSGSGGSGGSGSGSGSGSGSG (SEQ ID NO: 5); GSGSGGSGSGGSGGSGSGGSGGGSGSGGSGSGGSGSGGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSGSG (SEQ ID NO: 6); and the like.

When the complex is formed using an intermolecular interaction, the nucleic acid sequence recognition module and the epigenome modification enzyme may form a complex using a tag domain and a binding partner of the tag domain that can bind to the tag domain 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. Note that in the following description, an example is 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 used interchangeably.

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. Furthermore, the epigenome modification enzyme and the binding partner are, for example, directly or indirectly linked, i.e., form a fusion protein.

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.

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.

By configuring the nucleic acid sequence recognition module and the epigenome modification enzyme so that one nucleic acid sequence recognition module and multiple epigenome modification enzymes form a complex, epigenome modification can be performed more efficiently and epigenomes located farther from the target nucleic acid sequence within the target region can also be modified compared to 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, for example, be linked to multiple tag domains, and one binding partner may be linked to multiple epigenome modification enzymes.

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 contiguously. 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.

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 N- or C-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 both the nucleic acid sequence recognition module and the tag domain and the tag domains may 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 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.

When the epigenome modification enzyme and the binding partner are directly linked, the N- or C-terminal amino acid of the epigenome modification enzyme protein 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.

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 bound 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 to each other, 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.

When the nucleic acid sequence recognition module and the epigenome modification enzyme form a complex through 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, the tag domain can also be referred to as a peptide tag.

Examples of combinations of peptide epitopes and antibodies that recognize them include: a GCN (General Control Non-derepressible) 4 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; and the like (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).

A self-assembling split protein is a protein that, when divided into two, can reassemble to form the same structure as the original protein. In this case, a short peptide (small fragment) obtained by dividing the original protein into two 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 a 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.

The tag domain and 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.

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 peptide-protein domain pair can be strengthened by linking another inactive domain via a linker and improving the pair through evolutionary engineering. Using the pair obtained through this 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).

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.

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 other sources. 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 modules and epigenome modification enzymes 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 to match 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, for example. Data on codon usage in the host may be obtained from, for example, 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.

The nucleic acid encoding the nucleic acid sequence recognition module and the epigenome modification enzyme is configured so that the expression of the nucleic acid sequence recognition module and the epigenome modification enzyme is induced during gametogenesis. As described above, the nucleic acid sequence recognition module and the epigenome modification enzyme form a complex, bind to a nucleic acid sequence in a target region in the genomic DNA of the gamete being formed, and modify the epigenome of the target region. Therefore, the nucleic acid is preferably configured so that the induction period for expression of the nucleic acid sequence recognition module and the induction period for 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 to be induced to express during gamete formation 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 gamete formation.

The period during which expression is induced in gametogenesis can be, for example, any period until the precursor cells of the gametes differentiate into gametes. Specific examples of periods during which expression is induced in gametogenesis include, for example, the period from primordial germ cells to spermatogonia and spermatocytes until differentiation into spermatozoa. When the gametes are eggs, the period during which expression is induced in gametogenesis can be, for example, the period from primordial germ cells to oogonia and oocytes until differentiation into eggs. When the gametes are pollen, the period during which expression is induced in gametogenesis can be, for example, the period from pollen oocytes to pollen. When the gametes are embryo sacs, the period during which expression is induced in gametogenesis can be, for example, the period from embryo sac oocytes to embryo sacs.

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 gametogenesis-specific promoters include spermatogenesis-specific promoters, oogenesis-specific promoters, pollen formation-specific promoters, and embryo sac formation-specific promoters. Examples of gametogenesis-specific promoters include spermatogenesis-specific promoters, 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 as a specific example, the STRA8 promoter is the promoter of the STRA8 gene. The gametogenesis-specific promoter includes an oogenesis-specific promoter, and specific examples thereof include Gdf-9 promoter, Zp3 promoter, and Msx2 promoter.

・Sperm-specific promoter
Stra8: Patricia I Sadate-Ngatchou et.al., “Cre recombinase activity specific to postnatal, premeiotic male germ cells in transgenic mice”, Genesis, 2008 Dec;46(12):738-42
Pgk2: Tatsuo Kido et.al., “The testicular fatty acid binding protein PERF15 regulates the fate of germ cells in PERF15 transgenic mice”, Dev Growth Differ, 2005 Jan;47(1):15-24.
Dazl: Cory R Nicholas et.al., “Characterization of a Dazl-GFP germ cell-specific reporter”, Genesis, 2009 Feb;47(2):74-84
GSG2(haspin): Keizo Tokuhiro et.al., “The 193-base pair Gsg2 (haspin) promoter region regulates germ cell-specific expression bidirectionally and synchronously”, Biol Reprod, 2007 Mar;76(3):407-14
・Egg-specific promoter
Gdf-9: Zi-Jian Lan et.al., “Differential oocyte-specific expression of Cre recombinase activity in GDF-9-iCre, Zp3cre, and Msx2Cre transgenic mice”, Biol Reprod, 2004 Nov;71(5):1469-74
Zp3: M Lewandoski et.al., “Zp3-cre, a transgenic mouse line for the activation or inactivation of loxP-flanked target genes specifically in the female germ line”, 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

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

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").

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 nucleic acid is operably linked to the first expression vector so as to enable expression of the nucleic acid sequence recognition module, and the second nucleic acid is operably linked to the second expression vector 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 ligated to different expression vectors.

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

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, terminator, enhancer, polyadenylation signal sequence, and 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.

The expression vector may further include, for example, a coding sequence for a selectable marker. Examples of the selectable marker include a drug resistance marker, a fluorescent protein marker, an enzyme marker, and a cell surface receptor marker.

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

When the composition of the present invention contains multiple expression vectors, the composition of the present invention 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, etc.

<Method for producing parental lines of animals and plants 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.

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.

The target plants and animals include, for example, plant cells that form callus and can develop into individual plants; animal cells that can develop into individual animals such as fertilized eggs and animal embryos (embryos, blastocysts); etc. The plants and animals mentioned above exclude, for example, humans.

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 other cells. 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 liposomes include lipofectamine and cationic liposomes, and examples of 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.

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. The introduction method for integration into the genomic DNA can be a combination of the above-mentioned method for introduction into cells with a method using homologous recombination or a method using genome editing technology.

For methods of producing genetically modified animals 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 may be, for example, a method of introducing foreign DNA into the 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)).

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)).

When plant cells capable of developing into individual plants or animal cells capable of developing into individual animals are used as the target plant or plant, the first production method of the present invention preferably includes, after the introduction step, a step of generating individual plants or individual animals from the plant cells or the animal cells. The method for generating individual plants can be carried out, for example, by known methods of forming shoots from callus and regenerating individual plants. Furthermore, the method for generating individual animals can be carried out, for example, by transplanting the animal cells into a pseudopregnant mother and allowing them to develop and give birth, thereby obtaining individual animals as offspring.

The first manufacturing 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 and examining 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 selectable marker.

<Parent strains of animals and plants 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, wherein the animals and plants contain 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 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 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.

As described above, with regard to the parental lineages of animals and plants of the present invention, depending on the type of gamete in which the epigenome is modified, it is possible to produce parental lines used to produce animals and plants in which the epigenome of the target region has been modified, and parental lines used to maintain the parental lineages of animals and plants 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. It is preferable that the exogenous nucleic acid be inserted into the genomic DNA of the parental lineage, as this allows for stable maintenance of the parental lineage.

When the gamete in which the epigenome is modified is sperm, modification of the epigenome in the target region occurs during spermatogenesis in the male parent line. Therefore, when the gamete in which the epigenome is modified is sperm, the parent line used to produce an animal or plant with the epigenome modified 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 in which the epigenome is modified is sperm, it is preferable that the exogenous nucleic acid be operably linked to a spermatogenesis-specific promoter.

When the gamete in which the epigenome is 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 in which the epigenome is modified is an egg, the parent line used to produce an animal or plant with the epigenome 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. Furthermore, 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 in which the epigenome is modified is sperm, it is preferable that the exogenous nucleic acid be operably linked to a spermatogenesis-specific promoter.

If the exogenous nucleic acid is integrated into the genomic DNA of the parent lineage of an animal or plant, it is preferable that the parent lineage of an animal or plant 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.

<Parent strains of animals and plants 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 line 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 in which the epigenome has been modified, can be produced.

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

It is preferable that the epigenome of the target region of the gamete has been modified. In this case, the gamete can be isolated from a parental lineage animal or plant in which the epigenome of the target region in the genomic DNA of the gamete being formed 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.

When the gametes are used to produce animals or plants in which the epigenome of the target region has been modified, the gametes may or may not contain the exogenous nucleic acid, but preferably do not. Furthermore, when the gametes are used to produce animals or plants in which the epigenome has been modified, or to produce parent lines of animals or plants for maintenance, the gametes contain the exogenous nucleic acid.

<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 in which the epigenome has been modified from the resulting progeny individuals, wherein the first parent and/or the second parent are animals or plants of the parent lineage of the present invention. According to the second production method of the present invention, animals or plants in which the epigenome of the target region has been modified can be produced.

In the hybridization step, one of the first parent and the second parent may be an animal or plant of the parent lineage of the present invention, or both may be an animal or plant of the parent lineage of the present invention. By using an animal or plant of the parent lineage 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 lineage 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.

In the hybridization process, gametes of the first parent and/or the second parent may be used instead of the individual animal or plant as the first parent and/or the second parent.

In the hybridization process, hybridization between the first parent and the second parent can be carried out using known methods.

The second production method of the present invention may select from the progeny individuals those individuals that do not contain the exogenous nucleic acid. In this way, the second production method of the present invention can 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.

<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, wherein the animal or plant includes, 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 that is similar to an animal or plant in which the epigenome of the target region has not been modified and 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.

When the target region is the control 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.

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 that modifies the epigenome, and an epigenome modification enzyme capable of forming a complex with the nucleic acid sequence recognition module and capable of modifying the epigenome, 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, where 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 for the maintenance.

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 and kits were used according to their protocols.

[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.

(1) Target Region The Igf2 gene is an imprinted gene, and in chromosomes derived from male parents, the DMR region (H19-DMR) of the Igf2 gene is methylated, whereas in chromosomes derived from female parents, the DMR region (H19-DMR) of the Igf2 gene is unmethylated. Furthermore, the inventors have separately confirmed that when systemic demethylation of the H19-DMR causes abnormal methylation of the DMR region of the Igf2 gene in mice, i.e., when the H19-DMR is demethylated in chromosomes derived from male parents, the mice exhibit growth disorders, 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.

(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 (GSGSGGGSGSGSGGSGSGSG: 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, in the epigenome editing vector described in Reference 1, an anti-GCN4 single-chain antibody (scFv) is 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 an sgRNA capable of hybridizing to nine nucleic acid sequences (target nucleic acid sequences) in the 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 nucleic acid encoding 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 of the example (pStrPlATTET-gRNA2-H19DMRx9). Note that the epigenome editing vector of 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.

(3) Preparation of embryos
B6D2F1 female mice (8-10 weeks old, purchased from CLEA Japan) were administered 7.5 units of an ovulation inducer (SEROTROPIN, ASKA Pharmaceuticals) and, 48 hours later, 7.5 units of human chorionic gonadotropin (hCG; GONATROPIN, ASKA Pharmaceuticals) 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. After washing, the fertilized eggs were transferred into a drop of M16 medium (Sigma-Aldrich) containing penicillin and streptomycin at 37°C.

(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). The number of transplanted embryos was 20 to 25 per oviduct. Insertion of the epigenome editing vector of the Example into mice derived from the ICR mice was confirmed by examining 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 determine whether an amplified fragment derived from the epigenome editing vector of the example was obtained. As a result, it was confirmed that the epigenome editing vector of the example was inserted into the genomic DNA of 9 out of 41 individuals. Mice in which the insertion of the epigenome editing vector of the example was confirmed were backcrossed with B6 mice to establish three fertile recombinant mouse strains (441-2, 445-7, 445-14) and their sublines. The weight of newborn mice from each subline was measured. Sperm from the subline mice was collected and frozen at -80°C until DNA extraction.

(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). The RSB solution contained 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, extracted with phenol/chloroform, and then precipitated with ethanol to isolate DNA.

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 also lists 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 same procedure was followed except that wild-type B6 mice were used as controls. These 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 amount of DNA (mV μm)

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, mice with sperm-specific epigenome modification had significantly reduced methylation in sperm 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.

(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. The insertion of the epigenome editing vector into the genomic DNA of each F1 progeny was confirmed by PCR using the primers shown in Table 2.

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

The methylation rate was measured in the same manner as in Example 1(5) above, except that the genomic DNA was used instead of the isolated DNA. The results are shown in Figures 5 to 8.

Figure 5 is a graph showing the body weight of F1 progeny individuals derived from the male parent 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 of mice whose epigenomes were modified in a sperm-specific manner had reduced body weight compared to wild-type mice. In other words, it was presumed that the modification of the epigenome caused growth retardation, similar to Silver-Russell syndrome.

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

Figure 7 is a graph showing the methylation rates of target regions in F1 progeny derived from the male or female parent line. 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 represents the mouse lineage, and the vertical axis represents the methylation rate. As shown in Figures 7(A) and (B), F1 progeny derived from the female parent line exhibited a methylation rate comparable to that of wild-type mice. In contrast, F1 progeny derived from the male parent line exhibited a significantly reduced methylation rate compared to wild-type mice and F1 progeny derived from the female parent line. In other words, in the female parent line, the spermatogenesis-specific promoter was not activated, resulting in no epigenome modification and no change in methylation. In contrast, in the male parent line, the spermatogenesis-specific promoter was activated, and demethylases were induced and targeted, thereby modifying the epigenome of the target region.

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 F1 progeny of the male parent strain had a reduced methylation rate compared to wild-type mice. As shown in Figure 8(B), regardless of the presence or absence of insertion of an epigenome editing vector into the genomic DNA, the F1 progeny of the male parent strain had a reduced body weight 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.

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.

From the above, it has been 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.

The present invention has been described above with reference to embodiments and examples, but the present invention is not limited to the above embodiments and examples. Various modifications that would be understood by a person skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

This application claims priority based on Japanese Patent Application No. 2021-85099, filed on May 20, 2021, the entire disclosure of which is incorporated herein by reference.

<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 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 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 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;
comprising 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 express 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;
a Cas protein; and
Including,
5. The composition of any of claims 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;
a Cas protein; and
5. The composition of any one of claims 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 one 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 one of Appendices 1 to 8, wherein the epigenome-modifying enzyme is a base-modifying enzyme.
(Appendix 10)
The composition of any one of appendixes 1 to 9, wherein the epigenome-modifying enzyme is a methylase, demethylase, acetylase, deacetylase, kinase, phosphatase, ubiquitinase, and/or sumoylase.
(Appendix 11)
11. The composition of any of appendixes 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 Appendixes 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 manufacturing, 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 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 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 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 express 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 of 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;
a Cas protein; and
Including,
23. The plant or animal described in 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;
a Cas protein; and
24. The plant or animal described in any one of appendices 19 to 23, comprising:
(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 described in 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 appendix 19 to 27, wherein the epigenome-modifying enzyme is a methylase, demethylase, acetylase, deacetylase, kinase, dephosphorylase, 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 animal or plant 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 described in 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 described in 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 described in 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 described in any one of Appendices 19 to 38, wherein the parent line has no modification of the epigenome 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 of an animal or plant for maintenance,
40. Gametes isolated from an animal or plant of the parental lineage described in 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 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,
40. The method of claim 19, wherein the first parent and/or the second parent is an animal or plant of a parental lineage described in any one of appendices 19 to 39.
(Appendix 45)
45. The production method according to claim 44, comprising a 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 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 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 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 gamete-specific promoter that is active in 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.

As described above, the composition of the present invention makes it possible to produce 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 changes in the epigenome. Therefore, the present invention is extremely useful, for example, in the fields of life science and medicine.

Claims (49)

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. 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. The composition of claim 2, comprising multiple tag domains. the tag domain is a peptide epitope;
The composition of claim 2 , wherein the binding partner is an antibody against the peptide epitope. 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. The composition of claim 1 , wherein the nucleic acid sequence recognition module does not cleave both strands of the double-stranded genomic DNA. The composition of claim 1 , wherein the target region is a regulatory and/or promoter region of an imprinted gene. The composition of claim 1 , wherein the epigenome-modifying enzyme is a base-modifying enzyme. The composition of claim 1 , wherein the epigenome-modifying enzyme is a methylase, demethylase, acetylase, deacetylase, kinase, phosphatase, ubiquitinase, and/or sumoylase. 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. The composition of claim 1 , wherein the gametogenesis-specific promoter is a spermatogenesis-specific promoter or an oogenesis-specific promoter. 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. The composition of claim 1 , wherein the gametogenesis-specific promoter is a Gdf-9 promoter, a Zp3 promoter, and/or an Msx2 promoter. 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. 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. A method for producing parental lines of epigenome-modified animals and plants (excluding humans) , comprising:
A method of manufacturing, comprising the step of introducing a composition according to any one of claims 1 to 15 into a target animal or plant (excluding humans) . The manufacturing method described in claim 16, wherein the plant or animal is a non-human animal. 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;
In the formation of gametes, 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, in animals and plants (excluding humans) . 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. The animal or plant described in claim 19, comprising multiple tag domains. the tag domain is a peptide epitope;
The plant or animal of claim 19 , wherein the binding partner is an antibody against the peptide epitope. 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,
The animal or plant of claim 19 , wherein the Cas protein is linked to the tag domain. The animal or plant described in claim 18, wherein the nucleic acid sequence recognition module does not cleave both strands of the double strand of the genomic DNA. The animal or plant of claim 18 , wherein the target region is a regulatory region and/or promoter region of an imprinted gene. The animal or plant described in claim 18, wherein the epigenome-modifying enzyme is a base-modifying enzyme. 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. 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. The animal or plant of claim 18 , wherein the gametogenesis-specific promoter is a spermatogenesis-specific promoter or an oogenesis-specific promoter. The animal or plant of claim 18 , wherein the gametogenesis-specific promoter is a STRA8 promoter, a Pgk2 promoter, a Dazl promoter, and/or a Gsg2 promoter. The animal or plant of claim 18 , wherein the gametogenesis-specific promoter is a Gdf-9 promoter, a Zp3 promoter, and/or an Msx2 promoter. 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. 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 second expression vector is operably linked to the nucleic acid so that the epigenome modification enzyme can be expressed. The plant or animal of claim 18 , wherein the plant or animal is a non-human animal. The plant or animal of claim 18 , wherein the nucleic acid is inserted into genomic DNA. the gametogenesis-specific promoter is a spermatogenesis-specific promoter;
The parent line used in the production is a male parent line,
The plant or animal described in claim 18, wherein the parent line used for the maintenance is a female parent line. 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 described in claim 18, wherein the parent line used for the maintenance is a male parent line. The animal or plant described in claim 18, wherein the parent line has no modification of the epigenome of the target region in cells other than the gametes. A gamete used to produce an epigenome-modified animal or plant or a parent line of an animal or plant for maintenance,
38. Gametes isolated from an animal or plant of the parental lineage of any one of claims 18 to 37. The gamete described in claim 38, wherein the gamete is a sperm and/or an egg. The gamete described in claim 38, wherein 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. A gamete as described in claim 38, comprising the exogenous nucleic acid. 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. The production method described in claim 42, further comprising a step of selecting individuals that do not contain the nucleic acid from the progeny individuals. 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) . The plant or animal described in claim 44 , obtained by the manufacturing method described in claim 42. The animal or plant described in claim 44, wherein the animal or plant is a non-human animal. 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 animal or plant is an animal or plant (excluding humans) that 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. 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. The animal or plant described in claim 47 or 48, wherein the animal or plant is a non-human animal.