KR101896518B1 - Zinc finger nuclease for targeting CMAH gene and use thereof - Google Patents

Abstract

The present invention relates to a method for producing porcine cells in which a swine cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene is knocked out; A method for producing a pig in which a pig CMAH gene is knocked out; A method for producing pig-derived artificial organs or artificial tissues in which a pig CMAH gene is knocked out; A zinc finger nuclease that targets the CMAH gene; A polynucleotide encoding said zinc finger nuclease; A vector comprising said polynucleotide; A method of producing cells knocked out of the CMAH gene using the zinc finger nuclease; And an oocyte, a non-human animal, or a non-human animal-derived artificial organs-producing kit of a non-human animal in which the CMAH gene is knocked out. According to the present invention, zinc finger nuclease for the CMAH gene can be used to obtain a large amount of transfected cells for the production of a heterologous transplantable porcine from which the CMAH gene has been removed.

Description

Zinc finger nuclease targeting < RTI ID = 0.0 > CMAH < / RTI > gene and use thereof,

The present invention relates to a method for producing porcine cells in which a swine cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene is knocked out; A method for producing a pig in which a pig CMAH gene is knocked out; A method for producing pig-derived artificial organs or artificial tissues in which a pig CMAH gene is knocked out; A zinc finger nuclease that targets the CMAH gene; A polynucleotide encoding said zinc finger nuclease; A vector comprising said polynucleotide; A method of producing cells knocked out of the CMAH gene using the zinc finger nuclease; And an oocyte, a non-human animal, or a non-human animal-derived artificial organs-producing kit of a non-human animal in which the CMAH gene is knocked out.

The shortage of donor organs has become a stumbling block to the spread of allografts. To solve these problems, the development of animals suitable for xenotransplantation has been proposed, and pigs are considered to be the most outstanding candidates at present. Pigs are highly available in supply, and these organs are similar in size to humans. Moreover, animals that have been raised for centuries are relatively easy to handle. However, differences in cell surface antigens such as carbohydrate composition between pigs and human cells can lead to failure of xenotransplantation because of the tendency of the recipient patient to cause an acute immune response following organ transplantation.

The Halganutziu-Deicher antigen (N-glycolylneuraminic acid; NeuGC) is one of the major sialic acids present in all mammals except humans. Most healthy human beings have natural antibodies to NeuGC, which can lead to acute rejection, a major barrier in xenotransplantation. N-acetylneuraminic acid hydroxylase (cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) is an enzyme essential for NeuGC synthesis. Thus, genetic removal of CMAH from cells or organs from possible xenotransplant donors (e. G. Pigs) provides a way to avoid possible acute rejection that can be caused by natural human NeuGC antibodies at xenograft . Therefore, it is necessary to study a method capable of inhibiting the activity of the CMAH gene and a method of producing a transgenic animal having an inhibited activity of the CMAH gene with high efficiency.

Therefore, the present inventors developed a modified ZFN pair so that the pig CMAH gene can be recognized and cleaved in a cell, and when the ZFN is expressed in the cell, the mutation can be effectively induced in the CMAH gene to induce translation frame movement mutation Respectively. Further, cells prepared by knocking out CMAH gene using ZFN were prepared. Furthermore, the reporter construct containing the ZFN target sequence and the reporter gene can be used to efficiently select cells knocked out of the CMAH gene, and the blastocyst can be prepared from oocytes into which the CMAH gene knockout mutant cells have been nuclear-transplanted The present invention has been completed.

It is an object of the present invention to provide a method for producing pig cells in which swine cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene is knocked out.

Another object of the present invention is to provide a method for producing a pig in which a pig CMAH gene is knocked out.

It is still another object of the present invention to provide a method for producing pig-derived artificial organs or artificial tissues knocked out of a porcine CMAH gene.

Yet another object of the present invention is to provide a fusion protein comprising a zinc finger domain and a nucleotide cleavage domain, wherein the zinc finger domain is assembled by combining zinc finger modules that specifically recognize a specific target sequence in the CMAH gene, Which has an activity of cleaving the CMAH gene.

Yet another object of the present invention is to provide a polynucleotide encoding the zinc finger nuclease, and a vector containing the polynucleotide.

It is still another object of the present invention to provide a method for screening a zinc finger nucrease or a zinc finger nuclease, which method comprises cleaving a CMAH gene by zinc finger nuclease obtained by expressing the zinc finger nuclease or zinc finger nuclease-encoding polynucleotide, Gt; cell < / RTI >

Another object of the present invention is to provide a kit for the production of an artificial organ derived from an oocyte, a non-human animal, or a non-human animal other than a human having a CMAH gene knockout.

In one embodiment, the invention provides a nuclease that targets the CMAH gene.

In the present invention, " cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) " is an enzyme involved in biosynthesis of sialic acid. Sialic acid is a terminal component of the carbohydrate chain of glycoconjugates that is involved in ligand-receptor, cell-cell and cell-pathogen interactions. The two most common forms of sialic acid found in mammalian cells are N-acetylneuramic acid (NeuAc) and its hydroxylated derivative, N-glycolylneuraminic acid (N-glycolylneuraminic acid; NeuGc). NeuGc is not detected in normal human tissues while other mammals are abundant in sialic acid, and in fact, NeuGc is immunogenic in humans. The absence of NeuGc in humans can be attributed to a deletion in the human gene CMAH encoding cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH), an enzyme involved in NeuGc biosynthesis in humans. Sequences encoding mouse, pig, and chimpanzee hydroxylase are obtained by cDNA cloning and have high homology. However, the corresponding human cDNA has a deletion of 92 bp in the 5 'region, unlike these. The deletion corresponds to exon 6 of the mouse hydroxylase gene, which causes frameshift mutations in humans and premature termination of the polypeptide chain. As such, the human hydroxylase mRNA in the nodule is unable to code the active enzyme. Therefore, NeuGc is not detected in normal human tissues.

The nuclease according to the present invention comprises an artificial nuclease capable of recognizing and cleaving a specific position of DNA on a genome. The nuclease is a fusion between a domain recognizing a specific target sequence on the genome and a domain cleaved For example, a meganuclease, a transcription activator-like effector domain derived from a plant pathogenic gene, which is a domain recognizing a specific target sequence on the genome, and a cleavage domain, Fusion protein, or zinc-finger nuclease may be included without limitation.

Preferably, the nuclease is a fusion protein comprising a zinc finger domain and a nucleotide cleavage domain, wherein the zinc finger domain specifically recognizes a specific target sequence in a cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene Zinc finger modules, and is a zinc finger nuclease that targets the CMAH gene, which has activity to cleave the CMAH gene.

In the present invention, " zinc finger nuclease " refers to a fusion protein comprising a zinc finger domain and a nucleotide cleavage domain, and may include both known or commercially available zinc finger nuclease. In the present invention, the terms " zinc finger nuclease " and " ZFN "

Generally, the zinc finger domain of ZFN is located near the amino terminus (N-terminus) of the ZFN and the nucleotide cleavage domain (or truncated half domain) is located near the carboxy terminus (C-terminus) -Terminus at the C-terminus, and a nucleotide cleavage domain (or cleavage half domain) at the C-terminus.

Each monomeric zinc finger nuclease recognizes and binds 9 to 12 specific nucleotide sequences at 5- and 6-nucleotide spacing in a forward strand and a reverse strand. When two zinc finger nuclease are attached to a specific nucleotide sequence, the cleavage domain located at the carboxyl terminus forms a dimer, resulting in a double-strand break in the nucleotide sequence (Smith J. et al., Nucleic Acids Res ., 27: 674-681, 1999). Cells of multicellular animals, when subjected to such double-strand damage, rapidly undergo homologous recombination and non-homologous end joining, It is restored by a DNA repair system. Of these repair mechanisms, the heteroduplexes act as the main repair mechanism, often resulting in loss or insertion of nucleotide sequences because of the lack of homology or the cross-linking of microhomology alone (Kristoffer V. & Lawrence FP, Oncogene , 22: 5792-5812, 2003). That is, a variation occurs. In this manner, the gene can be knocked out using zinc finger nuclease.

In the present invention, " targeting CMAH gene " means specifically recognizing and cleaving a specific nucleic acid sequence of the CMAH gene. In the present invention, the zinc finger domain specifically recognizes a specific nucleic acid sequence of the CMAH gene, and cleavage may occur in the CMAH gene due to a nucleotide cleavage domain linked to the zinc finger domain by a peptide bond. The term " sequence " means a nucleotide sequence regardless of its length, which may be a linear, circular or branched DNA or RNA, or may be single-stranded or double-stranded.

The position of the CMAH target sequence of the zinc finger nuclease according to the present invention is not particularly limited, but may be located in the coding region 4 of the CMAH gene.

As used herein, the term " target sequence " or " target site " refers to a specific sequence or region capable of cleaving and cleaving a nuclease to a particular gene, wherein a specific recognition sequence or recognition site of the nuclease, It may be that the sequence or region to be cleaved is included.

The term " recognition sequence " or " recognition site " in the present invention may be a nucleic acid sequence that defines a portion of the nucleic acid to which the nuclease is capable of binding.

The zinc finger nuclease may comprise a pair of a first zinc finger nuclease and a second zinc finger nuclease wherein each zinc finger nuclease may independently comprise a zinc finger domain.

The zinc finger domain refers to a protein that binds to the DNA sequence of the CMAH gene in a sequence-specific manner through one or several zinc finger modules. The zinc finger domain may be designed to combine zinc finger modules to select sequences of the porcine CMAH gene.

The zinc finger module refers to an amino acid sequence in which the structure is stable within the domain during coordination with zinc ions.

The design of a zinc finger domain that recognizes a specific sequence of the CMAH gene can be performed using a computer algorithm developed by ToolGen, but is not limited thereto.

The zinc finger domains can be assembled by combining the zinc finger modules shown in Table 1.

The zinc finger domain has a structure in which two or more zinc finger modules for recognizing DNA 3 bp (base pair) are connected. Preferably, the zinc finger module can be connected to an amino acid linker, wherein the amino acid linker can be composed of 3 to 20 amino acids, preferably 3 to 9 amino acids.

The zinc finger domain that binds to a specific base sequence of the CMAH gene can be prepared by linking two or more zinc finger modules using an amino acid linker consisting of an appropriate number of amino acids. At this time, by appropriately adjusting the number of amino acids of the amino acid linker connecting between the zinc finger module and the module, the sequence of the CMAH gene on each of the zinc finger modules and the interval between the base sequences are controlled, A zinc finger domain having an activity of binding to the zinc finger domain can be produced. More preferably, two or more zinc finger modules described in Table 1 are combined and assembled.

Since each zinc finger module independently recognizes the DNA sequence of the CMAH gene, a zinc finger domain consisting of, for example, three or four modules can bind to the 9 or 12 bp sequence of the CMAH gene. Thus, in the case of a zinc finger nuclease acting as a dimer, a pair of zinc finger nuclease consisting of three or four zinc finger modules each can specifically recognize 18 to 24 bp. In order to prepare a zinc finger nuclease targeting the pig CMAH gene, zinc finger modules of appropriate types and numbers were selected in consideration of the target sites of the six zinc finger modules described in Table 2, A zinc finger domain having binding activity can be produced.

The zinc finger domain binding to a specific sequence of the CMAH gene according to the present invention includes zinc finger modules sequentially recognizing AAG, CAG, GAC and CGA, zinc finger modules recognizing CGA, GGA, TGG and TGG, May be sequentially included. Preferably, the first zinc finger domain of the first zinc finger nuclease sequentially includes zinc finger modules that recognize AAG, CAG, GAC, and CGA, respectively, and the second zinc finger domain of the second zinc finger nuclease, The domain may sequentially include zinc finger modules that recognize CGA, GGA, TGG, and TGG, respectively.

Accordingly, the target sequence in the CMAH gene of the zinc finger nuclease of the present invention includes at least one sequence selected from the group consisting of AAG, CAG, GAC, CGA, GGA and TGG, and AAG, CAG, GAC, CGA, GGA Or TGG may each independently be combined by a zinc finger module selected from the group consisting of SEQ ID NOS: 1 to 6 and include one or more of the selected zinc finger modules.

Preferably, the zinc finger domain may comprise a zinc finger module as shown in SEQ ID NO: 1, 2, 3 and 4, more preferably from the N-terminus of the fusion protein as SEQ ID NOs: 1, 2, 3 and 4 May be a zinc finger domain that is sequenced, more preferably, represented by the amino acid sequence of SEQ ID NO: 35.

Also preferably, the zinc finger domain may comprise a zinc finger module represented by SEQ ID NOS: 5, 5, 6 and 1, more preferably from the N-terminus of the fusion protein of SEQ ID NOS: 5, 5, 6 and 1 , And more preferably a zinc finger domain represented by the amino acid sequence of SEQ ID NO: 36.

The term " cleavage " in the present invention means cleaving the covalently linked backbone of the porcine CMAH gene nucleotide molecule, and " nucleotide cleavage domain " means a polypeptide sequence having enzymatic activity for nucleotide cleavage of such porcine CMAH gene .

The nucleotide cleavage domain can be obtained from an endonuclease or an exonuclease. Examples of endonuclease capable of obtaining a nucleotide cleavage domain include, but are not limited to, a restriction endonuclease, a regenerative endonuclease, and the like. These enzymes can be used as the origin of the nucleotide cleavage domain. Further, the nucleotide cleavage domain can not only cleave a single nucleotide sequence but also cleave the nucleotide sequence of a double bond according to the origin of the cleavage domain. In this sense, a cleavage domain that cleaves all double-stranded nucleotide sequences may be used as a cleavage half-domain.

The restriction endonuclease is present in many classes of species, capable of sequence-specific binding to DNA (recognition site), and capable of cleaving DNA near the binding site. Some restriction endonucleases, such as type IIs, have cleaved DNA fragments at sites where recognition sites have been cleaved and have cleaved binding domains and cleavage domains. For example, FokI , a type II enzyme, promotes double helix cleavage of DNA at a site that is 9 nucleotides apart from the recognition site in one spiral and 13 nucleotides away from the recognition site in the other spiral.

The nucleotide cleavage domain may be a type IIs restriction endonuclease derived from the Type IIs restriction endonuclease include, but are not limited to FokI, AarI, AceIII, AciI, AloI, BaeI, Bbr7I, CdiI, CjePI, EciI , Esp3I , FinI , MboI , SapI, or SspD51 , And preferably It can be FokI .

In addition, the zinc finger nuclease of the present invention may further comprise a nuclear localization signal sequence. The nucleotide position signal can be used as a means for transferring zinc finger nuclease into the nucleus to knock out the porcine CMAH gene by a zinc finger nuclease targeting the porcine CMAH gene of the present invention. Preferably, the nucleotide position signal may be a nucleotide position signal represented by the amino acid sequence (PPKKKRKV) of SEQ ID NO: 39.

Further, the zinc finger nuclease of the present invention may be a zinc finger nuclease represented by the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38.

In another aspect, the invention provides polynucleotides encoding said zinc finger nuclease.

The polynucleotide is a polymer of a nucleotide in which a monomer is linked in a long chain by a covalent bond and is a DNA having a certain length or more of deoxyribonucleic acid or RNA (ribonucleic acid) Lt; / RTI > is a polynucleotide that encodes a cysteine.

The polynucleotides can be delivered intracellularly in a known form, for example, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, etc., which have been used as conventional gene carriers a vector, a viral vector, a liposome, a polylysine, a polyethylenimine (PEI), a protamine, a histone, a polyester amine, and a variant thereof Non-viral vectors such as micelles, emulsions, and nanoparticles can be used as well as cationic polymers, and they can be efficiently delivered into cells using known intracellular mass transfer peptides and the like in addition to the vector system.

In another aspect, the present invention provides a vector comprising the polynucleotide.

As the means for expressing the zinc finger nuclease of the present invention by introducing the vector into the cell, a known expression vector such as a plasmid vector, a cosmid vector, and a bacteriophage vector may be used, and the vector may be an arbitrary vector Can be easily prepared by those skilled in the art according to known methods.

The vector of the present invention may be a recombinant vector in which a polynucleotide encoding a zinc finger nuclease according to the present invention is operably linked. Operably linked " refers to an expression control sequence that is linked to regulate transcription and translation of a polynucleotide sequence encoding a zinc finger nuclease, wherein a polynucleotide sequence is expressed under the control of an expression control sequence (including a promoter) And maintaining the correct decoding frame so that a zinc finger nuclease that is encoded by the polynucleotide sequence is generated.

In another aspect, the present invention provides cells knocked out of the CMAH gene and a method for producing the same.

The method of producing the cell comprises cleaving the CMAH gene in a cell by an artificial nuclease that specifically recognizes a specific target sequence in the CMAH gene.

The cell may be a cell in which one or two alleles of the intracellular CMAH gene are knocked out.

The term " knockout " in the present invention means inhibiting the expression of the porcine CMAH gene. In the present invention, a zinc finger nuclease that targets the porcine CMAH gene causes mutation in the porcine CMAH gene, .

The nuclease may be an artificial nuclease capable of recognizing and cleaving a specific position of DNA on the genome of the CMAH gene. The nuclease may include a nuclease that is fused with a domain that recognizes a specific target sequence on the genome and a cleavage domain. Examples of the nuclease include a meganuclease, a phytopathogenic gene that is a domain that recognizes a specific target sequence on a genome, A fusion protein with a transcription activator-like effector domain fused to a cleavage domain, or a zinc-finger nuclease may be included without limitation.

Preferably, the nuclease is a fusion protein comprising a zinc finger domain and a nucleotide cleavage domain, wherein the zinc finger domain is assembled by combining zinc finger modules that specifically recognize a specific target sequence in the CMAH gene, And can be a zinc finger nuclease that targets the CMAH gene, which has activity of cleaving the CMAH gene.

The target sequence of the nuclease may be located in exon 4 of the porcine CMAH gene.

In addition, the nuclease may be one or two or more pairs of zinc finger nuclease.

Wherein the nuclease is a zinc finger nuclease and a translational frame transfer mutation is formed by a non-homologous end joining (NHEJ) in a site cleaved by the zinc finger nuclease in the target sequence of the pig CMAH gene, The CMAH gene may be knocked out.

The zinc finger nuclease may sequentially include zinc finger modules for recognizing AAG, CAG, GAC, and CGA, or zinc finger modules sequentially including zinc finger modules for recognizing CGA, GGA, TGG, and TGG . Preferably, the first zinc finger domain of the first zinc finger nuclease sequentially includes zinc finger modules that recognize AAG, CAG, GAC, and CGA, respectively, and the second zinc finger domain of the second zinc finger nuclease, The domain may sequentially include zinc finger modules that recognize CGA, GGA, TGG, and TGG, respectively.

Accordingly, the target sequence in the CMAH gene of the zinc finger nuclease of the present invention includes at least one sequence selected from the group consisting of AAG, CAG, GAC, CGA, GGA and TGG, and AAG, CAG, GAC, CGA, GGA Or TGG may each independently be combined by a zinc finger module selected from the group consisting of SEQ ID NOS: 1 to 6 and include one or more of the selected zinc finger modules.

Preferably, the zinc finger domain may comprise a zinc finger module as shown in SEQ ID NO: 1, 2, 3 and 4, more preferably from the N-terminus of the fusion protein as SEQ ID NOs: 1, 2, 3 and 4 May be a zinc finger domain that is sequenced, more preferably, represented by the amino acid sequence of SEQ ID NO: 35.

Also preferably, the zinc finger domain may comprise a zinc finger module represented by SEQ ID NOS: 5, 5, 6 and 1, more preferably from the N-terminus of the fusion protein of SEQ ID NOS: 5, 5, 6 and 1 , And more preferably a zinc finger domain represented by the amino acid sequence of SEQ ID NO: 36.

The zinc finger nuclease may be a zinc finger nuclease represented by the amino acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38 or a zinc finger nuclease represented by the amino acid sequence of SEQ ID NO: 37 and an amino acid sequence of SEQ ID NO: 38 Lt; / RTI > may be a pair of zinc finger nuclease represented by < RTI ID = 0.0 >

The cell is a cell to knock out the CMAH gene by the zinc finger nuclease of the present invention, and is a prokaryotic cell such as E. coli; Eukaryotic cells such as yeast, fungi, protozoa, higher plants, insects; Amphibian cells; Fish cells; Mammalian cells such as CHO, HeLa, COS-1, HEK, HCT116, K562, BJ fibroblast such as cultured cells (in vitro), grafts and primary cultures (in vitro and in vivo) and in vivo cells; Or cells derived from human donors and blood separated from the patient and from various tissues, but are not limited thereto. Preferably pig-derived cells, more specifically, pig-derived primary fibroblasts, porcine ear tissue cells, and the like, but are not limited thereto.

Preferably, the method of knocking out the porcine CMAH gene of the present invention comprises the steps of: (a) expressing zinc finger nuclease according to the present invention in a cell or extracellularly and introducing the zinc finger nuclease into the cell; (b) a step of causing double-strand damage to intracellular porcine CMAH gene expressed or introduced in said cell by zinc finger nuclease; And (c) inducing a mutation in the porcine CMAH gene by the double-stranded injury.

In a specific embodiment, the method further comprises introducing a reporter construct comprising the target sequence and a reporter gene into a porcine-derived cell; And a porcine-derived cell in which the target sequence on the reporter construct has been cleaved by the nuclease, is separated according to the presence or absence of expression of the reporter gene, and a porcine-derived cell in which the genomic-phase pig CMAH gene is knocked out by the nuclease Further comprising the step of separating.

In introducing the reporter construct into the porcine-derived cells, the reporter construct comprises a target sequence recognized by the nuclease and a reporter gene, wherein the nuclease is bound to the target sequence to cleave the reporter construct And thus the expression of the reporter gene may be determined.

Such reporter constructs may be those described in PCT / KR2012 / 001367, the scope of which is incorporated herein by reference.

The reporter construct according to one embodiment of the present invention is characterized in that the reporter gene is not expressed unless the nuclease binds to the target sequence and cleaves the reporter construct. However, when the nuclease is bound to the target sequence and the reporter construct is cut, DNA can be recovered by homologous recombination (HR) or single strand annealing (SSA) mechanism or NHEJ present in cells or in vivo to express the reporter gene. For example, the reporter construct may have the configuration described in FIGS. 5, 10, and 15.

When nuclease is injected directly into fertilized eggs for the production of transgenic cells using nuclease, the conversion efficiency is lowered due to the mosaic phenomenon occurring during embryo development have. Therefore, replication technique by nuclear transfer is mainly used. In this case, mass production and high purity acquisition method of transformed cells are required, but mutant cells which are induced by artificial nucleases and genetically modified Because it is very difficult to distinguish wild-type cells phenotypically, there are many limitations to isolate mutant cells only.

Therefore, in the present invention, only a reporter gene-expressing cell was isolated using a reporter construct designed to determine the expression of a reporter gene depending on whether a nuclease is bound to a target sequence and a reporter construct is cleaved. According to one embodiment of the present invention, when the reporter construct described above is introduced into pig-derived cells and the step of separating according to the presence or absence of the reporter gene is included, the CMAH gene is mutated The cell sorting ratio was remarkably high. That is, by using the above-described method of the present invention, it is possible to obtain cells in which the genomic pig CMAH gene is knocked out in high purity.

In isolating mutated cells in which the target sequence has been modified by the nuclease, the mutations include not only local mutations but also mutations such as chromosomal rearrangements such as deletion, insertion, inversion, redundancy, translocation, It is not.

In another aspect, the present invention provides a method for identifying a target nucleic acid comprising: preparing a nuclease that specifically recognizes a specific target sequence; Introducing a reporter construct comprising the target sequence and a reporter gene into pig derived cells; Knocking out the genome-phase pig CMAH gene with the nuclease in the porcine-derived cells into which the reporter construct has been introduced; The pig-derived cells in which the target sequence on the reporter construct is cleaved by the nuclease are separated according to the presence or absence of expression of the reporter gene, and the pig-derived cells knocked out by the nuclease in the genomic phase pig CMAH gene are isolated ; And removing the nucleus from the nuclear transfer receptor porcine oocyte and transplanting the nucleus of the separated porcine derived cell, wherein the porcine CMAH gene is knocked out.

The term " nuclear transfer " as used herein means that the nucleus of a cell is implanted into an oocyte having already been removed from the nucleus, and an individual born with such a nuclear transfer embryo is implanted with a nuclear material, It is a completely genetically identical replicating entity because it has been transferred intact into the cytoplasm.

Methods for removing oocyte genetic material include physical methods, chemical methods, and centrifugation using Cytochalasin B (Tatham et al ., Hum . Reprod . , 11 (7): 1499-1503,1996). Somatic cells knocked out of the pig CMAH gene can be introduced into the nucleus-removed oocyte using cell membrane fusion, intracellular microinjection, or the like. The cell membrane fusion method is simple and suitable for large-scale modified production. The intracellular microinjection method has an advantage of maximizing the contact between the nucleus and the materials in the oocyte. The fusion of somatic cells with nucleated oocytes is recombined by changing the viscosity of the cell membrane through electrical stimulation and fusing. At this time, it is convenient to use an electric fusion machine capable of freely adjusting minute current and voltage.

In another aspect, the present invention provides a method for producing a pig in which a pig CMAH gene is knocked out.

The method comprising: preparing a nuclease that specifically recognizes a specific target sequence in a porcine CMAH gene; Introducing a reporter construct comprising the target sequence and a reporter gene into pig derived cells; Knocking out the genome-phase pig CMAH gene from the pig-derived cells with the nuclease; The pig-derived cells in which the target sequence on the reporter construct is cleaved by the nuclease are separated according to the presence or absence of expression of the reporter gene, and the pig-derived cells knocked out by the nuclease in the genomic phase pig CMAH gene are isolated ; Removing the nucleus from the nuclear transfer receptor porcine oocyte and transplanting the nucleus of the isolated porcine-derived cell to prepare a porcine oocyte cell knocking out the porcine CMAH gene; And preparing a pig from which the pig CMAH gene is knocked out from the porcine oocyte.

The present invention provides a method for preparing a porcine oocyte cell in which a porcine CMAH gene is knocked out by the above-described method, and then knocking out a porcine CMAH gene from the porcine oocyte cell.

The production of the transformed pig from the porcine oocyte can be carried out by methods known in the art. For example, a nuclear transplanted oocyte can be activated and generated to a transplantable stage and then implanted as a surrogate moth to produce transgenic pigs.

The pig may be a pig lacking the CMAH gene and not causing an immune rejection reaction when a heterologous organ is transplanted into a human.

As used herein, the term " transplantation " refers to the need to transfer from one individual (donor) to another (beneficiary) or from the donor site of the patient himself for the purpose of replacing a tissue or organ in which function is impaired or absent To move the organization or organ to the place. As tissue regeneration medicine develops, the patient's own cells (for example, stem cells, cells extracted from the organ or the like) that can grow at the site and produce organs can be used. Thus, transplanting the same human cells and / or tissues to themselves is known as autografting and the transplantation between two individuals of the same species is called allograft. Furthermore, transplantation of cells, tissues or organs from one species to another is also possible. This is called "xenograft or xenotransplantation" and "heterotransplantation". In humans, heterologous organ transplantation offers the potential to treat end stage organs, but is followed by medical and legal and ethical issues such as immune rejection. The best candidate animal for such heterologous organ transplantation is pigs. Pigs have organs similar in size to human organs, and are phylogenetically silent with humans, thus reducing the risk of cross-species disease transmission.

The zinc finger nuclease may induce double-strand damage to a desired site on the porcine CMAH gene when introduced into a cell. If double-strand damage occurs on the porcine CMAH gene, the cell uses its own repair mechanism Fix the damaged area. At this time, the cells are fixed by the non-homologous end joining (NHEJ) method, and errors or prone to insertions or deletions occur at the ends of the broken DNA strands ). Thus, mutations can be induced in the porcine CMAH gene and ultimately knockout of the porcine CMAH gene.

In another aspect, the present invention provides a method for producing pig-derived artificial organs or artificial tissues in which pig CMAH gene is knocked out.

The method comprising: preparing a nuclease that specifically recognizes a specific target sequence in a porcine CMAH gene; Introducing a reporter construct comprising the target sequence and a reporter gene into pig derived cells; Knocking out the genome-phase pig CMAH gene from the pig-derived cells with the nuclease; The pig-derived cells in which the target sequence on the reporter construct is cleaved by the nuclease are separated according to the presence or absence of expression of the reporter gene, and the pig-derived cells knocked out by the nuclease in the genomic phase pig CMAH gene are isolated ; Removing the nucleus from the nuclear transfer receptor porcine oocyte and transplanting the nucleus of the isolated porcine-derived cell to prepare a porcine oocyte cell knocking out the porcine CMAH gene; Preparing a pig knockout of a porcine CMAH gene from the porcine oocyte; And isolating pig-derived artificial organs or artificial tissues from which the pig CMAH gene has been knocked out.

The organ or tissue may be a state containing blood as well as an organ or tissue itself, and may include, without limitation, heart, stomach, small intestine, large intestine, kidney, liver, lung, pancreas and the like. Since the organs or tissues are derived from a pig knockout CMAH gene, the immune rejection reaction does not occur or can be alleviated when the organ or tissue is transplanted into a human or a non-human animal. Therefore, the organ or tissue of the present invention can be transplanted into an organ in need of organ or tissue transplantation, and it is possible to treat a disease according to the kind of organ desired. The individual requiring organ transplantation may be an animal or a human.

In another aspect, the present invention provides a method for screening a zinc finger nuclease, a polynucleotide encoding the zinc finger nuclease, a vector comprising the polynucleotide, And a reporter construct comprising a reporter gene comprising a specific target sequence in the CMAH gene recognized by the nuclease, and a reporter construct comprising a reporter construct in the CMAH gene, wherein the CMAH gene knockout kit comprises an egg, a non-human animal, to provide.

The animal is not particularly limited as a mammal, but is preferably a pig.

The present invention firstly developed a knockout cell of a porcine CMAH gene using artificial nucleases, and developed a zinc finger nuclease that can be applied to produce the cells with high efficiency. Also, by efficiently selecting and enriching the transformed cells, CMAH-inactivated cells can be obtained with high purity by the zinc finger nuclease, and in a blastocyst generated by nuclear transfer into oocytes, a mutant gene , We confirmed the possibility of producing transgenic pigs knocking out CMAH gene. Thus, it is possible to obtain a large amount of transfected cells for the production of a heterozygous transgenic pig in which the CMAH gene has been deleted.

BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing the amino acid sequence of CMAH 11 L4 ZFN. The HA tag and nuclear localization signal sequences included to detect their expression in cells are underlined. The alpha helix region of the zinc finger, which is expected to bind to the target sequence, is shown in bold.
2 is a diagram showing the amino acid sequence of CMAH 11 R4 ZFN. The HA tag and nuclear localization signal sequences included to detect their expression in cells are underlined. The alpha helix region of the zinc finger, which is expected to bind to the target sequence, is shown in bold.
Figure 3 shows the T7E1 assay results to confirm the CMAH 11 ZFN-mediated genome modification. The ZFN pair appeared on the bottom of the agarose gel. The generated DNA band
4 is a diagram showing the DNA sequence of the target gene region of the CMAH 11 ZFN pair. ZFN recognition elements are shown in capital letters. The deletions were indicated by bold and underlined nucleotides inserted in dotted lines. wt represents a wild-type sequence.
Figure 5 is a schematic representation of a reporter arrangement designed for magnetic classification. In the reporter constructs, the CMV promoter was used for intracellular expression, the RFP gene for confirming the gene injection efficiency, the target gene capable of binding with ZFN, and the H-2K ^k gene designed to be expressed when ZFN was functioning properly Respectively.
6 is a conceptual diagram for the development of a transformed cell line using a magnetic reporter selection method. Cells injected with ZFN and reporter constructs were labeled with magnetic spheres specific for H-2K ^k after 48 hours and labeled cells were selected using magnetic force.
FIG. 7 is a diagram showing fluorescence images of cells selected using a magnetic force. FIG. Intracellular induction of ZFN and reporter constructs was induced, and the intracellular RFP and GFP expression levels were compared between the cells separated and separated by magnetic force after magnetic labeling on day 2. The percentage of cells expressing RFP and GFP in the cells after separation was higher than that of the cells before separation.
Figure 8 shows the percentage of transformed cells increased and the mutant base sequence in the cells selected by the magnetic reporter construct. (a) shows the results of analysis of the proportion of cells in which the gene mutation occurred in the cells selected by magnetic labeling 48 hours after intracellular introduction of ZFN and reporter constructs by the T7E1 analysis method. A higher percentage of transformed cells was identified in selected cells (15%) compared to pre-selected cells (3.5%). (b) is the result of attempting nuclear transfer by taking the cells expressing GFP among selected cells. During in vitro fertilization, blastocysts (BL) were formed in 3 out of 40 cells, and when these cells were analyzed, it was confirmed that they were transformed at a high rate. (c) are two different mutant base sequences introduced from cells of the blastocyst (BL) stage.
FIG. 9 shows a transformed single cell clone using a magnetic reporter selection method. FIG. (a) shows the colonies was then affixed to the ZFN within the reporter construct in the cells after introduction of the magnetic cover screening after 48 hours the cells in 6-well plates at a density of 1.2 × 10 ³ cells / plate formed from a single cell culture 16 days to be. (b) is the result of T7E1 analysis to confirm the ratio of transformed cells to each single colony. (c) is the nucleotide sequence analysis result of the PCR product obtained from a single colony. Four clones out of the six clones were analyzed and confirmed to contain all the transfected sequences.
Figure 10 is a schematic representation of a reporter construct designed for hygromycin screening. A CMV promoter was used for expression in the reporter constructs, and a RFP gene for confirming gene injection efficiency, a target gene capable of binding with ZFN, and a hygromycin phosphotransferase designed to be expressed when ZFN worked properly Hygromycin phosphotransferase (HPT) and eGFP genes.
11 is an overall schematic diagram for the development of a transformed cell line using the hygromycin reporter selection method. On day 0, a pair of ZFN (36 μg) capable of knocking out the pig CMAH gene and a reporter construct (9 μg) for hygromycin screening were injected into porcine ear tissues through electrical stimulation, and 1 × 10 ⁶ cells Were dispensed into 100-mm dishes and cultured. On the second day after injection of the ZFN and reporter constructs, the number of cells was 3 × 10 ⁵ cells per culture dish and the hygromycin B was treated at a concentration of 300 μg / ml for 48 hours. On the fourth day, a new culture medium . At this time, the number of cells survived by the hygromycin treatment was 1.5 x 10 ⁴ cells. On day 7, an initial colony was formed, a complete colony was formed on day 18, and a colonies formed on day 22 were transferred to a new 96-well plate to prepare a transformed cell line.
FIG. 12 is a fluorescence image showing cells before and after hygromycin screening. FIG. The intracellular RFP and GFP expression levels of ZFN and reporter constructs were compared before and 2 days after hygromycin B treatment and after 4 days of no treatment or treatment. The ratio of RFP and GFP expressing cells was higher in the treated group than in the non - treated group.
FIG. 13 shows the results of T7E1 assay for cells selected by hygromycin B. FIG. The percentage of cells with mutated genes in selected cells treated with hygromycin B (300 μg / ml) for 2 days after intracellular introduction of ZFN and reporter constructs was analyzed by the T7E1 assay method. As a result, it was confirmed that the proportion of transformed cells in the selected cells was increased. That is, a higher proportion of transformed cells was identified in the treatment (12.1%) than the untreated control (3.1%).
Figure 14 shows a transgenic single cell clone obtained using the hygromycin B reporter selection method. (a) is a colony from a single cell formed by culturing selected cells for 30 days by treatment with hygromycin B (300 μg / ml) for 2 days from the second day after intracellular introduction of ZFN and reporter constructs. (b) is the result of T7E1 analysis to confirm the ratio of transformed cells to each single colony. (c) is the nucleotide sequence analysis result of the PCR product obtained from a single colony. Two clones out of 9 clones were analyzed and found to contain all the transfected sequences.
Figure 15 is a schematic representation of a reporter construct designed for screening using flow cytometry. The reporter constructs used the CMV promoter for intracellular expression, the RFP gene to confirm gene injection efficiency, the target gene that ZFN could bind to, and the eGFP gene designed to be expressed when ZFN worked properly.
16 shows FACS classification results using ZFN and reporter constructs. Cells injected with ZFN and reporter constructs were selected by FACS for cells expressing RFP and GFP simultaneously (1%) which were not observed in WT after 48 hours.
FIG. 17 is a fluorescence image of cells before and after FACS screening. FIG. The expression of RFP and GFP was confirmed by fluorescence microscopy of cells separated and isolated by FACS on day 3 after inducing intracellular introduction of ZFN and reporter constructs.
18 is a graph showing the results of T7E1 analysis on cells selected by FACS. Cells injected with ZFN and reporter constructs were screened by FACS after 48 hours for positive cells for RFP and GFP, and the percentage of cells with mutated genes was confirmed by T7E1 analysis. A high percentage of transformed cells were identified in selected cells (13%) compared to pre-selection cells (less than 0.5%).
19 is a diagram showing gene mutation of blastocysts developed from oocytes transplanted with CMAH knockout cells. Nuclear transplantation was performed on porcine oocytes using FACS sorting cells through ZFN and reporter constructs and in vitro fertilization was performed. It was confirmed that the mutation was introduced by the action of ZFN in the developed blastocysts.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  One: Zinc Finger  Production of plasmids containing nuclease

To develop a pair of zinc finger nuclease (ZFN) targeting the pig CMAH (cytidine monophospho-N-acetylneuraminic acid hydroxylase) gene, a computer algorithm developed by ToolGen, A potential ZFN target site in the DNA sequence of cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) exon 4 (coding region 4) was searched (Table 1).

The ZF target site pair can bind three or four zinc finger arrays on the opposite strand and can be separated into five or six nucleotides. Then, based on the search results, a pair of ZFN units for one specific target site were constructed by combining six zinc-finger modules by an exemplary module combination method (Table 2) These combined ZF modules were fused with the Fokl nucleic acid degrading enzyme domain to synthesize ZFN monomers. These pairs of ZFN monomers were used to test whether mutation could be induced on the porcine CMAH gene. The zinc finger modules (F1 to F4) of the pair of ZFN units used in the experiment and the amino acid sequence of the target site of the zinc finger are shown in Table 2, Fig. 1 and Fig. 2, respectively.

Example  2: pig CMAH  Gene-targeted ZFN Mutation detection in porcine fibroblasts treated with

Double strand breaks (DSBs) induced by ZFNs, which are gene scissors (artificial DNA restriction enzymes), are produced by an error-prone non-homologous end joining (NHEJ) Can be restored. The resulting DNA contains a small DNA sequence insertion / deletion (indel) mutation near the site of the DSB. Insertion / deletion mutations of DNA sequences of these specific sites can be searched in vitro by treating amplified DNA fragments with mismatch-sensitive T7 endonuclease I (T7E1) .

Primary pig fibroblasts were cultured in DMEM (Dulbecco's modified eagle medium) supplemented with 10% FBS and 2 ng / ml hbFGF and cultured in vitro using TransITLT1 reagent (Mirus Bio) to prepare CMAH 11 ZFN expression plasmid. 3 days after G-spin genome DNA extraction kit; genomic DNA was isolated from the transformed cells using the (G-spin ^TM Genomic DNA Extraction Kit Intron Bio). The sequence around the ZFN target site was amplified by PCR using a primer specific for the porcine CMAH gene exon 4 (Table 3). A PCR amplicon containing a ZFN target site was subjected to a mismatch-sensitive T7 endonuclease cleavage assay. Briefly, when the CMAH target site was cleaved by ZFN, the DNA DSB Can be recovered by an error-prone NHEJ (non-aseptic termination) mechanism and thus induce indels at the target site in the subpopulation of the transformed cells. For 15 minutes with 5 units of T7E1 followed by the addition of 2.5-fold of ethanol. To identify the mutated alleles, amplified CMAH treated with wild type alleles and T7E1 enzyme Exon 4 fragments were analyzed on 2% agarose gel electrophoresis. As a result, expression of CMAH 11 ZFN in primary porcine fibroblasts resulted in mutation at the target site located in exon 4 of CMAH gene It was confirmed that the introduction of effective (Fig. 3).

As shown in Figure 3, small fragments of amplified DNA from cells treated with the CMAH 11 ZFN pair targeting the porcine CMAH gene were cleaved by T7E1 and the ZFN pair generated a unique type of cleavage at the site they targeted (Fig. 3).

Example  3: pig CMAH  Gene-targeted ZFN Mutant DNA sequence analysis

Genomic DNA was isolated from transformed cells 3 days after transformation by the method described in Example 2 to determine the characteristics and the incidence rate of the mutation induced by CMAH 11 ZFN targeting the pig CMAH gene, Respectively. The ZFN target sequence was amplified by PCR using a primer specific for the exon 4 of the pig CMAH gene (Table 3) and cloned into the T-vector. The frequency and identity of the mutation at the CMAH 11 ZFN target site ). ≪ / RTI > As a result, it was confirmed that the frequency of the mutation induced by expression of CMAH 11 ZFN was 4%, and various mutations related to various insertions, deletions, or combinations thereof were identified (FIG. 4). All mutations identified in the present invention can lead to translational frameshifts and thus can easily lead to loss of functional CMAH protein expressed from mutated alleles.

Example  4: Magnetic-activating cell classification ( MACS ), The concentration of the target gene-modified cells

The magnetic-activated cell sorting method was used to classify and concentrate transformed cells of porcine CMAH gene.

Reporter construct was composed of mRFP gene, the target sequence, 2A- peptide sequence and the mouse MHC class I molecule H-2K ^k gene of artificial nucleases (Fig. 5). In the reporter system of the above structure, mRFP is expressed by the CMV promoter, whereas H-2K ^k is not expressed because it exists outside the frame when the artificial nucleases are not active. When DSB occurs in the target sequence by artificial nucleases, DNA damage is restored by NHEJ, but it involves frame-mobility mutagenesis. These mutations may be to allow the peptide 2A- and H-2K ^k may be present in the frame with mRFP, induce the expression of H-2K ^k functional protein. Cells were labeled with H-2K ^k -specific magnetic beads at 3 or 4 days after transfection with plasmids encoding reporter plasmids and nuclease and were isolated by magnetic force on a MACS column (Fig. 6).

Based on the experimental design above, primary pig fibroblasts were cotransfected with plasmids encoding 2 μg of reporter plasmid and 2 μg of CMAH 11 ZFN. The reporter plasmid was composed of the mRFP gene, the target sequence of CMAH 11 ZFN, the 2A-peptide sequence, and the mouse MHC class I molecule H-2K ^k gene. On the third day after transfection, cells were magnetically labeled and separated using MACSelect K ^k (miltenyi Biotech), from which genomic DNA was extracted.

First, mutation enrichment was identified by fluorescence using magnetic force. After inducing intracellular introduction of ZFN and reporter constructs, cells were trypsinized on day 2 and resuspended in PBE buffer. MACSelect microbeads; was added (K ^k MACSelect microbeads Miltenyi Biotech) to a single cell suspension was reacted for 15 minutes at 4 ℃. The magnetically labeled cells were selected by passing them through a column (MACS mini MS column; Miltenyi Biotech). As a result, as shown in FIG. 7, the proportion of cells expressing intracellular RFP and GFP after selection using magnetic beads was increased as compared with that before separation. The results are shown in Table 4. In addition, the proportion of cells in which the gene mutation occurred in the cells selected by magnetic beads was confirmed by T7E1 analysis, and the results are shown in FIG.

Example  5: Hygromycin ( hygromycin ) Concentration and classification of cells with gene modifications using reporter constructs

The reporter construct used the CMV promoter for intracellular expression, and the RFP gene for confirming the gene injection efficiency, the target gene capable of binding with ZFN, and the hygromycin phosphotransferase Hygromycin phosphotransferase (HPT) and eGFP gene (Fig. 10).

A pair of ZFNs (36 μg) capable of knocking out the porcine CMAH gene and the reporter construct (9 μg) for hygromycin screening were injected into the porcine ear tissues through electrical stimulation and then 1 × 10 ⁶ cells 100 mM culture dish. On the second day after injection of the ZFN and reporter constructs, the number of cells was 3 × 10 ⁶ per culture dish and the hygromycin B was treated at a concentration of 300 μg / ml for 48 hours. On the fourth day, a new culture . At that time, the number of surviving cells by hygromycin treatment was 1.5 x 10. ^On day 7, an initial colony was formed, and a complete colony was formed on day 18, and colonies formed on day 22 were transferred to a new 96- A transformed cell line transformed with the gene by cleavage was prepared (Fig. 11).

Intracellular introduction of ZFN and reporter constructs was induced, and the expression levels of intracellular RFP and GFP were compared before and 2 days after hygromycin B treatment and 4 days after treatment. The percentage of cells expressing RFP and GFP in the treatment groups was higher than before treatment or non-treatment (Table 5 and FIG. 12).

In addition, the percentage of cells in which the gene mutation occurred in the selected cells treated with hygromycin B (300 μg / ml) for 2 days from the second day after intracellular introduction of the ZFN and reporter constructs was analyzed by T7E1 assay . A higher proportion of transformed cells was identified in the treatment (12.1%) than the untreated control (3.1%) (Figure 13). The hygromycin B-treated cells were cultured for 30 days to form colonies from single cells (FIG. 14A). The ratio of transformed cells to each of the formed single colonies was analyzed by T7E1 analysis and the nucleotide sequence of the PCR product obtained from the single colonies was analyzed to confirm the transformation pattern. Analysis of two clones of 9 clones confirmed that all of them contained the transformed sequences at a high ratio (Fig. 14 b and c).

Example  6: FACS Classification of genetically mutated cells using

Reporter plasmids are designed to replicate in a reporter plasmid encoding a fusion protein of a monomeric red fluorescent protein (mRFP) -enhanced green fluorescent protein (eGFP), between the DNA sequences encoding the mRFP and eGFP An artificial nuclease target sequence was inserted to allow the eGFP sequence to fuse outside the frame of the mRFP sequence. The stop codon was inserted upstream of the eGFP sequence (Figure 15).

The reporter plasmid and ZFN were transfected into primary porcine adult ear fibroblasts and sorted by flow cytometry (Figure 16). As a result, mRFP was expressed by the CMV promoter, whereas functional eGFP was not expressed when the artificial nucleases were not active because they existed outside the frame. When DSB occurs in the target sequence by artificial nucleases, DNA damage is repaired by NHEJ, but this causes frame-mobility mutations that allow eGFP and mRFP to co-exist in the frame, expression of the mRFP-eGFP fusion protein was induced. Using this principle, we could enrich and classify cells whose genome was modified by artificial nucleases. As described above, it was confirmed that the cells before and after the selection using flow cytometry were counted by a glaucomicroscope to increase the ratio of RFP and GFP expression (that is, transformed cells) (FIG. 17) (Fig. 18). As a result, the proportion of transformed cells that were less than 0.5% before sorting increased to 13% for selected cells.

Example  7: CMAH  Gene knockout cells Nuclear-implanted  From egg Developed  Mutation-induced Blastocyst  formation

Among the cells selected by magnetic separation, GFP-expressing cells were taken and nuclear transfer was performed. After oocytes were fused with somatic cells, they were induced to induce cell division in vitro to form blastocysts. When the experiment was carried out in a total of 40 oocytes, three normally formed blastocysts were obtained. The gene sequence of the blastocyst was analyzed to identify the mutant sequence, which is shown in Fig.

<110> Toolgen, Inc. <120> Zinc finger nuclease for targeting CMAH gene and use thereof <130> PA120306 / KR &Lt; 150 > US 61 / 469,171 <151> 2011-03-30 <160> 41 <170> Kopatentin 2.0 <210> 1 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> QSHV ZF module <400> 1 Tyr Glu Cys Asp His Cys Gly Lys Ser Phe Ser Gln Ser Ser His Leu   1 5 10 15 Asn Val His Lys Arg Thr His              20 <210> 2 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> DSNR ZF module <400> 2 Tyr Arg Cys Lys Tyr Cys Asp Arg Ser Phe Ser Asp Ser Ser Asn Leu   1 5 10 15 Gln Arg His Val Arg Asn Ile His              20 <210> 3 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> rdne ZF module <400> 3 Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Arg Ala Asp Asn Leu   1 5 10 15 Thr Glu His Gln Arg Thr His              20 <210> 4 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> rdnq ZF module <400> 4 Phe Ala Cys Pro Glu Cys Pro Lys Arg Phe Met Arg Ser Asp Asn Leu   1 5 10 15 Thr Gln His Ile Lys Thr His              20 <210> 5 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> RDHT ZF module <400> 5 Phe Gln Cys Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu   1 5 10 15 Lys Thr His Thr Arg Thr His              20 <210> 6 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> QSHR ZF module <400> 6 Tyr Lys Cys Gly Gln Cys Gly Lys Phe Tyr Ser Gln Val Ser His Leu   1 5 10 15 Thr Arg His Gln Lys Ile His              20 <210> 7 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> QSHR2 ZF module <400> 7 Tyr Lys Cys Gly Gln Cys Gly Lys Phe Tyr Ser Gln Val Ser His Leu   1 5 10 15 Thr Arg His Gln Lys Ile His              20 <210> 8 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> RSHR zinc finger <400> 8 Tyr Lys Cys Met Glu Cys Gly Lys Ala Phe Asn Arg Arg Ser His Leu   1 5 10 15 Thr Arg His Gln Arg Ile His              20 <210> 9 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> DSCR zinc finger <400> 9 Tyr Thr Cys Ser Asp Cys Gly Lys Ala Phe Arg Asp Lys Ser Cys Leu   1 5 10 15 Asn Arg His Arg Arg Thr His              20 <210> 10 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> dghr zinc finger <400> 10 Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Asp Pro Gly His Leu   1 5 10 15 Val Arg His Gln Arg Thr His              20 <210> 11 <211> 23 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > QSNV3 zinc finger <400> 11 Tyr Lys Cys Asp Glu Cys Gly Lys Asn Phe Thr Gln Ser Ser Asn Leu   1 5 10 15 Ile Val His Lys Arg Ile His              20 <210> 12 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> DSAR2 zinc fimger <400> 12 Tyr Ser Cys Gly Ile Cys Gly Lys Ser Phe Ser Asp Ser Ser Ala Lys   1 5 10 15 Arg Arg His Cys Ile Leu His              20 <210> 13 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> ISNR zinc finger <400> 13 Tyr Arg Cys Lys Tyr Cys Asp Arg Ser Phe Ser Ser Ser Ser Asn Leu   1 5 10 15 Gln Arg His Val Arg Asn Ile His              20 <210> 14 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> KSNR zinc finger <400> 14 Tyr Gly Cys His Leu Cys Gly Lys Ala Phe Ser Lys Ser Ser Asn Leu   1 5 10 15 Arg Arg His Glu Met Ile His              20 <210> 15 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> QNTQ <400> 15 Tyr Thr Cys Ser Tyr Cys Gly Lys Ser Phe Thr Gln Ser Asn Thr Leu   1 5 10 15 Lys Gln His Thr Arg Ile His              20 <210> 16 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> QSNT zinc finger <400> 16 Tyr Glu Cys Val Gln Cys Gly Lys Gly Phe Thr Gln Ser Ser Asn Leu   1 5 10 15 Ile Thr His Gln Arg Val His              20 <210> 17 <211> 23 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > QSNR1 zinc finger <400> 17 Phe Glu Cys Lys Asp Cys Gly Lys Ala Phe Ile Gln Lys Ser Asn Leu   1 5 10 15 Ile Arg His Gln Arg Thr His              20 <210> 18 <211> 23 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > QSSR1 zinc finger <400> 18 Tyr Lys Cys Pro Asp Cys Gly Lys Ser Phe Ser Gln Ser Ser Leu   1 5 10 15 Ile Arg His Gln Arg Thr His              20 <210> 19 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> QTHQ zinc finger <400> 19 Tyr Glu Cys His Asp Cys Gly Lys Ser Phe Arg Gln Ser Thr His Leu   1 5 10 15 Thr Gln His Arg Arg Ile His              20 <210> 20 <211> 25 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > RDER2 zinc finger <400> 20 Tyr His Cys Asp Trp Asp Gly Cys Gly Trp Lys Phe Ala Arg Ser Asp   1 5 10 15 Glu Leu Thr Arg His Tyr Arg Lys His              20 25 <210> 21 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> VDYK zinc finger <400> 21 Phe His Cys Gly Tyr Cys Glu Lys Ser Phe Ser Val Lys Asp Tyr Leu   1 5 10 15 Thr Lys His Ile Arg Thr His              20 <210> 22 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> VSNV zinc finger <400> 22 Tyr Glu Cys Asp His Cys Gly Lys Ala Phe Ser Val Ser Ser Asn Leu   1 5 10 15 Asn Val His Arg Arg Ile His              20 <210> 23 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> WSNR zinc finger <400> 23 Tyr Arg Cys Glu Glu Cys Gly Lys Ala Phe Arg Trp Pro Ser Asn Leu   1 5 10 15 Thr Arg His Lys Arg Ile His              20 <210> 24 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> dgnv zinc finger <400> 24 Phe Gln Cys Arg Ile Cys Met Arg Asn Phe Ser Asp Ser Gly Asn Leu   1 5 10 15 Arg Val His Ile Arg Thr His              20 <210> 25 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> thse zinc finger <400> 25 Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Thr Ser His Ser Leu   1 5 10 15 Thr Glu His Gln Arg Thr His              20 <210> 26 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> tnse zinc finger <400> 26 Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Thr Lys Asn Ser Leu   1 5 10 15 Thr Glu His Gln Arg Thr His              20 <210> 27 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> hghe zinc finger <400> 27 Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser His Thr Gly His Leu   1 5 10 15 Leu Glu His Gln Arg Thr His              20 <210> 28 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> srta zinc finger <400> 28 Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Ser Arg Arg Thr Cys   1 5 10 15 Arg Ala His Gln Arg Thr His              20 <210> 29 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> qste zinc finger <400> 29 Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Gln Asn Ser Thr Leu   1 5 10 15 Thr Glu His Gln Arg Thr His              20 <210> 30 <211> 23 <212> PRT <213> Artificial Sequence <220> Rdnt zinc finger <400> 30 Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Arg Glu Asp Asn Leu   1 5 10 15 His Thr His Gln Arg Thr His              20 <210> 31 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> sadr zinc finger <400> 31 Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Ser Ala Asp Leu   1 5 10 15 Thr Arg His Gln Arg Thr His              20 <210> 32 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> tldr zinc finger <400> 32 Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Thr His Leu Asp Leu   1 5 10 15 Ile Arg His Gln Arg Thr His              20 <210> 33 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> skae zinc finger <400> 33 Tyr Lys Cys Pro Glu Cys Gly Lys Ser Phe Ser Ser Lys Lys Ala Leu   1 5 10 15 Thr Glu His Gln Arg Thr His              20 <210> 34 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> VSTR zinc finger <400> 34 Tyr Glu Cys Asn Tyr Cys Gly Lys Thr Phe Ser Val Ser Ser Thr Leu   1 5 10 15 Ile Arg His Gln Arg Ile His              20 <210> 35 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> ZF domain L4 <400> 35 Tyr Glu Cys Asp His Cys Gly Lys Ser Phe Ser Gln Ser Ser His Leu   1 5 10 15 Asn Val His Lys Arg Thr His Thr Gly Glu Lys Pro Tyr Arg Cys Lys              20 25 30 Tyr Cys Asp Arg Ser Phe Ser Asp Ser Ser Asn Leu Gln Arg His Val          35 40 45 Arg Asn Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly      50 55 60 Lys Ser Phe Ser Arg Ala Asp Asn Leu Thr Glu His Gln Arg Thr His  65 70 75 80 Thr Gly Glu Lys Pro Phe Ala Cys Pro Glu Cys Pro Lys Arg Phe Met                  85 90 95 Arg Ser Asp Asn Leu Thr Gln His Ile Lys Thr His             100 105 <210> 36 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> ZF domain R4 <400> 36 Phe Gln Cys Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu   1 5 10 15 Lys Thr His Thr Arg Thr His Thr Gly Glu Lys Pro Phe Gln Cys Lys              20 25 30 Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr His Thr          35 40 45 Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Gly Gln Cys Gly Lys      50 55 60 Phe Tyr Ser Gln Val Ser His Leu Thr Arg His Gln Lys Ile His Thr  65 70 75 80 Gly Glu Lys Pro Tyr Glu Cys Asp His Cys Gly Lys Ser Phe Ser Gln                  85 90 95 Ser Ser His Leu Asn Val His Lys Arg Thr His             100 105 <210> 37 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> ZFN L4 <400> 37 Tyr Glu Cys Asp His Cys Gly Lys Ser Phe Ser Gln Ser Ser His Leu   1 5 10 15 Asn Val His Lys Arg Thr His Thr Gly Glu Lys Pro Tyr Arg Cys Lys              20 25 30 Tyr Cys Asp Arg Ser Phe Ser Asp Ser Ser Asn Leu Gln Arg His Val          35 40 45 Arg Asn Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Pro Glu Cys Gly      50 55 60 Lys Ser Phe Ser Arg Ala Asp Asn Leu Thr Glu His Gln Arg Thr His  65 70 75 80 Thr Gly Glu Lys Pro Phe Ala Cys Pro Glu Cys Pro Lys Arg Phe Met                  85 90 95 Arg Ser Asp Asn Leu Thr Gln His Ile Lys Thr His Thr Gly Glu Lys             100 105 110 Gln Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu Leu Arg His         115 120 125 Lys Leu Lys Tyr Val Pro His Glu Tyr Ile Glu Leu Ile Glu Ile Ala     130 135 140 Arg Asn Pro Thr Gln Asp Arg Ile Leu Glu Met Lys Val Met Glu Phe 145 150 155 160 Phe Met Lys Val Tyr Gly Tyr Arg Gly Glu His Leu Gly Gly Ser Arg                 165 170 175 Lys Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr Gly             180 185 190 Val Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile         195 200 205 Gly Gln Ala Asp Ala Met Gln Ser Tyr Val Glu Glu Asn Gln Thr Arg     210 215 220 Asn Lys His Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr Pro Ser Ser 225 230 235 240 Val Thr Glu Phe Lys Phe Leu Phe Val Ser Gly His Phe Lys Gly Asn                 245 250 255 Tyr Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr Asn Cys Asn Gly             260 265 270 Ala Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met Ile Lys         275 280 285 Ala Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe Asn Asn Gly     290 295 300 Glu Ile Asn Phe Leu Asp 305 310 <210> 38 <211> 309 <212> PRT <213> Artificial Sequence <220> <223> ZFN R4 <400> 38 Phe Gln Cys Lys Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu   1 5 10 15 Lys Thr His Thr Arg Thr His Thr Gly Glu Lys Pro Phe Gln Cys Lys              20 25 30 Thr Cys Gln Arg Lys Phe Ser Arg Ser Asp His Leu Lys Thr His Thr          35 40 45 Arg Thr His Thr Gly Glu Lys Pro Tyr Lys Cys Gly Gln Cys Gly Lys      50 55 60 Phe Tyr Ser Gln Val Ser His Leu Thr Arg His Gln Lys Ile His Thr  65 70 75 80 Gly Glu Lys Pro Tyr Glu Cys Asp His Cys Gly Lys Ser Phe Ser Gln                  85 90 95 Ser Ser His Leu Asn Val His Lys Arg Thr His Thr Gly Glu Lys Gln             100 105 110 Leu Val Lys Ser Glu Leu Glu Glu Lys Lys Ser Glu Leu Arg His Lys         115 120 125 Leu Lys Tyr Val Pro His Glu Tyr Ile Glu Leu Ile Glu Ile Ala Arg     130 135 140 Asn Pro Thr Gln Asp Arg Ile Leu Glu Met Lys Val Met Glu Phe Phe 145 150 155 160 Met Lys Val Tyr Gly Tyr Arg Gly Glu His Leu Gly Gly Ser Arg Lys                 165 170 175 Pro Asp Gly Ala Ile Tyr Thr Val Gly Ser Pro Ile Asp Tyr Gly Val             180 185 190 Ile Val Asp Thr Lys Ala Tyr Ser Gly Gly Tyr Asn Leu Pro Ile Gly         195 200 205 Gln Ala Arg Glu Met Gln Arg Tyr Val Glu Glu Asn Gln Thr Arg Asn     210 215 220 Lys His Ile Asn Pro Asn Glu Trp Trp Lys Val Tyr Pro Ser Ser Val 225 230 235 240 Thr Glu Phe Lys Phe Leu Phe Val Ser Gly His Phe Lys Gly Asn Tyr                 245 250 255 Lys Ala Gln Leu Thr Arg Leu Asn His Ile Thr Asn Cys Asn Gly Ala             260 265 270 Val Leu Ser Val Glu Glu Leu Leu Ile Gly Gly Glu Met Ile Lys Ala         275 280 285 Gly Thr Leu Thr Leu Glu Glu Val Arg Arg Lys Phe Asn Asn Gly Glu     290 295 300 Ile Asn Phe Leu Asp 305 <210> 39 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Nuclear Localization Signal <400> 39 Pro Pro Lys Lys Lys Arg Lys Val   1 5 <210> 40 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CMAH ZFN forward primer <400> 40 cagaggtctg ccataacatc 20 <210> 41 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CMAH ZFN reverse primer <400> 41 tccatatgcc acaggttcag 20

Claims (35)

Comprising cleaving a CMAH gene in an isolated eukaryotic cell by an artificial nuclease that specifically recognizes a specific target sequence in a cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene,
Wherein the artificial nucleases are meganuclease, a fusion protein fused with a TAL operator domain and a cleavage domain, or a zinc finger nuclease,
Wherein the zinc finger nuclease comprises a pair of a first zinc finger nuclease and a second zinc finger nuclease,
Each zinc finger nuclease comprises a zinc finger domain and a nucleotide cleavage domain assembled by combining zinc finger modules that specifically recognize a specific target sequence in exon 4 of the CMAH gene,
Wherein one of the first zinc finger finger domain of the first zinc finger nuclease and the second zinc finger domain of the second zinc finger nuclease has the zinc finger modules represented by SEQ ID NOS: 1, 2, 3, and 4 sequentially , And the other comprises zinc finger modules represented by SEQ ID NOS: 5, 5, 6 and 1, sequentially. The method according to claim 1, wherein the CMAH gene is selected from the group consisting of the amino acid sequence of SEQ ID NO: 35 and the amino acid sequence of SEQ ID NO: 36, wherein one of the first zinc finger domain and the second zinc finger domain is represented by amino acid sequence SEQ ID NO: A method for producing a mutated cell. The method of claim 1, wherein the CMAH gene is selected from the group consisting of SEQ ID NO: 37 and SEQ ID NO: 38, wherein the first zinc finger domain and the second zinc finger domain are represented by the amino acid sequence of SEQ ID NO: 37 and the other is the amino acid sequence of SEQ ID NO: A method for producing a mutated cell. The method according to claim 1,
Introducing into the eukaryotic cell a reporter construct comprising the target sequence and the reporter gene; And
Separating the eukaryotic cells mutated by the nuclease of the target sequence on the reporter construct according to the presence or absence of expression of the reporter gene and separating the mutant cells of the genomic CMAH gene by the nuclease Wherein the CMAH gene is mutagenized. The method for producing a cell according to any one of claims 1 to 4, wherein the CMAH gene is mutated, wherein the expression of the CMAH gene is inhibited. The method for producing a cell according to any one of claims 1 to 4, wherein the CMAH gene is a porcine CMAH gene and the cell is a porcine cell. 5. The method according to any one of claims 1 to 4, wherein the artificial nucleases further comprise a nuclear localization signal sequence. Preparing a cell in which a CMAH gene is mutated by cleaving a CMAH gene in an isolated eukaryotic cell by an artificial nuclease that specifically recognizes a specific target sequence in a cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene; And
Nuclear transfer of the cell into an oocyte of an animal other than a human,
The artificial nuclease is a fusion protein comprising a meganuclease, a transcription activator-like effector domain and a cleavage domain, or a zinc finger nuclease,
Wherein the zinc finger nuclease comprises a pair of a first zinc finger nuclease and a second zinc finger nuclease,
Each zinc finger nuclease comprises a zinc finger domain and a nucleotide cleavage domain assembled by combining zinc finger modules that specifically recognize a specific target sequence in exon 4 of the CMAH gene,
Wherein one of the first zinc finger finger domain of the first zinc finger nuclease and the second zinc finger domain of the second zinc finger nuclease has the zinc finger modules represented by SEQ ID NOS: 1, 2, 3, and 4 sequentially And the other one sequentially containing the zinc finger modules represented by SEQ ID NOS: 5, 5, 6 and 1,
Wherein the CMAH gene is mutated. 9. The method according to claim 8, wherein the CMAH gene, wherein one of the first zinc finger domain and the second zinc finger domain is represented by the amino acid sequence of SEQ ID NO: 35 and the other is represented by the amino acid sequence of SEQ ID NO: A method for producing an animal, excluding mutated human. 9. The method according to claim 8, wherein the CMAH gene, wherein one of the first zinc finger domain and the second zinc finger domain is represented by the amino acid sequence of SEQ ID NO: 37 and the other is represented by the amino acid sequence of SEQ ID NO: A method for producing an animal, excluding mutated human. 9. The method according to claim 8, wherein the step of preparing the CMAH gene-
Introducing into the eukaryotic cell a reporter construct comprising the target sequence and the reporter gene; And
Separating the eukaryotic cells mutated by the nuclease of the target sequence on the reporter construct according to the presence or absence of expression of the reporter gene and separating the mutant cells of the genomic CMAH gene by the nuclease , Wherein the CMAH gene is mutated. 12. The method for producing an animal according to any one of claims 8 to 11, wherein the CMAH gene is mutated, wherein expression of the CMAH gene is inhibited. 12. The method for producing an animal according to any one of claims 8 to 11, wherein the CMAH gene is a porcine CMAH gene and the cell is a porcine cell. 12. The method according to any one of claims 8 to 11, wherein the artificial nuclease further comprises a nuclear localization signal sequence, wherein the CMAH gene is mutated. Preparing a cell in which a CMAH gene has been mutated by cleaving the CMAH gene in eukaryotic cells separated by an artificial nuclease that specifically recognizes a specific target sequence in a cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene;
Nuclear transfer of the cell into the oocyte of an animal other than a human to produce an animal other than a human to which the CMAH gene has been mutated; And
Isolating an artificial organ or artificial tissue from which the CMAH gene has been mutated,
The artificial nuclease is a fusion protein comprising a meganuclease, a transcription activator-like effector domain and a cleavage domain, or a zinc finger nuclease,
Wherein the zinc finger nuclease comprises a pair of a first zinc finger nuclease and a second zinc finger nuclease,
Each zinc finger nuclease comprises a zinc finger domain and a nucleotide cleavage domain assembled by combining zinc finger modules that specifically recognize a specific target sequence in exon 4 of the CMAH gene,
Wherein one of the first zinc finger finger domain of the first zinc finger nuclease and the second zinc finger domain of the second zinc finger nuclease has the zinc finger modules represented by SEQ ID NOS: 1, 2, 3, and 4 sequentially And the other one sequentially containing the zinc finger modules represented by SEQ ID NOS: 5, 5, 6 and 1,
A method for producing an artificial organ or artificial tissue in which the CMAH gene has been mutated. 16. The method according to claim 15, wherein the CMAH gene is selected from the group consisting of SEQ ID NO: 35 and SEQ ID NO: 36, wherein the first zinc finger domain and the second zinc finger domain are represented by the amino acid sequence of SEQ ID NO: A method for producing a mutated artificial organ or artificial tissue. 18. The method of claim 16, wherein the CMAH gene is selected from the group consisting of SEQ ID NO: 37 and SEQ ID NO: 38, wherein the first zinc finger domain and the second zinc finger domain are represented by the amino acid sequence of SEQ ID NO: 37 and the other is the amino acid sequence of SEQ ID NO: A method for producing a mutated artificial organ or artificial tissue. 16. The method according to claim 15, wherein the step of preparing the CMAH gene-
Introducing into the eukaryotic cell a reporter construct comprising the target sequence and the reporter gene; And
Separating the eukaryotic cells mutated by the nuclease of the target sequence on the reporter construct according to the presence or absence of expression of the reporter gene and separating the mutant cells of the genomic CMAH gene by the nuclease Wherein the CMAH gene is mutated. 19. The method for producing an artificial organ or artificial tissue according to any one of claims 15 to 18, wherein the CMAH gene is mutated, wherein the expression of the CMAH gene is inhibited. 19. The method for producing an artificial organ or artificial tissue according to any one of claims 15 to 18, wherein the CMAH gene is a CMAH gene of a pig, and the cell is a porcine cell. 19. The method according to any of claims 15 to 18, wherein the artificial nucleases further comprise a nuclear localization signal sequence, wherein the CMAH gene is produced by mutagenizing an artificial organ or artificial tissue Way. An artificial nuclease which specifically recognizes a specific target sequence in a cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene,
Wherein said artificial nucleases are meganuclease, a fusion protein fused with a TAL operator domain and a cleavage domain, or a zinc finger nuclease,
Wherein the zinc finger nuclease comprises a pair of a first zinc finger nuclease and a second zinc finger nuclease,
Each zinc finger nuclease comprises a zinc finger domain and a nucleotide cleavage domain assembled by combining zinc finger modules that specifically recognize a specific target sequence in exon 4 of the CMAH gene,
Wherein one of the first zinc finger finger domain of the first zinc finger nuclease and the second zinc finger domain of the second zinc finger nuclease has the zinc finger modules represented by SEQ ID NOS: 1, 2, 3, and 4 sequentially , And the other one comprises zinc finger modules sequentially represented by SEQ ID NOS: 5, 5, 6 and 1, respectively. The method of claim 22, wherein one of the first zinc finger domain and the second zinc finger domain is represented by the amino acid sequence of SEQ ID NO: 35 and the other is represented by the amino acid sequence of SEQ ID NO: 36. Cleaze. The method of claim 22, wherein one of the first zinc finger domain and the second zinc finger domain is represented by the amino acid sequence of SEQ ID NO: 37 and the other is represented by the amino acid sequence of SEQ ID NO: 38. Cleaze. 23. The method of claim 22,
Wherein the zinc finger nuclease further comprises a nuclear localization signal sequence. 26. A polynucleotide encoding the zinc finger nuclease of any one of claims 22 to 25. 26. A vector comprising the polynucleotide of claim 26. Mutating a CMAH gene by a zinc finger nuclease according to any one of claims 22 to 25 or a zinc finger nuclease obtained by expressing a polynucleotide encoding said zinc finger nuclease, Wherein the CMAH gene is mutated. 29. The method according to claim 28, wherein the cell is a porcine-derived cell, wherein the CMAH gene is mutated. 25. A non-human animal having a mutated CMAH gene, comprising the zinc finger nuclease of any one of claims 22 to 25, a polynucleotide encoding said zinc finger nuclease, or a vector comprising said polynucleotide An artificial organ preparation kit derived from egg, non-human animal, or non-human animal. 31. The kit of claim 30, wherein the animal is a pig. delete delete delete delete

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