Precise breeding methods for gene-edited non-human animals

Abstract

To provide a breeding method for making gene-edited non-human animals.SOLUTION: This method involves simultaneous reprogramming and gene-editing performed in somatic cells, and subsequent subcloning and genotyping conducted at the in vitro cell stage, to yield precisely gene-edited induced pluripotent stem cells (iPSCs), which are then used in somatic nuclear transfer (SCNT) to make a precisely gene-edited non-human animal embryo and a resultant gene-edited non-human animal.SELECTED DRAWING: None

Description

Technical Field

[0001] Related Applications This application claims the benefit of priority under 35 U.S.C. § 119 to U.S. Provisional Patent Application No. 63 / 319,920, filed on March 15, 2022, the entire content of which is incorporated herein by reference.

[0002] Technical Field The present invention relates to a breeding method for producing gene-edited non-human animals. In particular, the method of the present invention simultaneously performs reprogramming and gene editing in somatic cells, and then performs subcloning and genotyping at the in vitro cell stage to obtain accurately gene-edited induced pluripotent stem cells (iPSCs), and uses them in somatic cell nuclear transfer (SCNT) to produce accurately gene-edited non-human animal embryos and the resulting gene-edited non-human animals.

Background Art

[0003] Background of the Invention Takahashi and Yamanaka [1] reported that only four transcription factors, Oct4, Sox2, Klf4, and c-Myc (OSKM), are required for the induction of mouse induced pluripotent stem cells (iPSCs). The same group [2] and Yu et al. [3] produced human iPSCs using OSKM and the transcription factors Oct4 / Sox2 / Lin28 / Nanog, respectively. There are also research teams that reported the production of porcine iPSCs [4-6]. West et al. [7, 8] reported germline chimeric pigs produced from porcine iPSCs. Liu et al. used small molecule compounds together with porcine Oct4 and Klf4 to proliferate porcine iPSCs and confirmed pluripotency by forming teratomas [9]. There are also teams that reported the aggregation of morulas or cloned 4-cell embryos injected with iPSCs

[10] . Furthermore, porcine iPSCs were used as nuclear donors for somatic cell nuclear transfer (SCNT) to produce cloned early embryos [10-13] and live piglets [14, 15].

[0004] In this field, gene editing (GE) technologies based on zinc finger nucleases (ZFNs)

[17] , activator-like effector nucleases (TALENs)

[18] , and CRISPR / CAS9 (clustered regularly interspaced short palindromic repeats and the associated protein 9)

[19] have been developed. CRISPR / Cas9 technology is considered an inexpensive and correct method that is highly efficient, quick to implement, has low technical barriers, and is widely applicable to many organisms. Several modified forms of Cas9 are available, allowing for more precise nicking of single strands of target DNA or editing of single bases

[20] . To date, iPSC applications have been concentrated, particularly in human cell therapies for cancer or genetic diseases. Howden et al.

[21] were the first researchers to simultaneously reprogram and modify fibroblasts from adult patients with retinitis pigmentosa and children with severe combined immunodeficiency to create DNMT3B and Oct4 KI iPSCs with the green fluorescent protein (GFP) gene, in which only 3–5% of their iPSC subclones expressed GFP. Wang et al.

[22] created CHD8 hemizygote iPSCs using dermal fibroblasts from psychiatric patients to study autism spectrum disorder. Kim et al.

[23] created activated ossificans receptor type I (also known as ALK2) gene-corrected (ALK2 c.617G4 A) iPSCs using fibroblasts from patients with fibrodysplasia ossificans progressive (FOP) syndrome. Bell et al.

[24] established a rapid pipeline for developing targeted therapy models for rare neurodevelopmental disorders. Tidball et al.

[25] rapidly created human loss-of-function iPS cell lines. Melo et al.

[26] and Wen et al.

[27] generated CAPN1 mutant iPSCs and established high-level, accurate knock-in iPSCs using erythroblasts and peripheral blood mononuclear cells (PBMCs), respectively, readily available from a healthy 97-year-old woman, and performed genome editing on the bulk iPSC population without selection.Howden et al.

[28] developed a model of osteogenesis imperfecta (OI), a human 'brittle bone' disease, and established and modified a patient's COL1A1 c.3936 G>T iPSC line. Ye et al.

[29] used iPS cells derived from Alzheimer's disease (Alz) patients to carry three copies of the APP (amyloid precursor protein) gene and studied gene dose dysregulation in Alz disease. However, there are no reports of applying gene-edited iPSCs to breeding non-human animals.

[0005] Since the late 1980s, porcine reproductive and respiratory syndrome virus (PRRSV) has rapidly spread, devastating the global pig farming industry. In vivo, the virus exhibits very narrow cellular localization, targeting specific subsets of porcine monocytes / macrophages and infecting cells via heparan sulfate, sialoadhesin (CD169), and CD163 receptors

[30] . To date, CD163 on porcine macrophages is the most well-studied receptor involved in PRRSV infection

[31] . Studies such as CD163 knockout [32-35], deletion of exon 7 of the CD163 gene (the scavenger receptor cysteine-rich domain 5 (SRCR5) region of the CD163 protein) [36, 37], and partial deletion of exon 7 in the virion infection pocket

[38] have resulted in complete resistance to PRRSV infection in GE pigs without compromising their well-being [33, 37-41]. However, there are still fundamental limitations to the precision of gene sequences used to create non-human animals with desired gene editing and traits.

[0006] Therefore, there is a need to develop more accurate and efficient breeding techniques for creating genetically edited non-human animals, especially large livestock. [Overview of the project]

[0007] This invention provides a precise breeding method for gene-edited non-human animals. In particular, the method of this invention is characterized by simultaneously performing reprogramming and gene editing in somatic cells, followed by in vitro subcloning and genotyping at the cellular level to obtain precisely gene-edited iPSCs, which are then used in somatic cell nuclear transfer (SCNT) to produce precisely gene-edited non-human embryos and the resulting gene-edited non-human animals.

[0008] In one embodiment, the present invention relates to a method for producing gene-edited non-human embryos and resulting gene-edited non-human animals, (a) A process of inducing target gene editing in non-human mammalian somatic cells through gene editing, and simultaneously reprogramming those cells into induced pluripotent stem cells (iPS cells) to produce multiple gene-edited iPS cell candidates; (b) A step of subcloning a candidate gene-edited iPSC, determining its genotype, and obtaining a gene-edited iPSC subclone having a genome with the desired gene edit; (c) The process of creating a reconstituted embryo by transplanting a gene-edited iPSC subclone into an enucleated oocyte; and (d) A process of culturing the reconstituted embryo to the blastocyst stage to obtain a non-human gene-edited animal having the desired gene edit. This provides a method that includes [something].

[0009] In some embodiments, gene editing is CRISPR / Cas9-based gene editing.

[0010] In some aspects, the reprogramming factors include Klf4, c-Myc, Nanog, Oct4, Sox2, and SV40 large T antigen.

[0011] In some embodiments, somatic cells are simultaneously transfected with CRISPR / Cas9-based gene editing vectors and reprogramming vectors.

[0012] In some embodiments, CRISPR / Cas9-based gene editing vectors are, - A Cas9 vector containing nucleic acids encoding the Cas9 protein, and - One or more gRNA vectors, each containing nucleic acids encoding gRNA molecules for targeting a target gene with Cas9 to induce gene editing. Includes.

[0013] In some ways, the reprogramming vector is - A first reprogramming vector containing nucleic acids encoding Oct4, Sox2, Klf4, and Nanog; - A second reprogramming vector containing nucleic acid encoding c-Myc; and - A third reprogramming vector containing nucleic acid encoding the SV40 large T antigen. Includes.

[0014] In some embodiments, somatic cells and oocytes originate from the same species.

[0015] In some embodiments, somatic cells are fibroblasts.

[0016] In some embodiments, gene editing involves gene knock-in, gene knock-out, or partial deletion.

[0017] In some embodiments, the non-human animal is selected from a group consisting of sheep, cattle, deer, goats, monkeys, camels, and pigs.

[0018] In some embodiments, gene editing involves the knockout or partial deletion of the CD163 gene.

[0019] Specifically, the present invention is a method for providing a CD163 gene-edited pig. (a) A step of simultaneously transfecting a porcine somatic cell with a gene editing vector and a reprogramming vector, wherein gene editing of the CD163 gene is provided to generate a plurality of gene-edited porcine iPSC (piPSC) candidates; (b) A step of subcloning the gene-edited piPSC candidates and determining the genotype to obtain CD163 gene-edited piPSC subclones having a genome with gene editing of the CD163 gene; (c) A step of transplanting the iPSC subclone with the CD163 gene edited into an enucleated porcine egg to produce a reconstructed porcine embryo; and (d) A step of culturing the reconstructed porcine embryo until the blastocyst stage to obtain a pig having gene editing of the CD163 gene A method comprising the above steps is provided.

[0020] Particularly, the obtained pigs having gene editing of the CD163 gene can exhibit resistance to porcine reproductive and respiratory syndrome virus (PRRSV) infection.

[0021] Also provided is a method for conferring resistance to porcine reproductive and respiratory syndrome virus (PRRSV) infection in pigs, (a) A step of simultaneously transfecting a porcine somatic cell with a gene editing vector and a reprogramming vector, wherein the gene editing vector provides gene knockout or partial deletion of the CD163 gene to produce a plurality of CD163 gene-edited porcine iPSC (piPSC) candidates; (b) A step of subcloning the gene-edited piPSC candidates and performing genotyping to obtain a population of CD163 gene-edited piPSC subclones having a genome with gene knockout or partial deletion of the CD163 gene; (c) A step of transplanting each of the iPSC subclones into which the CD163 gene has been introduced into an enucleated porcine egg to produce a reconstructed porcine embryo; (d) A step of culturing the reconstructed porcine embryo until the blastocyst stage to obtain a plurality of CD163 gene-edited pigs; and (e) A step of selecting a pig line from multiple CD163 gene-edited pigs obtained in step (d) that shows resistance to PRSV compared to non-gene-edited pigs grown under the same conditions. This provides a method that includes [something].

[0022] Details of one or more aspects of the present invention are described below. Other features or advantages of the present invention may become apparent from the following detailed descriptions of some aspects and from the appended claims. [Brief explanation of the drawing]

[0023] The above summary and the following detailed description of the invention can be better understood in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, currently preferred embodiments are shown in the drawings. However, it should be understood that the present invention is not limited to the exact arrangements and means shown.

[0024] [Figure 1A-1D]Figures 1A-1D are schematic diagrams of recombinant Cas9 protein and single-stranded guide RNA expression vectors. Figure 1A shows a linear map of the pCX-Flag2-NLS1-Cas9-NLS2 plasmid DNA vector and the NLS1-Cas9-NLS2 recombinant protein. Figure 1B shows a linear map of the ppU6-(BsaI)2-gRNA plasmid DNA vector, as well as the DNA sequence (SEQ ID NO: 1) containing the pU6 promoter, BsaI cleavage site, and guide RNA coding region. Figure 1C shows a diagram illustrating the construction method of a single-stranded guide RNA (sgRNA) expression vector by a simple ligation reaction. Figure 1D shows the sequences of two protospacers, including protospacer 26 (forward sequence: SEQ ID NO: 8, reverse sequence: SEQ ID NO: 9) and protospacer 28 (forward sequence: SEQ ID NO: 12, reverse sequence: SEQ ID NO: 13), as well as the sequences of two primer pairs for constructing a spacer sgRNA expression vector, including primers pCD163-Sp26F (SEQ ID NO: 6), pCD163-Sp26R (SEQ ID NO: 7), pCD163-Sp28F (SEQ ID NO: 10), and pCD163-Sp28R (SEQ ID NO: 11) for cloning a CD163 single-stranded guide RNA vector. [Figure 2A-2B] Figures 2A and 2B show the gene editing sites for precisely removing exon 7 of the porcine CD163 gene. Figure 2A is a schematic diagram of the porcine CD163 gene, showing exons 6 through 8, including protospacers 26 (Sp26) and 28 (Sp28). Figure 2B shows the porcine genomic DNA sequence near CD163 exon 7 (sequence number 14). The Cas9 cleavage sites are indicated as “sgSL26 cut” and “sgSL28 cut”. [Figure 3A-3C]Figures 3A–3C show the production of porcine CD163 exon 7 edited / deleted (CD163ΔE7) induced pluripotent stem cells (piPSCs) by transfection with iPS cell inducers and CRISPR / Cas9 gene editing plasmid vectors via electroporation. Figure 3A shows the morphology of piPSCs after two electroporations (2EP), three electroporations (3EP), or four electroporations (4EP). Treated primary fibroblasts were established from pigs L259-10 and D529-16. Figure 3B shows primer pairs, F2 (SEQ ID NO: 15) and R2 (SEQ ID NO: 16), which are used in PCR amplification to amplify a DNA fragment containing two protospacers to confirm the gene deletion effect. The sequences on porcine DNA recognized by primers F2 and R2 are shown in Figure 2A. Candidate piPSCs were analyzed by genomic DNA PCR. Red indicates homologous piPSCs with double chromosome CD163ΔE7, and green indicates heterologous piPSCs. The numbers in parentheses indicate the number of candidates / number of analyses, and the average efficiency % for homologous CD163ΔE7. Figure 3C shows that PCR amplicons from homologous CD163ΔE7 piPSCs were further analyzed by PCR amplicon sequencing and confirmed to be CD163ΔE7. This clone lacked exon 7 from 23268 bp to 23753 bp in SEQ ID NO: 17 and showed a PCR amplicon of 454 bp in length. [Figure 4] Figure 4 shows porcine blastocysts cloned using CD163ΔE7 piPSC. The nucleus was removed from mature IVM oocytes, and CD163ΔE7 piPSC was directly microinjected into the cytoplasm. After culturing in PZM5 medium for 6-7 days, reconstituted porcine blastocysts were obtained (indicated by arrows). [Modes for carrying out the invention]

[0025] Detailed description of the invention The following description is intended to illustrate various aspects of the present invention. Thus, any specific aspects or modifications discussed herein should not be construed as limiting the scope of the invention. It will be apparent to those skilled in the art that various modifications or equivalents can be made without departing the scope of the invention.

[0026] To ensure that the present invention is clearly and readily understood, certain terms are first defined. Other definitions are provided in the detailed description. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains.

[0027] In this specification, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise. Thus, for example, a reference to “a certain component” includes multiple such components and their equivalents, as known to those skilled in the art.

[0028] The terms “comprise” or “comprising” are generally used in the sense of “include / including,” meaning to allow the presence of one or more features, components, or constituent elements. The terms “comprise” or “comprising” encompass the terms “consists” or “consisting of.”

[0029] As used herein, the terms “nucleic acid” or “polynucleotide” may mean a polymer containing nucleotide units. Polynucleotides include natural nucleic acids such as deoxyribonucleic acid (“DNA”) and ribonucleic acid (“RNA”), as well as nucleic acid analogs, including nucleic acid analogs having non-natural nucleotides. Polynucleotides can be synthesized, for example, using an automated DNA synthesizer. When a nucleotide sequence is represented as a DNA sequence (i.e., A, T, G, C), it may be understood that RNA sequences in which “T” is replaced by “U” (i.e., A, U, G, C) are also included. The term “cDNA” means DNA that is complementary to or identical to mRNA, either in single-stranded or double-stranded form.

[0030] As used herein, the term “polypeptide” refers to a polymer consisting of amino acid residues linked together via peptide bonds. The term “protein” generally refers to a relatively large polypeptide. The term “peptide” generally refers to a relatively short polypeptide (e.g., including up to 100 amino acid residues, 90 amino acid residues, 70 amino acid residues, 50 amino acid residues, 30 amino acid residues, 20 amino acid residues, or 10 amino acid residues).

[0031] As used herein, the term “coding” means a natural or resulting biological property in which a particular sequence of nucleotides in a polynucleotide (e.g., a gene, cDNA, or mRNA) functions as a template for the synthesis of other polymers and macromolecules in biological processes having either a given sequence of RNA transcripts (i.e., rRNA, tRNA, and mRNA) or a given sequence of amino acids. Therefore, a gene codes for a protein when the mRNA produced by that gene is transcribed and translated, resulting in the production of that protein in a biological system such as a cell. As used herein, a “coding sequence” or a sequence that “codes” an expression product such as RNA or polypeptide means a nucleotide sequence that, when expressed, leads to the production of that RNA or polypeptide, i.e., a nucleotide sequence that codes for the amino acid sequence of that polypeptide. A protein coding sequence may include a start codon (usually ATG) and a stop codon. As a result of the degeneracy of genetic coding, it will be understood by those skilled in the art that many different polynucleotides and nucleic acids can code for the same polypeptide. Furthermore, it is understood that, using conventional techniques, nucleotide substitutions can be made that do not affect the polypeptide sequence encoded by the polynucleotides described herein, in order to reflect the codon usage of any particular host organism in which the polypeptide is to be expressed. Thus, “nucleotide sequences encoding an amino acid sequence” means, unless otherwise specified, degenerate nucleotide sequences encompassing all nucleotide sequences encoding the same amino acid sequence.

[0032] As used herein, the term “gene” means a nucleic acid containing an open reading frame that codes for a polypeptide of interest, which includes both exon sequences and (if applicable) intron sequences. A gene can be a DNA sequence that, when placed under the control of appropriate regulatory sequences, is transcribed in vivo into RNA and possibly translated into a polypeptide. For example, a gene of interest may code for an RNA transcript, a native polypeptide (i.e., a polypeptide found in nature) or a fragment thereof, an artificial polypeptide, or a peptide fragment.

[0033] The terms “gene editing” and “genome editing,” as used interchangeably herein, refer to a type of genetic engineering that involves deleting, inserting, and / or replacing DNA in the genome of a target cell. Targeted gene editing (interchangeable with targeted genome editing) may include deletion, insertion, and / or replacement at one or more predetermined sites within the genome. When an endogenous sequence is deleted during gene editing, the endogenous gene containing the affected sequence may be knocked out or knocked down as a result of the sequence deletion. Therefore, endogenous gene expression can be disrupted using targeted gene editing. Gene editing can be achieved by specifically introducing double-strand breaks using a particular endonuclease. Examples of genome editing include zinc fingers, TALENs, and CRISPR / Cas9, which are known and available in the art, but CRISPR is preferred.

[0034] CRISPR / caspase-9 requires two main components: (1) caspase-9 endonuclease (Cas9) and (2) a crRNA-tracrRNA complex. When co-expressed, these two elements form a complex and are recruited to target DNA sequences containing the PAM and the seeding region near the PAM. The crRNA and tracrRNA can be combined to form a chimeric guide RNA (gRNA), which can guide Cas9 to the target-selection sequence. These two elements can be delivered to mammalian cells by transfection or transduction.

[0035] As used herein, the term “recombinant polynucleotide” means a polynucleotide or nucleic acid having sequences that are not naturally bound. Recombinant nucleic acids can exist in the form of a vector. A “vector” may contain a specific nucleotide sequence of interest and a regulatory sequence. A vector can be used to express a specific nucleotide sequence or to maintain a specific nucleotide sequence for replication, manipulation, or transfer between different locations (e.g., between different organisms). A vector can be introduced into a suitable host cell for the purposes described above. The term “operable ligation” may mean that a polynucleotide is ligated to an expression regulatory sequence in such a way that the expression of the polynucleotide is enabled when an appropriate molecule (such as a transcription factor) is bound to the expression regulatory sequence. The term “expression regulatory sequence” or “regulatory sequence” means a DNA sequence that controls the expression of an operationally ligated nucleic acid sequence in a host cell. Examples of vectors include, but are not limited to, plasmids, cosmids, phages, YACs, or PACs. Generally, in a vector, a specific nucleotide sequence is operable ligated to a regulatory sequence such that, when the vector is introduced into a host cell, the specific nucleotide sequence can be expressed in the host cell under the control of the regulatory sequence. Examples of regulatory sequences include, but are not limited to, promoter sequences (e.g., cytomegalovirus (CMV) promoter, Simianvirus 40 (SV40) initial promoter, lac promoter, T7 promoter, and alcohol oxidase gene (AOX1) promoter), start codons, replication origin, enhancers, operator sequences, secretion signal sequences (e.g., α-conjugation factor signals), and other regulatory sequences (e.g., Shine-Dalgano sequences, termination sequences, etc.). Preferably, the vector may further include marker sequences for subsequent screening methods (e.g., antibiotic resistance marker sequences).

[0036] As used herein, the terms “totipotent” or “totipotency” refer to the ability of a cell to divide and ultimately produce an entire organism, including extraembryonic tissues. For example, a fertilized egg is a totipotent stem cell that gives rise to all of the organism's embryonic and extraembryonic tissues. Specifically, the term “totipotency” can refer to the ability of a cell to progress through a series of divisions to a blastocyst. A blastocyst contains an inner cell mass (ICM) and outer layer cells called chorionic blasts. Chorioblasts ultimately form extraembryonic tissues such as the placenta and amnion. The cells in the ICM become pluripotent stem cells with the ability to proliferate indefinitely and, when properly induced, differentiate into various cell types within the body.

[0037] As used herein, the terms “pluripotent” or “pluripotency” refer to the ability of a cell to regenerate and differentiate into all cell lineages. For example, embryonic stem cells (ESCs) are a type of pluripotent stem cell (PSC) that can form cells from each of the three germ layers: ectoderm (stomach wall, gastrointestinal tract, lungs), mesoderm (muscle, bone, blood, genitourinary tract), and endoderm (epidermal tissue and nervous system). PSCs can give rise to a variety of fetal or adult cell types, including germ cells. However, because PSCs cannot contribute to extraembryonic tissues such as the placenta, they cannot create a fetus or adult animal when implanted in the uterus.

[0038] As used herein, the term “differentiation” refers to the developmental process by which cells differentiate and acquire specific characteristics in order to perform a particular function. Differentiation is a relative process. Mature somatic cells, such as osteoblasts (bone), chondrocytes (cartilage), adipocytes (fat), fibroblasts (skin), and hepatocytes (liver), may be terminally differentiated cells that have already lost the ability to differentiate into different cell types.

[0039] As used herein, the terms “reprogramming” and “dedifferentiation” refer to the process of enhancing the capabilities of a cell or dedifferentiating it to a less differentiated state. Specifically, a reprogrammed cell is a cell that is less differentiated than the same cell in an unreprogrammed state. Reprogramming can be induced using reprogramming factors or chemical substances.

[0040] As used herein, the terms “induced pluripotent stem cells” or “iPSCs” refer to pluripotent stem cells reprogrammed from adult tissue or differentiated cells (e.g., somatic cells). While iPS cells are thought to possess the same pluripotency as naturally occurring pluripotent stem cells (e.g., ESCs), the term iPS cells does not refer to cells that exist in nature. iPS cells can be created by inserting one or more specific genes into somatic cells or by stimulating them with chemical substances.

[0041] As used herein, the term "zygote" refers to a cell formed by the fusion of two gamete cells. It is the earliest stage of embryonic development. A zygote arises when two haploid cells, an egg (female gamete) and a sperm cell (male gamete), fertilize and fuse to form a single diploid cell.

[0042] As used herein, the term “embryo” means a mass of cells produced by one or more cell divisions of a zygote or activated oocyte in which a source of cell nuclei has been artificially incorporated.

[0043] As used herein, the term "morula" refers to a stage of embryonic development. Approximately 3 to 4 days later, the zygote forms a mulberry-shaped group of cells called a morula through a series of divisions, generally consisting of 12 to 32 cells (called blastomeres). Through cell differentiation and cavitation, the morula develops into a blastocyst. During blastocyst formation, the cells of the morula differentiate into the inner cell mass, which proliferates within the blastocyst, and the villous cells, which form the outer membrane of the blastocyst.

[0044] As used herein, the term "blastocyst" refers to a structure formed during the early development of mammals. A blastocyst is formed approximately 5 to 7 days after fertilization. Generally, it is a fluid-filled sphere of about 60-100 cells, consisting of (i) an outer layer of cells (ectoderm), (ii) an inner cell mass (ICM, which is the source of embryonic stem cells), and (iii) a fluid-filled cavity (blastocyst coel).

[0045] As used herein, “reconstructed embryo” refers to cells formed by inserting donor cells or the nuclei of donor cells into an enucleated oocyte, which corresponds to a zygote.

[0046] As used herein, the term “subcloning” means sequentially diluting cells in a stepwise manner, for example in a 96-well microlitter plate, in order to obtain a single colony.

[0047] As used herein, the term “genotyping” means determining the genetic information that a cell or organism has at one or more locations in the genome. For example, genotyping may include determining which allele(s) an organism has for a single nucleotide polymorphism (SNP) or a gene mutation such as an insertion, deletion, or substitution. For example, a particular nucleotide on the genome may be T in one organism and G in another. An organism with T at the polymorphic location will have the T allele, and an organism with G will have the G allele. In diploid organisms, an organism will have two copies of the sequence containing the polymorphic location, and may have a T allele and a G allele, or two copies of the T allele or two copies of the G allele. An organism with two copies of the T allele is homozygous for the T allele, an organism with two copies of the G allele is homozygous for the G allele, and an organism with one copy of each allele is heterozygous. In the specific examples described herein, genotyping may involve determining which allele(s) a porcine iPSC clone possesses at the location of exon 7 between protospacers 26 and 28 (from 23268 bp to 23753 bp) in the genomic DNA of CD163. For example, an iPSC clone with a full-length fragment at exon 7 between protospacers 26 and 28 possesses the full-length exon 7(F) allele, while one with a deletion at exon 7 between protospacers 26 and 28 (from 23268 bp to 23753 bp) possesses the deleted exon 7(D) allele. These iPSC clones may have homogeneous full-length exon 7 in both alleles, homogeneous exon 7 deletions in both alleles, or heterogeneous full-length exon 7 in one allele and exon 7 deletion in the other.

[0048] The present invention provides a method for producing gene-edited non-human animal embryos and the resulting gene-edited non-human animals, characterized by simultaneously performing reprogramming and gene editing in somatic cells, then performing subcloning and genotyping at the in vitro cell stage to obtain gene-edited iPSC subclones, transplanting these into enucleated oocytes, and generating gene-edited non-human animal embryos from them to produce gene-edited non-human animals.

[0049] The non-human animals of the present invention are generally non-human mammals. In some embodiments, the non-human mammals are primates, goats, sheep, pigs, dogs, cattle, or rodents.

[0050] The somatic cells of the present invention may be primary cells (unimmortalized cells), such as cells newly isolated from an animal, or cells derived from a cell line (immortalized cells). Differentiated somatic cells are suitable as starting cells for this method. Examples of somatic cells include, but are not limited to, fibroblasts, muscle cells, keratinocytes, and hepatocytes. These can be obtained by methods known in the art and from suitable organs or tissues containing living somatic cells, such as skin.

[0051] In this invention, somatic cells are reprogrammed into iPSCs. Reprogramming can be achieved by using multiple reprogramming factors. The reprogramming factors may include one or more gene products that can be introduced into cells by transfecting the cells with a recombinant vector containing a gene encoding the reprogramming factor. In this way, reprogramming of differentiated cells can be induced by expressing the reprogramming factor expressed as a product of a gene contained in the recombinant vector. In a particular embodiment, the reprogramming factor includes multiple gene products: Oct family genes, Klf family genes, Sox family genes, and Myc family genes. Examples of Oct family genes include Oct3 / 4, Oct1A, Oct6, etc. Examples of Klf family genes include Klf1, Klf2, Klf4, Klf5, etc. Examples of Myc family genes include c-Myc, N-Myc, L-Myc, etc. Examples of Sox family genes include Sox1, Sox2, Sox3, Sox7, Sox15, Sox17, Sox18, etc. In some embodiments, the reprogramming factor further includes one or more of the following genes: Nanog, SV40 large T antigen, Fbx15, ERas, Tc11, Grb2, Gdf3, Rex1, ECAT1, ECAT8, ECAT15-1, ECAT15-2, Stella, Stat3, and Sall4. In specific examples, the reprogramming factor includes Oct4, Sox2, Klf4, c-Myc, Nanog, and SV40 large T antigen. After reprogramming, iPSC candidates can be selected based on morphological features such as round shape, large nucleolus, and low cytoplasm, similar to ESCs. Generally, reprogrammed colonies are known to be dense, sharp, flat, and mitotically active due to their self-renewal. iPSC candidates can be further identified based on the expression of specific cell surface proteins, e.g., SSEA-4, alkaline phosphatase, and transcription factors, e.g., Oct4, Sox2, and Nanog.Various standard approaches are used, but are not limited to them. For example, at the RNA level, polymerase chain amplification, ribonuclease protection (RNase) assays, and Northern blot analysis are used, while at the protein level, enzyme-linked immunosorbent assay (ELISA), Western blotting, and intracellular staining are used.

[0052] In this invention, gene editing is performed simultaneously with reprogramming. Gene editing may include genetic engineering that involves inserting, deleting, and / or substituting nucleotides / nucleic acids(s) into DNA sequences such as the genome of a target cell. Targeted gene editing can perform insertions, deletions, and / or substitutions at pre-selected sites on the genome of a target cell. When the base sequence of an endogenous gene is edited, for example, by deletion, insertion, or substitution of nucleotides / nucleic acids(s), the endogenous gene containing the affected sequence may be knocked out by the alteration of its sequence. Therefore, targeted editing can be used to disrupt the expression or function of an endogenous gene. A “disrupted gene” means a gene that includes an insertion, deletion, or substitution to an endogenous gene such that the expression of a functional protein from the endogenous gene is reduced or inhibited, or the function of the protein is inhibited or impaired. In some embodiments, cells containing a disrupted gene do not express the protein encoded by that gene at a detectable level. In some embodiments, cells containing a disrupted gene may express a cleaved form of the protein encoded by that gene, which has a loss of function. Examples of genes that should be edited include, but are not limited to, CD163, PDX1, and RAG2 / IL2RG2.

[0053] In this invention, conventional gene editing methods can be used. A nuclease-independent approach is homologous recombination, which is led by homologous sequences flanking exogenous polynucleotides introduced into an endogenous sequence by the enzymatic machinery of the host cell. A nuclease-dependent approach utilizes the DNA repair mechanism by specifically introducing double-strand breaks (DSBs) using a specific rare-cut nuclease (e.g., an endonuclease). In some embodiments, gene disruption can be caused by deletion of a genomic sequence using two guide RNAs in CRISPR / Cas9 gene editing technology. Methods for creating genomic deletions in cells using CRISPR / Cas9 gene editing technology are known in the art.

[0054] In some embodiments, Cas9 endonuclease is used in the CRISPR method for producing gene-edited iPSCs as described herein. The Cas9 enzyme may be derived from Streptococcus pyogenes, but other Cas9 homologs may also be used. Wild-type Cas9 or modified Cas9 may be used. In some embodiments, Cas9 is modified to include two nuclear localization signals (NLS) and a FLAG tag. The CRISPR technique uses a genomic target nucleic acid that can guide the endonuclease to a specific target sequence in the target gene, thereby performing gene editing at the specific target sequence. The genomic target nucleic acid may be RNA. Genomic target RNA is referred to herein as “guide RNA” or “gRNA”. The guide RNA includes at least a spacer sequence that hybridizes to the target nucleic acid sequence in the gene to be edited, and a CRISPR repeat sequence. The spacer sequence in gRNA is a sequence that defines the target sequence of the target gene of interest. In some embodiments, the spacer sequence is 15 to 30 nucleotides long, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long. In some embodiments, the spacer sequence contains 20 nucleotides. The target sequence is a sequence adjacent to a PAM sequence in a target gene that is modified by an RNA-guided nuclease such as Cas9. In the CRISPR / Cas system described herein, the spacer sequence is designed to hybridize to a region of the target nucleic acid located at 5' of a PAM that is recognizable by the Cas9 enzyme used in the system. Each Cas9 enzyme has a specific PAM sequence that is recognized by the target DNA. For example, S. pyogenes recognizes a PAM containing the sequence 5'-NRG-3' within the target nucleic acid, where R contains A or G, N is any nucleotide, and N is located 3' immediately above the target nucleic acid sequence targeted by the spacer sequence.In some embodiments, the gRNAs described herein target the CD163 gene, for example, a site within exon 7 of the CD163 gene. Such gRNAs may contain a spacer sequence that is (fully or partially) complementary to the target sequence within exon 7 of the CD163 gene. Exemplary target sequences in the CD163 gene and exemplary gRNAs specific to the CD163 gene are illustrated in the following examples (Figures 1D and 2B). Delivery of RNA guide nucleases and gRNAs can be carried out by direct injection or by known methods, such as cell transfection using electroporation or chemical transfection.

[0055] Reprogramming and gene editing are performed simultaneously to generate gene-edited iPS cell candidates. Subsequently, subcloning and genotyping can be performed to obtain gene-edited iPS cell subclones having genomes with the desired gene edits. Genotyping can be performed using any method known in the art, such as oligonucleotide ligase assays, restriction fragment length polymorphism (RFLP), polymerase chain reaction (PCR), sequencing, and immunoassays. In some embodiments, primers specific to the target gene are designed and used to determine the genotype of the gene in the gene-edited iPSC candidate.

[0056] Next, a gene-edited iPSC subclone having a genome with the selected target gene edit is transplanted into an enucleated oocyte to produce a reconstituted embryo. In this invention, the recipient cell in the nuclear transfer step is an enucleated oocyte. An oocyte is an immature female germ cell that has not completed the maturation process necessary to form an egg (gamete). The oocytes of this invention can be isolated from the fallopian tube and / or ovary of a mammal. In one embodiment, oocytes are harvested by aspiration. Oocytes are usually matured in various media known to those skilled in the art before nucleation. In vitro maturation of oocytes is usually carried out in maturation medium until the oocyte reaches metaphase II or the first polar body disappears. Enucleation of mature oocytes can be carried out using methods known in the art, such as aspiration, physical removal, use of DNA-specific fluorescent dyes, exposure to ultraviolet light, and / or chemically assisted enucleation. A gene-edited iPSC having the predetermined gene edit is injected into an enucleated oocyte as a fertilized egg to form a reconstituted zygote. Subsequently, the zygote is activated with a chemical activator such as ionomycin or ethanol to form an embryo. The embryo is cultured through to the dehiscing stage (2-4 cells) and then to the blastocyst stage. The cultured embryo is transplanted into a host mammal, and the embryo develops into a genetically edited non-human animal.

[0057] In a particular embodiment, the method of the present invention produces CD163 gene-edited pigs by simultaneously transfecting porcine somatic cells with a gene-editing vector and a reprogramming vector, wherein the gene-editing vector provides gene editing of the CD163 gene, thereby generating a plurality of CD163 gene-edited porcine iPSC (piPSC) candidates; subcloning the gene-edited piPSC candidates and determining their genotypes to obtain CD163 gene-edited piPSC subclones having genomes with gene editing of the CD163 gene; transplanting the CD163 gene-edited piPSC subclones into enucleated porcine oocytes to generate reconstituted porcine embryos; and culturing the reconstituted porcine embryos to the blastocyst stage to obtain a plurality of CD163 gene-edited pigs. In a particular example, the gene editing of the CD163 gene is a knockout or exon 7 deletion of the CD163 gene. The pigs thus produced exhibit resistance to Porcine Reproductive and Respiratory Syndrome Virus (PRSV) infection. Furthermore, the method of the present invention includes the steps of analyzing the resistance of chimeric pigs to PRSV infection, and / or selecting a pig strain from among a plurality of gene-edited pigs that exhibits resistance to PRSV compared with a non-gene-edited pig control (counterpart) grown under the same conditions.

[0058] The present invention is further illustrated by the following embodiments, which are provided for demonstration purposes rather than for limiting purposes. Those skilled in the art will understand that, in light of the description herein, many modifications can be made to the particular embodiments described without departing from the spirit and scope of the invention, and similar or comparable results can still be obtained. [Examples]

[0059] Porcine fibroblasts were simultaneously electroporated with Yamanaka factor and a CRISPR / Cas9 editing vector to induce and achieve precisely gene-deficient or knockout (KO) porcine pluripotent stem cells (piPSCs) (CD163ΔE7 piPSCs or KO piPSCs). These piPSCs were subcloned and their genome sequences were evaluated to obtain precisely gene-edited piPSCs. In this study, CD163ΔE7 piPSCs were generated from livestock, and CD163ΔE7 blastocysts (BCs) were further generated by somatic cell nuclear transfer (SCNT), resulting in the development of pigs resistant to Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection.

[0060] 1. Materials and Methods 1.1 Construction of gene editing vectors 1.1.1 Cas9 Vectors The coding region of the Streptococcus pyrogenes strain A20 cas9 protein was chemically synthesized with human-optimized codons. The 24th amino acid E (Glu) of the standard strain SF370 was replaced with D (Asp) in strain A20. Daxx NLS was added to the C-terminus of Cas9. The pCX-Flag2-NLS1-Cas9-NLS2 vector (first KO vector) was constructed using the eukaryotic expression vector pCX-Flag2-NLS1-MCS-pA

[42] (Figure 1A).

[0061] 1.1.2 CD163 sgRNA vector The porcine type III promoter pU6, which corresponds to the mouse U6 promoter and uses RNA polymerase III, was cloned

[43] and used to construct a ppU6-(BsaI)2-gRNA vector

[42] (Figure 1B) for expressing single-stranded guide RNA. Generally, the ppU6-(BsaI)2-gRNA vector containing sequence number 1 is recognized and cleaved by BsaI, resulting in two adherent end overhangs. DNA sequence CGTCGN 19 GTTTTAGAGCTAGAAAT (Sequence ID 2) and TGCTATTTCTAGCTCTAAAACN 19 Primer pair of C (sequence number 3) (where N of sequence number 2)19 and N of sequence number 3 19 The two (which are complementary to each other) were annealed and ligated with a BsaI restriction enzyme-treated ppU6-(BsaI)2-gRNA vector to create a ppU6-SP-gRNA single-stranded guide RNA expression vector (Figure 1C, including the strand of SEQ ID NO: 4 and its complementary strand of SEQ ID NO: 5). Specifically, to construct the CD163 sgRNA vector, pCD163-Sp26 F (SEQ ID NO: 6) and pCD163-Sp26R (SEQ ID NO: 7) were annealed to each other as the first primer pair, and then ligated with a BsaI restriction enzyme-treated ppU6-(BsaI)2-gRNA vector to produce the ppU6-pCD163SP26-gRNA vector. Similarly, pCD163-SP28F (SEQ ID NO: 10) and pCD163-Sp28R (SEQ ID NO: 11) were annealed to each other as the second primer pair, and then ligated with a BsaI restriction enzyme-treated ppU6-(BsaI)2-gRNA vector to produce the ppU6-pCD163Sp28-gRNA vector (Figure 1D). Protospacer 26 contains the strand of SEQ ID NO: 8, and its complementary strand is SEQ ID NO: 9 (Figure 1D). Protospacer 28 contains the chain of sequence number 12, and its complementary chain is sequence number 13 (Figure 1D).

[0062] By transfecting the CD163 genomic DNA (SEQ ID NO: 14) with a vector containing pCX-Flag2-NLS1-Cas9-NLS2, ppU6-pCD163SP26-gRNA, and ppU6-pCD163SP28-gRNA, the DNA fragment between protospacer 26 and protospacer 28 can be removed. The Cas9-sgRNA complex recognizes the protospacer sequences Sp26 and Sp28, and can cut the porcine genomic DNA with "sgSL26 cut" and "sgSL28 cut," respectively (Figures 2A and 2B), thereby removing CD163 exon 7. As a result, cells with a genotype of CD163 exon 7 edited / deleted (CD163ΔE7) can be produced.

[0063] 1.2 Construction of a Reprogramming Vector The iPSC induction vectors for reprogramming, pCX-Oct4-2 A-Sox2-2A-Klf4-2A-NANOG (pCX-OSKN), pCX-cmyc, and pCX-Tag, were constructed based on previously described vectors

[16] , where the reprogramming genes are native for porcine Oct4, porcine Sox2, and SV40 large T antigen (Tag), and codon-optimized for porcine Klf4, porcine c-Myc, and human NANOG.

[0064] 1.3 Treatment methods using iPSC induction and gene editing 1.3.1 Establishment of Primary Fibroblast Cells Ear tissue was excised from newborn piglets, breeding pigs, or sows and preserved in Dulbecco's phosphate-buffered saline (D-PBS) supplemented with 10× penicillin / streptomycin / amphotericin (PSA; CORNING, USA). The tissue was packed with ice until brought back to the laboratory. After removing the surface hairs, it was sterilized with 75% ethanol and thoroughly washed twice with PBS containing 3× PSA. The tissue was finely cut with scissors, and 50-60 μL of tissue suspension was seeded into 6 cm dishes. The dishes were covered with coverslips to promote tissue adhesion to the dishes, and finally, 5 mL of DMEM containing 10× PSA was supplied for further incubation. Each tissue sample was seeded into a total of 10-15 dishes. After culturing for 7-14 days, contaminated dishes with yellowed culture medium were discarded. Dishes with well-developed primary fibroblasts around the tissue were detached with trypsin, re-seeded into 10cm dishes, and the fibroblasts were amplified for further use, such as in the production of gene-edited piPSCs or cryopreservation.

[0065] 1.3.2 Simultaneous generation of porcine iPS cells and gene editing of CD163 exon 7 deletion (ΔE7) Primary fibroblasts were simultaneously transfected with reprogramming vectors and gene editing vectors. The reprogramming vectors included pCX-Oct4-2A-Sox2-2A-Klf4-2A-NANOG (pCX-OSKN), pCX-cMyc, and pCX-TAG in a weight ratio of 2:1:1 (5.0 μg, 2.5 μg, and 2.5 μg per 100 μL). The gene editing vectors included ppU6-pCD163-Sp26-sgRNA, ppU6-pCD163-Sp28-sgRNA, and pCX-Flag2-NLS1-Cas9-NLS2 in a weight ratio of 1:1:2 (2.5 μg, 2.5 μg, and 5.0 μg per 100 μL). Transfection by electroporation (EP) was performed using the Neon transfection system (Gibcoo, USA) at a rate of 1 × 10⁶ 6 The procedure was performed with a 100 μL cell suspension. After a single 30 μs pulse of 1.2 kV / cm AC, cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 1× GlutaMAX, 1× non-essential amino acids, 1× β-mercaptoethanol, 1× penicillin / streptomycin, 50 μg / mL vitamin C (Vit.C), and 5.25 μg / mL Cellmaxin. After 7 days of culture, the cells were cultured in a mouse embryonic fibroblast (MEF) feeder, except that the second EP and culture were performed under the same conditions as the first EP. Around 7 days after the second EP, candidate piPSCs appeared and were picked up with a glass pipette, and the remaining cells underwent a third and / or fourth EP under the same conditions as the second EP. The picked-up candidate piPSC clones (Figure 3A) were subcloned for further genetic evaluation.

[0066] 1.3.3 Evaluation of Recombinant piPSCs Candidate piPSC clones were picked and dispersed in 10 μL of accutase (CORNING, USA) for 5 minutes, then dissociated using a glass pipette. After seeding in a 24-well culture dish on an MEF feeder, candidate subclones were grown to 80-90% confluence, and the same treatment was repeated. The cells were then cultured in a 12-well culture dish coated with 0.2% gelatin (without feeder cells). When the cell proliferation reached approximately 90% confluence, the candidates were harvested, and genomic DNA was extracted and evaluated. Genomic DNA was amplified by PCR using F2 (SEQ ID NO: 15) and R2 (SEQ ID NO: 16) primers.

[0067] [Table 1]

[0068] The PCR amplicons of wild-type and CD163ΔE7 genomic DNA were 940 bp and 454 bp, respectively. These homologous edited subclones were further analyzed by sequencing of the PCR products, and the homologous subclones were amplified for further use or storage.

[0069] 1.4 Somatic cell nuclear transfer (SCNT) 1.4.1 Collection of pig eggs Pig ovaries were collected from the slaughterhouse, stored in saline solution at 30-35°C, and returned to the laboratory within 2 hours. Cumulus oocyte complexes (COCs) were collected from 3-6 mm follicles using an 18-gauge needle / syringe. COCs containing at least three layers of cumulus cells were collected, washed with culture medium, and further subjected to in vitro maturation (IVM).

[0070] 1.4.2 COC of pigs in IVM IVM medium was prepared by adding 10 IU / mL pregnant mare serum gonadotropin (PMSG), 10 IU / mL human chorionic gonadotropin (hCG), 20 ng / mL epidermal growth factor (EGF), 20 ng / mL non-essential amino acids (AA), 10% porcine follicular fluid (pFF), 100 μg / mL cysteine ​​(ComaDex, USA), and 50 μg / mL vitamin C (Vit. C) to medium 199 (M199). After culturing in IVM medium for 20-22 hours, COCs were transferred to the same hormone-free IVM medium and cultured for a further 20-22 hours. All cumulus cells that excreted COCs were removed, and oocytes with a first polar body were selected for somatic cell nuclear transfer (SCNT).

[0071] 1.4.3 SCNT All IVM oocytes were chemically enucleated, and CD163ΔE7 piPSCs were directly injected into the cytoplasm of the oocytes. After reconstruction, the zygotes were chemically activated with 15 μM ionomycin for 5 minutes, and then further activated with 5 μM TPEN [N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine] for 15 minutes. All reconstituted embryos were further cultured for 16 hours in porcine zygote medium 5 (PZM5) supplemented with 500 nM scriptide to reprogram and enhance endogenous gene expression. Subsequently, all reconstituted embryos were cultured for 48 hours in PZM-5 containing 50 μg / mL vitamin C and 1 nM melatonin to reach the dehiscence stage (2-4 cells) and then to the blastocyst stage over 6-7 days.

[0072] 2. Results 2.1 Production and Analysis of Porcine CD163 Gene Exon 7 Deletion (CD163ΔE7) piPSCs After the second to fourth EP cycles, typical iPS cells with a dome-shaped morphology were obtained from pigs L259-10 and D529-16 (Figure 3A). The iPS cell candidates were further subcloned, and PCR was performed for genotyping. As a result, gene-edited piPSC subclones containing homogeneous exon 7 deletions in both alleles, and heterologous exon 7 deletions in only one of the two alleles, were obtained through the second to fourth EP cycles (Figure 3B). Further analysis of these subclones by PCR amplicon sequencing revealed that L-259-10-1, L-259-10-4 and L-259-10-12 (second EP), L259-10-2, L259-10-4, L259-10-6 and L259-10-9 (third EP), L259-10-10 and L259-10-11 (fourth EP), L529-16-1, L529-16-3, L529-16-4 and L529-16-10 (second EP), L529-16-6 (third EP) and L529-16-2 (fourth EP) (Figure 3C) were deleting exon 7 (from 23268 bp to 23753 bp). These CD163ΔE7 piPSCs were used as SCNTs to create reconstituted embryos.

[0073] 2.2 Creation of CD163ΔE7 blastocysts CD163ΔE7 piPSCs, used as nuclear donors, were enucleated using IVM and microinjected into the cytoplasm of mature oocytes. The resulting reconstituted zygotes were further cultured in PZM5 for 6-7 days to obtain CD163ΔE7 blastocysts (Figure 4), which could then be transplanted into the uterus of a recipient female to develop into gene-edited pigs.

[0074] 3. Conclusion In this study, we generated candidate gene-edited piPSCs by co-transfection with pCX-pOct4-2A-pSox2-2A-pKlf4-2A-hNANOG(pCX-OSKN), pCX-pcMyc, and pCX-TAG plasmid vectors, along with sgRNA and Cas9 plasmid vectors. These candidates were subcloned and screened by PCR and sequencing at the in vitro cell stage to obtain piPSCs with precisely edited CD163 genes. These were then used as nuclear donors for SCNTs to produce CD163ΔE7 blastocysts and pigs resistant to PRRSV infection. For example, PRRSV-resistant pigs can be produced by deletion of the CD163 exon 7 allele or by CD163 knockout.

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Claims

1. A method for producing gene-edited non-human embryos and / or resulting gene-edited non-human animals, (a) A process of gene editing non-human mammalian somatic cells to induce the desired gene editing, simultaneously reprogramming the cells into induced pluripotent stem cells (iPSCs), and producing multiple gene-edited iPSC candidates; (b) A step of subcloning the gene-edited iPSC candidate, determining its genotype, and obtaining a gene-edited iPSC subclone having a genome containing the desired gene edit; (c) The step of implanting the gene-edited iPSC subclone into an enucleated oocyte to produce a reconstituted embryo; and (d) A process of culturing the reconstituted embryo to the blastocyst stage and producing a non-human gene-edited animal with the desired gene editing. Methods that include...

2. The method according to claim 1, wherein the gene editing is CRISPR / Cas9-based gene editing.

3. The method according to claim 1, wherein the reprogramming factor comprises Klf4, c-Myc, Nanog, Oct4, Sox2, and SV40 large T antigen.

4. The method according to claim 1, wherein somatic cells are simultaneously transfected with a CRISPR / Cas9-based gene editing vector and a reprogramming vector.

5. CRISPR / Cas9-based gene editing vectors - A Cas9 vector containing nucleic acid encoding the Cas9 protein, and - A gRNA vector containing one or more nucleic acids, each encoding a gRNA molecule for targeting a target gene with Cas9 to induce gene editing. The method according to claim 4, including the method described in claim 4.

6. The aforementioned reprogramming vector - A first reprogramming vector containing nucleic acids encoding Oct4, Sox2, Klf4, and Nanog; A second reprogramming vector containing nucleic acid encoding -c-Myc; and - A third reprogramming vector containing nucleic acid encoding the SV40 large T antigen The method according to claim 4, including the method described in claim 4.

7. The method according to claim 1, wherein the somatic cells and oocytes are of the same species.

8. The method according to claim 1, wherein the somatic cell is a fibroblast.

9. The method according to claim 1, wherein the gene editing is gene knock-in, gene knock-out, or partial deletion.

10. The method according to claim 1, wherein the non-human animal is selected from the group consisting of sheep, cattle, deer, goats, monkeys, camels, and pigs.

11. The method according to claim 10, wherein the non-human animal is a pig.

12. The method according to claim 11, wherein the gene editing is a knockout or partial deletion of the CD163 gene.

13. A method for providing a CD163 gene-edited pig, (a) A step of simultaneously transfecting porcine somatic cells with a gene editing vector and a reprogramming vector, wherein the gene editing vector provides gene editing of the CD163 gene to generate multiple gene-edited porcine iPSC (piPSC) candidates; (b) A step of subcloning the gene-edited piPSC candidate and determining its genotype to obtain a CD163 gene-edited piPSC subclone having a genome containing gene editing of the CD163 gene; (c) A step of transplanting the CD163 gene-edited piPSC subclone into an enucleated pig oocyte to produce a reconstituted pig embryo; and (d) A process of culturing the reconstituted pig embryos to the blastocyst stage to obtain CD163 gene-edited pigs that have the CD163 gene edited. Methods that include...

14. The method according to claim 13, wherein the gene editing vector is a CRISPR / Cas9-based gene editing vector.

15. The method according to claim 13, wherein the reprogramming vector encodes one or more reprogramming factors selected from the group consisting of Klf4, c-Myc, Nanog, Oct4, Sox2, and SV40 large T antigen.

16. CRISPR / Cas9-based gene editing vectors - A Cas9 vector containing nucleic acid encoding the Cas9 protein, and - One or more gRNA vectors each containing nucleic acids encoding gRNA molecules for targeting the CD163 gene with Cas9 to induce gene knockout or partial deletion of the CD163 gene. The method according to claim 14, including the method described in claim 14.

17. The aforementioned reprogramming vector - A first reprogramming vector containing nucleic acids encoding Oct4, Sox2, Klf4, and Nanog; A second reprogramming vector containing nucleic acid encoding -c-Myc; and - A third reprogramming vector containing nucleic acid encoding the SV40 large T antigen The method according to claim 13, including the method described in claim 13.

18. The method according to claim 13, wherein the porcine somatic cells are porcine fibroblasts.

19. The method according to claim 13, wherein the gene editing of the CD163 gene is a knockout or exon 7 deletion of the CD163 gene.

20. The method according to claim 13, wherein gene-edited pigs exhibit resistance to porcine reproductive and respiratory syndrome virus (PRSV) infection.

21. A method for conferring resistance to porcine reproductive and respiratory syndrome virus (PRSV) infection to pigs, (a) A step of simultaneously transfecting porcine somatic cells with a gene editing vector and a reprogramming vector, wherein the gene editing vector provides a gene knockout or partial deletion of the CD163 gene to generate multiple gene-edited porcine iPSC (piPSC) candidates; (b) Subcloning candidate gene-edited piPSCs and determining their genotypes to obtain a population of CD163 gene-edited piPSC subclones having genomes with gene knockout or partial deletion of the CD163 gene; (c) The process of creating a reconstituted pig embryo by transplanting each of the CD163 gene-edited iPSC subclones into an enucleated pig oocyte; (d) A step of culturing reconstituted pig embryos to the blastocyst stage to obtain multiple CD163 gene-edited pigs; and (e) A step of selecting a pig line from multiple CD163 gene-edited pigs obtained in step (d) that shows resistance to PRSV compared to non-gene-edited pigs grown under the same conditions. Methods that include...

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