Application of knocking down CDYL gene in improving beef production performance

Abstract

The present invention relates to the field of genetic engineering technology, and particularly to the application of knocking down CDYL genes in improving the meat production performance of cattle. The present invention provides the application of knocking down CDYL genes in improving the differentiation ability of bovine muscle satellite cells and / or improving the meat production performance of cattle, and the gene ID of the CDYL gene is 539013. The present invention deletes the 404th base A upstream of the transcription start site of the CDYL gene by gene editing, thereby creating a CDYL gene-edited cattle with knocked-down gene. Compared with wild-type cattle, the myotube formation ability of muscle satellite cells in gene-edited cattle is significantly enhanced, the number of desmin-positive cells, a myotube marker, is significantly increased, the number of multinucleated myotubes is significantly increased, and the body weight and body size data (body height, rump height, body slant length, chest girth and abdominal girth) of gene-edited cattle are all greater than those of wild-type cattle. The present invention enriches the breeding genes for high-yield beef cattle and provides technical support for the cultivation of new gene-edited animals.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and particularly to the application of knocking down CDYL genes in improving the meat production performance of cattle. Background Art

[0002] High efficiency, high benefit and high output in the beef cattle breeding process are the core goals of the industry development. Traditional breeding mainly selects through four aspects: production performance measurement, crossbreeding, pedigree recording and body conformation evaluation. Compared with traditional breeding, gene editing breeding can greatly shorten the cultivation time of new varieties (lines) and accurately obtain the expected phenotypes. At present, gene editing technology has made great contributions to high yield, high quality, disease resistance and stress resistance in cattle breeding.

[0003] Chromatin domain Y-like ( CDYL ) gene is a transcriptional co-repressor, which contains a typical amino-terminal chromatin domain and a carboxyl-terminal enoyl-CoA hydratase / isomerase catalytic domain. CDYL It has been reported to be involved in negatively regulating histone crotonylation modification, further affecting the transcription level of key functional genes. In terms of specific function research, CDYL it was first reported as a regulator of spermatogenesis. In addition, CDYL it plays a key role in controlling the intrinsic excitability of neurons. However, CDYL the relationship between genes and animal meat production performance has not been reported.

[0004] To solve the above problems, the present invention provides the application of knocking down CDYL genes in improving the meat production performance of cattle. The present invention discovers that knocking down CDYL genes can improve the meat production performance of cattle.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides the application of knocking down CDYL genes in improving the differentiation ability of bovine muscle satellite cells and / or improving the meat production performance of cattle, and the CDYL gene has a gene ID of 539013 in the NCBI database.

[0007] Preferably, the improvement of the differentiation ability of bovine muscle satellite cells includes: improving the myotube formation ability of bovine muscle satellite cells, increasing the expression level of bovine desmin and increasing the number of bovine multinucleated myotubes; the meat production performance includes: one or more of body weight, body height, rump height, body slant length, chest girth and abdominal girth.

[0008] Preferably, the cattle are Simmental cattle.

[0009] Preferably, the method for knocking down CDYL the gene includes: deleting the CDYL base A at the 404th position upstream of the gene transcription start site by gene editing.

[0010] The present invention provides a biomaterial for knocking down CDYL the gene, including: one or more of pegRNA, a recombinant plasmid containing the pegRNA, a bovine fibroblast cell line positive for gene editing, and a bovine blastocyst positive for gene editing; the nucleotide sequence of the pegRNA is as shown in SEQ ID NO: 1; the bovine fibroblast cell line positive for gene editing is CDYL a cell line with knocked-down gene; the bovine blastocyst positive for gene editing is CDYL a bovine blastocyst with knocked-down gene; the CDYL gene has a gene ID of 539013 in the NCBI database.

[0011] Preferably, the expression elements contained in the recombinant plasmid include: pegRNA, nCas9, M-MLV reverse transcriptase, GFP, and EBNA1.

[0012] Preferably, the bovine fibroblast cell line positive for gene editing is a bovine fibroblast cell line transfected with the recombinant plasmid and CDYL with knocked-down gene; the bovine blastocyst is a bovine blastocyst obtained by injecting the bovine fibroblast cell line positive for gene editing into enucleated oocytes by somatic cell nuclear transfer method and culturing.

[0013] The present invention provides the application of the biomaterial described in the above technical solution in the preparation of gene-edited cattle with improved meat production performance.

[0014] The present invention provides a method for preparing gene-edited cattle with improved meat production performance by using the biomaterial described in the above technical solution, including the following steps:

[0015] Transplanting the bovine blastocyst positive for gene editing into the body of a recipient female cow to obtain gene-edited cattle.

[0016] Preferably, the recipient female cow is Simmental cattle.

[0017] Beneficial effects:

[0018] The present invention provides the application of knocking down CDYL the gene in improving the differentiation ability of bovine muscle satellite cells and / or improving the meat production performance of cattle, and the CDYL gene has a gene ID of 539013 in the NCBI database. The present invention discovers that knocking down CDYLGenes can improve the meat production performance of cattle. The results of the examples show that, through gene editing, the present invention deletes the CDYL base A at the 404th position upstream of the transcriptional start site of the gene, thereby creating CDYL gene-edited cattle with knocked-down genes. Compared with wild-type cattle, the myotube formation ability of muscle satellite cells in gene-edited cattle is significantly enhanced, the number of positive cells for desmin, a myotube marker, is significantly increased, the number of multinucleated myotubes is significantly increased, and the body weight and body size data (body height, rump height, body slant length, chest girth, and abdominal girth) of gene-edited cattle are all greater than those of wild-type cattle. The present invention enriches the breeding genes for high beef production and provides technical support for the cultivation of new gene-edited animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments.

[0020] Figure 1 Schematic diagram of the editing site for knocking down bovine CDYL gene expression;

[0021] Figure 2 Electrophoresis diagram of amplifying the core promoter sequence of bovine CDYL gene;

[0022] Figure 3 For CDYL genotype identification results of the gene-edited positive cell line;

[0023] Figure 4 For CDYL mRNA expression detection results of the gene in the mutant cell line;

[0024] Figure 5 For CDYL protein expression detection results in the mutant cell line;

[0025] Figure 6 For CDYL gene-edited cloned embryo diagram;

[0026] Figure 7 For CDYL gene-edited cattle diagram;

[0027] Figure 8 For CDYL protein expression detection results of gene-edited cattle;

[0028] Figure 9 For CDYL myogenic differentiation detection results of muscle satellite cells of gene-edited cattle;

[0029] Figure 10 For CDYL body weight and body size detection results of gene-edited cattle. Detailed implementation mode

[0030] The present invention provides the application of knocking down CDYL a gene in improving the differentiation ability of bovine muscle satellite cells and / or improving the meat production performance of cattle. The gene ID of the gene described in the present invention in the NCBI database is 539013. CDYL

[0031] As an implementation mode, the improvement of the differentiation ability of bovine muscle satellite cells includes: improving the myotube formation ability of bovine muscle satellite cells, increasing the expression level of desmin, and increasing one or more of multinucleated myotubes in cattle; the meat production performance includes: one or more of body weight, body height, rump height, body slant length, chest girth, and abdominal girth.

[0032] As an implementation mode, the cattle are Simmental cattle.

[0033] As an implementation mode, the method for knocking down CDYL the gene includes: deleting the 404th base A (position 49745768 on chromosome 23 of the bovine genome) upstream of the transcription start site of the CDYL gene (position 49745364 on chromosome 23 of the bovine genome) by gene editing. The present invention selects to delete the 404th base A upstream of the transcription start site of the CDYL gene. This editing site is located in the promoter region. By editing this editing site, the expression level of the CDYL gene can be knocked down, thereby creating a bovine fibroblast cell line with CDYL gene mutation and CDYL gene-edited cattle.

[0034] The present invention discovers that knocking down CDYL the gene can improve the meat production performance of cattle. Compared with wild-type cattle, the myotube formation ability of bovine muscle satellite cells with CDYL the gene knocked down is significantly enhanced, the number of desmin (Desmin) positive cells, which is a myotube marker, is significantly increased, the number of multinucleated myotubes is significantly increased, and the body weight and body measurement data (body height, rump height, body slant length, chest girth, and abdominal girth) are all greater than those of wild-type cattle. The present invention enriches the breeding genes for high-yield beef cattle and provides technical support for the cultivation of new gene-edited animals.

[0035] Based on the above advantages, the present invention provides a biological material for knocking down CDYL the gene, including: one or more of pegRNA, a recombinant plasmid containing the pegRNA, a bovine fibroblast cell line with positive gene editing, and a bovine blastocyst with positive gene editing; the nucleotide sequence of the pegRNA is as shown in SEQ ID NO:1; the bovine fibroblast cell line with positive gene editing is CDYL ​Gene - knocked - down cell line; the gene - edited positive bovine blastocyst is CDYL Gene - knocked - down bovine blastocyst; the CDYL gene has a gene ID of 539013 in the NCBI database.

[0036] As an embodiment, the expression elements contained in the recombinant plasmid include: pegRNA, nCas9 (H840A), M - MLV reverse transcriptase, GFP, and EBNA1; the gene - edited positive bovine fibroblast cell line is transfected with the recombinant plasmid and CDYL is a gene - knocked - down bovine fibroblast cell line. As an embodiment, the recombinant plasmid is composed of the ePE plasmid and the pegRNA inserted into the ePE plasmid; the pegRNA is inserted between the Nhe I and Hind Ⅲ restriction enzyme sites of the ePE plasmid; the ePE plasmid and its construction method are disclosed in Chinese Patent CN119120578A.

[0037] As an embodiment, the bovine blastocyst is a bovine blastocyst obtained by injecting the gene - edited positive bovine fibroblast cell line into enucleated oocytes by somatic cell nuclear transfer method and culturing.

[0038] The biological material provided by the present invention is based on the ePE plasmid and inserted with the CDYL pegRNA sequence of the bovine gene editing site to obtain a recombinant plasmid, and then the recombinant plasmid is transfected into bovine fibroblasts, and a CDYL gene - edited fibroblast cell line is successfully obtained. Further, a CDYL gene - edited bovine with improved meat - producing performance is obtained by somatic cell nuclear transfer.

[0039] Based on the above advantages, the present invention provides the application of the biological material described in the above technical solution in the preparation of gene - edited bovines with improved meat - producing performance. As an embodiment, the recipient cows of the gene - edited bovines are Simmental cows.

[0040] Based on the above advantages, the present invention provides a method for preparing gene - edited bovines with improved meat - producing performance using the biological material described in the above technical solution, including the following steps:

[0041] Transplanting the gene - edited positive bovine blastocyst into the body of a recipient cow to obtain a gene - edited cow.

[0042] As an embodiment, the recipient cow is a Simmental cow.

[0043] To further illustrate the present invention, the application of knocking down the CDYL gene in improving the meat - producing performance of bovines provided by the present invention will be described in detail below with reference to the drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0044] The main reagents used in the following examples:

[0045] Collagenase IV used for isolating bovine fetal fibroblasts was purchased from Sigma; DMEM, FBS, PBS, and Trypsase used for cell culture were all purchased from Gibco; primers were synthesized by Shanghai Sangon Biotech Co., Ltd.; the fluorescence quantitative PCR kit was purchased from Novoprotein Scientific Inc.; antibodies used for immunoblotting: CDYL antibody was purchased from Santa Cruz, and α-Tubulin antibody was purchased from Proteintech.

[0046] The main instruments used in the following examples:

[0047] CO 2 Incubator (Thermo, USA); fluorescence quantitative PCR instrument (Roche, Switzerland); exposure instrument (Tanon, China); flow cytometer (SONY, Japan); micromanipulator (Nikon, Japan).

[0048] Example 1 CDYL Construction of gene editing plasmid

[0049] 1.1. According to the bovine CDYL gene editing site ( Figure 1 ), pegRNA-CDYL-1 was designed and synthesized, and its nucleotide sequence is shown in SEQ ID NO:1, specifically as follows:

[0050] 5'-gctagcTATTTAGAGGTGAAGCTTTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTAAACACCCCAAAGCTTCACCTCTAAATAaagctt-3'; among them, 5'-gctagc-3' is the Nhe I restriction site, and 5'-aagctt-3' is the Hind Ⅲ restriction site.

[0051] 1.2. Construction of recombinant plasmid

[0052] The ePE plasmid was digested with restriction enzymes Nhe I and Hind Ⅲ at 37 °C for 1.5 h, and the linearized fragment was recovered by gel cutting; the ePE plasmid and its construction method are disclosed in Chinese Patent CN119120578A.

[0053] After mixing the synthesized pegRNA-CDYL-1 double-stranded fragment in Step 1.1 with the recovered linearized fragment and ligating overnight at 16 °C, it was transformed into DH5α competent cells, spread and grown on an LB plate containing ampicillin, and then single colonies were picked for expanded culture and sequencing. The sequencing primers are as follows:

[0054] U6-promoter: 5'-CCGTAACTTGAAAGTATTTCG-3' (SEQ ID NO:2);

[0055] After culturing the positive clones, the recombinant plasmid (named ePE-CDYL-Bos) was extracted using a plasmid endotoxin-free midiprep kit (TIANGEN, China) for subsequent cell transfection.

[0056] Example 2 Construction of gene-edited positive cell line

[0057] 2.1. Isolation and culture of bovine fetal fibroblasts

[0058] The bovine fibroblasts used in this example were obtained from wild Chinese Simmental cattle fetuses (about three months old) at the Grassland Livestock Germplasm Innovation and Breeding Base. The primary fibroblasts were isolated and cultured by the tissue adherence method. The fetal tissue was washed 3 times with PBS containing double antibiotics, soaked in 75% alcohol for 5 s and then quickly transferred into PBS, washed 3 times and then cut into pieces of 1 - 3 mm 3 , and evenly spread on a culture dish, and cultured in an inverted manner in an incubator at 38.5 °C and 5% CO 2 for 1 - 2 h. After the tissue blocks adhered to the culture dish, complete culture medium was added. When the cell growth reached 80% - 90% confluence, subculture and cryopreservation were carried out.

[0059] 2.2. Cell transfection

[0060] One day before transfection, the primary bovine fibroblasts were seeded into a six-well plate. When the cells reached 70% - 80% confluence, transfection could be carried out. The transfection steps were strictly operated according to the instructions of the Lipofectamine 2000 (Invitrogen, USA) kit. Specifically, 6 μg of the recombinant plasmid ePE-CDYL-Bos in Example 1 was used to transfect bovine fibroblasts.

[0061] 2.3. Flow sorting of gene-edited positive cells

[0062] 36 - 48 h after transfection, it was seeded into a 96-well plate by flow sorting method to ensure single-cell seeding into each well. The monoclonal cells were further amplified. When the density reached 80% in the six-well plate, half of the cells were taken to extract genomic DNA for identification, and the remaining cells were cryopreserved for somatic cell cloning.

[0063] 2.4. Identification of monoclonal cells

[0064] According to the DNA sequence of the bovine CDYL promoter region containing the editing site in Example 1, PCR primers were designed, and the specific sequences are as follows:

[0065] Bos-CDYL-1F: 5'-ACAACTGAGGAAACTTAA-3' (SEQ ID NO:3);

[0066] Bos-CDYL-1R: 5'-GAAAAGTACAGGTACACG-3' (SEQ ID NO:4);

[0067] DNA of edited cells and unedited cells was extracted, and the sequences containing CDYL the editing site were amplified respectively. After purification of the PCR products ( Figure 2 ), first-generation sequencing was performed, and the results showed that the edited cell line ( Figure 3 ) was successfully obtained.

[0068] Example 3 Detection of gene mRNA and protein levels in mutant cells CDYL 3.1. Real-time fluorescence quantitative PCR

[0069] According to the HiPure total RNA Mini Kit (Magen, China) instruction manual, total RNA of edited cells was extracted, and the RNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo, USA).

[0070] Total RNA was reverse transcribed into cDNA using GoScript

[0071] Reverse Transcription Mix, Oligo(dT) (Promega, USA). TM Primers were designed using Primer Premier 5.0 software, and the specific sequences are as follows:

[0072] Bos-CDYL-2F: 5'-GACCCGACTGGAACAAAGTGT-3' (SEQ ID NO:5);

[0073] Bos-CDYL-2R: 5'-CTTTCAACCTCGTACAGCTCCT-3' (SEQ ID NO:6);

[0074] Bos-CDYL-2R: 5'-CTTTCAACCTCGTACAGCTCCT-3' (SEQ ID NO:6);

[0075] PCR system: 80 ng cDNA, 10 μM forward primer, 10 μM reverse primer, SYBR green master mix (TaKaRa, China) and RNase-free water, with a total volume of 20 μL.

[0076] The reaction system includes: 30 s at 95 °C, 5 s at 95 °C, 34 s at 60 °C, for a total of 40 cycles.

[0077] GAPDH As an internal reference, the primer sequences are as follows:

[0078] Bos-GAPDH-F: 5'-TGGTGAAGGTCGGAGTGAAC-3' (SEQ ID NO:7);

[0079] Bos-GAPDH-R: 5'-ATGGCGACGATGTCCACTTT-3' (SEQ ID NO:8);

[0080] The results showed that compared with unedited cells, the mRNA expression level in mutant cells CDYL was significantly decreased ( Figure 4 ).

[0081] 3.2. Immunoblotting

[0082] The edited cells were lysed with RIPA Buffer (Beyotime, China), and the protein concentration was measured using the BCA Protein Assy Kit (Beyotime, China). Protein samples were separated by 10% SDS-PAGE and transferred to a membrane.

[0083] The primary antibody was incubated overnight at 4 °C, the secondary antibody was incubated for 1 h at 37 °C, and after extensive washing with 1×TBST, it was developed using the ECL chemiluminescence detection kit (Biosharp, China).

[0084] The results showed that compared with unedited cells, the protein expression of CDYL in mutant cells was significantly decreased ( Figure 5 ).

[0085] Example 4 Obtaining gene-edited cattle by somatic cell nuclear transfer

[0086] 4.1. Oocyte collection and in vitro maturation

[0087] Ovaries of Chinese Simmental cattle were collected from a local abattoir and stored in physiological saline containing penicillin and streptomycin at a temperature of 20 - 25 °C. Cumulus-oocyte complexes (COCs) were collected from antral follicles with diameters of 3 to 8 mm using a needle. They were washed three times with a buffer (M199 medium containing 1% FBS) under a stereomicroscope and then placed in an in vitro maturation medium for oocyte maturation. The culture conditions were 38.5 °C and 5% CO 2 2 saturation humidity environment. After 22 h of in vitro maturation, cumulus cells were removed in 1 mg / mL hyaluronidase, and mature oocytes that had extruded the first polar body were selected. The in vitro maturation medium was based on M199 medium and also contained only 10% FBS, 20 ng / mL epidermal growth factor, 1 μg / mL β-estradiol, 1 μg / mL gonadotropin, and 0.1 IU / mL luteinizing hormone.

[0088] 4.2. Somatic cell nuclear transfer, fusion, activation, and embryo culture

[0089] Before somatic cell nuclear transfer (SCNT), oocytes were exposed to 7.5 μg / mL Hoechst 33342 for 5 min and then placed in M199 medium containing 7.5 μg / mL cytochalasin B for somatic cell nuclear transfer operations. Under a brief (1 - 2 s) ultraviolet light irradiation, the orientation of the oocyte nucleus was located and removed through a glass micropipette (18 - 20 μm). Then CDYL gene-edited positive cells were injected into the enucleated oocytes and allowed to recover in the operating drop for 30 min. The cloned embryos were then electrofused with a direct current pulse of 1.8 kV / cm. The electrofused cloned embryos were incubated in 5 μmol / L ionomycin for 5 min and then transferred to 10 μg / mL cycloheximide (CHX) for 5 h. Finally, the cloned embryos were transferred to SOFaa medium and cultured in a humid environment at 38.5 °C with 5% CO 2 2. Fresh SOFaa medium was replaced every 48 h until the cloned embryos developed to the blastocyst stage ( Figure 6 ).

[0090] 4.3. Embryo transfer

[0091] First, the recipient cows were synchronized estrus. A progesterone vaginal sponge (CIDR) was placed in the recipient cows. After 7 days, it was removed and prostaglandin (PG) was injected, and the estrus status of the recipient cows was observed. Nine CDYL gene-edited cloned blastocysts were transferred into 9 estrus recipient cows. Pregnancy was determined by rectal ultrasound EVO scanner on the 35th day after transplantation.

[0092] Example 5 Detection of CDYL Protein Level in Edited Cattle

[0093] 5.1. Western Blot

[0094] The method was the same as step 3.2 in Example 3.

[0095] The results showed that compared with wild-type cattle, the expression of CDYL protein in edited cattle ( Figure 7 ) was significantly decreased ( Figure 8 ).

[0096] Example 6 CDYL Detection of the Differentiation Ability of Muscle Satellite Cells Derived from Mutant Cattle

[0097] Muscle satellite cells of mutant cattle and wild-type cattle were isolated and cultured, and induced to differentiate into myoblasts (for the method, see

Li Shuang, Liu Dan, Fu Yuying et al. Podocan Promotes Differentiation of Bovine Skeletal Muscle Satellite Cells by Regulating the Wnt4-β-Catenin Signaling Pathway.[J]. Front Physiol, 2019, 10: 1010.

[0098] Example 7 Detection of Body Weight and Body Dimensions of Edited Cattle

[0099] For CDYL gene mutant cattle and wild-type cattle, their growth indexes were detected. The results are shown in Figure 10 , where * is P < 0.05, ** is P < 0.01. The body weight and body dimension data (body height, rump height, body slant length, chest girth and abdominal girth) of mutant cattle from birth to 6 months of age were all greater than those of wild-type cattle.

[0100] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments according to these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Knockdown CDY Application of genes in improving the differentiation ability of cattle muscle satellite cells and / or improving cattle meat production performance, the CDY The gene ID of the gene in the NCBI database is 539013; the cattle are Simmental cattle; and the purpose of improving the differentiation ability of cattle muscle satellite cells is non-therapeutic.

2. The use according to claim 1, characterized in that: The method for improving the differentiation ability of cattle muscle satellite cells includes: improving the ability of cattle muscle satellite cells to form myotubes, increasing the expression of cattle desmin and increasing cattle multinucleated myotubes; the meat production performance includes: one or more of body weight, body height, cross height, body oblique length, chest circumference and abdominal circumference.

3. The use according to claim 1, characterized in that: The knockdown CDY Genetic methods include: deletion through gene editing CDY The A base 404 upstream of the gene transcription start site.

4. Application of biomaterials in the preparation of gene-edited cattle with improved meat production performance; The cattle is Simmental cattle; the biological material comprises: one or more of a recombinant plasmid, a gene-editing positive bovine fibroblast cell line and a gene-editing positive bovine blastocyst; the expression elements contained in the recombinant plasmid include: pegRNA, nCas9, M-MLV reverse transcriptase, GFP and EBNA1; the nucleotide sequence of the pegRNA is shown in SEQ ID NO: 1; the gene-editing positive bovine fibroblast cell line is CDY Gene knockdown cell line; the gene editing positive bovine blastocyst is CDY Gene knockdown bovine blastocyst; CDY The gene ID of the gene in the NCBI database is 539013.

5. The use according to claim 4, characterized in that: The gene editing positive bovine fibroblast cell line is transfected with the recombinant plasmid and CDY A bovine fibroblast cell line with gene knockdown; the gene-editing-positive bovine blastocyst is a bovine blastocyst obtained by injecting the gene-editing-positive bovine fibroblast cell line into an enucleated oocyte using a somatic cell nuclear transplantation method.

6. The use according to claim 4 or 5, characterized in that: The recombinant plasmid consists of an ePE plasmid and pegRNA inserted into the ePE plasmid.

7. A method for preparing gene-edited cattle with improved meat production performance using biological materials, characterized in that: The biological material comprises: one or more of a recombinant plasmid, a gene-editing positive bovine fibroblast cell line and a gene-editing positive bovine blastocyst; the expression elements contained in the recombinant plasmid comprise: pegRNA, nCas9, M-MLV reverse transcriptase, GFP and EBNA1; the nucleotide sequence of the pegRNA is shown in SEQ ID NO: 1; the gene-editing positive bovine fibroblast cell line is CDY Gene knockdown cell line; the gene editing positive bovine blastocyst is CDY Gene knockdown bovine blastocyst; CDY The gene ID of the gene in the NCBI database is 539013, and the method comprises the following steps: The gene-edited bovine blastocysts that are positive for gene editing are transplanted into recipient cows to obtain gene-edited cows; the recipient cows are Simmental cows.

8. The method according to claim 7, characterized in that The gene editing positive bovine fibroblast cell line is transfected with the recombinant plasmid and CDY A bovine fibroblast cell line with gene knockdown; the gene-editing-positive bovine blastocyst is a bovine blastocyst obtained by injecting the gene-editing-positive bovine fibroblast cell line into an enucleated oocyte using a somatic cell nuclear transplantation method.

9. The method according to claim 7 or 8, characterized in that: The recombinant plasmid consists of an ePE plasmid and pegRNA inserted into the ePE plasmid.

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