A method for knocking out cattle FBXO40 gene and its application

Abstract

The invention discloses a knockout cattle FBXO40 The method and application of gene knockout in cattle using CRISPR / Cas12i system FBXO40 Gene, CRISPR / Cas12i system targeting cattle FBXO40 Exon 5 of gene, cattle FBXO40 The nucleotide sequence of the fifth exon of the gene is shown in SEQ ID No. 1. The present invention provides a method for editing the gene of cattle using CRISPR / Cas12i gene editing technology. FBXO40 The method of achieving precise gene knockout and the construction of stably inherited monoclonal cell lines and embryos with gene knockout can provide experimental reference for the study of the molecular mechanism of bovine skeletal muscle development and can also be applied to the breeding of new high-yield beef cattle breeds.

Description

Technical Field

[0001] The present invention belongs to the field of animal bioengineering technology and specifically relates to a knockout bovine FBXO40 Genetic methods and their applications. Background Art

[0002] In recent years, with the improvement of living standards and the upgrading of consumption structure, people's demand for beef is increasing. Breeding new breeds of high-yield beef cattle is an important direction for the development of the beef cattle industry. Finding the key regulatory factors of skeletal muscle development in beef cattle and clarifying the regulatory mechanism of skeletal muscle development in beef cattle can provide an important theoretical basis for the selection and matching of high-yield beef cattle and the development of breeding materials for bioengineering. The development of animal skeletal muscle involves a variety of molecular mechanisms, including IGF1 The Insulin-likegrowth factor-1 gene plays an important regulatory role. IGF1 interacts with the transmembrane protein IGF1R (Insulin-likegrowth factor 1 receptor), induces phosphorylation of IRS1 (Insulin receptor substrate), thereby activating the PI3K / Akt (Phosphatidylinositol 3-kinase / protein kinase B) pathway and regulating the proliferation and differentiation of myoblasts.

[0003] F-box proteins are key components of the SCF (Skp1-Cullin1-F-box protein) complex, which exert E3 ubiquitin ligase activity and participate in the regulation of cell cycle and signal transduction functions. FBXO40 (F-box only protein 40) is a muscle-specific expression gene, and its expression is significantly increased in the skeletal muscle of patients with myasthenia gravis. FBXO40 The gene targets IRS1 for ubiquitination and degradation, leading to inactivation of the IGF1 / IRS1 / PI3K / Akt pathway and inhibition of muscle development. FBXO40 After knocking down the FBXO40 gene, the protein expression of IRS1 was significantly upregulated. At the same time, after inducing differentiation, the diameter of myotubes increased by about 50%. FBXO40 Related reports on genes in cattle, including their expression patterns and biological functions.

[0004] The clustered regularly interspaced short palindromic repeats (CRISPR) / Cas system was originally discovered in bacteria and archaea. It is an adaptive immune system that provides resistance to viruses by cutting exogenous nucleic acids. The CRISPR / Cas system recognizes target DNA through sgRNA and uses Cas protein to cut the target DNA, thereby forming double-strand breaks (DSB). After the DNA forms a double-strand break, it can be repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). NHEJ will produce random mutations (insertions / deletions) at the break, resulting in changes in gene function. Therefore, designing an sgRNA based on the gene sequence to be edited can perform site-specific DNA cutting and achieve precise gene editing. CRISPR / Cas9 is currently the most commonly used gene editing technology, but its editing efficiency is low in mammals. Summary of the invention

[0005] In view of the above prior art, the present invention provides a knockout bovine FBXO40 Genetic methods and their applications can efficiently and accurately FBXO40 Gene knockout.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a knockout bovine FBXO40 Genetic method, using CRISPR / Cas12i system to knock out cattle FBXO40 Gene, CRISPR / Cas12i system targeting cattle FBXO40 Exon 5 of gene, cattle FBXO40 The nucleotide sequence of the fifth exon of the gene is shown in SEQ ID No.1.

[0007] Based on the above technical solution, the present invention can also be improved as follows.

[0008] Furthermore, the CRISPR / Cas12i system targets nucleotides 178 to 197 from the 5' end in SEQ ID No. 1.

[0009] Further, knockout cattle FBXO40 The genetic method comprises the following steps:

[0010] (1) synthesizing the sense strand and antisense strand of the crRNA corresponding to the DNA sequence, wherein the nucleotide sequences of the sense strand and the antisense strand are shown in SEQ ID No. 6 and SEQ ID No. 7;

[0011] (2) Annealing the sense strand and the antisense strand to obtain double-stranded DNA;

[0012] (3) Constructing a vector expressing Cas12i protein and using double-stranded DNA to construct a vector expressing crRNA;

[0013] (4) Introduce the vector expressing Cas12i protein and the vector expressing crRNA into the recipient cells and screen to obtain FBXO40 Monoclonal cell lines with homozygous knockout of a gene.

[0014] Furthermore, the vector for expressing Cas12i protein is pCAG-Cas12i, and the vector for transcribing crRNA is pUC19-U6-Cas12i.

[0015] Furthermore, the recipient cells are bovine fetal fibroblasts.

[0016] Further, knockout cattle FBXO40 Genetic methods for preparing FBXO40 Knockout cattle breed.

[0017] further, FBXO40 The preparation of gene knockout cattle breeds includes the following steps: FBXO40 The monoclonal cell line with homozygous gene knockout is used as the nuclear transplant donor cell, the oocyte is used as the nuclear transplant recipient cell, and the monoclonal embryo is obtained through somatic cell nuclear transplantation technology.

[0018] Further, knockout cattle FBXO40 Genetic methods are used for animal germplasm improvement.

[0019] The beneficial effects of the present invention are as follows: the CRISPR / Cas12i system is a Type V CRISPR / Cas system, which has the advantages of high targeting efficiency, strong specificity, and small protein size compared to the CRIPSR / Cas9 system. In gene editing of large agricultural animals, the CRISPR / Cas12i system exhibits significant advantages such as long knockout fragments and more PAMs (Protospacer adjacent motifs), and is more suitable for animal gene knockout experiments. FBXO40 The site of the fifth exon of the gene can be edited using the CRISPR / Cas12i gene editing system. FBXO40 The gene can achieve a cutting efficiency of more than 40%, which can FBXO40 The preparation and production of gene editing materials are facilitated. FBXO40 The gene-edited cell line is a bovine fetal fibroblast cell line, which can be used to prepare cloned embryos through nuclear transplantation technology, and then perform embryo transplantation to produce gene-edited living cattle. The gene-edited cell lines of the present invention are FBXO40The knockout fragment of the homozygous gene knockout monoclonal cell line is short and effective. If the knockout fragment is too long, it may cause biosafety problems in the subsequent preparation of living animals. The knockout fragments of the cell line of the present invention are all less than 20 bp and can form terminators, which effectively terminate the transcription and translation of DNA into protein, thereby causing it to lose its biological function.

[0020] The present invention uses CRISPR / Cas12i gene editing system to target cattle FBXO40 The site in the fifth exon of the gene was used to create a precise knockout cow FBXO40 Gene exon 5 editing vectors, cell lines and embryos to achieve bovine FBXO40 Inactivation of gene function at the cellular and embryonic levels, cell lines can be used for cloning embryo preparation and FBXO40 The study of the biological function mechanism of genes in cattle promotes the development of related scientific research; embryos can be used to prepare live materials of gene-edited cattle, and can also be used to breed new breeds of high-yield beef cattle. The gene editing sites, vectors, cell lines and embryos provided by the present invention can be used for: (1) preparing live materials of high-yield beef cattle, (2) studying the molecular mechanism of bovine skeletal muscle development, (3) preparing commercial high-quality embryo materials, (4) FBXO40 Research on the mechanism of action of genes in animal skeletal muscle development. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the pCAG-Cas12i plasmid map;

[0022] Figure 2 is the pUC19-U6-Cas12i plasmid map;

[0023] Figure 3 for FBXO40 Comparison of mRNA expression levels of genes in various bovine tissues;

[0024] Figure 4 For cattle FBXO40 Schematic diagram of gene structure and crRNA design site;

[0025] Figure 5 Electrophoresis diagram for T7E1 restriction enzyme digestion to verify the efficiency of editing vector cutting;

[0026] Figure 6 Electrophoresis diagram for T7E1 digestion to verify the cutting efficiency of pUC19-FBXO40-crRNA3 editing vector;

[0027] Figure 7 The sequencing result diagram of the pUC19-FBXO40-crRNA3 editing vector cutting efficiency verification for TA cloning;

[0028] Figure 8It is the sequence map of the monoclonal cell line with homozygous mutation;

[0029] Fig. 9 This is the sequencing result of 41# cell line;

[0030] Fig.10 This is the microscopic examination picture of 41# cell line;

[0031] Fig.11 This is the sequencing result of 90# cell line;

[0032] Fig.12 This is the microscopic examination picture of 90# cell line;

[0033] Fig.13 This is the sequencing result of 140# cell line;

[0034] Fig.14 This is the microscopic examination picture of 140# cell line;

[0035] Fig.15 Diagram of gene-edited cloned embryos. DETAILED DESCRIPTION

[0036] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0037] Plasmid pCAG-Cas12i (map as shown Figure 1 ) and plasmid pUC19-U6-Cas12i (map as shown Figure 2 The reference patent "Engineering Cas12i nuclease, effector protein and its use" (Li Wei, Zhou Qi, Chen Yangcan, et al. Engineered Cas12i nuclease, effector protein and its use: 202280035920.X [P]. Application date: 2022.5.25, application publication date: 2024.01.02). The heart, liver, spleen, lung, kidney, longissimus dorsi muscle, and subcutaneous fat of cattle were taken from Pingliang Red Bull. Other reagents were purchased directly from the market unless otherwise specified. Cell biology experimental methods and molecular biology experimental methods were conventional experimental methods.

[0038] The ear tissue of 40-day-old Huaxi cattle fetus was prepared according to conventional methods [Cell Experiment Guide (Volume 1), written by DL Spector et al.; translated by Huang Peitang et al. Beijing: Science Press, 2001.2; Chapter 1, Section 4, pages 27-31.] to obtain a bovine fetal fibroblast cell line; the ear tissue of 6-month-old Huaxi cattle was prepared according to conventional methods to obtain a bovine ear fibroblast cell line.

[0039] Example 1

[0040] FBXO40 Gene expression patterns in cattle:

[0041] ox FBXO40 The gene sequence was obtained from the NCBI website, and the Gene ID was 613597. The RNA of bovine heart, liver, spleen, lung, kidney, longissimus dorsi muscle, and subcutaneous fat was extracted by Trizol method and reverse transcribed into cDNA. B-Actin was used as an internal reference. FBXO40 RT-qPCR (Real-time quantitative reverse transcription PCR) experiments were performed to detect the mRNA expression levels in various tissues.

[0042] The results are as follows Figure 3 As shown (A and B in the figure indicate that there are statistically significant differences between different treatments), FBXO40 The gene is highly expressed in the heart tissue and longissimus dorsi muscle tissue of cattle, which is consistent with that of mice and humans. FBXO40 The sequence of exon 5 of the gene is 81.64% similar to that of mouse (NCBI Gene ID: 207215, SEQ ID No. 14);

[0043] ox FBXO40 The sequence of exon 5 of the gene is as follows:

[0044]

[0045] mouse Fbxo40 The sequence of exon 3 of the gene is as follows:

[0046]

[0047] Example 2

[0048] FBXO40 Construction of gene editing vector pUC19-FBXO40-crRNA:

[0049] S1. Cattle FBXO40 The gene will be transcribed and translated into FBXO40 protein (mainly corresponding to exon 5). The inactivation of this region will cause the SCF complex to lose the specific targeting function of FBXO40 protein, blocking the ubiquitination degradation of IRS1 protein mediated by FBXO40 protein, thereby increasing the expression level of IRS1 protein, activating the IGF1 / IRS1 / PI3K / Akt pathway, and promoting the development of bovine skeletal muscle. FBXO40 The 5th exon region of the gene is the target region, e.g. Figure 4 As shown in the figure (Exon1-Exon6 represents exon 1-exon 6, PAM represents the Protospaceradjacent motif in the CRISPR / Cas12i system, 5'UTR and 3'UTR represent the 5' untranslated region and 3' untranslated region), a total of 6 crRNAs with PAM of 5'-TTN-3' were designed using the website (http: / / www.rgenome.net / cas-designer / ); 6 crRNAs targeting bovine FBXO40 The DNA sequence containing the linker corresponding to the crRNA sequence of the gene is as follows (SEQ ID No. 2-13):

[0050] crRNA1-F: 5'-acggAAGCATCTCTCACAGTGTCT-3' (SEQ ID No. 2);

[0051] crRNA1-R: 5'-aaaaAGACACTGTGAGAGATGCTT-3' (SEQ ID No. 3);

[0052] crRNA2-F: 5'-acggCCCGGTGATAAACTGCCACC-3' (SEQ ID No. 4);

[0053] crRNA2-R: 5'-aaaaGGTGGCAGTTTATCACCGGG-3' (SEQ ID No. 5);

[0054] crRNA3-F: 5'-acggGAGCAGGTTCCATGCCTCAA-3' (SEQ ID No. 6);

[0055] crRNA3-R: 5'-aaaaTTGAGGCATGGAACCTGCTC-3' (SEQ ID No. 7);

[0056] crRNA4-F: 5'-acggCAGATGCTTGGCCAGCTTGT-3' (SEQ ID No. 8);

[0057] crRNA4-R: 5'-aaaaACAAGCTGGCCAAGCATCTG-3' (SEQ ID No. 9);

[0058] crRNA5-F: 5'-acggTTTCAGAGTCCACGTTTGGC-3' (SEQ ID No. 10);

[0059] crRNA5-R: 5'-aaaaGCCAAACGTGGACTCTGAAA-3' (SEQ ID No. 11);

[0060] crRNA6-F: 5'-acggGTAACAGGCTGCTCTCTGTC-3' (SEQ ID No. 12);

[0061] crRNA6-R: 5'-aaaaGACAGAGAGCAGCCTGTTAC-3' (SEQ ID No. 13).

[0062] S2. Synthesize the oligonucleotide chains of the 6 crRNA forward and reverse sequences with adapters and anneal them. The annealing system is CutSmart® Buffer: 2 μL, F sequence: 9 μL, R sequence: 9 μL, and react at 99 ℃ for 10 min. Bsa I endonuclease digestion of pUC19-U6-Cas12i original plasmid, the digestion system is: CutSmart® Buffer: 10 μL, plasmid: 10 μg, Bsa I endonuclease: 10 μL, ddH 2 O was added to 100 μL and reacted at 37 °C for 4 h. Afterwards, the annealed crRNA was connected to the digested plasmid in the following system: 2×solution I: 5 μL, annealed product: 4 μL, plasmid: 1 μL, and reacted at 16 °C for 3 h.

[0063] S3. Transform the ligation product into Escherichia coli Fast-T1 competent cells, add 1 mL of liquid LB medium containing ampicillin (Amp) and place in a 37 ℃ shaker for 4 hours to recover. Then centrifuge the bacterial solution and discard the supernatant. Resuspend it in 100 μL of liquid LB medium and spread it on a solid LB medium plate containing Amp and culture it at 37 ℃ overnight.

[0064] S4. On the second day, single clones were picked and streaked on the plate, cultured at 37 °C for 8 h, and then part of each line was added to 1 mL of liquid LB medium containing Amp, shaken at 37 °C for 4 h, and the bacterial solution was sequenced using universal primer HU6-F. The bacteria with successful recombinant plasmid construction verified by sequencing were picked and added to 50 mL of liquid LB medium containing Amp and placed in a 37 °C shaker overnight. The next day, the pUC19-FBXO40-crRNA1-6 recombinant plasmid was extracted using OMEGA's plasmid extraction kit (D6950-02) and the concentration was detected, and stored in a -20 °C refrigerator.

[0065] The sequence of universal primer HU6-F is as follows: 5′-GACTATCATATGCTTACCGT-3′ (SEQ ID No. 15).

[0066] Example 3

[0067] FBXO40 Verification of gene editing vector efficiency:

[0068] S1. Bovine ear fibroblasts were revived to 6-well plates and cultured in complete medium (DMEM + 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin) for 24 h. The pUC19-FBXO40-crRNA1-6 plasmid and pCAG-Cas12i plasmid were co-transfected into the cells using Lipo 3000 liposome transfection reagent. The plasmid dosage in the transfection was pUC19-FBXO40-crRNA1-6: 2μg, pCAG-Cas12i: 1μg. After 48 h of culture, the cells were digested with trypsin, the supernatant was discarded after centrifugation, and the cells were resuspended with 200 μL of resuspension solution (DMEM + 1% FBS), and the collection solution (DMEM + 15% FBS) was prepared.

[0069] S2. The resuspended cells were sorted using a flow cytometer, and the fluorescent cells were sorted into the collection solution using a BD FACS Aria SORP flow cytometer. The cell genome was extracted using a genome extraction kit, and the target region was amplified by nested PCR. The PCR system was 1 μL genome / template, 0.5 μL forward and reverse primers, 12.5 μL KODONE PCR Mix, ddH 2 O to make up to 50 μL;

[0070] The primer sequences for nested PCR are as follows:

[0071] Outer primer-F: 5′-TGGTGGTTGATCATGTGAAT-3′ (SEQ ID No. 16);

[0072] Outer primer-R: 5′-GGCAAGTCCTGTCTTTTCTA-3′ (SEQ ID No. 17);

[0073] Inner primer-F: 5′-AGCAGTCACTTGTTTTCTCT-3′ (SEQ ID No. 18);

[0074] Inner Primer-R: 5'-CTTGACTTAGCTCTACCACC-3' (SEQ ID No. 19).

[0075] The amplification procedure of nested PCR is: first amplify the outer band, and then use the outer band as a template to amplify the inner band. The amplification procedure is: 98 ℃ 5 min; 98 ℃ 10 s, 55 ℃ 5 s, 68 ℃ outer primer 15 s, inner primer 1 s, 35 cycles; 68 ℃ 5 min, 4 ℃ storage.

[0076] S3. Take 15 μL of PCR product for touchdown annealing: 95 ℃ for 10 min, then cool from 80 ℃ to 20 ℃ at a rate of -10 ℃ / min, and store at 4 ℃. T7E1 enzyme can recognize mismatched regions in double-stranded DNA for cutting, and these regions are usually caused by incorrect repair of DNA after Cas protein cutting. Therefore, the annealed product is digested by T7E1 to verify its cutting efficiency. The digestion system is: annealed product 15 μL, 10×NEB buffer2: 2.5 μL, T7E1 enzyme: 0.5 μL, ddH 2 The reaction conditions were: 37 °C for 1 h, 75 °C for 5 min, and then agarose gel electrophoresis was performed.

[0077] Agarose gel electrophoresis results Figure 5As shown in the figure, M represents Marker, cr1-cr6 represent the treatment of plasmids pUC19-FBXO40-crRNA1-6, and NC1-NC6 represent the blank controls corresponding to the treatment of plasmids pUC19-FBXO40-crRNA1-6; the results show that among the 6 crRNAs, crRNA3 has the most obvious cutting effect, so pUC19-FBXO40-crRNA3 is used for subsequent experiments.

[0078] S4. Transfect pUC19-FBXO40-crRNA3 again according to the above steps (S1-S3) and extract the genome for PCR. After T7E1 digestion, the agarose gel electrophoresis results are as follows Figure 6 As shown in the figure, M represents Marker, crRNA represents the treatment of plasmid pUC19-FBXO40-crRNA3, and NC represents blank control; ImageJ v1.54g software was used to analyze the gray value of the band to calculate the cutting efficiency, and the calculation formula was cutting efficiency = gray value of 2 bands formed after cutting / total gray value of 3 bands * 100%, and the final calculated cutting efficiency was 47.60%.

[0079] S5. Use a column recovery kit to purify the remaining PCR product in step S4. Connect the purified product to the pMD19-T vector for TA cloning. After transformation and plating, pick a single clone for colony PCR (50 μL system, the amount of primers and enzymes is the same as before, use a sterile pipette tip to pick a single colony and put it into the system, and the amplification procedure is the same as before), and send it to the company for sequencing using the universal primer M13;

[0080] The primer sequence of universal primer M13 is as follows:

[0081] M13-F: 5'-GTAAAACGACGGCCAGT-3' (SEQ ID No. 20);

[0082] M13-R: 5'-GTCATAGCTGTTTCCTG-3' (SEQ ID No. 21).

[0083] Sequencing showed that 13 were successful, 6 of which had mutations, and the calculated cutting efficiency was 46.15%. Figure 7 As shown, the efficiency is similar to that calculated by T7E1 enzyme cutting, verifying its high cutting efficiency.

[0084] Example 4

[0085] FBXO40 Preparation of monoclonal cell lines with homozygous gene knockout:

[0086] S1. Resuscitate bovine fetal fibroblasts into a T25 culture flask and add complete medium for 48 h. Digest the cells with trypsin, centrifuge at 1000×g for 5 min, and discard the supernatant. Resuspend the cells with the Nucleofector electroporation transfection solution in the Nucleofector transfection kit to obtain a cell suspension.

[0087] S2. 2 μg of each of the pUC19-FBXO40-crRNA3 plasmid and pCAG-Cas12i plasmid selected in Example 3 were mixed with the cell suspension and electroporated using the U023 program. The electroporated cells were transferred to a T25 culture flask and cultured in complete medium for 48 h.

[0088] S3. Digest the electroporated cells with trypsin, and sort the fluorescent cells using flow cytometry. Directly inject the fluorescent single cells into each well of a 96-well plate filled with complete medium. Then cultivate the cells, subculture the cells with good growth status to a 48-well plate, and so on until they are subcultured to a 6-well plate, and the cell confluence is above 80%.

[0089] S4. Digest the cells with trypsin, take some cells from each monoclonal cell line, add freezing solution (DMEM+20% FBS+10% dimethyl sulfoxide), and freeze them in a liquid nitrogen tank for later use. Inoculate the remaining cells into a 12-well plate. When the cell confluence is above 80%, use a genome extraction kit to extract the cell genome, and use the aforementioned nested PCR amplification method to amplify the target region, and sequence the PCR products. The sequencing result is a single peak, and the monoclonal cell line with a changed genotype is a homozygous edited monoclonal cell line. Among the 160 monoclonal cell lines cultured, there are 14 homozygous edited monoclonal cell lines, 10 of which are frameshift mutations. Their sequence information is as follows. Figure 8 As shown in the figure, #18, #38, #41, #55, #65, #69, #90, #103, #140, and #158 are the numbers of 10 frameshift mutation monoclonal cell lines.

[0090] Example 5

[0091] Nuclear transfer preparation FBXO40 Homozygous knockout cloned embryos:

[0092] S1. FBXO40 Homozygous gene knockout monoclonal cell lines were used as nuclear transplant donor cells. The three cell lines in Example 4 were selected as nuclear transplant donor cells.

[0093] S2. The first donor cell is a homozygous mutant #41 cell line, the mutation type is: missing cow FBXO40The 5th exon of the gene (SEQ ID No. 1) is from the 192th to the 208th position at the 5' end, a total of 17 bp of nucleotides, so that it forms a terminator at the 102nd amino acid, thereby achieving the purpose of knocking out the gene. The sequencing results and microscopic examination of the #41 cell line are shown in Fig. 9 and Fig.10 shown.

[0094] S3. The second donor cell is a homozygous mutant #90 cell line, the mutation type is: missing cow FBXO40 The 5th exon of the gene (SEQ ID No. 1) is from the 197th to the 201st position at the 5' end, a total of 5 bp of nucleotides, so that it forms a terminator at the 106th amino acid, thereby achieving the purpose of knocking out the gene. The sequencing results and microscopic examination of the #90 cell line are shown in Fig.11 and Fig.12 shown.

[0095] S4. The third donor cell is a homozygous mutant #140 cell line, the mutation type is: missing cow FBXO40 The 5th exon of the gene (SEQ ID No. 1) from the 192th to the 202th position at the 5' end, a total of 11 bp of nucleotides, forms a terminator at the 65th amino acid, achieving the purpose of knocking out the gene. The sequencing results and microscopic examination of the #140 cell line are shown in the figure below. Fig.13 and Fig.14 shown.

[0096] The above-mentioned cell lines all selected cell lines with shorter deletion fragments to avoid subsequent gene safety issues that may be caused by the deletion of large gene fragments.

[0097] S5. Obtain adult cow ovaries from slaughterhouses, take follicles with a diameter of 2-8 mm, and recover cumulus-oocyte complexes (COCs) with regular morphology and dense structure from these follicles. Next, place COCs in a four-well cell culture plate in maturation medium (liquid M199 medium + 10% FBS + 0.01 U / mL of bovine follicle-stimulating hormone, 0.01 U / mL of bovine luteinizing hormone + 1 μg / mL estradiol), culturing 50-60 COCs per well. Subsequently, place in a 38.5 ℃ and 5% CO 2 The oocytes were cultured in an incubator with a concentration of 100 μg / mL for 18-20 h to mature. Afterwards, the mature oocytes were transferred to a centrifuge tube containing a hyaluronidase solution at a concentration of 1 mg / mL, shaken for 2-3 min, and moderately blown with a glass tube to achieve complete separation of cumulus cells and oocytes. Finally, oocytes with complete morphology, uniform cytoplasm distribution, and successful expulsion of the first polar body were selected as recipient oocytes.

[0098] S6. Move the oocyte with the first polar body into a micromanipulation droplet (liquid M199 culture medium + 10% FBS + 7.5 μg / mL cytochalasin B), use a glass needle to make a tiny incision in the zona pellucida above the polar body, and then use a glass tube with an inner diameter of 20μm to suck out the first polar body and the chromosomes in the oocyte below it. Then, place the oocyte without the first polar body and chromosomes in liquid M199 culture medium containing 20% ​​FBS and wash three times.

[0099] S7. Starve the donor cells for 2-4 days and digest them. Then select cells with uniform shape and size and place them into the zona pellucida of the recipient oocyte through the incision. Place the reconstructed oocyte in Zimmerman solution (containing 0.3 M mannitol, 0.1 M MgSO 4 , 0.05M CaCl 2 , 0.5mM HEPES, 0.05g / 100mL BSA aqueous solution, pH adjusted to 7.2, and filtered through a 0.22μm filter membrane) for equilibration for 3-5 min. After equilibration, place the oocyte in a fusion tank, rotate the oocyte so that the contact surface between the donor cell and the oocyte is perpendicular to the electric field, and use a BTX ECM-2001 fusion instrument for fusion under the conditions of a DC pulse field strength of 2.5 kv / cm, a pulse time of 10 μs, a pulse number of 2 times, and a pulse interval of 1 s. After that, quickly transfer the reconstructed embryo to the culture medium (liquid M199 medium + 10% FBS) for 30 min. Place the reconstructed embryo in an aqueous solution containing 5 μmol / L ionomycin for 4 min, transfer to an aqueous solution containing 1.9 mmol / L 6-DMAP for 4 h, and then transfer to a CR1aa culture medium containing 5% FBS and place it in a 5% CO 2 Incubator at 38.5℃ for 7 days, and the FBXO40 Homozygous knockout cloned embryos (e.g. Fig.15 as shown).

[0100] Although the specific implementation of the present invention is described in detail in conjunction with the embodiments, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A Knockout Cow FBXO40 A genetic method, characterized in that: Knockout of cattle using CRISPR / Cas12i system FBXO40 Genes targeted by the CRISPR / Cas12i system in cattle FBXO40 The fifth exon of the gene, the bovine FBXO40 The nucleotide sequence of the fifth exon of the gene is shown in SEQ ID No. 1, and the CRISPR / Cas12i system targets nucleotides 178 to 197 from the 5' end of SEQ ID No.

1.

2. The knockout cattle according to claim 1 FBXO40 A genetic method, characterized in that The following steps are involved: (1) synthesizing the sense strand and antisense strand of the crRNA corresponding DNA sequence, wherein the nucleotide sequences of the sense strand and antisense strand are shown in SEQ ID No. 6 and SEQ ID No. 7; (2) annealing the sense strand and the antisense strand to obtain double-stranded DNA; (3) constructing a vector expressing Cas12i protein, and using the double-stranded DNA to construct a vector expressing crRNA; (4) Introducing the vector expressing Cas12i protein and the vector expressing crRNA into recipient cells, and screening to obtain FBXO40 Monoclonal cell lines with homozygous knockout of a gene.

3. The knockout cattle according to claim 2 FBXO40 A genetic method, characterized in that: The vector expressing the Cas12i protein is pCAG-Cas12i, and the vector expressing crRNA is pUC19-U6-Cas12i.

4. The knockout cattle according to claim 2 FBXO40 A genetic method, characterized in that: The recipient cells are bovine fetal fibroblasts.

5. The knockout cattle according to any one of claims 1 to 4 FBXO40 Application of a genetic method, characterized in that: The knockout cattle FBXO40 Genetic methods for preparing FBXO40 Knockout cattle breed.

6. The use according to claim 5, characterized in that: Said FBXO40 The preparation of gene knockout cattle breeds comprises the following steps: FBXO40 The monoclonal cell line with homozygous gene knockout is used as the nuclear transplant donor cell, the oocyte is used as the nuclear transplant recipient cell, and the monoclonal embryo is obtained through somatic cell nuclear transplantation technology.

7. The knockout bovine according to any one of claims 1 to 4 FBXO40 Application of a genetic method, characterized in that: The knockout cattle FBXO40 Genetic methods are used for animal germplasm improvement.

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