CN110468133B - Method for knocking out pig GOT1 gene by using CRISPR/Cas9 system - Google Patents

Method for knocking out pig GOT1 gene by using CRISPR/Cas9 system Download PDF

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CN110468133B
CN110468133B CN201910763752.6A CN201910763752A CN110468133B CN 110468133 B CN110468133 B CN 110468133B CN 201910763752 A CN201910763752 A CN 201910763752A CN 110468133 B CN110468133 B CN 110468133B
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成志敏
蔡春波
李步高
张宁芳
张万锋
郭晓红
高鹏飞
曹果清
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Shanxi Agricultural University
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Abstract

The invention relates to the technical field of biology, in particular to a method for knocking out a pig GOT1 gene by using a CRISPR/Cas9 system. The invention provides sgRNA of a specific targeted pig GOT1 gene, a CRISPR/Cas9 gene knockout vector containing the sgRNA, a method for carrying out pig GOT1 gene knockout by using the vector and a detection method of pig GOT1 gene. The CRISPR/Cas9 knockout vector provided by the invention can realize higher pig GOT1 gene knockout efficiency, remarkably improves the construction efficiency of GOT1 gene knockout cells and GOT1 gene knockout pigs, and provides an effective method and a foundation for the functional research and application of pig GOT1 genes.

Description

Method for knocking out pig GOT1 gene by using CRISPR/Cas9 system
Technical Field
The invention relates to the technical field of biology, in particular to sgRNA of a specific target pig GOT1 gene, a CRISPR/Cas9 gene knockout vector containing the sgRNA, a method for carrying out pig GOT1 gene knockout by using the vector and a detection method for the pig GOT1 gene knockout.
Background
GOT1 is a Pyridoxal phosphate (PLP) -dependent transaminase, participates in the reversible reaction of Aspartate (Asp) and alpha-Ketoglutarate (alpha-Ketoglutarate, alpha-KG) to Glutamate (Glutamate) and Oxaloacetate (OAA) in the cytoplasmic matrix, and plays an important role in maintaining the redox balance of cells, promoting the proliferation of tumor cells and regulating the metabolism of amino acids.
Currently, studies on the GOT1 gene are mainly focused on mice, and relatively few studies are conducted on mammals such as pigs. The pig GOT1 gene is knocked out by using a CRISPR/Cas9 system, the function of the pig GOT1 gene is studied in vitro and in vivo, and the method has important significance for studying the function of the GOT1 gene in the pig development process and the mechanism of the function.
CRISPR/Cas9 is an adaptive immune defense formed during long-term evolution of bacteria and archaea, and can be used to fight invading viruses and foreign DNA. The CRISPR/Cas9 gene editing technology is a technology for carrying out specific DNA modification on a target gene, and the technology is also a method for comparing the front edges of the gene editing at present. The gene editing technology based on CRISPR/Cas9 shows great application prospect in the application field of gene therapy of a series of diseases (such as hematopathy, tumor and other genetic diseases). The development of an efficient CRISPR/Cas9 system knockout method of the pig GOT1 gene has important significance for improving the knockout efficiency of the pig GOT1 gene and the construction efficiency of GOT1 gene knockout cells or animals.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a method for efficiently knocking out a pig GOT1 gene by using a CRISPR/Cas9 system and a method for efficiently detecting the pig GOT1 gene knocking out.
In order to achieve the purpose, the technical scheme of the invention is as follows:
in a first aspect, the present invention provides sgrnas that specifically target the porcine GOT1 gene, targeting the sequences from position 517 to position 536 of the porcine GOT1 gene.
According to the invention, a large number of screening discovers that the sequence from 517 th site to 536 th site on the pig GOT1 gene is used as a target sequence, so that the knockout efficiency is higher.
Aiming at the target sequence on the pig GOT1 gene, the invention designs specific sgRNA, and further finds that the sgRNA corresponding to the nucleotide sequence shown in SEQ ID NO.3 can realize higher targeting efficiency and GOT1 gene knockout efficiency.
Preferably, the nucleotide sequence of the sgRNA coding gene specifically targeting the pig GOT1 gene is shown in SEQ ID No. 3.
SEQ ID NO.3：sgRNA-3：5’-ACATTCGGTCCTATCGCTAT-3’。
In a second aspect, the invention provides a CRISPR/Cas9 gene knockout vector containing the sgRNA specifically targeting the porcine GOT1 gene.
As a preferred embodiment of the present invention, the CRISPR/Cas9 gene knockout vector is a pHS-CR054 vector to which the sgRNA specifically targeting the porcine GOT1 gene is linked.
In a third aspect, the invention also provides a construction method of the CRISPR/Cas9 gene knockout vector, which comprises the following steps: and connecting the sgRNA of the specific targeted pig GOT1 gene with a pHS-CR054 vector to obtain the CRISPR/Cas9 gene knockout vector.
Preferably, the construction method comprises the following steps:
(1) digesting the vector pHS-CR054 by BsmBI enzyme to obtain a linearized vector;
(2) annealing the sgRNA sequence fragment with BsmBI enzyme digestion sites to form a fragment containing the cohesive end of the BsmBI enzyme digestion sites;
(3) and (3) connecting the annealing product obtained in the step (2) with the linearized vector obtained in the step (1) to construct a pHS-CR054 vector connected with the sgRNA of the specific targeted pig GOT1 gene.
Further preferably, in the step (1), the reaction system of enzyme digestion is as follows: vector pHS-CR 054500 ng, restriction enzyme BsmBI 1. mu.L, NEB 3.1Buffer 1. mu.L, ddH2The content of O is filled to 10 mu L. The reaction sequence of the enzyme digestion is as follows: 60min at 55 ℃; 80 ℃ for 20 min.
Further preferably, in the step (2), the reaction system of annealing is as follows: PNK buffer 2. mu.L, upstream sequence 100. mu.M, downstream sequence 100. mu.M, ddH2The content of O is filled to 20 mu L. The reaction sequence for annealing was as follows: 95 ℃ for 5 min; slowly cooling at 95-25 deg.C, and reducing the temperature by 2 deg.C per minute.
Further preferably, in the step (3), the attached reaction system is as follows: 1. mu.L of annealing product, 1. mu.L of enzyme digestion product, 1. mu.L of T4DNA Ligase, 1. mu.L of 10 XBuffer, ddH2The content of O is filled to 10 mu L. Reaction scheme of ligationThe sequence is as follows: 16 ℃ overnight.
In a fourth aspect, the invention further provides application of the sgRNA specifically targeting the pig GOT1 gene or the CRISPR/Cas9 gene knockout vector containing the sgRNA in pig GOT1 gene knockout.
In a fifth aspect, the present invention provides a method for preparing a GOT1 gene knockout cell, comprising: introducing a CRISPR/Cas9 gene knockout vector containing sgRNA of the specific targeted pig GOT1 gene into cells to knock out the GOT1 gene.
The CRISPR/Cas9 gene knockout vector can be introduced into cells by conventional biological methods such as transfection.
According to the method for preparing the GOT1 gene knockout cell, after the CRISPR/Cas9 gene knockout vector containing the sgRNA of the specific targeted pig GOT1 gene is introduced into a cell, a positive cell can be screened by adopting a conventional method such as PCR (polymerase chain reaction) and the like.
The invention obtains the pig cell with the GOT1 gene knockout by utilizing the method for preparing the GOT1 gene knockout cell.
In a sixth aspect, the invention provides a method for preparing a GOT1 gene knockout pig, which is to realize the knockout of a pig GOT1 gene by using the CRISPR/Cas9 gene knockout vector.
In a sixth aspect, the invention provides a specific primer pair for detecting the expression level of the pig GOT1 gene, wherein the sequence of the specific primer pair is shown as SEQ ID NO. 4-5.
In a seventh aspect, the present invention provides a method for detecting expression level of GOT1 gene in pig, which comprises the following steps:
(1) extracting total RNA of cells to be detected, and performing reverse transcription to obtain cDNA;
(2) and (3) amplifying the GOT1 gene by using the cDNA in the step (2) as a template and using a specific primer pair shown as SEQ ID NO.4-5 and fluorescent quantitative PCR (qPCR), and detecting the expression level of the GOT1 gene.
Preferably, in the step (2), the fluorescent quantitative PCR amplification includes 18S rDNA as an internal reference gene.
Further preferably, the 18S rDNA fluorescent quantitative PCR amplification primer sequence is shown in SEQ ID NO. 6-7.
Preferably, in the step (2), the reaction system of the qPCR is as follows: 2 XGoTaq qPCR Master Mix 10. mu.L, RNase-free water 7.4. mu.L, 10. mu.M upstream primer 0.3. mu.L, 10. mu.M downstream primer 0.3. mu.L, cDNA template 2.0. mu.L.
The reaction procedure for the qPCR was as follows: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s, and 45 cycles; melting curves were prepared at 95 ℃ for 15s, 65 ℃ for 1min, 95 ℃ for 30min, and 60 ℃ for 15 s.
Preferably, in the step (2), the amplification curve and the melting curve obtained by the qPCR real-time monitoring are analyzed. The expression level of GOT1 gene was calculated by using the control group as external reference (the external reference was set to eliminate the test error of different batches). The expression difference of the GOT1 gene in different treatments is compared by independent sample T test analysis, the result is expressed by mean value + -standard error, P <0.05 indicates that the difference is significant, and P <0.01 indicates that the difference is extremely significant. All statistical analyses were done using SPSS version 20.0 software.
The beneficial effects of the invention at least comprise:
the porcine GOT1 gene is knocked out by using a CRISPR/Cas9 knocking out system. The sgRNA capable of efficiently targeting the pig GOT1 gene is obtained by specific selection of a pig GOT1 gene target site and specific design of the sgRNA aiming at the target site, and the knockout efficiency of the pig GOT1 gene is obviously improved. Experiments prove that the CRISPR/Cas9 knockout vector provided by the invention can realize higher pig GOT1 gene knockout efficiency, and remarkably improves the construction efficiency of GOT1 gene knockout cells and GOT1 gene knockout pigs.
The specific primer for detecting the expression level of the pig GOT1 gene can quickly and accurately perform quantitative analysis on the expression level of the pig GOT1 gene.
The method for knocking out the pig GOT1 gene and the method for detecting the expression level of the pig GOT1 gene have strong practicability, and provide an effective method and a foundation for functional research and application of the pig GOT1 gene.
Drawings
FIG. 1 is a map of the pHS-CR054 vector in example 2 of the present invention; the position of BsmBI cleavage sites is indicated, with two BsmBI cleavage sites at 3058bp and 3741 bp.
Fig. 2 is a sequencing result of a sgRNA sequence with a BsmBI enzyme cutting site inserted into a CRISPR/Cas9 gene knockout vector containing 3 sgrnas of specifically targeted porcine GOT1 genes in example 2 of the present invention; wherein GOT1_ KO1 is a CRISPR/Cas9 gene knockout vector containing sgRNA-1, GOT1_ KO2 is a CRISPR/Cas9 gene knockout vector containing sgRNA-2, and GOT1_ KO3 is a CRISPR/Cas9 gene knockout vector containing sgRNA-3.
FIG. 3 is a graph showing the result of microscopic observation of PK15 cells transfected in example 3 of the present invention; among them, GOT1_ KO1, GOT1_ KO2 and GOT1_ KO3 are transfected cells carrying pHS-CR054 vectors of sgRNA-1, sgRNA-2 and sgRNA-3, respectively.
FIG. 4 shows the result of measuring the mRNA expression level of GOT1 gene 48h after transfection in example 3; wherein NC is an empty vector, GOT1_ KO1, GOT1_ KO2 and GOT1_ KO3 are pHS-CR054 vectors carrying sgRNA-1, sgRNA-2 and sgRNA-3 respectively; indicates significant difference (P <0.05) and indicates very significant difference (P < 0.01).
FIG. 5 shows the result of Western blotting detection of GOT1 protein 48h after transfection in example 3; wherein, Control is an empty vector Control, and GOT1_ KO1, GOT1_ KO2 and GOT1_ KO3 are pHS-CR054 vectors carrying sgRNA-1, sgRNA-2 and sgRNA-3 respectively; indicates significant difference (P <0.05) and indicates very significant difference (P < 0.01).
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
According to the invention, the nucleotide sequence of the pig GOT1 gene is downloaded from a database, based on the analysis of the pig GOT1 gene sequence, the screening of the target site of the pig GOT1 gene and the design and screening of sgRNA aiming at the target site are carried out according to the design principle of identifying the target site by CRISPR/Cas9, sgRNA-3 with the highest knockout efficiency is finally obtained by screening (shown as SEQ ID NO.3), and a CRISPR/Cas9 knockout vector containing the sgRNA is constructed, so that the efficient knockout of the pig GOT1 gene is realized. In the following examples, the effects of 3 sgrnas designed according to the present invention for different target sites are described as examples.
Example 1 acquisition of sgrnas specifically targeting pig GOT1 gene
The sgRNA-1 targets a sequence from 518 th to 537 th of a pig GOT1 gene, the sgRNA-2 targets a sequence from 919 th to 938 th of a pig GOT1 gene, and the sgRNA-3 targets a sequence from 517 th to 536 th of a pig GOT1 gene. The sequences of sgRNA-1, sgRNA-2 and sgRNA-3 are as follows:
SEQ ID NO.1：sgRNA-1：5’-CATTCGGTCCTATCGCTATT-3’；
SEQ ID NO.2：sgRNA-2：5’-AGAAGATCGTGCGAGTGACG-3’；
SEQ ID NO.3：sgRNA-3：5’-ACATTCGGTCCTATCGCTAT-3’。
for subsequent ligation to the vector, BsmBI cleavage site sequences were added to both ends of the sgRNA, and the resulting double-stranded DNA sequences are shown in table 1.
Table 13 double stranded DNA sequences corresponding to sgrnas
Figure BDA0002171230070000071
Example 2 construction of CRISPR/Cas9 gene knockout vector containing sgRNA specifically targeting pig GOT1 gene
The present example provides a CRISPR/Cas9 knockout vector containing sgRNA specifically targeting pig GOT1 gene and a construction method thereof.
The construction method of the CRISPR/Cas9 gene knockout vector containing sgRNA of the specific target pig GOT1 gene comprises the following steps:
1. construction of linearized vectors
Recovering and amplifying the strain carrying pHS-CR054 vector (pHS-CR054 vector map is shown in FIG. 1, and is purchased from Beijing Synbiotic Gene technology Co., Ltd.), and extracting plasmid according to the instruction of OMEGA plasmid extraction kit.
The extracted product is cut by restriction enzyme BsmBI, and the reaction system of the enzyme cutting is as follows: restriction enzyme BsmBI 1. mu.L, pHS-CR054 vector plasmid 500ng, NEB 3.1Buffer 1. mu.L, ddH2The content of O is filled to 10 mu L. The reaction procedure of enzyme digestion is as follows: enzyme digestion is carried out for 1h at 55 ℃, and inactivation is carried out for 20min at 80 ℃.
2. Annealing of sgRNA sequences
Fragments of the sgRNA sequence carrying the BsmBI cleavage site, shown in table 1 as 3, were annealed separately to form fragments containing sticky ends of the BsmBI cleavage site. The reaction system of annealing is as follows: PNK buffer 2. mu.L, upstream sequence 100. mu.M, downstream sequence 100. mu.M, ddH2The content of O is filled to 20 mu L. The reaction sequence for annealing was as follows: 95 ℃ for 5 min; slowly cooling at 95-25 deg.C, and reducing the temperature by 2 deg.C per minute.
3. Connection of
And (3) connecting the sgRNA annealing product obtained in the step (2) with the linearized vector pHS-CR054 obtained in the step (1). The ligation reaction system is as follows: mu.L of annealing product, 1. mu.L of digestion product, 1. mu.L of T4DNA Ligase, 1. mu.L of 10 XBuffer, and 10. mu.L of ddH2O, and the ligation reaction program is as follows: 16 ℃ overnight.
4. Transformation of
And (3) adding 5 mu L of the connection product in the step 3 into 50 mu L of DH5 alpha competent cells which are just thawed, uniformly mixing, carrying out ice bath for 30min, carrying out heat shock at 42 ℃ for 45s, immediately putting the cells on ice, standing for 2min, adding 950 mu L of LB liquid culture medium preheated at 37 ℃, carrying out shake culture at 37 ℃ for 45min, centrifuging the bacterial liquid at a low speed for 2-3min, pouring off part of supernatant, blowing and precipitating the rest supernatant by using a gun head, coating 100 mu L of the bacterial liquid on a plate of ampicillin, positively placing the bacterial liquid in a constant-temperature incubator at 37 ℃ for 1h, and carrying out inverted culture for 12 h.
5. Screening positive bacterial colony and sequencing identification
And (4) selecting the single colony obtained by culturing in the step (4), carrying out colony PCR, and detecting by agarose gel electrophoresis. And (3) shaking the qualified bacterial colonies, and sending 1mL of bacterial liquid to Huada Gene company for sequencing primer sequences as follows: CAGGAAGAGGGCCTATTTCCC are provided. And (4) performing bacterium preservation and plasmid extraction on the bacterium liquid sample with correct sequencing. The correctly sequenced pHS-CR054 vectors containing 3 sgRNAs were named GOT1_ KO1 (pHS-CR054 vector containing sgRNA-1), GOT1_ KO2 (pHS-CR054 vector containing sgRNA-2), and GOT1_ KO3 (pHS-CR054 vector containing sgRNA-3), respectively, and the sequencing results are shown in FIG. 2.
Example 3 knockout and detection of pig GOT1 Gene
The present example provides a method for knocking out pig GOT1 gene and a method for detecting expression level of pig GOT1 gene by using CRISPR/Cas9 gene knock-out vectors GOT1_ KO1, GOT1_ KO2 and GOT1_ KO3 of sgRNA containing a specific target pig GOT1 gene constructed in example 2.
1. Knockout of pig GOT1 gene
(1) Cell culture and Collection
PK15 cells were revived using complete medium and after 3 passages, transfection was performed until the cells entered logarithmic growth phase. The day before transfection, 5X 10 cells were added5～8×105Cells were seeded into 6-well plates and transfected when cell density reached 60% -80% with 2mL antibiotic-free medium per well.
(2) Transfection
The CRISPR/Cas9 gene knockout vectors GOT1_ KO1, GOT1_ KO2 and GOT1_ KO3 containing sgRNA specifically targeting pig GOT1 gene constructed in example 2 were transfected with Lipofectamine 2000 as per the requirements specified by Lipofectamine 2000, and cells were harvested after 48 h; NC plasmid (empty plasmid) was used as a control. Microscopic observations of transfected PK15 cells are shown in FIG. 3.
2. qPCR detection of pig GOT1 gene expression level
And (2) carrying out qPCR detection on the positive cells obtained in the step (1), and specifically comprising the following steps:
(1) cellular RNA extraction and cDNA Synthesis
Total RNA of cells to be detected is extracted by adopting a TaKaRa RNAioso Plus kit, and the extraction method refers to the kit instruction. The integrity of total RNA was determined by agarose gel electrophoresis detection; total RNA concentration was determined by NanoDrop 1000 micro uv spectrophotometer.
(2) Primer design
A primer pair capable of detecting pig GOT1 gene knockout is designed according to a pig GOT1 gene mRNA sequence (GenBank: EU822301.1) published by NCBI, and a pair of primers GOT1-1 capable of realizing high-efficiency pig GOT1 gene detection is obtained through screening. The sequence of primer pair GOT1-1 is as follows:
upstream primer, GOT 1-1F: 5'-CATCCTGCGAGTCCTTTC-3', respectively;
downstream primer, GOT 1-1R: 5'-CGGTCAGCCATTGTCTTC-3', respectively;
designing a primer pair 18S according to the 18S gene sequence published by NCBI, wherein the sequence of the primer pair 18S is as follows:
upstream primer, 18S-F: 5'-ATGCCAGAGTCTCGTTCGTTAT-3', respectively;
downstream primer, 18S-R: 5'-CGGACAGGATTGACAGATTGAT-3' are provided.
(3) qPCR amplification
And (3) performing qPCR amplification by using the cell cDNA obtained in the step (1) as a template and using the primer pair GOT1-1 and the primer pair 18S obtained in the step (2).
The reaction system of qPCR is: GoTaq qPCR Master Mix (2X) 10. mu.L, RNase-free water 7.4. mu.L, 10. mu.M forward primer 0.3. mu.L, 10. mu.M reverse primer 0.3. mu.L, cDNA template 2.0. mu.L. The qPCR reaction system is prepared by adopting a mixed sample adding method, namely the total amount of various reaction components is calculated according to the quantity of various components required by each reaction system and the quantity of PCR reactions required by 1 reaction, the reaction components are added into 1.5mL centrifuge tubes for removing RNA enzyme, the mixture is subjected to instantaneous centrifugation after being fully and uniformly mixed, the mixture is subpackaged into 8 tubes special for fluorescent quantitative PCR, template cDNA is added respectively, fluorescent quantitative PCR amplification is carried out after instantaneous centrifugation, and the whole operation process is protected from light as much as possible.
The reaction program for qPCR was: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 30s, and 45 cycles; melting curves were prepared at 95 ℃ for 15s, 65 ℃ for 1min, 95 ℃ for 30min, and 60 ℃ for 15 s.
(4) Data analysis of qPCR
And analyzing an amplification curve and a melting curve of the qPCR result. The results show that each gene amplification curve is S-shaped and reaches the platformPhase, indicated as efficient amplification; and the melting curve peak value of each gene is single, which indicates that the amplification product is specific, no primer dimer and other non-specific amplifications, and the subsequent result analysis can be carried out. According to the CT values of different treatment groups, a control group of NC plasmids (empty plasmids) is used as an external parameter (the external parameter is set to eliminate test errors of different batches), and a relative quantitative method is adopted to calculate the relative expression quantity of the pig GOT1 gene knockout, namely 2-△△CT。
(5) Drawing
Based on the calculation of relative quantification, the significance of differences was examined using SPSS version 20.0 software and plotted by GraphPad Prism 5, with the results shown in fig. 4, where the number of individual replicates was 3 and the qPCR loading technique replicate 4. The results show that knocking out GOT1 gene down significantly reduced its transcription level compared to the control group; in the 3 sgRNA-mediated porcine GOT1 gene knockout, the transcription level of GOT1 gene in sgRNA-3-mediated knockout cells is the lowest, which indicates that sgRNA-3 has excellent porcine GOT1 gene knockout efficiency, and can realize high-efficiency knockout of porcine GOT1 gene.
3. Western blotting detection of pig GOT1 gene expression level
Western blotting is adopted to further verify the expression level of the pig GOT1 gene, and the specific method is as follows:
total proteins of cells of the knockout group and the corresponding blank control group are extracted, 200ng of the proteins are sampled and subjected to SDS polyacrylamide gel electrophoresis. And exposing the strips by using a LICOR instrument after membrane conversion, sealing, incubation of primary antibody and secondary antibody, and calculating and analyzing the optical density value of the strips by using Image Studio software of the instrument. The results are shown in fig. 5, in the deletion of 3 sgRNA-mediated porcine GOT1 genes, the expression level of GOT1 gene in sgRNA-3-mediated deleted cells was the lowest, which indicates that sgRNA-3 has excellent porcine GOT1 gene deletion efficiency, and can realize high-efficiency deletion of porcine GOT1 gene.
According to the review, the sgRNA (SEQ ID NO.3) of the special targeted pig GOT1 gene and the CRISPR/Cas9 gene knockout vector containing the sgRNA can realize high-efficiency pig GOT1 gene knockout; meanwhile, the specific primer of the pig GOT1 gene can be used for realizing the rapid and accurate detection of the expression level of the GOT1 gene; the invention provides an effective method and a foundation for the functional research and the application of the pig GOT1 gene.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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Claims (3)

1. The CRISPR/Cas9 knockout vector for the specific targeting pig GOT1 gene is a pHS-CR054 vector connected with a coding gene of sgRNA of the specific targeting pig GOT1 gene, the sgRNA targets the sequence from the 517 th position to the 536 th position of the pig GOT1 gene, and the nucleotide sequence of the coding gene is shown as SEQ ID NO. 3.
2. Use of the CRISPR/Cas9 gene knock-out vector of claim 1 in porcine GOT1 gene knock-out.
3. A method for preparing a GOT1 gene knockout cell, which is characterized in that the CRISPR/Cas9 gene knockout vector of claim 1 is introduced into a cell to knock out a GOT1 gene.
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