CN112553200A - Preparation method and application of targeting vector for knocking out pig UGT2C1 gene - Google Patents
Preparation method and application of targeting vector for knocking out pig UGT2C1 gene Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a targeting vector for knocking out pig UGT2C1 gene, wherein the method comprises the following steps: (1) designing a sgRNA guide sequence according to a pig UGT2C1 gene; (2) annealing the positive strand sgRNA sequence and the negative strand sgRNA sequence to form double-stranded DNA; connecting the double-stranded DNA with a linearized pGK1.1 vector to obtain a positive targeting vector; (3) and (3) carrying out mixed electrotransfection on the target cell IPEC-J2 by using the positive targeting vector to obtain UGT2C1 gene knockout IPEC-J2 cells. According to the invention, the UGT2C1 gene sequence in the genome of the IPEC-J2 cell line is knocked out by using the CRISPR/Cas9 technology, and the built UGT2C1 gene knocked-out IPEC-J2 cell can provide a more direct and effective research model for deeply disclosing the action mechanism of the UGT2C1 gene in disease resistance and the application of the UGT2C1 gene in pig disease resistance breeding.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to an application technology of CRISPR/Cas9, and also relates to a construction technology of a pig small intestine epithelial cell line (IPEC-J2) gene knockout model.
Background
The CRISPR/Cas9 is a high-efficiency nucleic acid cutting tool, and based on the high-efficiency nucleic acid cutting capability and simple operation of CRISPR/Cas9, the CRISPR technology is widely applied to site-directed modification of genes such as pigs, rabbits, cynomolgus monkeys, rats, mice and the like, and in addition, the CRISPR/Cas9 system is also applied to modification research of a plurality of plant genomes. The CRISPR/Cas9 can be used as an emerging genome editing technology to effectively research animal growth and immune system related genes, and further has important significance for research on animal disease treatment and perfection of gene biological functions.
The porcine small intestinal epithelial cell line (IPEC-J2) is a good porcine intestinal epithelial model cell, has strong similarity with primary porcine small intestinal epithelial cells, and is an ideal in-vitro model for researching porcine intestinal diseases. Zearalenone (ZEA) is a class of mycotoxins that exert estrogenic effects and pose serious risks to the animal body or human health. In recent years, it has been found that exposure to specific mycotoxins causes altered gut function and induces chronic gut inflammation in humans. Transcriptome analysis is carried out on ZEA-induced IPEC-J2 cells at the early stage of the subject group, and UGT2C1 gene expression level is found to be remarkably reduced, which indicates that the cells possibly participate in cytotoxicity regulation caused by ZEA induction. The UGT2C1 gene belongs to the glucuronic acid transferase family (UPT), the UPT is a membrane protein combined in endoplasmic reticulum, and the enzyme can catalyze glucuronic acid to perform glucuronic acid combination reaction with a large number of exogenous and endogenous chemical substances, and is an important detoxification way. A knockout carrier of the pig UGT2C1 gene and a small intestine epithelial cell model knocked out by the UGT2C1 gene are established, and important bases are provided for deeply researching UGT2C1 gene functions and disclosing a ZEA pathogenic mechanism.
Disclosure of Invention
The invention aims to provide a CRISPR/Cas9 knockout vector capable of targeting pig UGT2C1 gene and a construction method of the CRISPR/Cas9 knockout vector for knocking the UGT2C1 gene in a pig small intestine epithelial cell line (IPEC-J2).
The method can be realized by the following technical scheme:
a group of sgRNA guide sequences for targeted knockout of pig UGT2C1 gene based on CRISPR/Cas9 technology comprise sgRNA1 and sgRNA 2:
sgRNA1:CTTGGTGAAGGTACCAGGA(SEQ ID NO.1)；
sgRNA2:TCAGCCTTGATGCGCTGCT(SEQ ID NO.2)。
a double-stranded DNA for targeted knockout of pig UGT2C1 gene based on CRISPR/Cas9 technology comprises (1) to (2) groups of double-stranded DNAs:
(1)sgRNA1F:5’-CACCGCTTGGTGAAGGTACCAGGA-3’(SEQ ID NO.3)，sgRNA1R:5’-AAACTCCTGGTACCTTCACCAAGC-3’(SEQ ID NO.4)；
(2)sgRNA2F:5’-CACCGTCAGCCTTGATGCGCTGCT-3’(SEQ ID NO.5)，sgRNA2R:5’-AAACAGCAGCGCATCAAGGCTGAC-3’(SEQ ID NO.6)。
a pig UGT2C1 gene knockout vector based on CRISPR/Cas9 technology target knockout is constructed by the following steps:
(1) target site design and sgRNA sequence synthesis: designing the sgRNA guide sequences according to CDS sequences of transcripts of pig UGT2C1 genes, adding CACC to the 5 ' end of each sgRNA guide sequence, adding CACC if the first base at the 5 ' end of the sgRNA is not G to form a positive strand sgRNA sequence, and adding AAAC to the 5 ' end of a reverse complementary sequence of each sgRNA guide sequence to form a negative strand sgRNA sequence;
(2) vector construction: annealing the positive strand sgRNA sequence and the negative strand sgRNA sequence to form two groups of double-stranded DNA; and respectively connecting each group of double-stranded DNA with a linearized pGK1.1 vector to obtain a connection product, carrying out colony PCR screening on the connection product, and carrying out amplification culture and plasmid extraction to obtain two positive targeting vectors, namely the knockout vector for targeted knockout of the pig UGT2C1 gene.
A cell line for specifically and targeted knockout of pig UGT2C1 gene based on CRISPR/Cas9 technology is obtained by transferring the two positive targeting vectors into IPEC-J2 cell in a mixed mode, and screening and verifying the cell line.
A method for targeted knockout of UGT2C1 gene in porcine small intestine epithelial cell line based on CRISPR/Cas9 technology, comprising the steps of:
(1) target site design and sgRNA sequence synthesis: designing the sgRNA guide sequences according to CDS sequences of transcripts of pig UGT2C1 genes, adding CACC to the 5 ' end of each sgRNA guide sequence, adding CACC if the first base at the 5 ' end of the sgRNA is not G to form a positive strand sgRNA sequence, and adding AAAC to the 5 ' end of a reverse complementary sequence of each sgRNA guide sequence to form a negative strand sgRNA sequence;
(2) vector construction: annealing the positive strand sgRNA sequence and the negative strand sgRNA sequence to form two groups of double-stranded DNA; connecting each group of double-stranded DNA with a linearized pGK1.1 vector to obtain a connection product, performing colony PCR screening on the connection product, and performing amplification culture and plasmid extraction to obtain two positive targeting vectors, namely knockout vectors for targeted knockout of pig UGT2C1 genes;
(3) cell transfection: and (3) mixing the two positive targeting vectors to electrically transfect a target cell IPEC-J2, and performing screening verification and culture to obtain the pig UGT2C1 gene targeted knockout IPEC-J2 cell.
As a preferred technical scheme, the process of performing colony PCR screening on the ligation products in the step (2) of the method comprises the following steps: transforming the ligation product into escherichia coli for culture, selecting positive colonies, designing a colony PCR primer group according to the sgRNA guide sequence, and performing PCR screening verification to obtain two positive targeting vectors; the colony PCR primer group comprises two pairs of primer pairs, wherein the forward primer of each primer pair is a universal primer, the reverse primer is two sgRNA primers, and the specific sequence of each primer pair is as follows:
F1：CATATGCTTACCGTAACTTGAAAG(SEQ ID NO.7)
R1：CTTGGTGAAGGTACCAGGA(SEQ ID NO.1)
F2：CATATGCTTACCGTAACTTGAAAG(SEQ ID NO.8)
R2：TCAGCCTTGATGCGCTGCT(SEQ ID NO.2)。
as a preferred technical solution, the screening and verifying process in step (3) of the above method is:
(I) two positive targeting vectors are mixed to electrically transfect a target cell IPEC-J2, the genomic DNA of the cell is extracted by puromycin drug screening, a high-specificity primer is designed near a knockout target site for PCR amplification, and the sequence of the primer is as follows:
UGT2C1-seqF1:5’-CCGCCCTTCGCACCTCAT-3’(SEQ ID NO.9)；
UGT2C1-seqR1:5’-AGCCACCAACTCCCCACA-3’(SEQ ID NO.10)；
UGT2C1-seqF2:5’TCATACCAAAATCCCCTT-3’(SEQ ID NO.11)；
UGT2C1-seqR2:5’-TGCTGTAATACTCATCCC-3’(SEQ ID NO.12)；
(II) T7 Endonuclease I enzyme digestion PCR product identification effective targeting positive clone;
(III) sequencing and verifying the DNA PCR product of UGT2C1 gene knockout cells screened by T7 Endonuclease I enzyme digestion to obtain UGT2C1 gene knockout IPEC-J2 cells.
The sgRNA guide sequence, the double-stranded DNA, the knockout vector or the cell line are used for breeding pig for disease resistance. The method is applied to the breeding of pig disease resistance.
The detailed steps of the technical scheme of the invention are as follows:
the invention discloses a method for knocking out UGT2C1 gene sequence in IPEC-J2 cell line genome by CRISPR/Cas9 technology, which comprises the following steps:
1) annealing reverse complementary sequences of sgRNA sequences 5'-CTTGGTGAAGGTACCAGGA-3' and 5'-TCAGCCTTGATGCGCTGCT-3' with CACCG added at the 5 'end and an AAAC added at the 5' end to form double-stranded DNA;
2) connecting the double-stranded DNA with a linearized knockout vector pGK2.1 to obtain a connection product;
3) the primer is used for:
F1：CATATGCTTACCGTAACTTGAAAG
R1：CTTGGTGAAGGTACCAGGA
F2：CATATGCTTACCGTAACTTGAAAG
R2：TCAGCCTTGATGCGCTGCT
performing colony PCR screening on the ligation product, and obtaining a positive targeting vector through amplification culture and plasmid extraction;
4) transfecting the IPEC-J2 cell with the positive targeting vector, extracting cell genome DNA through puromycin drug screening, designing a primer with high specificity near a knockout target site, and performing PCR amplification;
5) screening T7 Endonuclease I enzyme digestion PCR products to obtain 2 sgRNAs for effectively knocking UGT2C1 genes out;
6) sequencing and verifying a PCR product to obtain the UGT2C1 gene knockout IPEC-J2 cell.
According to the invention, the UGT2C1 gene sequence in the genome of the IPEC-J2 cell line is knocked out by using the CRISPR/Cas9 technology, and the built UGT2C1 gene knocked-out IPEC-J2 cell can provide a more direct and effective research model for deeply disclosing the action mechanism of the UGT2C1 gene in disease resistance and the application of the UGT2C1 gene in pig disease resistance breeding.
In the technology of specifically targeted knockout of pig UGT2C1 gene by using CRISPR-Cas9, the design of sgRNA guide sequence plays a key role, sgRNA is not well designed, and cells screened by later monoclone have no knockout efficiency, so the sequence of sgRNA is one of important invention points of the invention. The invention screens a guide sequence of sgRNA which can efficiently knock out pig UGT2C1 gene from a plurality of possible sequences of sgRNA through experiments.
The invention has the beneficial effects that:
(1) the IPEC-J2 cell line knocked out by UGT2C1 is constructed by using a CRISPR/Cas9 technology, and can be used as an ideal cell model for pig intestinal disease research;
(2) the gene knockout is carried out by using the CRISPR/Cas9 technology, and UGT2C1 gene can be knocked out more effectively than technical means such as gene silencing and interference. UGT2C1 belongs to the family of glucuronyl transferase, but the specific gene function is not known, and the establishment of a UGT2C1 knockout cell line is helpful for deep exploration of the function;
(3) gene sequencing detection shows that UGT2C1 gene sequence is knocked out, so that the loss of gene function can be caused, and the method is an ideal UGT2C1 gene knocked-out IPEC-J2 cell model.
Drawings
FIG. 1 is a colony PCR gel electrophoresis test chart.
FIG. 2 is a diagram of colony PCR product sequencing peaks. A is a sequencing peak diagram of a corresponding targeting vector of sgRNA 1; b is a graph of sequencing peaks of the sgRNA2 corresponding to the targeting vector.
FIG. 3 is a gel electrophoresis assay of PCR amplification of knock-out target sites.
FIG. 4 is a gel electrophoresis detection chart of DNA PCR products of T7 Endonuclease I enzyme digestion screening UGT2C1 gene knockout cells.
FIG. 5 is a sequence peak diagram of DNA PCR products of T7 Endonuclease I enzyme digestion screening UGT2C1 gene knockout cells.
FIG. 6 shows the comparative analysis result of the UGT2C1 gene knockout cell DNA sequence and the UGT2C1 gene knockout cell DNA sequence.
FIG. 7 is a photograph (magnification 100) of pig UGT2C1 gene knock-out IPEC-J2 cell.
FIG. 8 is a determination of the activity of UGT2C1 gene knockout IPEC-J2 cells.
Detailed Description
The present invention will be described in detail with reference to specific examples. From the following description and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.
Example 1
1. Target site design and sgRNA sequence synthesis:
obtaining a CDS sequence of a transcript of a pig UGT2C1 Gene (Gene ID:100624700) according to an NCBI database, finding out the 5' end of the CDS region for knocking out a target site, and designing two sgRNA guide sequences by using CRISPR Design (http:// CRISPR. mit. edu /):
sgRNA1:CTTGGTGAAGGTACCAGGA
sgRNA2:TCAGCCTTGATGCGCTGCT
the synthesis of the primers needs to add CACC in the forward primers of two sgRNA sequences, and if the first base at the 5' end of the sgRNA sequence is not G, CACC is added; and designing two reverse complementary sequences of the sgRNA, and adding a base AAAC at the 5' end. The two pairs of sgRNA sequences are as follows:
sgRNA1F:5’-CACCGCTTGGTGAAGGTACCAGGA-3’
sgRNA1R:5’-AAACTCCTGGTACCTTCACCAAGC-3’
sgRNA2F:5’-CACCGTCAGCCTTGATGCGCTGCT-3’
sgRNA2R:5’-AAACAGCAGCGCATCAAGGCTGAC-3’
2. construction of targeting vectors:
(1) annealing the positive and negative strand sgRNA sequences to form a double-stranded DNA sequence, wherein the reaction system is as follows:
the reaction system is centrifuged instantly and incubated at 95 ℃ for 3min, and then cooled naturally for 20 min.
(2) The enzyme digestion linearization is carried out on the knocking-out vector pGK2.1 by BbsI enzyme, and the reaction system is as follows:
and (3) incubating at 37 ℃ for 3h, and purifying and recovering the linearized vector pGK2.1 by using the kit.
(3) Connecting the double-stranded DNA sequence formed by annealing with a linearized vector pGK2.1, wherein the reaction system is as follows:
the above system was centrifuged instantaneously and incubated at 16 ℃ for 30min to obtain a ligation product.
(4) The DH5 alpha competence is placed on ice to be thawed, then 10 mu L of the ligation product is added, the mixture is mixed evenly and lightly, and the mixture is placed on ice to be incubated for 5 min; thermally shocking at 42 deg.C for 45sec, rapidly taking out, cooling on ice for 2 min; 500ml of non-resistant LB liquid medium was added to the tube, 300ml of the supernatant was discarded, the remaining 200ml of LB was resuspended, spread evenly on ampicillin-containing plates, and cultured overnight at 37 ℃ by inversion.
Designing colony PCR primers, wherein the upstream is a VSP universal primer, the downstream is two sgRNA primers, and the colony PCR primers have the following sequences:
F1：CATATGCTTACCGTAACTTGAAAG
R1：CTTGGTGAAGGTACCAGGA
F2：CATATGCTTACCGTAACTTGAAAG
R2：TCAGCCTTGATGCGCTGCT
selecting positive colonies for PCR screening verification, and FIG. 1 is a colony PCR gel electrophoresis detection diagram; sequencing the PCR product of the positive colony, and as shown in FIG. 2, the gene sequence of the positive targeting vector is consistent with the pGK2.1 vector and the sgRNA sequence. The positive targeting vector is subjected to amplification culture and plasmid extraction by adopting a conventional method and is used for subsequent experiments.
3. Cell transfection:
about 5X 10 of the morphology is preferably in the logarithmic growth phase6Placing the pig small intestine epithelial cells (IPEC-J2) into a 15mL centrifuge tube, centrifuging at 1000rpm for 5min, discarding supernatant, then re-suspending the cells with 210 μ L PBS, transferring into a 1.5mL centrifuge tube, adding the two positive plasmids into the centrifuge tube, wherein the concentration of the plasmids is required to be 1 μ g/μ L, and gently mixing. Transferring the mixed liquid by using a special electric transfer gun head, transferring the mixed liquid into an electric shock cup after the liquid level is raised, covering an electric shock cup cover and placing the electric shock cup cover on an electric transfer instrument after the liquid level is raised, carrying out 650V electric transfer for 30ms, and transferring the cell suspension into a six-hole plate culture medium.
Knockout of UGT2C1 gene:
after 72 hours of electroporation, the cells were diluted in 10 96-well plates by limiting dilution using a drug sieve with puromycin at a concentration of 3. mu.g/ml and CO at 37 ℃ in a2After one week of static culture in the incubator, the growth of the monoclonal cells was observed, and after about two weeks, the monoclonal cells were transferred to a 48-well plate for expanded culture. When the cells grow to half the area of the 48-well plate, 10 cells are taken2～104Individual cells, using Genloci DNA extraction kit(Cat: GP0155) and the procedures provided for it extract cellular genomic DNA.
To this end, knockout of the UGT2C1 gene in porcine small intestine epithelial cells has been accomplished.
PCR amplification of the DNA extracted in step 4:
according to the UGT2C1 Gene complete sequence (Gene ID:100624700), a primer with high specificity is designed near the knockout target site, and the sequence is as follows:
UGT2C1-seqF1:5’-CCGCCCTTCGCACCTCAT-3’；
UGT2C1-seqR1:5’-AGCCACCAACTCCCCACA-3’；
UGT2C1-seqF2:5’TCATACCAAAATCCCCTT-3’；
UGT2C1-seqR2:5’-TGCTGTAATACTCATCCC-3’；
the PCR amplification reaction system is as follows:
the PCR reaction program is 95 ℃ for 5 min; 30sec at 95 ℃, 30sec at 60 ℃, 30sec at 72 ℃, 35 cycles; 10min at 72 ℃. PCR products of 652bp and 518bp in length were obtained.
FIG. 3 is a gel electrophoresis assay of PCR amplification of knock-out target sites. Wherein M is 2000bp DNA Marker, and 1-1, 1-2, 2-1 and 2-2 are PCR product bands. As can be seen from fig. 3: the primer designed near the knockout target site can successfully amplify the target sequence.
Screening and verification of UGT2C1 gene knockout cells:
(1) t7 Endonuclease I enzyme digestion screening UGT2C1 gene knockout cell
Carrying out enzyme digestion detection on the PCR products with the lengths of 652bp and 518bp by using T7 Endonuclease I enzyme for identifying incompletely-paired DNA, and configuring a reaction system as follows:
after 3 hours of reaction at 37 ℃ and 2. mu.l of 10 × loading Buffer was added to the 10. mu.l reaction system, detection was performed by agarose gel electrophoresis. FIG. 4 shows the results of enzyme digestion and screening UGT2C1 gene knockout cells by T7 Endonuclease I, wherein M represents D2000 DNA Marker; 1-1 sgRNA1 PCR product which is not cut by enzyme; 1-2, carrying out enzyme digestion on an sgRNA1 PCR product; 2-1 represents the sgRNA2 PCR product without enzyme digestion; 2-2 represents the sgRNA2 PCR product after digestion. As can be seen from fig. 4, both sgRNA1 and sgRNA2 can effectively knock down the UGT2C1 gene, and the knock-out efficiency analysis performed according to gray scale values shows that the UGT2C1 gene targeting efficiencies of 54.8% and 56.1% in cells corresponding to 1 and 2, respectively.
(2) Sequencing verification of UGT2C1 gene knockout cell DNA PCR products:
sequencing verification is carried out on UGT2C1 gene knockout cell DNA PCR products screened by T7 Endonuclease I enzyme digestion, and as shown in a sequencing peak diagram of figure 5, cells transfected with sgRNA1 and sgRNA2 targeting vectors are determined to be UGT2C1 gene knockout cells. The DNA of UGT2C1 gene knockout cell is sequenced after TA cloning, and is compared with the DNA sequence of UGT2C1 gene knockout cell, and the DNA sequence of the knockout cell is mutated into deletion of 18bp and 16bp (figure 6).
FIG. 6 shows TA clone sequencing analysis of UGT2C1 knockout sequence. The first row is a wild sequence of UGT2C1 gene without knockout, and the second row is a sequence of UGT2C1 gene knockout. By contrast, the UGT2C1 gene knockout IPEC-J2 cell is successfully constructed by adopting the method, and the 18bp (SEQ ID NO.13) and 14bp (SEQ ID NO.14) sequence table of the deletion fragment is as follows:
deletion fragment 1: CTCCTGGTACCTTCACCA (SEQ ID NO.13)
Deletion fragment 2: CCTATGTTCCTAGC (SEQ ID NO. 14).
Expanded culture of UGT2C1 gene knockout IPEC-J2 cells:
UGT2C1 knockout IPEC-J2 cells are placed in DMEM culture solution containing 10% fetal calf serum and 5% CO at 37 DEG C2The expansion culture in the incubator can obtain a large amount of IPEC-J2 with UGT2C1 gene knockoutCells, as shown in FIG. 7.
Assay of the Activity of UGT2C1 Gene knockout IPEC-J2 cells
2X 10 of UGT2C1 gene knockout IPECJ2 cells and common IPEC-J2 cells3cells/well Density were plated in 96-well plates and DMEM (10% FBS in) medium was added, CO at 37 ℃2And incubation in an incubator for 24h and 48 h. Cell viability was determined using the Cell Counting Kit-8 according to the manufacturer's protocol, and absorbance at 450nm was measured on a Tecan Infinite 200 microplate reader. The experiment was repeated 3 times as shown in fig. 8.
Sequence listing
<110> Yangzhou university
<120> preparation method and application of targeting vector for knocking out pig UGT2C1 gene
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
cttggtgaag gtaccagga 19
<210> 2
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tcagccttga tgcgctgct 19
<210> 3
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
caccgcttgg tgaaggtacc agga 24
<210> 4
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
aaactcctgg taccttcacc aagc 24
<210> 5
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
caccgtcagc cttgatgcgc tgct 24
<210> 6
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
aaacagcagc gcatcaaggc tgac 24
<210> 7
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
catatgctta ccgtaacttg aaag 24
<210> 8
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
catatgctta ccgtaacttg aaag 24
<210> 9
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
ccgcccttcg cacctcat 18
<210> 10
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
agccaccaac tccccaca 18
<210> 11
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
tcataccaaa atcccctt 18
<210> 12
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tgctgtaata ctcatccc 18
<210> 13
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
ctcctggtac cttcacca 18
<210> 14
<211> 14
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cctatgttcc tagc 14
Claims (9)
1. A group of sgRNA guide sequences for targeted knockout of pig UGT2C1 gene based on CRISPR/Cas9 technology are characterized in that the sgRNA guide sequences comprise sgRNA1 and sgRNA 2:
sgRNA1:CTTGGTGAAGGTACCAGGA；
sgRNA2:TCAGCCTTGATGCGCTGCT。
2. a double-stranded DNA for targeted knockout of pig UGT2C1 gene based on CRISPR/Cas9 technology is characterized by comprising (1) to (2) groups of double-stranded DNAs:
(1)sgRNA1F:5’-CACCGCTTGGTGAAGGTACCAGGA-3’，sgRNA1R:5’-AAACTCCTGGTACCTTCACCAAGC-3’；
(2)sgRNA2F:5’-CACCGTCAGCCTTGATGCGCTGCT-3’，sgRNA2R:5’-AAACAGCAGCGCATCAAGGCTGAC-3’。
3. a pig UGT2C1 gene knockout vector based on CRISPR/Cas9 technology targeted knockout is characterized in that the vector is constructed by the following steps:
(1) target site design and sgRNA sequence synthesis: designing sgRNA guide sequences according to the CDS sequence of a transcript of a pig UGT2C1 gene, adding CACC to the 5 ' end of each sgRNA guide sequence, adding CACCG if the first base at the 5 ' end of each sgRNA is not G to form a positive strand sgRNA sequence, and adding AAAC to the 5 ' end of the reverse complementary sequence of each sgRNA guide sequence to form a negative strand sgRNA sequence;
(2) vector construction: annealing the positive and negative strand sgRNA sequences to form two sets of double stranded DNA as claimed in claim 2; and respectively connecting each group of double-stranded DNA with a linearized pGK1.1 vector to obtain a connection product, carrying out colony PCR screening on the connection product, and carrying out amplification culture and plasmid extraction to obtain two positive targeting vectors, namely the knockout vector for targeted knockout of the pig UGT2C1 gene.
4. A cell line for specifically and targeted knockout of pig UGT2C1 gene based on CRISPR/Cas9 technology is characterized in that the cell line is obtained by mixedly transferring two positive targeting vectors described in claim 3 into IPEC-J2 cell and carrying out screening and verification.
5. A method for targeted knockout of UGT2C1 gene in porcine small intestine epithelial cell line based on CRISPR/Cas9 technology, which is characterized by comprising the following steps:
(1) target site design and sgRNA sequence synthesis: designing sgRNA guide sequences according to the CDS sequence of a transcript of a pig UGT2C1 gene, adding CACC to the 5 ' end of each sgRNA guide sequence, adding CACCG if the first base at the 5 ' end of each sgRNA is not G to form a positive strand sgRNA sequence, and adding AAAC to the 5 ' end of the reverse complementary sequence of each sgRNA guide sequence to form a negative strand sgRNA sequence;
(2) vector construction: annealing the positive and negative strand sgRNA sequences to form two sets of double stranded DNA as claimed in claim 2; connecting each group of double-stranded DNA with a linearized pGK1.1 vector to obtain a connection product, performing colony PCR screening on the connection product, and performing amplification culture and plasmid extraction to obtain two positive targeting vectors, namely knockout vectors for targeted knockout of pig UGT2C1 genes;
(3) cell transfection: and (3) mixing the two positive targeting vectors to electrically transfect a target cell IPEC-J2, and performing screening verification and culture to obtain the pig UGT2C1 gene targeted knockout IPEC-J2 cell.
6. The method of claim 5, wherein the step (2) of performing colony PCR screening on the ligation products comprises: transforming the ligation product into escherichia coli for culture, selecting positive colonies, designing a colony PCR primer group according to the sgRNA guide sequence, and performing PCR screening verification to obtain two positive targeting vectors; the colony PCR primer group comprises two pairs of primer pairs, wherein the forward primer of each primer pair is a universal primer, the reverse primer is two sgRNA primers, and the specific sequence of each primer pair is as follows:
F1：CATATGCTTACCGTAACTTGAAAG
R1：CTTGGTGAAGGTACCAGGA
F2：CATATGCTTACCGTAACTTGAAAG
R2：TCAGCCTTGATGCGCTGCT。
7. the method of claim 5, wherein the screening and verifying process in step (3) is as follows:
(I) two positive targeting vectors are mixed to electrically transfect a target cell IPEC-J2, the genomic DNA of the cell is extracted by puromycin drug screening, a high-specificity primer is designed near a knockout target site for PCR amplification, and the sequence of the primer is as follows:
UGT2C1-seqF1:5’-CCGCCCTTCGCACCTCAT-3’；
UGT2C1-seqR1:5’-AGCCACCAACTCCCCACA-3’；
UGT2C1-seqF2:5’TCATACCAAAATCCCCTT-3’；
UGT2C1-seqR2:5’-TGCTGTAATACTCATCCC-3’；
(II) T7 Endonuclease I enzyme digestion PCR product identification effective targeting positive clone;
(III) sequencing and verifying the DNA PCR product of UGT2C1 gene knockout cells screened by T7 Endonuclease I enzyme digestion to obtain UGT2C1 gene knockout IPEC-J2 cells.
8. Use of the sgRNA guide sequence of claim 1, the double-stranded DNA of claim 2, the knock-out vector of claim 3, or the cell line of claim 4 in breeding for swine resistance.
9. The method of claim 5, wherein the method is used in breeding pigs for disease resistance.
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