SgRNA for targeted knockout of pig ATP11C gene, sgRNA composition, vector and application

Abstract

The invention discloses sgRNA for targeted knockout of a pig ATP11C gene, an sgRNA composition, a carrier and application, and belongs to the technical field of biology. And the sgRNA is any one of sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5 and sgRNA6, and the sgRNA is any one of the sgRNA1, the sgRNA2, the sgRNA3, the sgRNA4, the The sgRNA for specifically knocking out the pig ATP11C gene in a targeted manner is designed aiming at a pig ATP11C gene target spot, so that the pig ATP11C gene is efficiently and accurately specifically knocked out, the pig kidney fibroblasts with the ATP11C gene stably knocked out are obtained, proliferation of the pig kidney fibroblasts is remarkably inhibited after the ATP11C gene is knocked out, and the pig kidney fibroblasts with the ATP11C gene stably knocked out are inhibited. The invention lays a solid foundation for producing cloned pigs with related disease model characteristics through a somatic cell cloning technology, and further provides a favorable experimental material for researches such as treatment of ATP11C-related diseases and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a sgRNA, an sgRNA composition, a vector and an application thereof for targeted knockout of the porcine ATP11C gene. Background Art

[0002] ATP11C is a member of the P4-ATPase family, an enzyme that transports lipids across cell membranes. The gene encoding ATP11C is highly conserved and widely expressed in different species. ATP11C is involved in the process of flipping phospholipids from the outer layer of the lipid bilayer to the inner layer, which is essential for maintaining the asymmetry of the membrane and various cellular processes, and thus plays a role in intercellular signaling. In addition, ATP11C is involved in the excretion of drugs, affects tumorigenesis, and is essential for the activation and proliferation of immune cells. ATP11C has been shown to be associated with the occurrence of a variety of diseases, especially in autoimmune diseases and tumors. In autoimmune diseases, ATP11C, due to its role in regulating the lipid composition of immune cell membranes, leads to enhanced autoimmune responses, thereby causing diseases such as systemic lupus erythematosus and rheumatoid arthritis. Studies have found that the absence or dysfunction of ATP11C affects the activation of T cells and B cells and promotes the development of pathological conditions. In terms of tumors, the expression of ATP11C is associated with the invasiveness and metastasis of a variety of cancers. Its role in the tumor microenvironment may affect cell survival and proliferation, making tumor cells more resistant to chemotherapeutic drugs. Therefore, ATP11C plays a key role in both normal physiological functions and disease occurrence.

[0003] CRISPR / Cas9 technology is a powerful gene editing tool derived from the bacterial immune system. It uses guide RNA (gRNA) to guide the Cas9 protein to cut the target DNA, generating double-strand breaks. The cell then repairs the DNA through non-homologous end joining or homologous recombination, thereby achieving gene inactivation or precise insertion. This technology is widely used in basic research, disease model creation, gene therapy, and crop improvement. Due to its high efficiency, precision, and simplicity, it is rapidly changing the research and application landscape of gene editing.

[0004] Pigs are ideal for studying human diseases because of their high similarity to humans in physiological structure and genome. Pigs are large in size, which facilitates surgery and intervention, and their immune system is similar to that of humans, making them suitable for infection and vaccine research. In addition, pigs have a moderate growth cycle and can reproduce quickly, making them suitable for long-term research. Pigs can be used to study a variety of disease models, such as cardiovascular disease, diabetes, and neurodegenerative diseases, providing an important platform for basic research and preclinical research.

[0005] At present, there are few reports on the efficient and precise knockout of the pig ATP11C gene using CRISPR / Cas9 technology. Summary of the invention

[0006] The purpose of the present invention is to provide a sgRNA, sgRNA composition, vector and application for targeted knockout of pig ATP11C gene. In the present invention, a sgRNA for specifically targeting and knocking out pig ATP11C gene is designed for the pig ATP11C gene target, thereby efficiently and accurately knocking out the pig ATP11C gene specifically, and obtaining pig kidney fibroblasts with stable knockout of ATP11C gene, thereby providing favorable experimental materials for research on the treatment of diseases related to ATP11C.

[0007] In a first aspect, the present invention provides an sgRNA for targeted knockout of the pig ATP11C gene, wherein the sgRNA is any one of sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5, and sgRNA6; wherein the nucleotide sequence of sgRNA1 is as shown in SEQ ID NO: 1, the nucleotide sequence of sgRNA2 is as shown in SEQ ID NO: 2, the nucleotide sequence of sgRNA3 is as shown in SEQ ID NO: 3, the nucleotide sequence of sgRNA4 is as shown in SEQ ID NO: 4, the nucleotide sequence of sgRNA5 is as shown in SEQ ID NO: 5, and the nucleotide sequence of sgRNA6 is as shown in SEQ ID NO: 6.

[0008] In the present invention, sgRNA1 targets the sequence from positions 94466 to 94485 of the pig ATP11C gene, sgRNA2 targets the sequence from positions 94119 to 94138 of the pig ATP11C gene, sgRNA3 targets the sequence from positions 83879 to 83898 of the pig ATP11C gene, sgRNA4 targets the sequence from positions 83460 to 83479 of the pig ATP11C gene, sgRNA5 targets the sequence from positions 124184 to 124203 of the pig ATP11C gene, and sgRNA6 targets the sequence from positions 124103 to 124122 of the pig ATP11C gene; the above sgRNAs have high targeting efficiency and knockout efficiency.

[0009] In a second aspect, the present invention provides a sgRNA composition for targeted knockout of the pig ATP11C gene, the sgRNA composition comprising at least two of the above-mentioned sgRNA1, the above-mentioned sgRNA2, the above-mentioned sgRNA3, the above-mentioned sgRNA4, the above-mentioned sgRNA5, and the above-mentioned sgRNA6.

[0010] In the present invention, the inventors further studied and found that the use of sgRNA compositions can further improve the targeting efficiency and knockout efficiency.

[0011] In some embodiments, the sgRNA composition consists of sgRNA1 and sgRNA2, or consists of sgRNA3 and sgRNA4, or consists of sgRNA5 and sgRNA6.

[0012] In a third aspect, the present invention provides a CRISPR / Cas9 recombinant vector for targeted knockout of the pig ATP11C gene, comprising the above-mentioned sgRNA or any one of the above-mentioned sgRNA compositions.

[0013] In the present invention, the CRISPR / Cas9 recombinant vector can be conventionally selected according to actual use needs. For example, the PX459 (pSpCas9(BB)-2A-Puro) vector is preferred in the present invention.

[0014] In a fourth aspect, the present invention provides a method for constructing a CRISPR / Cas9 recombinant vector for targeted knockout of the pig ATP11C gene as described above, comprising the following steps: using BbSⅠ to cut the CRISPR / Cas9 vector to obtain a linearized vector fragment; annealing the sense chain and antisense chain of the above-mentioned sgRNA with a BbSⅠ restriction site, or the sense chain and antisense chain of the sgRNA in any of the above-mentioned sgRNA compositions, to obtain an annealed fragment; operably connecting the linearized vector fragment to the annealed fragment to obtain a CRISPR / Cas9 recombinant vector.

[0015] In some embodiments, the nucleotide sequences of the sense chain and antisense chain of sgRNA1 with a BbSⅠ cleavage site are shown in SEQ ID NOs: 7-8, respectively, the nucleotide sequences of the sense chain and antisense chain of sgRNA2 with a BbSⅠ cleavage site are shown in SEQ ID NOs: 9-10, respectively, the nucleotide sequences of the sense chain and antisense chain of sgRNA3 with a BbSⅠ cleavage site are shown in SEQ ID NOs: 11-12, respectively, the nucleotide sequences of the sense chain and antisense chain of sgRNA4 with a BbSⅠ cleavage site are shown in SEQ ID NOs: 13-14, respectively, the nucleotide sequences of the sense chain and antisense chain of sgRNA5 with a BbSⅠ cleavage site are shown in SEQ ID NOs: 15-16, respectively, and the nucleotide sequences of the sense chain and antisense chain of sgRNA6 with a BbSⅠ cleavage site are shown in SEQ ID NOs: 17-18, respectively.

[0016] In a fifth aspect, the present invention provides a kit for targeted knockout of the pig ATP11C gene, comprising the above-mentioned sgRNA, any one of the above-mentioned sgRNA compositions, the above-mentioned CRISPR / Cas9 recombinant vector, or the CRISPR / Cas9 recombinant vector constructed by the above-mentioned construction method.

[0017] The kit provided by the present invention also includes a transfection reagent and a knockout gene identification reagent. The knockout gene identification reagent preferably includes a PCR amplification primer pair, a PCR amplification reagent, and the like.

[0018] In a sixth aspect, the present invention provides the use of the above-mentioned sgRNA, any of the above-mentioned sgRNA compositions, the above-mentioned CRISPR / Cas9 recombinant vector, or the CRISPR / Cas9 recombinant vector constructed by the above-mentioned construction method in knocking out the pig ATP11C gene.

[0019] In the present invention, sgRNA has high targeting efficiency and knockout efficiency.

[0020] In the seventh aspect, the present invention provides a method for preparing a cell line for targeted knockout of the pig ATP11C gene, comprising the following steps: transfecting the above-mentioned CRISPR / Cas9 recombinant vector or the CRISPR / Cas9 recombinant vector constructed by the above-mentioned construction method into a cell line, and obtaining a cell line for targeted knockout of the ATP11C gene through positive screening.

[0021] In the present invention, transfection of the CRISPR / Cas9 recombinant vector into a cell line can be performed by conventional methods, such as electroporation, and positive screening can be performed by conventional methods.

[0022] In some embodiments, the cell line comprises porcine kidney fibroblasts.

[0023] In the present invention, the cell line can be routinely selected according to actual use needs, and is preferably porcine kidney fibroblasts.

[0024] In an eighth aspect, the present invention provides the use of the above-mentioned sgRNA, any of the above-mentioned sgRNA compositions, the above-mentioned CRISPR / Cas9 recombinant vector, or the CRISPR / Cas9 recombinant vector constructed by the above-mentioned construction method in inhibiting pig cell proliferation.

[0025] The beneficial effect of the present invention is that, different from the prior art, the present invention targets the pig ATP11C gene target, designs sgRNA that specifically targets and knocks out the pig ATP11C gene, thereby efficiently and accurately knocking out the pig ATP11C gene, and obtaining pig kidney fibroblasts with stable knockout of the ATP11C gene, which lays a solid foundation for producing cloned pigs with relevant disease model characteristics through somatic cell cloning technology, and further provides powerful experimental materials for research on disease treatment related to ATP11C. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The map of the PX459 (pSpCas9(BB)-2A-Puro) vector of the present invention; wherein there are two BbSⅠ restriction sites at 245bp and 267bp; Figure 2 The figure is a sequencing result diagram of the sgRNA sequence with the BbSⅠ restriction site inserted into the CRISPR / Cas9 recombinant vector containing 6 sgRNAs specifically targeting the porcine ATP11C gene in Example 2 of the present invention; wherein SG1 is a pSpCas9(BB)-2A-Puro vector containing sgRNA1, SG2 is a pSpCas9(BB)-2A-Puro vector containing sgRNA2, SG3 is a pSpCas9(BB)-2A-Puro vector containing sgRNA3, SG4 is a pSpCas9(BB)-2A-Puro vector containing sgRNA4, SG5 is a pSpCas9(BB)-2A-Puro vector containing sgRNA5, and SG6 is a pSpCas9(BB)-2A-Puro vector containing sgRNA6; Figure 3 This is a diagram of the qPCR detection results of the ATP11C gene in the cell line with targeted knockout of the porcine ATP11C gene in Example 4 of the present invention; wherein NC is an untransfected cell, SG1,2 is a cell containing SG1 and SG2, SG3,4 is a cell containing SG3 and SG4, and SG5,6 is a cell containing SG5 and SG6; Indicates significant difference (P<0.05), Indicates extremely significant difference (P<0.01); Figure 4 The results of western blot detection of the expression level of ATP11C gene in the cell line with targeted knockout of porcine ATP11C gene in Example 4 of the present invention are shown; wherein, Figure A is a western blot detection result diagram, and Figure B is a protein expression result diagram; NC is an untransfected cell, SG1,2 is a cell containing SG1 and SG2, SG3,4 is a cell containing SG3 and SG4, and SG5,6 is a cell containing SG5 and SG6; Indicates significant difference (P<0.05), Indicates extremely significant difference (P<0.01); Figure 5 The EdU staining result diagram of the effect of knocking out the porcine ATP11C gene on cell proliferation in Example 5 of the present invention, wherein Figure A is the staining result diagram, and Figure B is the EdU positive cell content result diagram; the scale of the image is 100 μm; Indicates significant difference (P<0.05), Indicates that the difference is extremely significant (P<0.01). DETAILED DESCRIPTION

[0027] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] The experimental methods without specific conditions in the examples are usually carried out according to conventional experimental methods in the field of molecular biology, including but not limited to the experimental methods described in Molecular Cloning: A Laboratory Manual by MR Green, Molecular Biology by Robert F. Weaver, etc., or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and biological materials used in the examples can be obtained from commercial channels unless otherwise specified.

[0029] In the present invention, the PX459 (pSpCas9(BB)-2A-Puro) vector plasmid is preserved by the Hubei Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Institute of Animal Husbandry and Veterinary Medicine, Hubei Academy of Agricultural Sciences, and the plasmid is extracted according to the instructions of the small-scale ultrapure plasmid extraction kit provided by Beijing Tiangen Biotechnology Co., Ltd.

[0030] Example 1 Design and acquisition of specific sgRNA for porcine ATP11C gene Targeting sequences of sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5, and sgRNA6 were designed for sequences at positions 94466 to 94485, positions 94119 to 94138, positions 83879 to 83898, positions 83460 to 83479, positions 124184 to 124203, and positions 124103 to 124122 of the porcine ATP11C gene, and their nucleotide sequences are shown below: sgRNA1: 5′-GTAGGGGCATATAACCCTAA-3′ (SEQ ID NO: 1); sgRNA2: 5′-AGCATGTAATGTCTCGTATG-3′ (SEQ ID NO: 2); sgRNA3: 5′-TACCACCATTAGTCTACAAT-3′ (SEQ ID NO: 3); sgRNA4: 5′-GTCAGCAACTAAGAGCTTAG-3′ (SEQ ID NO: 4); sgRNA5: 5′-TGCTTTTGTCAAAATACGGTA-3′ (SEQ ID NO: 5); sgRNA6: 5′-TCATTGAATGCTGCATAGAT-3′ (SEQ ID NO: 6).

[0031] To facilitate subsequent connection with the vector, BbSⅠ restriction site sequences were added to both ends of the above sgRNA, and the resulting double-stranded DNA sequences had the following nucleotide sequences: Sense strand of sgRNA1: 5′-CACCGTAGGGGCATATAACCCTAA-3′ (SEQ ID NO: 7); Antisense strand of sgRNA1: 5′-AAACTTAGGGTTATATGCCCCTAC-3′ (SEQ ID NO: 8); Sense strand of sgRNA2: 5′-CACCGAGCATGTAATGTCTCGTATG-3′ (SEQ ID NO: 9); Antisense strand of sgRNA2: 5′-AAACCATACGAGACATTACATGCTC-3′ (SEQ ID NO: 10); Sense strand of sgRNA3: 5′-CACCGTACCACCATTAGTCTACAAT-3′ (SEQ ID NO: 11); Antisense strand of sgRNA3: 5′-AAACATTGTAGACTAATGGTGGTAC-3′ (SEQ ID NO: 12); Sense strand of sgRNA4: 5′-CACCGTCAGCAACTAAGAGCTTAG-3′ (SEQ ID NO: 13); Antisense strand of sgRNA4: 5′-AAACCTAAGCTCTTAGTTGCTGAC-3′ (SEQ ID NO: 14); Sense strand of sgRNA5: 5′-CACCGTGCTTTGTCAAAATACGGTA-3′ (SEQ ID NO: 15); Antisense strand of sgRNA5: 5′-AAACTACCGTATTTTGACAAAGCAC-3′ (SEQ ID NO: 16); Sense strand of sgRNA6: 5′-CACCGTCATTGAATGCTGCATAGAT-3′ (SEQ ID NO: 17); Antisense strand of sgRNA6: 5′-AAACATCTATGCAGCATTCAATGAC-3′ (SEQ ID NO: 18).

[0032] The double-stranded DNA sequences of the above-mentioned sgRNAs were all commissioned to be synthesized by biological companies.

[0033] Example 2 Construction of porcine ATP11C gene knockout vector The method for constructing a CRISPR / Cas9 recombinant vector for targeted knockout of the porcine ATP11C gene comprises the following steps: 1) Use restriction endonuclease BbSⅠ to digest the pSpCas9(BB)-2A-Puro vector (vector map as shown in Figure 1 As shown), a linearized vector fragment was obtained; The enzyme digestion reaction system is: pSpCas9(BB)-2A-Puro vector 1.5μL (1000ng), restriction endonuclease BbSⅠ 1μL, Green Buffer 10x 1μL, ddH2O to 20μL; the enzyme digestion reaction procedure is as follows: 37°C, 30min.

[0034] 2) annealing the sense strand and antisense strand of sgRNA1, the sense strand and antisense strand of sgRNA2, the sense strand and antisense strand of sgRNA3, the sense strand and antisense strand of sgRNA4, the sense strand and antisense strand of sgRNA5, and the sense strand and antisense strand of sgRNA6 synthesized in Example 1, respectively, to form annealed fragments containing sticky ends of the BbSⅠ restriction site; The annealing reaction system was as follows: 2 μL NEB buffer, 1 μL sense chain sequence, 1 μL antisense chain sequence, and ddH2O was added to 20 μL. The annealing reaction procedure was as follows: 95°C, 5 min; 37°C, 1 h.

[0035] 3) Connect the sgRNA annealed fragment obtained in step 2 with the linearized vector fragment obtained in step 1) to obtain a connection product; The ligation reaction system was as follows: 3 μL of annealed fragment, 2 μL of linearized vector fragment, 1 μL of T4 DNA Ligase, 2 μL of Buf T4 Ligase, and ddH2O was added to make up to 20 μL. The ligation reaction procedure was as follows: 22°C, 1 h.

[0036] 4) Dilute the ligation product sample obtained in step 30 to a 10 mmol / L working solution, prepare a 20 mL system, and transform it into E. coli DH5α competent cells. Mix 5 mL of the ligation product and 50 mL of the DH5α competent cells, place on ice for 30 min, heat shock at 42°C for 90 s, and immediately place on ice for 2 min; add 1 mL of antibiotic-free culture medium, and resuscitate in a constant temperature shaker at 37°C, 200 rpm / min for 45 min; then suspend and mix the sample, spread it on an LB plate containing ampicillin (100 ng / mL Amp), and place it in a 37°C constant temperature incubator and invert overnight culture for 16 h.

[0037] 5) Pick the single colony obtained in step 4), perform colony PCR, and detect it by agarose gel electrophoresis; shake the qualified colony, send the bacterial solution to the company for sequencing detection using U6 primer, expand the positive single clone with correct sequencing, and extract the plasmid; the pSpCas9(BB)-2A-Puro vectors containing 6 sgRNAs with correct sequencing were named SG1 (pSpCas9(BB)-2A-Puro vector containing sgRNA1), SG2 (pSpCas9(BB)-2A-Puro vector containing sgRNA2), SG3 (pSpCas9(BB)-2A-Puro vector containing sgRNA3), SG4 (pSpCas9(BB)-2A-Puro vector containing sgRNA4), SG5 (pSpCas9(BB)-2A-Puro vector containing sgRNA5), SG6 (pSpCas9(BB)-2A-Puro vector containing sgRNA6), and their sequencing results are as follows: Figure 2 shown.

[0038] from Figure 2 It can be seen that the pig ATP11C gene knockout vector was successfully constructed.

[0039] Example 3 Preparation of cell lines for targeted knockout of porcine ATP11C gene This embodiment provides the preparation of a cell line for targeted knockout of the porcine ATP11C gene, specifically comprising the following steps: 1) Cell plating: porcine kidney fibroblasts were seeded in a six-well plate containing cell culture medium and grown to a density of about 80% before electroporation transfection; 2) Cleaning the electrophoresis cup: rinse the electrophoresis cup several times with alcohol, scrape the inside of the electrophoresis cup repeatedly with the electrophoresis gun tip until there is no cell residue, then add DPBS to clean the electrophoresis cup and pour out the remaining residual liquid, finally rinse the electrophoresis cup with electrophoresis solution and dry it in a 37℃ oven; 3) Digestion of cells: When the pig kidney fibroblasts grow to about 80% confluence and have good morphology, use 0.25% trypsin to digest the pig kidney fibroblasts (normal cell culture six-well plates are generally digested at 200mL~500mL / well of 0.25% trypsin. When the cells become round and out of contact and some cells fall off and suspend under a microscope, immediately add medium containing serum to terminate digestion). Repeatedly blow and beat to completely detach the adherent pig kidney fibroblasts and disperse them into single pig kidney fibroblasts; 4) Collect and rinse cells: Collect the digested and dispersed porcine kidney fibroblast suspension into a 1.5 mL sterile centrifuge tube, resuspend and rinse the cells with DPBS, centrifuge at 1500 rpm for 3 min, discard the supernatant, add electroporation buffer (KCl 120 mM, CaCl2 0.15 mM, Hepes 25 mM, EDTA 2 mM, anhydrous MgCl2 5 mM and K2HPO4 10 mM, pH 7.6) to re-rinse the cells, centrifuge again, centrifuge at 1500 rpm for 3 min, and discard the supernatant; according to the 100 mL system (cell volume is 1×10 6 5 mg of recombinant plasmid (prepared in Example 2, divided into three groups, the first group: SG1 and SG2 mixed at a ratio of 1:1; the second group: SG3 and SG4 mixed at a ratio of 1:1; the third group: SG5 and SG6 mixed at a ratio of 1:1) were added to a suitable amount of electroporation buffer, and the porcine kidney fibroblasts were resuspended by gently blowing to mix them evenly; 5) Transfection of cells by electroporation: When the porcine kidney fibroblasts are digested, turn on the electroporator in advance, and set the voltage, electric shock time, pulse number and other parameters to pre-run it. Transfer the cell electroporation mixture into a sterile clean electroporation cup of appropriate size, and use electroporation at 220V / cm, 3ms, one pulse to transfect the porcine kidney fibroblasts for 48h. After the electroporation is completed, culture the cells with DMEM medium containing 12% FBS; obtain the electrotransfected cells.

[0040] Example 4 Detection of cell lines with targeted knockout of porcine ATP11C gene 4.1 qPCR detection of ATP11C gene in cell lines with targeted knockout of porcine ATP11C gene The electroporated cells obtained in Example 3 were subjected to qPCR detection, which specifically included the following steps: 1) Cell RNA extraction and cDNA synthesis: When the cells in the 6-well plate (cells containing SG1 and SG2, recorded as SG1,2; cells containing SG3 and SG4, recorded as SG3,4; cells containing SG5 and SG6, recorded as SG5,6) have grown to 80-90%, the RNA of the cells to be tested is extracted by Ttizol precipitation method and the RNA concentration is measured and diluted to 300μg / μL. At the same time, RNA of non-transfected cells is extracted; then reverse transcribed into cDNA using RNA reverse transcription kit (Vazyme).

[0041] 2) Primer design: Based on the porcine ATP11C gene mRNA sequence in NCBI (accession number: NC_010461.5), a primer pair capable of detecting porcine ATP11C gene knockout was designed. After screening, a primer pair capable of achieving efficient porcine ATP11C gene detection was obtained, and its nucleotide sequence is shown below: ATP11C-1F: 5′-GCAGTGGAAGACAAGCTACAGGATC-3′ (SEQ ID NO: 19); ATP11C-1R: 5′-CCAGTAAGCACCCAGACTTTCAGAC-3′ (SEQ ID NO: 20); GAPDH was used as the internal reference gene, and its nucleotide sequence is shown below: GAPDH-F: 5′-TCGGAGTGAACGGATTTGGC-3′ (SEQ ID NO: 21); GAPDH-R: 5′-TGACAAGCTTCCCGTTCTCC-3′ (SEQ ID NO: 22).

[0042] 3) qPCR amplification: Using the cell cDNA obtained in step 1) as a template, and using the primer pair in step 2) to perform qPCR amplification, wherein the qPCR reaction system and reaction procedure are as follows: The reaction system of qPCR was: 2×Universal SYBR Green 10 μL, 10 μM upstream primer 0.4 μL, 10 μM downstream primer 0.4 μL, cDNA template 1.0 μL, ddH2O 8.2 μL.

[0043] The qPCR reaction procedure was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 5 s, annealing at 60°C for 30 s, and 40 cycles; the instrument automatically set the melting curve.

[0044] qPCR test results Figure 3 shown.

[0045] from Figure 3 It can be seen that compared with the control group (NC, untransfected cells), the expression level of ATP11C gene in the experimental groups (cells containing SG1 and SG2; cells containing SG3 and SG4; cells containing SG5 and SG6) was significantly downregulated, and the expression level of ATP11C gene in cells containing SG1 and SG2 was the lowest. The results showed that the combination of sgRNA1 and sgRNA2 can efficiently knock out the pig ATP11C gene.

[0046] 4.2 Western blot detection of ATP11C gene expression level in cell lines with targeted knockout of porcine ATP11C gene The cells after electroporation obtained in Example 3 were subjected to Western blotting detection, which specifically included the following steps: 1) Protein sample preparation: After removing the cell culture medium, wash the cells once with DPBS, then digest the cells with trypsin and add culture medium to terminate the digestion process, then centrifuge to remove the supernatant; add 100 µL of the prepared RIPA lysis buffer (containing 1% PMSF protease inhibitor) stored at -20°C and thawed before use to the cell pellet, lyse the cells on ice and place for 30 minutes (vortex and shake for 1 minute every 10 minutes); then, centrifuge at 12000 rpm for 15 minutes at 4°C, collect the supernatant and transfer it to a new 1.5 mL centrifuge tube; use the BCA protein assay kit to determine the protein concentration, and use the lysis buffer to adjust all samples to the same concentration; then, add 5×SDS-PAGE Loading Buffer, heat at 95°C for 10 minutes, and after the samples cool to room temperature, aliquot and store at -80°C.

[0047] 2) SDS-PAGE gel electrophoresis: First, make sure the glass plate is clean and dry, check that there is no dust on the surface, and then install the electrophoresis tank correctly; use distilled water to check if the system is leaking, let it stand for a few minutes, if the water level does not drop, it proves that the installation is correct and you can continue the experiment; then carefully inject the pre-prepared separation gel, make sure the gel surface is flat, if necessary, add anhydrous ethanol to help flatten the gel surface; leave it for about 30 minutes until the separation gel is completely solidified, then discard the anhydrous ethanol and use absorbent paper to remove the residue; slowly add the prepared concentrated gel on the upper layer of the separation gel, and avoid bubbles when inserting an appropriate amount of comb; leave it for about 15 minutes until the concentrated gel is completely solidified; after the gel is loaded in the electrophoresis tank, add an appropriate amount of electrophoresis buffer and slowly remove the comb; according to the experimental requirements, load the protein sample into the corresponding well; at the beginning of the electrophoresis process, run at a constant voltage of 80V for about 30 minutes until the protein marker is separated; then increase the voltage to 120V and continue electrophoresis for about 1.5 hours. The migration of the target protein is determined by observing the position of the marker. When the target protein reaches the predetermined position, the electrophoresis process is stopped.

[0048] 3) Transfer: Take out the glass plate from the electrophoresis tank, clean it, and cut the top corner of the glass plate with a plastic cutter. Gently pry open the glass plate with the glue plate, so that the glue remains on the glass slide; remove the concentrated gel and excess separation gel, leaving the protein maker area and the target protein area; cut a piece of PVDF membrane of similar size and soak it in high-purity methanol solution for about 30 seconds; the sandwich structure of the transfer membrane is: sponge, filter paper, PVDF membrane, glue, filter paper, sponge. After assembly, install the transfer clip into the tank in the correct direction, inject an appropriate volume of transfer solution, and place the entire transfer device in a 4°C refrigerator. Transfer the membrane at a constant current of 200mA for about 2 hours.

[0049] 4) Blocking: After transfer, remove the PVDF membrane and block it with 5% skimmed milk powder on a shaker at room temperature for 2 hours or at 4°C overnight.

[0050] 5) Primary antibody incubation: After blocking, wash the skimmed milk powder on the surface of the PVDF membrane with 1×TBST, cut the target protein bands according to the protein maker, and place the bands and the corresponding primary antibodies in an incubation box and incubate on a shaker at room temperature for 2 hours or overnight.

[0051] 6) Secondary antibody incubation: After the primary antibody incubation, the primary antibody was recovered and washed 4 times with 1× TBST, 5 times each time. Min; add the corresponding secondary antibody and incubate on a shaker at room temperature for 1 h; after the secondary antibody incubation, wash 4 times with 1× TBST, 5 min each time.

[0052] 7) Development: Add the prepared ECL working solution (make sure to prepare it before use); then immediately use a gel imaging instrument to scan the immunoblot, control the exposure intensity and exposure time until a suitable band image is displayed, and use ImageJ to perform grayscale analysis on the Western Blot bands.

[0053] Western blot results Figure 4 shown.

[0054] from Figure 4 It can be seen that compared with the control group (NC, non-transfected cells), the expression level of ATP11C protein in the experimental groups (cells containing SG1 and SG2; cells containing SG3 and SG4; cells containing SG5 and SG6) was significantly downregulated, and the expression level of ATP11C protein was lowest in cells containing SG1 and SG2. The results showed that the combination of sgRNA1 and sgRNA2 can efficiently knock out the pig ATP11C gene, resulting in a significant decrease in the expression level of ATP11C protein.

[0055] Example 5 Effect of knockout of porcine ATP11C gene on cell proliferation The cells containing SG1 and SG2 (ATP11C-KO) obtained in Example 3 were subjected to EdU staining (Biyuntian C0075S) when the cell confluence was 80%, and the untransfected control group was also subjected to the same treatment to observe the effect of knocking out the ATP11C gene on the proliferation of porcine kidney fibroblasts, specifically, comprising the following steps: 1. EdU labeling, fixation, washing and permeabilization of cultured porcine kidney fibroblasts 1) Culture an appropriate number of cells in a 6-well plate. After the cells have been cultured overnight and have returned to normal, perform the required drug treatment or other stimulation treatments. 2) Prepare 2X EdU working solution: dilute EdU (10mM) with cell culture medium at a ratio of 1:500 to obtain 2X EdU working solution (20μM); 3) Add an equal volume of 2X EdU working solution (20 μM) preheated at 37°C to a 6-well plate, so that the final concentration of EdU in the 6-well plate becomes 1X; 4) Continue to incubate the cells for 2 hours; 5) After EdU labeling of cells, remove the culture medium and add 1 ml of fixative (4% paraformaldehyde) and fix at room temperature for 15 minutes; 6) Remove the fixative and wash each well of cells three times with 1 ml of washing solution, each time for 3-5 minutes; 7) Remove the washing solution and use 1 ml of permeabilization solution (0.3% TritonX-100 in PBS) per well and incubate at room temperature for 10-15 minutes; 8) Remove the permeabilization solution and wash the cells 1-2 times with 1 ml of washing solution per well, each time for 3-5 minutes.

[0056] 2. EdU Detection 1) Prepare Click Additive Solution: Dissolve one tube of Click Additive in deionized water and mix until all dissolved. This is the Click Additive Solution. 2) Prepare Click reaction solution: the system of one well of a 6-well plate is: Click Reaction Buffer 430μL, CuSO4 20μL, Azide 5551μL, Click Additive Solution 50μL; 3) Remove the washing solution from the previous step; 4) Add 0.5 ml of Click reaction solution to each well and gently shake the culture plate to ensure that the reaction mixture can evenly cover the sample; 5) Incubate at room temperature in the dark for 30 minutes; 5) Aspirate the Click reaction solution and wash three times with washing solution, each time for 3-5 minutes.

[0057] 3. Nuclear Staining 1) Prepare 1X Hoechst 33342 solution: dilute Hoechst 33342 (1000X) in PBS at a ratio of 1:1000. 2) After removing the washing solution from the previous step, add 1 ml of 1X Hoechst 33342 solution to each well and incubate at room temperature in the dark for 10 minutes; 3) Wash three times with detergent, 3-5 minutes each time.

[0058] EdU staining results Figure 5 shown.

[0059] from Figure 5 It can be seen that knocking out the ATP11C gene significantly inhibited the proliferation of pig kidney fibroblasts.

[0060] In summary, the present invention targets the pig ATP11C gene target and designs sgRNA that specifically targets and knocks out the pig ATP11C gene, thereby efficiently and accurately knocking out the pig ATP11C gene, and obtaining pig kidney fibroblasts with stable knockout of the ATP11C gene. Moreover, knocking out the ATP11C gene significantly inhibits the proliferation of pig kidney fibroblasts, which provides favorable experimental materials for the study of the function of the pig ATP11C gene and the treatment of diseases related to ATP11C.

[0061] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0062] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A sgRNA for targeted knockout of the porcine ATP11C gene, characterized in that: The sgRNA is any one of sgRNA1, sgRNA2, sgRNA3, sgRNA4, sgRNA5, and sgRNA6; Wherein, the nucleotide sequence of the sgRNA1 is shown as SEQ ID NO: 1, the nucleotide sequence of the sgRNA2 is shown as SEQ ID NO: 2, the nucleotide sequence of the sgRNA3 is shown as SEQ ID NO: 3, the nucleotide sequence of the sgRNA4 is shown as SEQ ID NO: 4, the nucleotide sequence of the sgRNA5 is shown as SEQ ID NO: 5, and the nucleotide sequence of the sgRNA6 is shown as SEQ ID NO:

6.

2. A sgRNA composition for targeted knockout of the porcine ATP11C gene, characterized in that: The sgRNA composition comprises at least two of the sgRNA1, the sgRNA2, the sgRNA3, the sgRNA4, the sgRNA5, and the sgRNA6 according to claim 1.

3. The sgRNA composition according to claim 2, characterized in that The sgRNA composition consists of the sgRNA1 and the sgRNA2, or consists of the sgRNA3 and the sgRNA4, or consists of the sgRNA5 and the sgRNA6.

4. A CRISPR / Cas9 recombinant vector for targeted knockout of the porcine ATP11C gene, characterized in that: The invention comprises the sgRNA of claim 1 or the sgRNA composition of any one of claims 2 to 3.

5. A method for constructing a CRISPR / Cas9 recombinant vector for targeted knockout of the porcine ATP11C gene as claimed in claim 4, characterized in that: The steps include: Use BbSⅠ to digest the CRISPR / Cas9 vector to obtain a linearized vector fragment; Annealing the sense strand and antisense strand of the sgRNA according to claim 1 with a BbSⅠ restriction site, or the sense strand and antisense strand of the sgRNA in the sgRNA composition according to any one of claims 2 to 3, to obtain annealed fragments; The linearized vector fragment is operably linked to the annealed fragment to obtain a CRISPR / Cas9 recombinant vector.

6. A kit for targeted knockout of porcine ATP11C gene, characterized in that: The invention comprises the sgRNA according to claim 1, the sgRNA composition according to any one of claims 2 to 3, the CRISPR / Cas9 recombinant vector according to claim 4, or the CRISPR / Cas9 recombinant vector constructed by the construction method according to claim 5.

7. Use of the sgRNA according to claim 1, the sgRNA composition according to any one of claims 2 to 3, the CRISPR / Cas9 recombinant vector according to claim 4, or the CRISPR / Cas9 recombinant vector constructed by the construction method according to claim 5 in knocking out the porcine ATP11C gene.

8. A method for preparing a cell line for targeted knockout of porcine ATP11C gene, characterized in that: The steps include: The CRISPR / Cas9 recombinant vector according to claim 4 or the CRISPR / Cas9 recombinant vector constructed by the construction method according to claim 5 is transfected into a cell line, and a cell line with targeted knockout of the ATP11C gene is obtained through positive screening.

9. The preparation method according to claim 8, characterized in that: The cell line comprises porcine kidney fibroblasts.

10. Use of the sgRNA according to claim 1, the sgRNA composition according to any one of claims 2 to 3, the CRISPR / Cas9 recombinant vector according to claim 4, or the CRISPR / Cas9 recombinant vector constructed by the construction method according to claim 5 in inhibiting pig cell proliferation.

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