A method for constructing a pig SCD gene family member double gene mutant cell, a mutant cell line and a mutant animal

Abstract

The application provides a construction method of a pig SCD gene family member double-gene mutant cell, a mutant cell line and a mutant animal, and relates to the technical field of biomedicine. A gene editing target site is separated from a pig SCD1 gene, and two gene editing target sites are separated from a pig SCD5 gene, which can be applied to gene editing. Statistical results show that the targeting efficiency of the above sites is 90% and 82% respectively. The provided pig SCD1 and SCD5 double-gene editing sites provide effective targets for precise editing of the pig stearoyl-CoA desaturase gene family members, and simultaneously provide a pig SCD1 and SCD5 double-gene mutant cell model, which provides reliable research materials for lipid metabolism and pork quality improvement of pigs, and provides a new strategy for research and development of excellent pork quality pig breeds.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biomedical technology, and particularly relates to a construction method of a pig SCD gene family member double-gene mutant cell, a mutant cell line and a mutant animal. BACKGROUND

[0002] So far, gene editing technology has been widely applied in animal gene function research and livestock breeding. Gene editing mainly modifies, knocks out and inserts target genes to affect their expression and function. Currently, there are mainly zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology and clustered regularly interspaced short palindromic repeat (CRISPR) technology, especially the discovery and wide application of CRISPR-Cas9 gene editing technology, which can simply and efficiently complete base deletion, insertion and replacement modification in the target genome. Compared with ZFNs and TALEN technology, the construction and use of CRISPR-Cas9 system are more convenient, precise, efficient, short-period and low-cost, and it has great potential to edit and modify multiple genes at the same time.

[0003] Adipose deposition refers to the formation process of adipose tissue, which is formed by mature adipocytes through enlargement, proliferation and aggregation. Most of the space of adipocytes is occupied by lipid droplets (LD), which is a fat storage organelle composed of hydrophobic core of triacylglycerol (TAG) and cholesterol ester, and can be formed in almost all cells. In the core of LD, primary neutral lipids are sterol esters and TAG, and the main form is TAG (Guo et al., 2009b). LD includes lipases, membrane transport and structural proteins, participates in lipid synthesis, and separates the hydrophobic core from the aqueous cytosol by containing various proteins in the phospholipid monolayer. Glycerol triacetate is the most abundant type of fat, which is mainly composed of saturated fatty acids (SFAs) and unsaturated fatty acids (UFAs) containing 16 or 18 carbon atoms. Different proportions of SFAs and UFAs, especially stearic acid and oleic acid, can regulate cell membrane fluidity and signal transduction, and then affect cell growth and differentiation.

[0004] Mammalian stearoyl-CoA desaturase (SCD) is a ubiquitously expressed fatty acid Δ9 desaturase, which is a rate-limiting enzyme for the conversion of SFA to MUFA. Five SCD isoforms have been identified, but in pigs, only SCD1 and SCD5 isoforms have been found so far, and the two isoforms have 66.3% nucleic acid similarity and 61.7% amino acid similarity, both of which show similar Δ9 desaturase activity, especially their specific catalysis of 16:0 and 18:0 to form 16:1 and 18:1, which is an important target for fatty acid metabolism control. The disorder of the two fatty acid metabolisms mediated by SCD is related to several common diseases in humans, including obesity, diabetes, fatty liver, and cardiovascular and tumor diseases (Richard et al., 2009). Studies have shown that SCD-deficient mice can resist obesity and fatty acid degeneration caused by high-carbohydrate and high-fat diets (Ntmabi et al., 2002).

[0005] The pig SCD1 gene is located on chromosome 14, contains 6 exons, and encodes 359 amino acids. It is mainly expressed in adipose tissue, brain, liver and muscle tissue, and can be used as a potential biomarker for intramuscular fat deposition, and its genetic markers can be used for selecting the optimal fatty acid profile of pork. SCD5, also known as ACOD4 and FADS4, is located on chromosome 8, contains 5 exons, and encodes 332 amino acids. It is expressed in the brain, kidney and muscle tissue, and has been proven to play an important role in fatty acid composition and fat deposition (Ren et al., 2020).

[0006] Although some studies have partially explored the compensatory effect between SCD1 and SCD5, they are still limited to the functional study of single gene members, and the overall biological function of the gene family is still lacking. Since SCD5 has been found to exist only in several vertebrates including humans, the study of SCD5 is greatly limited, and the existing research is not sufficient to fully explain its specific function. SUMMARY

[0007] The purpose of the present application is to provide a method for constructing a pig SCD gene family member double-gene mutant cell, a mutant cell line and a mutant animal, and a cell model with SCD1 and SCD5 double-gene mutations, which provides research materials and methods for lipid metabolism and pork quality improvement.

[0008] The application provides a method for preparing a double gene mutant cell of a porcine stearoyl-CoA desaturase gene family member, taking an editing site of a porcine stearoyl-CoA desaturase gene family member SCD1 and SCD5 as a target, wherein the editing site of the SCD1 is located at 462-484 bp of NC_010456.5, and the editing site of the SCD5 is located at 12-34 bp and / or 323-345 bp of NC_010450.4.

[0009] Preferably, the editing site of the SCD1 comprises a sequence shown in SEQ ID No. 1.

[0010] The editing site of the SCD5 comprises a sequence shown in SEQ ID No. 2 and / or SEQ ID No. 3.

[0011] The application provides a group of sgRNAs designed for the editing sites of the porcine stearoyl-CoA desaturase gene family members SCD1 and SCD5, including sgRNA1-F and sgRNA1-R designed for the SCD1, wherein the nucleotide sequence of the sgRNA1-F is shown in SEQ ID No. 4, and the nucleotide sequence of the sgRNA1-R is shown in SEQ ID No. 5.

[0012] The sgRNA2-F, sgRNA2-R, sgRNA3-F and sgRNA3-R designed for the SCD5, wherein the nucleotide sequence of the sgRNA2-F is shown in SEQ ID No. 6, the nucleotide sequence of the sgRNA2-R is shown in SEQ ID No. 7, the nucleotide sequence of the sgRNA3-F is shown in SEQ ID No. 8, and the nucleotide sequence of the sgRNA3-R is shown in SEQ ID No. 9.

[0013] The editing site of the SCD1 is located at 462-484 bp of NC_010456.5, and the editing site of the SCD5 is located at 12-34 bp and / or 323-345 bp of NC_010450.4.

[0014] The application also provides a group of Cas9gRNA expression vectors for preparing a double gene mutant cell of a porcine stearoyl-CoA desaturase gene family member, wherein the Cas9gRNA expression vectors respectively contain editing sites of porcine stearoyl-CoA desaturase gene family members SCD1 and SCD5.

[0015] The editing site of the SCD1 is located at 462-484 bp of NC_010456.5, and the editing site of the SCD5 is located at 12-34 bp and / or 323-345 bp of NC_010450.4.

[0016] Preferably, the pSpCas9(BB)-2A-Puro vector is used as the backbone.

[0017] The application also provides a method for preparing a porcine stearoyl-CoA desaturase gene family member double gene mutant cell line, comprising the following steps: (1) annealing sgRNA1-F and sgRNA1-R, sgRNA2-F and sgRNA2-R, and sgRNA3-F and sgRNA3-R in the sgRNA respectively, and then connecting into a linearized pSpCas9(BB)-2A-Puro vector to obtain Cas9gRNA expression vectors PX459-SCD1-sgRNA1, PX459-SCD5-sgRNA2, and PX459-SCD5-sgRNA3;

[0018] (2) transforming the Cas9gRNA expression vectors PX459-SCD1-sgRNA1, PX459-SCD5-sgRNA2, and PX459-SCD5-sgRNA3 into E. coli DH5α competent cells respectively, and extracting plasmids respectively;

[0019] (3) mixing the three plasmids extracted in step (2) and co-transfecting porcine somatic cells, and screening positive cells to obtain the porcine stearoyl-CoA desaturase gene family member double gene mutant cell line.

[0020] Preferably, the porcine somatic cells in step (3) comprise porcine kidney epithelial cells.

[0021] The application also provides a porcine stearoyl-CoA desaturase gene family member double gene mutant cell line prepared by the above method.

[0022] Preferably, the mutation comprises deletion and / or insertion.

[0023] The application also provides application of the sgRNA, the Cas9gRNA expression vector, or the porcine stearoyl-CoA desaturase gene family member double gene mutant cell line in breeding improved animals.

[0024] Beneficial effects: the application provides a method for preparing a pig stearoyl-CoA desaturase gene family member double gene mutation cell, and specifically discloses a pig mammal stearoyl-CoA desaturase gene family member SCD1 and SCD5 gene editing site in a pig genome and application thereof. The application separates one gene editing target site from a pig SCD1 gene and two gene editing target sites from a pig SCD5 gene, and the above sites are located in the coding region 1st exon of the gene and can be specifically recognized by Cas9 endonuclease, thereby mediating a double-strand break, and making the gene mutate through a non-homologous end joining mode under the action of a self-repairing system. Statistical results show that the targeting efficiency of the above sites is 90% and 82% respectively. The provided pig SCD1 and SCD5 double gene editing sites provide an effective target for precise editing of the pig stearoyl-CoA desaturase gene family member, and also provide a pig SCD1 and SCD5 double gene mutation cell model, which provides reliable research materials for lipid metabolism and pig breed meat quality improvement, and also provides a new strategy for research and development of excellent meat quality pig breeds. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0026] Figure 1 It is a schematic diagram of the gene editing target site on the SCD1 and SCD5 genes, the SCD1 gene is a sgRNA guided single site point editing, and the SCD5 gene is a double site point editing guided by two sgRNAs;

[0027] Figure 2 It is a schematic diagram of part of the PX459 plasmid element including the insertion site of sgRNA in the PX459 plasmid, and BbsI is a linearization enzyme cutting site;

[0028] Figure 3 It is a PX459 plasmid linearization electrophoresis diagram;

[0029] Figure 4 It is a recombinant plasmid sequencing detection result diagram;

[0030] Figure 5Figure 1-3 are SCD1 and SCD5 gene PCR electrophoretograms of monoclonal cell lines; M is DL100 bp DNA Marker, 4 is blank control, and 5 is positive control in the SCD1 gene PCR electrophoretogram; M is DL2000 bp DNA Marker, 4 and 5 are heterozygote positive controls, and 6 is blank control in the SCD5 gene PCR electrophoretogram;

[0031] Figure 6 Figure 1-3 are SCD1 and SCD5 gene PCR electrophoretograms of monoclonal cell lines; M is DL100 bp DNA Marker, 4 is blank control, and 5 is positive control in the SCD1 gene PCR electrophoretogram; M is DL2000 bp DNA Marker, 4 and 5 are heterozygote positive controls, and 6 is blank control in the SCD5 gene PCR electrophoretogram;

[0032] Figure 7 Figure 1-3 are SCD1 and SCD5 gene PCR electrophoretograms of monoclonal cell lines; M is DL100 bp DNA Marker, 4 is blank control, and 5 is positive control in the SCD1 gene PCR electrophoretogram; M is DL2000 bp DNA Marker, 4 and 5 are heterozygote positive controls, and 6 is blank control in the SCD5 gene PCR electrophoretogram;

[0033] Figure 8 Figure 1-3 are SCD1 and SCD5 gene PCR electrophoretograms of monoclonal cell lines; M is DL100 bp DNA Marker, 4 is blank control, and 5 is positive control in the SCD1 gene PCR electrophoretogram; M is DL2000 bp DNA Marker, 4 and 5 are heterozygote positive controls, and 6 is blank control in the SCD5 gene PCR electrophoretogram; DETAILED DESCRIPTION

[0034] The application provides a method for preparing a porcine stearoyl-CoA desaturase gene family member double gene mutation cell, taking an editing site of porcine stearoyl-CoA desaturase gene family members SCD1 and SCD5 as a target, the editing site of SCD1 is located at 462-484 bp of NC_010456.5, and the editing site of SCD5 is located at 12-34 bp and / or 323-345 bp of NC_010450.4.

[0035] The pig SCD1 and SCD5 double gene editing site provided by the application is preferably a DNA sequence of three 23 deoxyribonucleotides, the first sequence is 5'-AAGTAATGGCCCCCAGACCGCGG-3'(SEQ ID No.1), the site is accurately located at 462-484 of the SCD1 gene (NC_010456.5) of chromosome 14, the coordinate of the chromosome is 111462022-111462044, and is located in the first exon of the SCD1 gene. Figure 1 The second sequence is 5'-CTTAGGCCACGGTGAACGCCTGG-3'(SEQ ID No.2), the site is accurately located at 12-34 of the SCD5 gene (NC_010450.4) of chromosome 8, the coordinate of the chromosome is 135507358-135507380, Figure 1) and the third segment sequence is 5'-CGCGGCAGGACATCGTCTGGAGG-3' (SEQ ID No. 3), the accurate location of the site in the pig genome is 323-345 of SCD5 gene of chromosome 8, the coordinates of the chromosome are 135507669-135507691 Figure 1 Both the second and third segment sequences are located in the first exon of SCD5 gene. The version number of the pig genome referred to in the present application is preferably NCBI:txid9823.

[0036] The present application provides a group of sgRNAs designed for editing sites of pig stearoyl-CoA desaturase gene family members SCD1 and SCD5, including sgRNA1-F and sgRNA1-R designed for SCD1, the nucleotide sequence of the sgRNA1-F is shown as SEQ ID No. 4, and the nucleotide sequence of the sgRNA1-R is shown as SEQ ID No. 5;

[0037] sgRNA2-F, sgRNA2-R, sgRNA3-F and sgRNA3-R designed for SCD5, the nucleotide sequence of the sgRNA2-F is shown as SEQ ID No. 6, the nucleotide sequence of the sgRNA2-R is shown as SEQ ID No. 7, the nucleotide sequence of the sgRNA3-F is shown as SEQ ID No. 8, and the nucleotide sequence of the sgRNA3-R is shown as SEQ ID No. 9;

[0038] The editing site of the SCD1 is located at 462-484 bp of NC_010456.5, and the editing site of the SCD5 is located at 12-34 bp and / or 323-345 bp of NC_010450.4.

[0039] The present application preferably adds CACCG at the 5' end of the sense strand of the complementary sequence of the sgRNA (sgRNA1, sgRNA2 and sgRNA3) corresponding to the above three editing target sites (SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3), and adds AAAC enzyme cutting site at the 5' end of the template strand, so that the oligonucleotide chain is complementary to the cohesive end of the vector plasmid, thereby obtaining the sgRNA sequence corresponding to each editing target site, as shown in Table 1.

[0040] Table 1 oligo sequence

[0041]

[0042]

[0043] The application also provides a set of Cas9gRNA expression vectors for preparing double-gene mutant cells of porcine stearoyl-CoA desaturase gene family members, wherein the Cas9gRNA expression vectors respectively contain editing sites of porcine stearoyl-CoA desaturase gene family members SCD1 and SCD5.

[0044] The editing site of the SCD1 is located at 462-484 bp of NC_010456.5, and the editing site of the SCD5 is located at 12-34 bp and / or 323-345 bp of NC_010450.4.

[0045] Preferably, the application uses a pSpCas9(BB)-2A-Puro (PX459 for short) vector as a skeleton to construct Cas9gRNA expression vectors PX459-SCD1-sgRNA1, PX459-SCD5-sgRNA2 and PX459-SCD5-sgRNA3 which respectively insert editing target sites of porcine stearoyl-CoA desaturase family (SCD) members SCD1 and SCD5.

[0046] The application does not have special limitations on the construction method of the Cas9gRNA expression vector, and preferably includes annealing sgRNA1-F and sgRNA1-R, sgRNA2-F and sgRNA2-R, and sgRNA3-F and sgRNA3-R in the sgRNA above, respectively, and then connecting them into a linearized pSpCas9(BB)-2A-Puro vector to obtain Cas9gRNA expression vectors PX459-SCD1-sgRNA1, PX459-SCD5-sgRNA2 and PX459-SCD5-sgRNA3. Preferably, the application anneals the sgRNA pairs respectively, and then inserts them into the linearized PX459 vector, wherein the linearization preferably includes enzyme digestion of the PX459 vector by BbsI.

[0047] The application also provides a method for preparing a double-gene mutant cell line of porcine stearoyl-CoA desaturase gene family members, which includes the following steps: (1) annealing sgRNA1-F and sgRNA1-R, sgRNA2-F and sgRNA2-R, and sgRNA3-F and sgRNA3-R in the sgRNA above, respectively, and then connecting them into a linearized pSpCas9(BB)-2A-Puro vector to obtain Cas9gRNA expression vectors PX459-SCD1-sgRNA1, PX459-SCD5-sgRNA2 and PX459-SCD5-sgRNA3;

[0048] (2) Cas9gRNA expression vectors PX459-SCD1-sgRNA1, PX459-SCD5-sgRNA2 and PX459-SCD5-sgRNA3 are respectively transformed into E. coli DH5α competent cells, and plasmids are respectively extracted;

[0049] (3) The three plasmids obtained in step (2) are mixed and co-transfected into porcine somatic cells, and a positive cell line of double gene mutation of the porcine stearoyl-CoA desaturase gene family is obtained.

[0050] The content in step (1) of the present application is the same as the construction method of the Cas9gRNA expression vector described above, and will not be repeated here.

[0051] After obtaining the Cas9gRNA expression vector, the Cas9gRNA expression vectors PX459-SCD1-sgRNA1, PX459-SCD5-sgRNA2 and PX459-SCD5-sgRNA3 are respectively transformed into E. coli DH5α competent cells, and plasmids are respectively extracted. The method of transformation is not particularly limited, and preferably includes heat shock method. After the transformation, recovery is performed, and the single colony is selected and cultured in LB medium containing Amp. The bacterial liquid is sent to the company for sequencing detection with U6 primers. The positive single colony with correct sequencing is cultured and the plasmid is extracted.

[0052] After obtaining the plasmid, the three plasmids obtained in step (2) are mixed and co-transfected into porcine somatic cells, and a positive cell line of double gene mutation of the porcine stearoyl-CoA desaturase gene family is obtained.

[0053] The ratio of the three plasmids is preferably 1:1:1 (total concentration of 10 μg per 100 μL system). The method of transfection preferably includes electroporation. The type of porcine somatic cells is not particularly limited, and porcine kidney epithelial cells (PK-15) are used in the examples, but it cannot be recognized as the entire protection scope of the present application.

[0054] The present application also provides a porcine stearoyl-CoA desaturase gene family double gene mutant cell line constructed by the above method.

[0055] The mutation of the application preferably comprises deletion and / or insertion. In an embodiment of the application, 50 cell lines are randomly selected from 92 positive monoclonal cell lines for detection by using the above technical solution, wherein 23 cell lines have AGAC base deletion in the SCD1 gene, 7 cell lines have CCGCGGG base deletion in the SCD1 gene, and another 15 cell lines have A, CC or hetero peak increase in the SCD1 gene. According to statistics, the SCD1 gene of a total of 45 cell lines is edited, and the editing efficiency reaches 90%. In the above 50 monoclonal cell lines, the SCD5 gene of a total of 41 cell lines is edited, and the editing efficiency is 82%, wherein 27 homozygous cell lines are present at the designed target site, and the shearing at the designed target site is achieved, realizing the gene site-directed deletion guided by two sgRNAs. The SCD1 and SCD5 genes of a total of 38 cells in the 50 monoclonal cell lines are edited, and the double-gene editing efficiency reaches 76%, wherein 3 clones are homozygous effective double-gene deletion cell lines.

[0056] The application also provides the use of the above sgRNA or the above Cas9gRNA expression vector or the above pig stearoyl-CoA desaturase gene family member double-gene mutant cell line in breeding improved animals.

[0057] The double-gene mutation technology provided by the application provides a reliable means and material for lipid metabolism and pork quality improvement of pigs, and has a very important role for the research of excellent economic traits of livestock and the breeding of high-quality pork varieties.

[0058] In order to further illustrate the application, the construction method of the pig SCD gene family member double-gene mutant cell, mutant cell line and mutant animal provided by the application is described in detail below in combination with the drawings and examples, but they should not be understood as limiting the scope of protection of the application.

[0059] Example 1

[0060] Construction of Cas9gRNA expression vectors PX459-SCD1-sgRNA1, PX459-SCD5-sgRNA2 and PX459-SCD5-sgRNA3 respectively inserted with pig stearoyl-CoA desaturase family (SCD) member SCD1 and SCD5 editing target sites

[0061] (1) Sources of main reagents and materials

[0062] The pSpCas9(BB)-2A-Puro vector plasmid was preserved by the Institute of Animal Husbandry and Veterinary Medicine, Hubei Academy of Agricultural Sciences / Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Hubei Province; the single nucleotide chain, primers used in the experiment and DH5a competent cells were purchased from Shanghai Sangon Biotech Co., Ltd. The BbsI restriction endonuclease was purchased from Fermentas. The recombinant vector was prepared according to the instructions of the small-scale plasmid extraction kit provided by Beijing Tiangen Biotech Co., Ltd. The DNA gel recovery kit was purchased from Qiagen Company. The sequencing was completed by Shanghai Genechem Co., Ltd.

[0063] The Cas9 gRNA expression vector is based on the PX459 vector as a skeleton, and then the required guide sequence is inserted according to the fixed oligo design mode. The guide sequence and the sequencing primer are shown in Table 1, which are synthesized by Shanghai Genechem Co., Ltd. in dry powder form, transported, and stored. The primer is diluted with sterilized ddH2O to a 10 μmol / L solution and stored at -20°C for standby use.

[0064] (2) Operation steps

[0065] sgRNAs (sgRNA1, sgRNA2 and sgRNA3) corresponding to three editing target sites (SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3) were designed, and CACCG was added at the 5' end of the sense strand of the two complementary sequences, and AAAC enzyme cutting site was added at the 5' end of the template strand, so that the oligonucleotide chain was complementary to the cohesive end of the vector plasmid. The above oligonucleotide chain was sent to the company for synthesis.

[0066] The synthesized sgRNA sample was diluted to 10 μmol / L working solution, and a 20 μL system was prepared: 10×NEB Buffer 2 μL, oligo sgRNA F 2 μL, oligo sgRNA R 2 μL and ddH2O 14 μL. After mixing and short centrifugation, react at 95°C for 5 min, and naturally anneal at room temperature.

[0067] The PX459 plasmid shown in Figure 2 was subjected to enzyme digestion with BbsI, and a 20 μL system was prepared: 10×FD Buffer 2 μL, BbsI (10 U / μL) 2 μL, PX459 plasmid (1000 ng / μL) 2 μL and ddH2O 14 μL. Linearization was performed at 37°C for 40 min, and the target fragment was recovered after agarose gel electrophoresis (gel concentration was 1%, electrophoresis voltage was 120V, and time was 35 min), and the results are shown in Figure 3 , and the band size after enzyme digestion was correct.

[0068] The annealing product and the enzyme digestion product were respectively prepared into a 20 μL system: 2x Solution I 10 μL, PX459 enzyme digestion product (50 ng / μL) 2 μL, annealing product (2 μmol / L) 5 μL and ddH2O 3 μL. The ligation was performed at 37 °C for 2 h.

[0069] The ligation product was transformed into E. coli DH5α competent cells. 10 μL of the ligation product and 10 μL of DH5α competent cells were mixed, incubated in an ice bath for 30 min, and then immediately placed on ice for 2 min after heating at 42 °C for 90 s.

[0070] 1 mL of antibiotic-free LB medium was added, and the cells were recovered at 37 °C on a constant temperature shaker at 200 rpm / min for 45 min.

[0071] The cells were centrifuged at 8000 rpm / min for 1 min on a table centrifuge, and then 900 μL of LB medium was removed. The remaining 100 μL of sample was suspended and mixed, and then plated on an LB plate containing ampicillin (100 ng / ml Amp) and incubated at 37 °C in a constant temperature incubator overnight for 16 h.

[0072] A single colony was picked and cultured in LB medium containing 100 ng / ml Amp, and the bacterial solution was sent to the company for sequencing detection using U6 primers (primer sequence see Table 1). The sequencing results are shown in Figure 4 As shown in Table 1, the three pairs of sgRNA were integrated into PX459 at the corresponding sites, and the PX459-SCD1-sgRNA1 sequencing map corresponds to sgRNA1-R in Table 1, the PX459-SCD5-sgRNA2 sequencing map corresponds to sgRNA2-R in Table 1, and the PX459-SCD5-sgRNA3 sequencing map corresponds to sgRNA3-R in Table 1.

[0073] Example 2

[0074] Screening of porcine transgenic cell lines after electroporation transfection

[0075] (1) Sources of main reagents and materials

[0076] Wild-type PK-15 (porcine kidney epithelial cells) was preserved by the Hubei Provincial Key Laboratory of Animal Embryo Engineering and Molecular Breeding, Institute of Animal Husbandry and Veterinary Medicine, Hubei Academy of Agricultural Sciences; fetal bovine serum and DMEM high-glucose medium were purchased from Gibco Company. Antibiotics were purchased from Sigma. DMSO was purchased from Invitrogen. The electroporation buffer was prepared in the laboratory. The BTX2001 cell electrofusion instrument was purchased from the United States BTX Company.

[0077] (2) Operation steps

[0078] Cell plating:

[0079] PK15 cells were inoculated in cell culture six-well plates and grown to about 80% confluence before electroporation transfection.

[0080] Digest the cells:

[0081] When the cells grow to about 80% confluence and have a good morphology, digest the cells with 0.25% trypsin (normal cell culture six-well plates are generally digested with 200 μL-500 μL / well of 0.25% trypsin, and when the cells are rounded and partially detached under a microscope, add serum-containing medium to terminate digestion), repeatedly blow to completely detach the adherent cells and disperse them into single cells.

[0082] Collect and rinse the cells

[0083] Collect the cell suspension after digestion and blowing 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, CaCl20.15 mM, Hepes 25 mM, EDTA 2 mM, anhydrous MgCl25 mM, and K2HPO410 mM, pH 7.6) to resuspend and rinse the cells, centrifuge again at 1500 rpm for 3 min, discard the supernatant. Add 5 μg of recombinant plasmid to the appropriate amount of electroporation buffer in a 100 μL system (cell amount is 1 x 10 6 ) and resuspend the cells by gently blowing to mix them evenly.

[0084] Transfect the cells by electroporation

[0085] Start the electroporator in advance when the cells are digested, and set the voltage, pulse time, and pulse number parameters for pre-running. Place the cell electroporation mixture in a sterile clean electroporation cup of appropriate size, and transfect the cells by electroporation at 220 V / cm, 3 ms, and one pulse. After electroporation, culture the cells in DMEM medium containing 12% FBS.

[0086] Screen and culture the cells

[0087] Culture the cells for 48 h after electroporation to restore normal growth, culture and screen the cells with 12% FBS / DMEM containing 2 μg / mL Puromycin, and replace the new screening medium every day to make the cells single, and pick single clones and expand the culture after the screened single cells proliferate into cell clusters.

[0088] Example 3

[0089] Identify the transgenic homozygous cell line and statistically analyze the targeting efficiency

[0090] (1) Main reagents and material sources

[0091] PowerPol 2x PCR Mix was purchased from Wuhan Aibotek Biotechnology Co., Ltd.; mammalian genomic DNA extraction kit was purchased from Beijing Tiangen Biotech Co., Ltd.; nucleic acid concentration detector was a product of Thermo, USA. Sequencing was completed by Shengong Bioengineering (Shanghai) Co., Ltd.

[0092] The pair of specific primers for detecting positive clone SCD1 were SCD1-F and SCD1-R, and the expected length of the original PCR product was about 368 bp. The pair of specific primers for detecting SCD5 were SCD5-F and SCD5-R, and the expected length of the original PCR product was about 473 bp, and the length of the band after deletion was 162 bp.

[0093] Primers were synthesized by Shengong Bioengineering (Shanghai) Co., Ltd., and were divided, transported and stored in the form of dry powder. The primers were diluted with sterilized ddH2O to a solution of 10 μmol / L, and stored at -20℃ for standby.

[0094] (2) Operation steps

[0095] Genomic DNA of the single clone cell line was extracted according to the instructions of the mammalian genomic DNA extraction kit of Tiangen Biotech Co., Ltd., and its concentration was detected by a nucleic acid concentration detector and its integrity was detected by agarose gel electrophoresis. Finally, the concentration was adjusted to 500 ng / μL for standby for PCR detection.

[0096] 25 μL PCR reaction system: PowerPol 2x PCR Mix 12.5 μL, upstream primer (10 μmol / L) 0.5 μL, downstream primer (10 μmol / L) 0.5 μL, DNA template (500 ng / μL) and ddH2O 10.5 μL. After adding the above ingredients on ice and mixing thoroughly, the following program was performed on the PCR instrument: 98℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 60℃ annealing for 30 s, 72℃ extension for 10 s, 32 cycles; 72℃ final extension for 5 min; 4℃ storage.

[0097] 5 μL of the PCR reaction product was electrophoresed in a 2% agarose gel at a voltage of 120 V for 30 min, and the band size was observed in a gel imager. The results are shown in Figure 5 , and the band size was as expected. The remaining product was sent to Shengong Bioengineering (Shanghai) Co., Ltd. for sequencing to confirm the accurate sequence of gene editing, and the results are shown in Figure 6 and Figure 7As shown, the SCD1 gene of the monoclonal cell line is gene edited at the expected site, resulting in multiple mutant types, respectively, deletion of 7 bp of CCGCGGG, deletion of 4 bp of AGAC, and addition of CC base and addition of A base, all of which can cause frame shift mutation, which is effective editing; the SCD5 gene in the monoclonal cell line is gene edited at the two sgRNA-guided preset sites, a total of 311 bp is deleted, which can cause frame shift mutation, which is effective editing.

[0098] Statistical gene editing efficiency:

[0099] After the single cell line after Puromycin screening is proliferated into a cell mass, a single clone thereof is picked and cultured (from a 48-well plate to a 24-well plate, then to a 12-well plate and a six-well plate). When the cells are subcultured to a 12-well plate or a 24-well plate, the genomic DNA thereof is extracted after trypsin digestion for PCR detection, and the remaining part of the cells is continuously cultured. If the identification result is a positive clone, the cells are expanded and stored, and if the identification result is a negative clone, the cells are destroyed.

[0100] As shown in the results, Figure 8 92 monoclonal cell lines were selected in the experiment, 50 cell lines were randomly selected for detection, 23 cell lines of SCD1 gene deleted AGAC base, 7 cell lines of SCD1 gene deleted CCGCGGG base, and another 15 cell lines of SCD1 gene appeared to increase base A, CC or hetero peak, etc. After statistics, 45 cell lines of SCD1 gene occurred base editing, the editing efficiency reached 90%. Among the above 50 monoclonal cell lines, 41 cell lines of SCD5 gene occurred base editing, the editing efficiency was 82%, of which 27 were homozygous cell lines, and all appeared to be cut at the designed target site, realizing the gene site-directed deletion guided by two sgRNAs. Among the 50 monoclonal cell lines, 38 cells of SCD1 and SCD5 genes occurred base editing, the double gene editing efficiency reached 76%, of which 3 clones were effective double gene deletion cell lines.

[0101] Table 2 targeting efficiency statistics

[0102]

[0103] Although the above embodiment has made a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. A method for preparing a porcine stearoyl-CoA desaturase gene family member double gene mutant cell, characterized in that, The editing site of a porcine stearoyl-CoA desaturase gene family member SCD1 and SCD5 is taken as a target to design sgRNA for gene editing, wherein the editing site of the SCD1 is located at 462~484bp of NC_010456.5, and the editing site of the SCD5 is located at 12~34bp and 323~345bp of NC_010450.4; The editing site of the SCD1 comprises a sequence shown in SEQ ID No.

1. The editing site of the SCD5 comprises a sequence shown in SEQ ID No. 2 and / or SEQ ID No.

3. The nucleotide sequences of sgRNA1-F and sgRNA1-R designed for the SCD1 are shown in SEQ ID No. 4-SEQ ID No.

5. The nucleotide sequences of sgRNA2-F, sgRNA2-R, sgRNA3-F and sgRNA3-R designed for the SCD5 are shown in SEQ ID No. 6-SEQ ID No.

9.

2. A set of sgRNAs designed against editing sites of porcine stearoyl-CoA desaturase gene family members SCD1 and SCD5, characterized in that, The nucleotide sequences of sgRNA1-F and sgRNA1-R designed for the SCD1 are shown in SEQ ID No. 4-SEQ ID No.

5. The nucleotide sequences of sgRNA2-F, sgRNA2-R, sgRNA3-F and sgRNA3-R designed for the SCD5 are shown in SEQ ID No. 6-SEQ ID No.

9. The editing site of the SCD1 is located at 462~484bp of NC_010456.5, and the editing site of the SCD5 is located at 12~34bp and 323~345bp of NC_010450.

4.

3. A set of Cas9 gRNA expression vectors for preparing double gene mutant cells of members of the porcine stearoyl-CoA desaturase gene family, characterized in that The Cas9 gRNA expression vector contains sgRNA designed for the editing site of a porcine stearoyl-CoA desaturase gene family member SCD1 and SCD5; The editing site of the SCD1 is located at 462~484bp of NC_010456.5, and the editing site of the SCD5 is located at 12~34bp and 323~345bp of NC_010450.

4. The editing site of the SCD1 comprises a sequence shown in SEQ ID No.

1. The editing site of the SCD5 comprises a sequence shown in SEQ ID No. 2 and / or SEQ ID No.

3. The nucleotide sequences of sgRNA1-F and sgRNA1-R designed for the SCD1 are shown in SEQ ID No. 4-SEQ ID No.

5. The nucleotide sequences of sgRNA2-F, sgRNA2-R, sgRNA3-F and sgRNA3-R designed for the SCD5 are shown in SEQ ID No. 6-SEQ ID No.

9.

4. The Cas9 gRNA expression vector of claim 3, wherein, The pSpCas9 (BB)-2A-Puro vector is taken as a backbone.

5. A method for preparing a porcine stearoyl-CoA desaturase gene family member double gene mutant cell line, characterized in that, The method comprises the following steps: (1) annealing sgRNA1-F and sgRNA1-R, sgRNA2-F and sgRNA2-R, and sgRNA3-F and sgRNA3-R in the sgRNA of claim 2 respectively, and then connecting into the linearized pSpCas9 (BB)-2A-Puro vector to obtain Cas9gRNA expression vectors PX459-SCD1-sgRNA1, PX459-SCD5-sgRNA2 and PX459-SCD5-sgRNA3; (2) transforming the Cas9gRNA expression vectors PX459-SCD1-sgRNA1, PX459-SCD5-sgRNA2 and PX459-SCD5-sgRNA3 into E. coli DH5α competent cells respectively, and extracting plasmids respectively; (3) mixing the three kinds of plasmids obtained in step (2) and co-transfecting porcine somatic cells, and screening positive cells to obtain the porcine stearoyl-CoA desaturase gene family member double gene mutant cell line.

6. The method of claim 5, wherein, The porcine somatic cells in step (3) comprise porcine kidney epithelial cells.

7. The porcine stearoyl-CoA desaturase gene family member double gene mutant cell line obtained by the method of claim 5 or 6.

8. The porcine stearoyl-CoA desaturase gene family member dual-gene mutant cell line according to claim 7, characterized in that, The mutation comprises deletion and / or insertion.

9. Use of the sgRNA of claim 2 or the Cas9gRNA expression vector of claim 3 or 4 or the porcine stearoyl-CoA desaturase gene family member double gene mutant cell line of claim 7 or 8 in breeding improved animals.

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