Pig hbb gene site-directed modification system and application

Abstract

The application discloses a pig HBB gene site-directed modification system and application. The application provides a system for site-directed modification of a pig HBB gene, the first carrier, the second carrier and the third carrier contained in the system can express gene editing proteins and sgRNA, three target sites of the HBB gene are effectively cleaved, a site-directed modification fragment of a donor DNA is used to replace a to-be-site-directed modification fragment of the target site, and accurate deletion of bases from 176 to 179 of a CDS sequence of the HBB gene is realized. 41‑42(‑CTTT) The model accurately simulates the most common beta thalassemia mutation genotype beta of Chinese population, provides a precise model for analyzing the pathogenesis of beta thalassemia of Chinese population and developing a new treatment method.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene editing, in particular to a pig HBB gene site-directed modification system and application. BACKGROUND

[0002] Beta thalassemia is a common recessive genetic autosomal blood disease, mainly due to deletion or mutation of beta globin gene, resulting in synthesis disorder of beta globin chain, imbalance of alpha and non-alpha globin chain synthesis, and hemolytic anemia. According to the existing epidemiological survey data, the beta thalassemia gene carrying rate of Guangdong population is as high as 2.54%, and the beta thalassemia gene carrying rate of Guangxi population is as high as 6.78%-7.97%. In Southeast Asian countries and provinces in southern China, the beta globin gene CD41-42(-CTTT) mutation accounts for more than 36% of beta thalassemia mutations, and its homozygote shows severe thalassemia. A suitable animal model is an important means to study the mechanism of beta thalassemia and the effect of gene therapy. Although the beta thalassemia animal model has developed rapidly, most of these animal models are based on the IVS-2-654 mutation which is more common in foreign countries, and do not include the main mutation site CD41-42(-CTTT) of the beta globin gene in the Chinese population. Therefore, it is particularly important to establish a disease model that accurately simulates the mutation of human beta CD41-42(-CTTT) gene, for the mechanism of beta thalassemia and the development of new treatment methods in China. Pigs are very good animal models for human diseases, and are very close to humans in terms of organ size, physiology, and pathology. The application value of using pigs to prepare a beta thalassemia disease model is much higher than that of small animals such as mice, rats, and rabbits, and has more important reference value for treating human beta thalassemia. SUMMARY

[0003] The purpose of the present application is to provide a pig HBB gene site-directed modification system and application.

[0004] In order to achieve the purpose of the present application, in a first aspect, the present application provides a CRISPR / Cas vector targeting pig HBB gene, wherein the nucleotide sequence of the sgRNA action site is selected from at least one of the sequences shown in SEQ ID NO: 1, 2 or 3.

[0005] Preferably, the nucleotide sequence of the sgRNA action site is selected from any two of SEQ ID NO: 1, 2 or 3.

[0006] In a second aspect, the present application provides a pig HBB gene site-directed modification system, comprising the CRISPR / Cas vector and donor DNA.

[0007] The donor DNA contains a porcine HBB gene exon 2 site-directed modification fragment, wherein the site-directed modification fragment refers to deletion of bases 176-179 of the porcine HBB gene.

[0008] Further, the CRISPR / Cas vector comprises a gene editing protein expression cassette and an sgRNA expression cassette.

[0009] The gene editing protein can be selected from Cas9, Cas9n, Cpf1 or C2c2, preferably Cas9.

[0010] The backbone vector of the CRISPR / Cas vector can be selected from pX330, pX260, pX334, pX335, pX458, pX459, pX461, pX462, pX551 or pX552, preferably pX458.

[0011] Further, when the nucleotide sequence of the sgRNA action site is as shown in SEQ ID NO: 1, the nucleotide sequence of the donor DNA is as shown in SEQ ID NO: 4.

[0012] Further, when the nucleotide sequence of the sgRNA action site is as shown in SEQ ID NO: 2, the nucleotide sequence of the donor DNA is as shown in SEQ ID NO: 5.

[0013] Further, when the nucleotide sequence of the sgRNA action site is as shown in SEQ ID NO: 3, the nucleotide sequence of the donor DNA is as shown in SEQ ID NO: 6.

[0014] In a third aspect, the present application provides any of the following applications of the system:

[0015] (a) constructing a HBB gene site-directed modification cell line;

[0016] (b) preparing a human p-thalassemia cell line model;

[0017] (c) constructing a human beta-thalassemia pig model.

[0018] In a fourth aspect, the present application provides a method for preparing a porcine HBB gene site-directed modification cell, comprising introducing the system into a target cell to obtain a HBB gene site-directed modification cell.

[0019] Preferably, the target cell is a porcine fibroblast cell, more preferably a porcine fetal fibroblast cell.

[0020] Preferably, the method for introduction comprises electroporation or liposome transfection.

[0021] Preferably, the HBB gene site-directed modified cell is obtained by screening and identification after the introduction operation.

[0022] Preferably, the screening comprises screening the monoclonal cells by flow sorting.

[0023] Preferably, the identification comprises sequencing identification.

[0024] In a fifth aspect, the present application provides a pig HBB gene site-directed modified cell prepared according to the method.

[0025] In a sixth aspect, the present application provides a method for preparing a pig with HBB gene site-directed modification, comprising the steps of: transplanting the pig HBB gene site-directed modified cell into a pig oocyte whose nucleus has been removed to obtain a recombined cloned embryo; and transplanting the recombined cloned embryo into a sow to obtain the pig with HBB gene site-directed modification through pregnancy.

[0026] Preferably, the method further comprises the step of identifying the genetically edited pig after the genetically edited pig is born.

[0027] Preferably, the identification comprises sequencing identification.

[0028] The purposes of the present application can also be further achieved by the following technical measures.

[0029] The present application provides a system for HBB gene site-directed modification, comprising a first vector, a second vector, a third vector and a donor DNA.

[0030] The first vector comprises a gene editing protein expression cassette and a first sgRNA (SEQ ID NO: 1) expression cassette.

[0031] The second vector comprises a gene editing protein expression cassette and a second sgRNA (SEQ ID NO: 2) expression cassette.

[0032] The third vector comprises a gene editing protein expression cassette and a third sgRNA (SEQ ID NO: 3) expression cassette.

[0033] The first sgRNA, the second sgRNA and the third sgRNA are respectively targeted to three target sites of the HBB gene.

[0034] The donor DNA contains a HBB gene 2nd exon site-directed modification fragment, and the site-directed modification fragment is used to replace the HBB gene site-directed modification fragment.

[0035] The HBB gene site-directed modification is to delete the 176th-179th base of the pig HBB gene sequence.

[0036] Furthermore, the nucleotide sequence encoding the first sgRNA is shown in SEQ ID NO: 1;

[0037] Preferably, the nucleotide sequence encoding the second sgRNA is the one shown in SEQ ID NO: 2;

[0038] Preferably, the nucleotide sequence encoding the third sgRNA is the one shown in SEQ ID NO: 3.

[0039] Furthermore, the donor DNA is the nucleotide sequence shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

[0040] Furthermore, the gene-editing protein includes Cas9, Cas9n, Cpf1, or C2c2, preferably Cas9.

[0041] Furthermore, both the first carrier and the second carrier independently include pX330, pX260, pX334, pX335, pX458, pX459, pX461, pX462, pX551 or pX552, preferably pX458.

[0042] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0043] This invention provides a system for site-specific modification of the porcine HBB gene. The system contains a first vector, a second vector, and a third vector that can express gene-editing proteins and sgRNA, effectively digesting three target sites of the HBB gene. The system utilizes the site-specific modification fragment of donor DNA to replace the target fragment, achieving precise deletion of bases 176–179 of the HBB gene sequence. This precisely simulates the most common β-thalassemia mutant genotype in the Chinese population. 41-42(-CTTT) The model provides a precise model for elucidating the pathogenesis of β-thalassemia in the Chinese population and developing novel treatment methods. Attached Figure Description

[0044] Figure 1 This is a schematic diagram of pig HBB gene targeting in Example 1 of the present invention. The agaa part is the targeting region.

[0045] Figure 2 The sequencing results are those of sgRNA ligated to pX458 vector in Example 1 of this invention.

[0046] Figure 3 This serves as a verification of the sgRNA editing efficiency in Example 1 of the present invention. The editing efficiency of sgRNA-1 was 31%, sgRNA-2 was 29%, sgRNA-7 was 29%, and no editing occurred with the other sgRNAs.

[0047] Figure 4 Sequencing results of the Donor vector provided for Example 2 of the present application. The dark shaded area is the sequence upstream of the deletion fragment, and the light shaded area is the sequence downstream of the deletion fragment. The bases in the box are synonymous substitutions, which prevent the sgRNA from editing the Donor vector.

[0048] Figure 5 A schematic diagram for constructing pig embryonic fibroblast (PEF) cells in which the HBB gene is precisely deleted by 4 bases in Example 2 of the present application.

[0049] Figure 6 Growth of the monoclonal cells after flow sorting in Example 3 of the present application.

[0050] Figure 7 Genotypes of the monoclonal cells obtained in Example 3 of the present application. DETAILED DESCRIPTION

[0051] The present application provides a system for site-directed modification of the HBB gene, which contains a first vector, a second vector, a third vector and a donor DNA, wherein the first vector comprises a gene editing protein expression cassette and a first sgRNA expression cassette, the second vector comprises a gene editing protein expression cassette and a second sgRNA expression cassette, the third vector comprises a gene editing protein expression cassette and a third sgRNA expression cassette, the first sgRNA, the second sgRNA and the third sgRNA are respectively targeted to three target sites of the HBB gene; the donor DNA contains a modification fragment of the HBB gene, which is used to replace the site-directed modification fragment of the HBB gene, and the site-directed modification of the HBB gene is to delete the 176th-179th bases of the pig HBB gene sequence.

[0052] In the system, the first sgRNA, the second sgRNA, the third sgRNA or a combination of any two sgRNAs can target the target fragment, the gene editing protein can perform enzyme cutting on the target site, and the donor DNA can be used to realize sequence recombination. The system can delete the 176th to 179th bases of the pig HBB gene sequence without changing other amino acids of the HBB. The donor DNA is a replacement template for the target sequence to be modified. Under the guidance of the first sgRNA, the second sgRNA or the third sgRNA which specifically recognizes the sequence near the 738th amino acid site of the HBB gene, the gene editing protein performs enzyme cutting on the target fragment and guides the donor DNA sequence to replace the original homologous fragment in the cell, so as to achieve the purpose of deleting the 176th to 179th bases of the HBB gene sequence. The system provided by the application can accurately delete the 176th to 179th bases of the HBB gene, and can avoid destroying or changing the normal expression of the remaining amino acids of the HBB gene, thereby accurately simulating the p CD41 -42(-CTTT) mutation.

[0053] It should be noted that the gene editing protein can effectively perform enzyme cutting in various cells and guide sequence recombination after enzyme cutting, and has the advantages of wide application range, high enzyme cutting efficiency and the like. The type of the gene editing protein is not limited, as long as it can realize the function of genome editing. The first sgRNA, the second sgRNA and the third sgRNA can realize the precise targeting function of the gene editing protein to the sequence near the 176th to 179th sites of the HBB gene, and the specific sequence is not limited. As long as the sequence can realize the precise targeting function, the donor DNA can replace the target fragment to realize sequence recombination, and the specific sequence is not limited. As long as the 176th to 179th sites can be deleted, the 176th to 179th sites of the HBB gene are deleted.

[0054] In a preferred embodiment, the nucleotide sequence encoding the first sgRNA is shown in SEQ ID NO: 1, the nucleotide sequence encoding the second sgRNA is shown in SEQ ID NO: 2, and the nucleotide sequence encoding the third sgRNA is shown in SEQ ID NO: 3. The targeting of this scheme is stronger, and the modification is more accurate.

[0055] In preferred embodiments, the gene editing protein comprises Cas9, Cas9n, Cpfl or C2c2, preferably Cas9. The first vector, the second vector and the third vector each independently comprises pX330, pX260, pX334, pX335, pX458, pX459, pX461, pX462, pX551 or pX552, preferably pX458. Cas9, pX458 is universal and has high versatility and product maturity, and using it as a gene editing vector skeleton can achieve higher enzyme cutting efficiency.

[0056] In preferred embodiments, the first vector, the second vector and the third vector of the present application are each a recombinant plasmid comprising a gene editing vector skeleton and a sequence encoding sgRNA, wherein the gene editing vector skeleton can be a CRISPR plasmid, a TALEN plasmid or a zinc finger plasmid, preferably a CRISPR plasmid.

[0057] In some specific embodiments, the nucleotide sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 are respectively connected to the vector skeleton, and the first vector, the second vector and the third vector are obtained by screening positive clones.

[0058] The present application also provides the use of the above-mentioned system in (a)-(c) as follows:

[0059] (a) constructing a cell line for site-directed modification of HBB gene;

[0060] (b) preparing a human beta thalassemia cell line model;

[0061] (c) constructing a human beta thalassemia pig model.

[0062] The above-mentioned system provided by the present application can achieve site-directed modification of HBB gene. Using the system, a cell line for site-directed modification of HBB gene can be constructed.

[0063] The present application also provides a method for preparing a cell for site-directed modification of HBB gene and the prepared cell, the method comprising introducing the system of the present application into a target cell to obtain a cell for site-directed modification of HBB gene. The target cell is preferably a pig fibroblast, and more preferably a pig fetal fibroblast. Compared with other cells, a pig fetal fibroblast has higher cloning efficiency. The method for introduction is preferably electroporation or lipofection, and more preferably electroporation, which has higher transfection efficiency.

[0064] In a preferred embodiment, the HBB gene site-directed modified cells are obtained by screening and identification after the system is introduced into the target cells. The screening method is preferably screening the monoclonal cells by flow sorting, and the monoclonal cells are identified whether they are the cells with the deletion of the 176th-179th sites of the HBB gene, and the identification method is preferably sequencing identification.

[0065] In some embodiments, the DNA of the monoclonal cells can be extracted, PCR amplification is performed using the primers shown in SEQ ID NO: 7-8, and the amplification product is sequenced to confirm whether the cells achieve the precise modification.

[0066] The above-mentioned HBB gene site-directed modified cells can be further used to prepare gene edited pigs. The cells are transplanted into enucleated oocytes to obtain recombined cloned embryos, the recombined cloned embryos are transplanted into a mother to be pregnant, and the gene edited pigs with the HBB gene site-directed modification are obtained.

[0067] In a preferred embodiment, the gene edited pigs need to be identified after birth, preferably sequencing identification.

[0068] In some embodiments, the DNA of the gene edited pigs can be extracted, PCR amplification is performed using the primers shown in SEQ ID NO: 7-8, and the amplification product is sequenced to confirm whether the pigs achieve the precise modification.

[0069] The following examples are used to illustrate the present application, but are not used to limit the scope of the present application. If not specifically indicated, the examples are all according to the conventional experimental conditions, such as Sambrook et al. Molecular Cloning: a Laboratory Manual (2001), or according to the conditions suggested by the manufacturer's instructions.

[0070] The main reagents used in the following examples are as follows:

[0071] Collagenase type IV used for isolating porcine fetal fibroblasts was purchased from Sigma;

[0072] DMEM, FBS, PS, NEAA, Glutamine, Trypsase used for cell culture were all purchased from Gibco;

[0073] The cell and ear tissue DNA extraction kit was purchased from Tiangen Biochemical Technology Co., Ltd.;

[0074] The primers were synthesized by Beijing Chengke Biological Technology Co., Ltd.;

[0075] KOD FX PCR enzyme used for PCR was purchased from TOYOBO.

[0076] Main instruments:

[0077] CO2 incubator (Thermo Scientific, 3131 / 3111);

[0078] Fluorescence inverted microscope (LEICA, DMI66B);

[0079] PCR instrument (BIO-RID, C1000 Touch);

[0080] Gel imaging system (BIO-RID, Universal Hood II);

[0081] Micro-manipulation system (Eppendorf, Celltram vario);

[0082] Cell flow sorter (Aria III).

[0083] Construction and activity detection of vectors in Example 1

[0084] 1. Taking pig HBB gene as the target sequence, sgRNA was scored by using sgRNA analysis tool CRISPOR (crispor.tefor.net), and sgRNA close to the 176-179 sites and with higher score was selected, as follows:

[0085] sg-1: 5'-TTGTCTACCCCTGGACTCAG-3' (SEQ ID NO: 1);

[0086] sg-2: 5'-TGACGGCATCGGCATTGGAC-3' (SEQ ID NO: 2);

[0087] sg-3: 5'-CTCGAAGAACCTCTGAGTCC-3';

[0088] sg-4: 5'-CGAAGAACCTCTGAGTCCAG-3';

[0089] sg-5: 5'-GTGAATGTGGACGAAGTTGG-3';

[0090] sg-6: 5'-GGTATCCAGGGCTTCAGGAG-3';

[0091] sg-7: 5'-GGCTGCTGGTTGTCTACCCC-3' (SEQ ID NO: 3).

[0092] To facilitate the connection with the vector backbone, the above-mentioned 7 sgRNA sequences are added with a linker sequence and a complementary oligonucleotide primer is synthesized.

[0093] 2, construct the vector, respectively named pX458-HBB-sgRNA-1 to pX458-HBB-sgRNA-7:

[0094] The oligonucleotide primer synthesized in step 1 is treated at 98℃ for 10 min, and then naturally cooled to room temperature for annealing.

[0095] The pX458 backbone vector containing the Cas9 sequence is digested with restriction enzyme Bbs I at 37℃ for 2h, and the linearized fragment is recovered by gel cutting.

[0096] Mix the above-mentioned annealed double-stranded fragment with the linear vector fragment, and connect at 16℃ for 1h, then transform into the competent cells of Top10 or DH5α, spread and grow on the LB plate containing ampicillin, then pick single colonies for expansion culture and sequencing. The sequencing primer is as follows:

[0097] U6-FWD: 5'-GAGGGCCTATTTCCCATGATT-3'.

[0098] The positive clones are cultured to obtain pX458-HBB-sgRNA-1 to pX458-HBB-sgRNA-7 plasmids, which are used for subsequent cell transfection. The plasmid extraction uses Endo-Free Plasmid Maxi Kit.

[0099] 3, cell transfection

[0100] The primary porcine fetal fibroblasts are resuscitated into a 6cm dish one day before transfection, and when the cells reach 70-80% confluence, the cell transfection is performed. The transfection steps are strictly performed according to the Basic Primary 20 Fibroblasts Nucleofector Kit (Lonza) kit instructions. Specifically, 5μg of recombinant plasmid obtained by Example 1 is used to transfect porcine fetal fibroblasts by electroporation, and 7 kinds of transfected cells are obtained.

[0101] The schematic diagram of porcine HBB gene targeting is shown in Figure 1 , and the sequencing results of sgRNA connecting pX458 vector are shown in Figure 2 .

[0102] 4, activity detection

[0103] 48h after electroporation, cells were collected, and genomic DNA was extracted, PCR amplified, and the activity of sgRNA was detected by mismatch endonuclease T7EnI.

[0104] PCR amplification was performed on the extracted genomic DNA using the primer pair HBB-2F-737: 5'-CTGCAGAGTCCCCAGCTATG-3' (SEQ ID NO: 7) and HBB-2R-737: 5'-ATTCAAGCCTCACCCTGTGG-3' (SEQ ID NO: 8), and after denaturation and annealing of the amplification product, T7EnI enzyme was added for enzyme digestion. After 1h of enzyme digestion, gel electrophoresis was performed for detection.

[0105] The results show that Figure 3 sgRNA-1, sgRNA-2 and sgRNA-7 effectively edited the target region of the HBB gene, with an editing efficiency of 31% for sgRNA-1, 29% for sgRNA-2 and 29% for sgRNA-7, and the remaining sgRNAs did not edit the target region of the HBB gene.

[0106] Example 2 Donor DNA sequence design

[0107] The donor DNA was designed according to the first sgRNA, the second sgRNA and the third sgRNA sequence, as follows:

[0108] The HBB-sg1-Donor sequence is shown as SEQ ID NO: 4;

[0109] The HBB-sg2-Donor sequence is shown as SEQ ID NO: 5;

[0110] The HBB-sg7-Donor sequence is shown as SEQ ID NO: 6.

[0111] The above sequences were connected to the pUC57 vector as double-stranded donor sequences, and sequencing verification was performed Figure 4 When the double-stranded Donor sequence replaces the wild-type sequence, the 176th to 179th sites of the pig HBB gene are deleted. The precise mutation mode diagram of the pig HBB gene is shown in Figure 5 .

[0112] Example 3 Establishment of pig fetal fibroblasts with precise deletion of the 176th to 179th sites of the HBB gene and genotype verification

[0113] 1. Preparation of pig fetal fibroblasts

[0114] The 35-day-old pig embryos were removed from the head, tail, limbs, internal organs and bones, and the blood was cleaned. The fetus was cut for 30 min with a bent eye scissors to ensure that it was cut into small pieces. The cut fetus was sucked into a 15 mL centrifuge tube with a blue gun head of the scissors, 5 mL complete medium was added, and after natural settlement for several minutes, the upper solution was removed, and a few drops of fetal bovine serum were added to the lower tissue block. A 15 cm glass Pasteur pipette was bent at a sharp end 1 cm, and the tissue block was sucked out and spread on two T75 culture bottles with the bottom up. 15 mL of complete medium was added to the opposite side, and after 6-8 h, the culture bottle was carefully turned over, and the tissue block was immersed in the culture medium. The liquid was changed every two days, and when the cells grew to cover the T75 culture bottle, they were frozen for use. Among them, the pig embryos were taken from the experimental base pig farm of the Beijing Institute of Animal Husbandry and Veterinary Medicine of the Chinese Academy of Agricultural Sciences.

[0115] 2. Cell transfection

[0116] The primary porcine fetal fibroblasts were resuscitated into a 10 cm dish one day before transfection. When the cells reached 70-80% confluence, they were co-transfected with 5 μg pX458-HBB-sgRNA-1 plasmid or 5 μg pX458-HBB-sgRNA-2 plasmid or 5 μg pX458-HBB-sgRNA-7 plasmid and 5 μg corresponding Donor plasmid (HBB-sg1-Donor or HBB-sg2-Donor or HBB-sg7-Donor) into porcine fetal fibroblasts. The transfection steps were strictly in accordance with the Basic Primary Fibroblasts Nucleofector Kit (Lonza) kit instructions.

[0117] 3. Screening of positive monoclonal cells

[0118] After 36 h of electroporation, the cells were collected and sorted into 96-well plates by flow cytometry and cultured, and the culture medium was changed every 3 days. The sorted cells were cultured for about 10 days, and the cells in the 96-well plate were observed to be full, and then the full monoclonal cells were subcultured into 48-well plates. When the cells in the 48-well plate were full, part of the cells were used to extract genomic DNA to identify the genotype.

[0119] 4. Identification of positive monoclonal cells

[0120] The monoclonal cells picked were identified: the extracted genomic DNA was used as a template, and the extracted DNA genome was amplified with the upstream and downstream primers shown in nucleotide sequences HBB-2F-737: 5'-CTGCAGAGTCCCCAGCTATG-3' (SEQ ID NO: 7) and HBB-2R-737: 5'-ATTCAAGCCTCACCCTGTGG-3' (SEQ ID NO: 8), to obtain a 737 bp fragment. The amplification conditions were 94°C for 5 min; 98°C for 30 s, 62.6°C for 30 s, 68°C for 100 s, 34 cycles; 72°C for 5 min. The band was observed by 2% agarose gel electrophoresis, and the PCR product was sent to Beijing Tianyi Huiyuan Company for sequencing. According to the sequencing, the pig fibroblasts with accurate deletion of the 176th-179th site of the HBB gene were screened and used as donor cells for nuclear transfer.

[0121] 5. Experimental results

[0122] The sequencing results showed that a plurality of pig fibroblasts with accurate deletion of the 176th-179th site of the HBB gene were successfully obtained in this embodiment. Figure 6 The sequencing results of the positive cells are shown in Figure 7 .

[0123] Example 4: Preparation of gene-edited pigs with accurate deletion of the 176th-179th site of the HBB gene by somatic cell nuclear transfer technology

[0124] The homozygous knockout positive cells obtained in Example 3 were used as nuclear transfer donor cells, and in vitro matured 40 h young pig oocytes were used as nuclear transfer recipient cells. The nuclear transfer donor cells were moved into the enucleated oocytes, and after electric fusion and activation, recombined cloned embryos were constructed. The cloned recombined embryos in good development state were selected and surgically transplanted into the uterus of a naturally estrous multiparous white female pig for pregnancy. The steps of the surgical embryo transfer were as follows: the recipient female pig was induced for anesthesia by intravenous injection of a Zoletil anesthetic, and the injection dose was 5 mg / kg of body weight. After anesthesia, the recipient female pig was moved to a surgical frame and fixed in a supine position, and was anesthetized by a breathing machine (the isoflurane concentration was 3%-4%). A surgical incision about 8 cm long was made on the middle line of the recipient female pig's abdomen, and the ovary, oviduct and uterus were exposed. A glass tube for embryo transfer was used to enter about 5 cm along the oviduct umbrella, and the cloned recombined embryos in good development state were transplanted to the ampulla-isthmus junction of the oviduct. After embryo transplantation, the technical personnel observed the recipient female pig regularly, and used a B-mode ultrasonic wave to check the pregnancy of the recipient female pig.

[0125] After the piglets were born, ear tissue was cut and genomic DNA was extracted, and the nucleotide sequences shown in SEQ ID NO: 7-8 were used for PCR amplification, and the PCR amplification product was sequenced to detect the genotype.

[0126] While the application has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various modifications or changes can be made therein without departing from the spirit and scope thereof. Accordingly, it is intended that all such modifications and changes be included within the scope of the application as claimed.

Claims

1. A pig HBB gene site-directed modification system characterized in that, The CRISPR / Cas vector targeting the pig HBB gene and the donor DNA; The CRISPR / Cas vector targeting the pig HBB gene, the nucleotide sequence encoding the sgRNA thereof is selected from at least one of the sequences shown as SEQ ID NO: 1, 2 or 3; The donor DNA contains a pig HBB gene exon 2 site-directed modification fragment, wherein the site-directed modification fragment refers to deletion of the 176th-179th base of the pig HBB gene; When the nucleotide sequence encoding the sgRNA is shown as SEQ ID NO: 1, the nucleotide sequence of the donor DNA is shown as SEQ ID NO: 4; and / or, When the nucleotide sequence encoding the sgRNA is shown as SEQ ID NO: 2, the nucleotide sequence of the donor DNA is shown as SEQ ID NO: 5; and / or, When the nucleotide sequence encoding the sgRNA is shown as SEQ ID NO: 3, the nucleotide sequence of the donor DNA is shown as SEQ ID NO:

6.

2. The system of claim 1, wherein, The CRISPR / Cas vector targeting the pig HBB gene, the nucleotide sequence encoding the sgRNA thereof is selected from any two of SEQ ID NO: 1, 2 or 3.

3. The system of claim 1 or 2, wherein, The CRISPR / Cas vector comprises a gene editing protein expression cassette and an sgRNA expression cassette.

4. The system of claim 3, wherein, The gene editing protein is selected from Cas9, Cas9n, Cpfl or C2c2; and / or, The backbone vector of the CRISPR / Cas vector is selected from pX330, pX260, pX334, pX335, pX458, pX459, pX461, pX462, pX551 or pX552.

5. The system of claim 4, wherein, The gene editing protein is Cas9; and / or, The backbone vector of the CRISPR / Cas vector is pX458.

6. Use of the system of any one of claims 1-5 in constructing a cell line with site-directed modification of the HBB gene. The cell line is a pig fibroblast cell.

7. A method for preparing a pig HBB gene site-directed modified cell, characterized in that, The method comprises introducing the system of any one of claims 1-5 into a target cell to obtain a cell with site-directed modification of the HBB gene; and the target cell is a pig fibroblast cell.

8. The method of claim 7, wherein, The pig fibroblast cell is a pig fetal fibroblast cell.

9. The method according to claim 7 or 8, characterized in that, The method of introduction comprises electroporation or lipofection. The method of introduction comprises electroporation or lipofection.

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