Pig HAT1 gene modification system and its application

Abstract

The present invention provides a porcine HAT1 gene modification system and its applications. The present invention provides a system for porcine HAT1 gene modification. The system contains a first vector and a second vector that can express a gene editing tool protein and sgRNA, effectively cleaving the two target sites of the HAT1 gene. The modified fragment of the donor DNA is replaced with the target fragment to be modified, achieving precise modification of the HAT1 gene. This system provides precise cell and animal models for studying the function of the HAT1 gene.

Description

Technical Field

[0001] The present invention relates to the field of gene editing technology, and specifically to a porcine HAT1 gene modification system and application. Background Art

[0002] The HAT1 gene regulates the acetylation of various histone and non-histone proteins, playing an important role in epigenetic regulation. It participates in biological processes such as chromatin remodeling, gene transcriptional activation, DNA replication and damage repair, embryonic development, and nuclear reprogramming. Previous studies have shown that HAT1 gene expression is closely correlated with the developmental potential of porcine cloned embryos, with those with high HAT1 expression exhibiting greater developmental potential. Therefore, constructing HAT1 gene-modified cell or animal models is crucial for further understanding the function of the HAT1 gene and promoting the developmental potential of porcine cloned embryos. Summary of the Invention

[0003] The purpose of the present invention is to provide a porcine HAT1 gene modification system and application.

[0004] To achieve the purpose of the present invention, in a first aspect, the present invention provides a CRISPR / Cas vector targeting the porcine HAT1 gene, wherein the nucleotide sequence of the sgRNA action site is shown in SEQ ID NO: 1 and / or 2.

[0005] Preferably, the nucleotide sequence of the sgRNA action site is selected from the combination of SEQ ID NO: 1 and 2.

[0006] Among them, the accession number of the porcine HAT1 gene in GenBank is 100622317.

[0007] In a second aspect, the present invention provides a porcine HAT1 gene modification system, comprising the CRISPR / Cas vector and donor DNA.

[0008] The donor DNA contains a modified fragment of exon 6 of the porcine HAT1 gene, and the amino acid sequence encoded by the modified fragment contains a mutation from T to A in the 188th amino acid of the porcine HAT1 protein.

[0009] Furthermore, the CRISPR / Cas vector comprises a gene editing protein expression cassette and an sgRNA expression cassette.

[0010] The gene editing protein can be selected from Cas9, Cas9n, Cpf1 or C2c2, etc., preferably Cas9.

[0011] The backbone vector of the CRISPR / Cas vector can be selected from pX330, pX260, pX334, pX335, pX458, pX459, pX461, pX462, pX551 or pX552, etc., preferably pX458.

[0012] Preferably, the nucleotide sequence of the donor DNA is shown in SEQ ID NO: 3.

[0013] In a third aspect, the present invention provides any of the following applications of the porcine HAT1 gene modification system:

[0014] (a) Construction of HAT1 gene-modified cell lines;

[0015] (b) Construction of a HAT1 gene-modified pig model.

[0016] In a fourth aspect, the present invention provides a method for preparing porcine HAT1 gene-modified cells, comprising introducing the porcine HAT1 gene modification system into target cells to obtain HAT1 gene-modified cells.

[0017] Preferably, the target cells are porcine fibroblasts, more preferably porcine fetal fibroblasts;

[0018] Preferably, the introduction method includes electroporation or lipofection.

[0019] Preferably, after the introduction operation, cells modified with the porcine HAT1 gene are obtained through screening and identification.

[0020] Preferably, the screening comprises screening monoclonal cells by flow sorting.

[0021] Preferably, said identification comprises sequencing identification.

[0022] In a fifth aspect, the present invention provides porcine HAT1 gene-modified cells prepared according to the method.

[0023] In a sixth aspect, the present invention provides a method for preparing a gene-edited pig modified with the HAT1 gene, wherein the pig HAT1 gene-modified cells are transplanted into an enucleated pig oocyte to obtain a recombinant cloned embryo, and the recombinant cloned embryo is transplanted into a sow for pregnancy to obtain a gene-edited pig modified with the HAT1 gene.

[0024] Preferably, the method also includes a step of identifying the gene-edited pigs after they are born.

[0025] Preferably, said identification comprises sequencing identification.

[0026] The purpose of the present invention can be further achieved by adopting the following technical measures.

[0027] The present invention provides a system for HAT1 gene modification, comprising a first vector, a second vector, and a donor DNA.

[0028] The first vector includes a gene editing protein expression cassette and a first sgRNA (SEQ ID NO: 1) expression cassette.

[0029] The second vector includes a gene editing protein expression cassette and a second sgRNA (SEQ ID NO: 2) expression cassette.

[0030] Among them, the first sgRNA and the second sgRNA target two target sites of the HAT1 gene respectively.

[0031] The donor DNA contains a modified fragment of exon 6 of the HAT1 gene, and the modified fragment is used to replace the fragment to be modified in the HAT1 gene.

[0032] The HAT1 gene is modified to replace the threonine (T) amino acid at position 188 of the pig HAT1 protein with alanine (A).

[0033] Furthermore, the first sgRNA is encoded by the nucleotide sequence shown in SEQ ID NO: 1.

[0034] Furthermore, the second sgRNA is encoded by the nucleotide sequence shown in SEQ ID NO: 2.

[0035] Furthermore, the donor DNA is the nucleotide sequence shown in SEQ ID NO: 3.

[0036] Furthermore, the gene editing protein includes Cas9, Cas9n, Cpf1 or C2c2, preferably Cas9.

[0037] Furthermore, the first vector and the second vector independently include pX330, pX260, pX334, pX335, pX458, pX459, pX461, pX462, pX551 or pX552, preferably pX458.

[0038] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0039] The present invention provides a system for pig HAT1 gene modification. The first vector or the second vector contained in the system can express a gene editing protein and sgRNA, effectively enzymatically cut the two target sites of the HAT1 gene, and use the modified fragment of the donor DNA to replace the fragment to be modified at the target site to achieve HAT1 gene sequence modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1This is the sequencing result of the sgRNA connected to the pX458 vector in Example 1 of the present invention.

[0041] Figure 2 This is the Sanger sequencing result of the target site after transfection of the candidate sgRNA in Example 1 of the present invention.

[0042] Figure 3 The sgRNA-R2 gene editing efficiency was analyzed using ICE software in Example 1 of the present invention, and the gene editing efficiency was 42%.

[0043] Figure 4 The sgRNA-L3 gene editing efficiency was analyzed using ICE software in Example 1 of the present invention, and the gene editing efficiency was 36%.

[0044] Figure 5 Schematic diagram of the sequence structure of HAT1-ssODN in Example 3 of the present invention.

[0045] Figure 6 Schematic diagram of precise modification of the HAT1 gene in Example 3 of the present invention.

[0046] Figure 7 This is the mutation caused at the pig HAT1 gene target site when sgRNA-R1 and sgRNA-L1 are transfected in pairs in Example 5 of the present invention.

[0047] Figure 8 This is the mutation caused at the pig HAT1 gene target site when sgRNA-R2 and sgRNA-L2 are transfected in pairs in Example 5 of the present invention.

[0048] Figure 9 This is the mutation caused at the pig HAT1 gene target site when sgRNA-R2 and sgRNA-L3 are transfected in pairs in Example 5 of the present invention. DETAILED DESCRIPTION

[0049] The present invention provides a system for HAT1 gene modification, which comprises a first vector, a second vector, and 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, and the first sgRNA and the second sgRNA respectively target two target sites of the HAT1 gene; the donor DNA comprises a modified fragment of the HAT1 gene, and the modified fragment is used to replace the threonine (T) amino acid at position 188 of the porcine HAT1 protein with alanine (A).

[0050] In this system, the first sgRNA, the second sgRNA, or a combination of the first and second sgRNAs can target the target fragment, guiding the gene editing protein to perform enzymatic cleavage of the target site, thereby knocking out the HAT1 gene. Alternatively, the first sgRNA, the second sgRNA, or a combination of the first and second sgRNAs can be used with donor DNA to achieve precise modification of the HAT1 gene. This system replaces the threonine (T) amino acid at position 188 of the porcine HAT1 protein with alanine (A) without changing other amino acids in HAT1. The donor DNA serves as a replacement template for modifying the target sequence. Under the guidance of the first sgRNA, the second sgRNA, or the combination of the first and second sgRNAs, which specifically recognize exon 6 of the HAT1 gene, the gene editing protein performs enzymatic cleavage of the target fragment and guides the donor DNA sequence to replace the original homologous fragment in the cell, thereby achieving the purpose of replacing the threonine (T) amino acid at position 188 of the HAT1 gene protein with alanine (A).

[0051] It should be noted that gene editing proteins can be effectively cleaved in a variety of cells and guide sequence recombination after cleavage, with the advantages of a wide range of applications and high cleavage efficiency. There is no limitation on the type of gene editing protein, as long as the genome editing function can be achieved. The first sgRNA and the second sgRNA can achieve gene editing protein targeting the sequence near the 188th amino acid site encoding the HAT1 protein. There is no limitation on the specific sequence, as long as the precise targeting function can be achieved. The donor DNA replaces the target fragment to achieve sequence recombination, specifically replacing the 188th threonine (T) amino acid of the HAT1 protein with alanine (A).

[0052] In a preferred embodiment, the first sgRNA is encoded by the nucleotide sequence shown in SEQ ID NO: 1, and the second sgRNA is encoded by the nucleotide sequence shown in SEQ ID NO: 2. This approach has stronger targeting and more precise modification.

[0053] In a preferred embodiment, the gene editing protein comprises Cas9, Cas9n, Cpf1, or C2c2, preferably Cas9. The first and second vectors independently comprise pX330, pX260, pX334, pX335, pX458, pX459, pX461, pX462, pX551, or pX552, preferably pX458. Cas9 and pX458 are widely applicable, highly versatile, and have a high degree of product maturity. Using them as the backbone of gene editing vectors can achieve higher enzymatic cleavage efficiency.

[0054] In a preferred embodiment, the first vector and the second vector of the present invention are both recombinant plasmids, which include a gene editing vector backbone and a sequence encoding sgRNA, wherein the gene editing vector backbone can be a CRISPR plasmid, a TALEN plasmid or a zinc finger plasmid, preferably a CRISPR plasmid.

[0055] In some specific embodiments, the nucleotide sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 are respectively ligated into a vector backbone, and positive clones are screened to obtain the first vector and the second vector.

[0056] The present invention also provides applications of the above system in the following (a)-(b):

[0057] (a) Construction of HAT1 gene-modified cell lines;

[0058] (b) Construction of a HAT1 gene-modified pig model.

[0059] The system provided by the present invention can achieve HAT1 gene modification and construct a HAT1 gene-modified cell line.

[0060] The present invention also provides a method for preparing HAT1 gene-modified cells and the resulting cells. The method comprises introducing the system of the present invention into target cells to obtain HAT1 gene-modified cells. The target cells are preferably porcine fibroblasts, more preferably porcine fetal fibroblasts, as porcine fetal fibroblasts have a higher cloning efficiency than other cells. The introduction method is preferably electroporation or lipofection, more preferably electroporation, which has a higher transfection efficiency.

[0061] In a preferred embodiment, the system is introduced into target cells and then screened and identified to obtain HAT1 gene-modified cells. Screening is preferably performed by flow cytometry to screen monoclonal cells and identify whether the monoclonal cells have a threonine (T) at position 188 of the HAT1 protein replaced with an alanine (A). Identification is preferably performed by sequencing.

[0062] In some embodiments, DNA of monoclonal cells can be extracted and PCR amplified using primers shown in SEQ ID NOs: 4-5. The amplified products can be sequenced to confirm whether the cells have been precisely modified.

[0063] The above-mentioned HAT1 gene-modified cells can be further used to prepare gene-edited pigs. The cells are transplanted into enucleated oocytes to obtain recombinant cloned embryos, and the recombinant cloned embryos are transplanted into the mother's body for pregnancy to obtain gene-edited pigs modified with the HAT1 gene.

[0064] In a preferred embodiment, gene-edited pigs need to be identified after birth, preferably by sequencing.

[0065] In some embodiments, the DNA of the gene-edited pig can be extracted and PCR amplified using the primers shown in SEQ ID NO: 4-5, and the amplified products can be sequenced to confirm whether the pig has been precisely modified.

[0066] The following examples are provided to illustrate the present invention but are not intended to limit the scope of the invention. Unless otherwise specified, the examples were performed according to conventional experimental conditions, such as those in Sambrook et al. (Sambrook J & Russell DW, Molecular Cloning: A Laboratory Manual, 2001), or according to the conditions recommended by the manufacturer's instructions.

[0067] The main reagents used in the following examples are:

[0068] Collagenase type IV used to isolate porcine fetal fibroblasts was purchased from Sigma;

[0069] DMEM, FBS, PS, NEAA, Glutamine, and Trypsase used in cell culture were purchased from Gibco;

[0070] Kits for extracting DNA from cells and ear tissues were purchased from Tiangen Biochemical Technology Co., Ltd.;

[0071] Primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.;

[0072] KOD FX PCR enzyme used for PCR was purchased from TOYOBO.

[0073] The main instruments are as follows:

[0074] CO2 incubator (Thermo Scientific, 3131 / 3111);

[0075] Fluorescence inverted microscope (LEICA, DMI66B);

[0076] PCR instrument (BIO-RID, C1000 Touch);

[0077] Gel imaging system (BIO-RID, Universal Hood II);

[0078] Micromanipulation system (Eppendorf, Celltram vario);

[0079] Cell flow sorter (Aria III).

[0080] Example 1 Construction of vector and design of activity detection

[0081] 1. Using the 6th exon of the porcine HAT1 gene (Gene ID: 100622317) as the target sequence, we searched for a suitable sgRNA sequence based on the N20NGG (N is any of the four bases A, T, G, and C) sequence rule. We then screened five candidate sgRNAs based on the GC content of the sequence, sequence complementarity, and whether the 5′ end starts with G. The details are as follows:

[0082] sgRNA-R1: 5′-GAACTGCTAGTTTTATTGACG-3′;

[0083] sgRNA-R2: 5′-GTTGACGTGGATGATGAAAGA-3′ (SEQ ID NO: 1);

[0084] sgRNA-L1: 5′-GTTCAATAAACCACATCAAAA-3′;

[0085] sgRNA-L2: 5′-GGCTTCAGACCTTTTTGATG-3′;

[0086] sgRNA-L3: 5′-GCTTTCGAGAATATCATGAA-3′ (SEQ ID NO: 2);

[0087] In order to facilitate connection with the vector backbone, linker sequences were added to the above five sgRNA sequences and complementary oligonucleotide primers were synthesized.

[0088] 2. Construct vectors and name them pX458-HAT1-sgRNA-1 to pX458-HAT1-sgRNA-5:

[0089] The oligonucleotide primers synthesized in step 1 were treated at 98° C. for 10 min, and then naturally cooled to room temperature for annealing.

[0090] The pX458 backbone vector containing the Cas9 sequence was digested with restriction endonuclease Bbs I at 37°C for 2 h, and the linearized fragment was recovered by gel cutting.

[0091] The annealed double-stranded fragments were mixed with the linearized vector fragments and ligated at 16°C for 1 hour. The cells were then transformed into Top10 or DH5α competent cells and plated on LB plates containing ampicillin for growth. Single colonies were then picked for expansion and sequencing. The sequencing primers are as follows:

[0092] U6-FWD: 5′-GAGGGCCTATTTCCCATGATT-3′.

[0093] The positive clones were cultured and extracted to obtain plasmids pX458-HAT1-sgRNA-1 to pX458-HAT1-sgRNA-5 ( Figure 1 ) for subsequent cell transfection. Plasmid extraction was performed using the Endo-Free Plasmid Maxi Kit.

[0094] 3. Cell transfection

[0095] The day before transfection, primary porcine fetal fibroblasts were revived and plated in 6 cm dishes. Cell transfection was performed when the cells reached 70-80% confluence. Transfection procedures were performed strictly according to the instructions for the Basic Primary 20 Fibroblasts Nucleofector Kit (Lonza). Specifically, 5 μg of each recombinant plasmid obtained in Example 1 was transfected into porcine fetal fibroblasts by electroporation to obtain five types of transfected cells.

[0096] 4. Activity Detection

[0097] 48 h after electroporation, cells were collected and the cell genome was extracted. The extracted cell genome was used as a template for PCR amplification using primers consisting of HAT1-1F-603 (5′-ACTCGGTTCTCAGTCCAACA-3′, SEQ ID NO: 4) and HAT1-1R-603 (0) 5′-TCTCAAGGTCTCCCAAAGCA-3′, SEQ ID NO: 5), and the amplified products were subjected to Sanger analysis.

[0098] The sequencing results showed that ( Figure 2 ), in the samples transfected with sgRNA-R2 and sgRNA-L3, sgRNA effectively edited the target region of the HAT1 gene, resulting in overlapping peaks in the Sanger sequencing results. The other sgRNAs failed to effectively edit the target region. Further analysis of the Sanger sequencing results using ICE software (https: / / ice,synthego,com / ) showed that the efficiency of sgRNA-R2 in causing Indel mutations in PEF cells was 42% ( Figure 3 ), the efficiency of sgRNA-L3 causing Indel mutation in PEF cells was 36% ( Figure 4 The remaining sgRNAs alone failed to effectively edit the target region.

[0099] Example 2 Construction of HAT1 gene knockout pig fetal fibroblasts

[0100] 1. Preparation of porcine fetal fibroblasts

[0101] 35-day-old pig embryos were removed from their heads, tails, limbs, internal organs, and bones, and all blood was removed. The fetuses were sheared continuously for 30 minutes with curved ophthalmic scissors to ensure thorough mincing. The minced fetal tissue was then pipetted into a 15-mL centrifuge tube using the blue tip of the scissors. 5 mL of complete culture medium was added, and the solution above was removed after natural sedimentation for several minutes. A few drops of fetal bovine serum were added to the underlying tissue block. The tissue was then aspirated using a 15-cm glass Pasteur pipette with a 1-cm bend at the tip. The tissue was then spread flat in two T75 culture flasks, bottoms facing up, and 15 mL of complete culture medium was added to the opposite side. After 6-8 hours, the culture flasks were carefully flipped over and the tissue blocks immersed in the culture medium. The medium was changed every two days. Once the cells had filled the T75 culture flasks, they were frozen for later use. Pig embryos were obtained from the pig farm at the experimental base of the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0102] 2. Cell transfection

[0103] The day before transfection, primary porcine fetal fibroblasts were revived and plated in 10 cm dishes. Cell transfection was performed when the cells reached 70-80% confluence. 5 μg of pX458-HAT1-sgRNA-R2 plasmid or 5 μg of pX458-HAT1-sgRNA-L3 plasmid was transfected into porcine fetal fibroblasts. The transfection steps were strictly performed according to the instructions of the Basic Primary Fibroblasts Nucleofector Kit (Lonza).

[0104] 3. Screening of positive monoclonal cells

[0105] 36 hours after electroporation, cells were collected and sorted using a flow cytometer into single cells in 96-well plates for culture, with the culture medium replaced every three days. After approximately 10 days of culture, the sorted cells were observed to be confluent in the 96-well plates. Confluent monoclonal cells were then subcultured into 48-well plates. Once the 48-well plates were confluent, a portion of the cells was harvested for genomic DNA extraction and genotyping.

[0106] 4. Identification of positive monoclonal cells

[0107] The selected cell clones were identified by amplifying the extracted genomic DNA using the upstream and downstream primers HAT1-1F-603 (5′-ACTCGGTTCTCAGTCCAACA-3′, SEQ ID NO: 4) and HAT1-1R-603 (5′-TCTCAAGGTCTCCCAAAGCA-3′, SEQ ID NO: 5) as templates, yielding a 603 bp fragment. Amplification conditions included 94°C for 5 minutes, followed by 34 cycles of 98°C for 30 seconds, 62.6°C for 30 seconds, and 68°C for 100 seconds, and finally 72°C for 5 minutes. Bands were observed by 2% agarose gel electrophoresis, and the PCR products were sequenced by Beijing Tianyi Huiyuan Biotechnology Co., Ltd. Based on the sequencing results, cells harboring frameshift mutations in the HAT1 gene were screened for subsequent HAT1 gene expression verification.

[0108] Example 3 Construction of pig fetal fibroblasts with precise mutation of HAT1 gene T188A

[0109] 1. Preparation of porcine fetal fibroblasts

[0110] 35-day-old pig embryos were removed from their heads, tails, limbs, internal organs, and bones, and all blood was removed. The fetuses were sheared continuously for 30 minutes with curved ophthalmic scissors to ensure thorough mincing. The minced fetal tissue was then pipetted into a 15-mL centrifuge tube using the blue tip of the scissors. 5 mL of complete culture medium was added, and the solution above was removed after natural sedimentation for several minutes. A few drops of fetal bovine serum were added to the underlying tissue block. The tissue was then aspirated using a 15-cm glass Pasteur pipette with a 1-cm bend at the tip. The tissue was then spread flat in two T75 culture flasks, bottoms facing up, and 15 mL of complete culture medium was added to the opposite side. After 6-8 hours, the culture flasks were carefully flipped over and the tissue blocks immersed in the culture medium. The medium was changed every two days. Once the cells had filled the T75 culture flasks, they were frozen for later use. Pig embryos were obtained from the pig farm at the experimental base of the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0111] 2. Donor DNA sequence design

[0112] Single-stranded oligodeoxynucleotides (ssODN) were designed based on the sgRNA position and alanine codon sequence. Figure 5 ), the HAT1-ssODN sequence is shown in SEQ ID NO: 3; the designed HAT1-ssODN was synthesized by chemical synthesis method for subsequent experiments.

[0113] 3. Cell transfection

[0114] The day before transfection, primary porcine fetal fibroblasts were revived and plated in 10 cm dishes. Cell transfection was performed when the cells reached 70-80% confluence. 5 μg of pX458-HAT1-sgRNA-R2 plasmid or 5 μg of pX458-HAT1-sgRNA-L3 plasmid was co-transfected with 2 μg of the single-stranded donor template HAT1-ssODN into porcine fetal fibroblasts. The transfection steps were strictly performed according to the instructions of the Basic Primary Fibroblasts Nucleofector Kit (Lonza).

[0115] 4. Screening of positive monoclonal cells

[0116] 36 hours after electroporation, cells were collected and sorted using a flow cytometer into single cells in 96-well plates for culture, with the culture medium replaced every three days. After approximately 10 days of culture, the sorted cells were observed to be confluent in the 96-well plates. Confluent monoclonal cells were then subcultured into 48-well plates. Once the 48-well plates were confluent, a portion of the cells was harvested for genomic DNA extraction and genotyping.

[0117] 5. Identification of positive monoclonal cells

[0118] The selected cell clones were identified by amplifying the extracted genomic DNA using the upstream and downstream primers HAT1-1F-603 (5′-ACTCGGTTCTCAGTCCAACA-3′, SEQ ID NO: 4) and HAT1-1R-603 (5′-TCTCAAGGTCTCCCAAAGCA-3′, SEQ ID NO: 5) as templates. A 603 bp fragment was amplified using the following amplification conditions: 94°C for 5 minutes; 98°C for 30 seconds, 62.6°C for 30 seconds, and 68°C for 100 seconds, for 34 cycles; and 72°C for 5 minutes. Bands were observed by 2% agarose gel electrophoresis, and the PCR products were sequenced by Beijing Tianyi Huiyuan Co., Ltd. Sequencing revealed cells harboring a precise mutation at codon 188 of the HAT1 gene.

[0119] Schematic diagram of precise modification of HAT1 gene is shown in Figure 6 Specifically, the threonine (T) amino acid at position 188 of the porcine HAT1 protein is replaced with alanine (A).

[0120] Example 4 Preparation of HAT1 gene T188A precise mutation gene editing pigs

[0121] Positive cells with the precise T188A mutation in the HAT1 gene obtained in Example 3 were used as nuclear transplant donor cells, and young porcine oocytes matured in vitro for 40 hours were used as nuclear transplant recipient cells. The nuclear transplant donor cells were transplanted into enucleated oocytes, and after electrofusion and activation, recombinant cloned embryos were constructed. Selected cloned recombinant embryos with good development were surgically transplanted into the uterus of naturally estrous multiparous Large White sows for gestation. The surgical embryo transfer steps were as follows: the recipient sow was anesthetized by intravenous injection of Zoletil anesthetic at a dose of 5 mg / kg body weight. After anesthesia, the recipient sow was moved to a surgical stand and restrained in a supine position under ventilator anesthesia (isoflurane concentration of 3% to 4%). A surgical incision approximately 8 cm long was made in the recipient sow's abdomen midline to expose the ovary, fallopian tube, and uterus. A glass embryo transfer tube was inserted approximately 5 cm along the fimbria of the fallopian tube, and the cloned recombinant embryo with good development was transplanted into the uterus of the ampulla-isthmus junction of the fallopian tube. After embryo transfer, technicians regularly observe and use B-ultrasound to check the pregnancy status of the recipient sows.

[0122] After the piglets were born, ear tissues were harvested and genomic DNA was extracted. PCR amplification was performed using the nucleotide sequence shown in SEQ ID NO: 4-5 above, and the genotype was determined by sequencing the PCR amplification products.

[0123] The experimental results show that the sgRNA of the present invention can be used to efficiently obtain a gene-edited pig model with precise mutation of the HAT1 gene.

[0124] Example 5 Targeting the porcine HAT1 gene using paired sgRNA

[0125] 1. Cell transfection

[0126] The primary porcine fetal fibroblasts were revived to a 6 cm dish the day before transfection, and cell transfection was performed when the cells reached 70-80% confluence. The transfection step was performed in strict accordance with the instructions of the Basic Primary 20 Fibroblasts Nucleofector Kit (Lonza). Specifically, the recombinant plasmids obtained in Example 1 were electroporated according to the combination of pX458-HAT1-sgRNA-R1 + pX458-HAT1-sgRNA-L1, pX458-HAT1-sgRNA-R2 + pX458-HAT1-sgRNA-L2, and pX458-HAT1-sgRNA-R2 + pX458-HAT1-sgRNA-L3 to obtain 3 types of transfected cells.

[0127] 2. Activity Detection

[0128] 48 hours after electroporation, cells were collected and the cell genome was extracted. Using the extracted cell genome as a template, PCR amplification was performed with a primer pair consisting of HAT1-1F-603 (5′-ACTCGGTTCTCAGTCCAACA-3′, SEQ ID NO: 4) and HAT1-1R-603 (5′-TCTCAAGGTCTCCCAAAGCA-3′, SEQ ID NO: 5). The amplified product was ligated into a T vector and transformed into DH5α Escherichia coli competent cells. 100 μl of the transformed Escherichia coli culture was spread on an LB solid culture plate containing ampicillin and cultured overnight at 37°C. 20 single colonies were selected for Sanger sequencing.

[0129] Analyze the sequencing results, remove the colony samples that failed sequencing, and compare the insert sequences of the colony samples that successfully sequenced.

[0130] Sequencing and alignment results showed that when sgRNA-R1 and sgRNA-L1 were transfected in pairs ( Figure 7 ), there were 2 mutant colonies among 19 colonies, indicating that the mutation efficiency of the target site by paired transfection of sgRNA-R1 and sgRNA-L1 was about 11%; when paired transfection of sgRNA-R2 and sgRNA-L2 ( Figure 8 ), there were 6 mutant colonies among 16 colonies, indicating that the mutation efficiency of the target site was about 37% when paired transfection of sgRNA-R2 and sgRNA-L2 was performed; when paired transfection of sgRNA-R2 and sgRNA-L3 was performed ( Figure 9 ), there were 10 mutant colonies among 18 colonies, indicating that the mutation efficiency of paired transfection of sgRNA-R2 and sgRNA-L3 on the target site was approximately 56%.

[0131] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A CRISPR / Cas vector targeting the porcine HAT1 gene, characterized in that: The vector contains sgRNA, and the nucleotide sequence of the sgRNA is shown in SEQ ID NO: 1 and / or 2.

2. A product for pig HAT1 gene modification, characterized in that: Comprising the CRISPR / Cas vector of claim 1 and donor DNA; The donor DNA contains a modified fragment of exon 6 of the porcine HAT1 gene, and the amino acid sequence encoded by the modified fragment contains a mutation of the 188th amino acid of the porcine HAT1 protein from T to A.

3. The product according to claim 2, characterized in that The CRISPR / Cas vector comprises a gene editing protein expression cassette and an sgRNA expression cassette.

4. The product according to claim 3, characterized in that The gene editing protein is Cas9.

5. The product according to claim 3, characterized in that The backbone vector of the CRISPR / Cas vector is selected from pX330, pX260, pX334, pX335, pX458, pX459, pX461, pX462, pX551 or pX552.

6. The product according to any one of claims 3 to 5, characterized in that The nucleotide sequence of the donor DNA is shown in SEQ ID NO:

3.

7. Any of the following uses of the product according to any one of claims 4 to 6: (a) Construction of HAT1 gene-modified cell lines; (b) Construction of a HAT1 gene-modified pig model.

8. A method for preparing porcine HAT1 gene-modified cells, characterized in that: The method comprises introducing the product according to any one of claims 4 to 6 into target cells to obtain HAT1 gene-modified cells.

9. Porcine HAT1 gene-modified cells prepared according to the method of claim 8.

10. A method for preparing gene-edited pigs modified with the HAT1 gene, characterized in that: The cells according to claim 9 are transplanted into enucleated pig oocytes to obtain recombinant cloned embryos, and the recombinant cloned embryos are transplanted into sows for pregnancy to obtain gene-edited pigs with modified HAT1 gene.

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