SgRNA specifically recognizing pig kit gene, coding DNA, kit and application thereof

Abstract

The present invention provides a sgRNA that specifically recognizes the porcine KIT gene, its encoding DNA, a kit, and applications, relating to the field of genetic engineering technology. The nucleotide sequence responsible for recognizing the target fragment region in the sgRNA is the sequence shown in SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3. This sgRNA enables the CRISPR / Cas9 gene editing system to delete redundant mutant copies of the KIT gene in the porcine genome, allowing the KIT gene to normally express the biologically active KIT protein, thereby changing the pig's coat color.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to an sgRNA that specifically recognizes the porcine KIT gene, its encoding DNA, a kit, and its applications. Background Technology

[0002] The KIT gene has been confirmed as the major gene regulating dominant white coat coloration in Large White pigs. The KIT gene is located on chromosome 8, short arm, region 1.2 (8p12). The normal pig KIT gene is a single copy, located between the PDGFRA and KDR genes, with a full length greater than 200 kb, consisting of 21 exons and a coding sequence of 2919 bp. The normal single-copy KIT gene exhibits a black phenotype. The KIT gene plays a crucial role in the normal migration and survival of melanocyte precursors. Large White and Landrace pigs, in addition to carrying the normal KIT gene (KIT1), also carry multiple full-length copies containing mutations (KIT2, KIT3, ..., KITn; n≥2). In these mutant copies, one G base in intron 17 is replaced with an A, causing the mRNA precursor to not splice properly to form normal mRNA. This interferes with the expression of normal KIT protein, affecting the normal migration and survival of melanocytes, resulting in the white coat trait in Large White and Landrace pigs.

[0003] The CRISPR (clustered regularly interspaced short palindromic repeats) / Cas (CRISPR-associated proteins) system is an effective acquired immune mechanism in bacteria and archaea. It cleaves and degrades exogenous DNA, including bacteriophages and exogenous plasmids, through sequence-specific RNA-mediated cleavage. CRISPR clusters are a family of specialized DNA repetitive sequences widely distributed in the genomes of bacteria and archaea. Based on the gene origin of the Cas site and the different Cas proteins involved, the CRISPR / Cas system is classified into type I, type II, and type III. The CRISPR / Cas9 gene editing system is an artificially modified version of the type II CRISPR / Cas acquired immune system found in bacteria and archaea. This system consists of the Cas9 protein and a short single-stranded RNA (sgRNA). The sgRNA recognizes the target DNA sequence, and the Cas9 protein cleaves the double-stranded DNA at the target site recognized by the sgRNA, thereby degrading foreign nucleic acid molecules. The CRISPR / Cas type II system has been widely used in various species, including mice, rats, pigs, cattle, sheep, monkeys, zebrafish, rice, wheat, and Arabidopsis thaliana. The CRISPR / Cas system is an effective tool for gene function research, plant and animal genome editing, breeding new plant and animal varieties, and creating bioreactors and disease models. Therefore, this paper proposes a method for editing the porcine KIT gene using the CRISPR / Cas system, which can alter the pig's phenotype and facilitate further research on the function of the porcine KIT gene.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The first objective of this invention is to provide an sgRNA that specifically recognizes the porcine KIT gene, wherein the sgRNA enables the CRISPR / Cas9 gene editing system to delete redundant mutated copies of the KIT gene on the porcine genome, thereby enabling the KIT gene to express the biologically active KIT protein normally.

[0006] A second objective of the present invention is to provide a DNA molecule containing a sequence encoding the above-mentioned sgRNA.

[0007] A third objective of this invention is to provide an application of the above-mentioned sgRNA or DNA molecule that specifically recognizes the porcine KIT gene.

[0008] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0009] According to one aspect of the present invention, the present invention provides an sgRNA that specifically recognizes the porcine KIT gene, wherein the nucleotide sequence in the sgRNA responsible for recognizing the target fragment region is the sequence shown in SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3.

[0010] Preferably, the nucleotide sequence in the sgRNA responsible for recognizing the target fragment region is the sequence shown in SEQ ID NO.2.

[0011] According to another aspect of the invention, the invention also provides a DNA molecule containing a sequence encoding the sgRNA.

[0012] Preferably, the DNA molecule contains at least one of the sequences shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6.

[0013] Preferably, the DNA molecule further contains a sequence encoding Cas9;

[0014] Preferably, the DNA molecule includes a vector that simultaneously encodes the sgRNA and Cas9;

[0015] Preferably, the sequence of the vector simultaneously encoding the sgRNA and Cas9 is shown in SEQ ID NO.8.

[0016] According to another aspect of the invention, the invention also provides a kit comprising the sgRNA or a DNA molecule containing a sequence encoding the sgRNA.

[0017] According to another aspect of the present invention, the present invention also provides a method for gene editing of the KIT gene in the pig genome, the method comprising using the sgRNA-mediated Cas9 gene editing of the pig KIT gene in pig-derived biological materials.

[0018] Preferably, the gene editing method includes introducing a gene editing element into porcine biological material, wherein the gene editing element comprises either (a) or (b):

[0019] (a) the sgRNA or the DNA molecule encoding the sgRNA; and Cas9-related elements, the Cas9-related elements including DNA encoding Cas9, mRNA encoding Cas9, or a Cas9 protein molecule;

[0020] (b) An expression vector that simultaneously encodes the sgRNA and Cas9.

[0021] Preferably, the porcine-derived biomaterial includes porcine somatic cells or porcine fertilized eggs;

[0022] Preferably, the porcine somatic cells include porcine fibroblasts;

[0023] Preferably, the gene editing element is introduced into porcine somatic cells using the calcium phosphate method, lipid transfection method, lentivirus transfection method, or electroporation method, with electroporation method being preferred.

[0024] Preferably, the gene editing element is introduced into a pig zygote using a zygote microinjection method.

[0025] According to another aspect of the invention, the invention also provides the application of the sgRNA, the DNA molecule, the kit, or the method in altering pig coat color;

[0026] Preferably, changing the pig's coat color includes changing the pig's coat color to black, where the pig's original coat color is white;

[0027] Preferably, the pig includes a Large White pig.

[0028] According to another aspect of the present invention, the present invention also provides a method for changing the color of pig hair, comprising introducing the sgRNA or the DNA molecule into pig-derived biological material.

[0029] Preferably, the method includes introducing the gene-editing element into porcine somatic cells, screening for KIT gene-edited positive somatic cells, and then constructing a reconstructed embryo by somatic cell nuclear transfer; the somatic cells preferably include fibroblasts;

[0030] Preferably, the screening includes PCR identification of somatic cells with the introduced gene editing element using a first primer pair and a second primer pair; the upstream and downstream primers of the first primer pair are located upstream and downstream of the 3-5 breakpoint, respectively; the second primer pair is used to amplify the 17 introns of the KIT gene; somatic cells with no amplification product from the first primer pair and with an amplification product from the second primer pair are KIT gene-editing positive somatic cells;

[0031] Preferably, the sequences of the first primer pair are as shown in SEQ ID NO.9 and SEQ ID NO.10;

[0032] Preferably, the second primer pair sequence is as shown in SEQ ID NO.11 and SEQ ID NO.12.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] The present invention provides a specific sgRNA that recognizes the porcine KIT gene. The nucleotide sequence responsible for recognizing the target fragment region is shown in SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3. The sgRNA provided by the present invention can recognize the target sequence downstream of the coding region of the porcine KIT gene, as shown in SEQ ID NO.7. Through the mediation of the sgRNA, Cas9 can target and delete redundant mutant copies of the KIT gene in the porcine genome, enabling the KIT gene to express the biologically active KIT protein normally. Normally, pigs containing redundant mutant copies of the KIT gene cannot properly splice the KIT gene mRNA precursor to form normal mRNA, thus interfering with the expression of normal KIT protein and affecting the normal migration and survival of melanocytes. Deleting redundant mutant copies of the KIT gene in the porcine genome can change the coat color and other traits of pigs, providing good breeding material for the improvement of pig breeds in my country. Attached Figure Description

[0035] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0036] Figure 1 A diagram illustrating how removing redundant mutated copies of the KIT gene from the Large White pig genome alters coat color.

[0037] Figure 2 Fluorescence graphs for efficiency detection of three sgRNAs;

[0038] Figure 3 Flow cytometry plots for efficiency detection of three sgRNAs;

[0039] Figure 4 Electrophoresis images of PCR amplification of 3-5 breakpoint sites and 17 introns in Large White and Duroc pigs;

[0040] Figure 5 Sequencing peak diagrams of PCR products from 17 introns in Large White and Duroc pigs;

[0041] Figure 6 This is an electrophoresis image of PCR for monoclonal detection;

[0042] Figure 7 This is a sequencing peak diagram of PCR products from 17 introns detected by monoclonal assay. Detailed Implementation

[0043] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the embodiments can be obtained commercially.

[0044] According to one aspect of the present invention, a sgRNA specifically recognizing the porcine KIT gene is provided. The sgRNA is a guide RNA, an important component of the CRISPR gene knockout / knock-in system, which binds to the Cas9 protein and guides the Cas9 enzyme to target and cleave genomic DNA. The nucleotide sequence in the sgRNA responsible for recognizing the target fragment region is the sequence shown in SEQ ID NO.1, SEQ ID NO.2, or SEQ ID NO.3. This sgRNA can target a sequence downstream of the coding region of the porcine KIT gene, as shown in SEQ ID NO.7. Through the mediation of the sgRNA, Cas9 can target and delete redundant mutated copies of the KIT gene in the porcine genome, enabling the KIT gene to express the biologically active KIT protein normally. In addition to the sequence responsible for recognizing the target fragment region, the sgRNA provided by the present invention may also contain sequences with other functions, including but not limited to Cas9 nuclease recruitment sequences and linker units connecting various functional units. Other sequences can be designed according to conventional selections in the art, and the present invention does not limit this. In some preferred embodiments, sgRNA with the nucleotide sequence shown in SEQ ID NO.2, which is responsible for recognizing the target fragment region, is used, and experiments have shown that it has higher activity.

[0045] According to another aspect of the present invention, a DNA molecule containing a sequence encoding the sgRNA is also provided. It is understood that, in addition to the DNA molecule encoding the sgRNA, it may also contain DNA elements with other functions, such as, but not limited to, promoters, terminators, or marker genes; or DNA molecule portions used as vector portions; for example, when a DNA molecule containing the sgRNA encoding the above-mentioned sgRNA is constructed in a vector, the DNA molecule includes a region encoding the sgRNA, promoter and terminator regions, and a region serving as a vector portion. The DNA molecule may also contain coding sequences for encoding other functional elements in the CRISPR / Cas9 system, such as a region encoding the Cas9 protein. The DNA molecule encoding the sgRNA sequence described in this invention can be a sense strand, an antisense strand, or a double-stranded DNA molecule; the present invention does not limit this.

[0046] In some preferred embodiments, the DNA molecule contains at least one of the sequences shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6. Optionally, the DNA molecule contains only one of the sequences shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6; alternatively, the DNA molecule contains both the sequences shown in SEQ ID NO. 4 and SEQ ID NO. 5; alternatively, the DNA molecule contains all three sequences shown in SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6.

[0047] In some optional embodiments, the DNA molecule includes a vector simultaneously encoding the sgRNA and Cas9. This vector, once introduced into a cell, can transcribe the sgRNA and transcribe and express the Cas9 protein, thereby enabling sgRNA-mediated Cas9 protein editing of the porcine KIT gene. The sequence of the vector simultaneously encoding the sgRNA and Cas9 is preferably as shown in SEQ ID NO. 8.

[0048] According to one aspect of the present invention, a kit is also provided, the kit comprising the aforementioned sgRNA that specifically recognizes the porcine KIT gene, or a DNA molecule encoding the aforementioned sgRNA. This kit can be used for gene editing of the porcine KIT gene, or for gene editing of multiple genes including the porcine KIT gene. The kit may also comprise conventional reagents for gene editing, and matching primers for screening positive gene editing results. In some optional embodiments, the kit comprises the aforementioned sgRNA and reagents for storing the sgRNA, as well as DNA encoding Cas9, RNA encoding Cas9, or a Cas9 molecule. In some optional embodiments, the kit comprises a vector simultaneously encoding the sgRNA and Cas9, and matching primers for screening positive gene editing results.

[0049] According to another aspect of the present invention, the present invention also provides a method for gene editing of the KIT gene in the porcine genome, the method comprising using the sgRNA described above to mediate Cas9 knockout of the porcine KIT gene in porcine biological material. The gene editing method provided by the present invention uses the aforementioned sgRNA, which is capable of recognizing the region downstream of the coding region of the porcine KIT gene as shown in SEQ ID NO. 7, deleting redundant mutated copies of the KIT gene on the porcine genome, thereby enabling the KIT gene to express biologically active KIT protein.

[0050] In some preferred embodiments, the preparation method includes introducing a gene-editing element into porcine biological material, which may include porcine somatic cells or porcine zygotes. Optionally, the gene-editing element is introduced into porcine somatic cells using the calcium phosphate method, lipid transfection method, lentivirus transfection method, or electroporation method; optionally, the gene-editing element is introduced into porcine zygotes using zygote microinjection. The gene-editing element includes the following (a) or (b):

[0051] (a) the sgRNA or the DNA molecule encoding the sgRNA; and Cas9-related elements, the Cas9-related elements including DNA encoding Cas9, mRNA encoding Cas9, or a Cas9 protein molecule; the DNA encoding Cas9 and the RNA encoding Cas9 may optionally contain functional elements independently, examples of which include, but are not limited to, promoters, terminators, enhancers, and marker genes, as well as a portion of the DNA molecule used as a vector; the Cas9 protein molecule may be a natural Cas9 molecule or a molecularly modified Cas9 protein or a fusion protein, and the Cas9-related elements are only required to perform Cas9 splicing functions, which is not limited in this invention.

[0052] (b) An expression vector that simultaneously encodes the sgRNA and Cas9; after being introduced into cells, the vector can transcribe the sgRNA and transcribe and express the Cas9 protein to achieve sgRNA-mediated Cas9 protein editing of the porcine KIT gene.

[0053] According to another aspect of the present invention, the present invention also provides the application of the sgRNA, a DNA molecule containing a sequence encoding the sgRNA, the kit, or the method for gene editing the KIT gene in the pig genome in changing pig coat color. The sgRNA provided by the present invention enables the CRISPR / Cas9 gene editing system to delete redundant mutated copies of the KIT gene in the pig genome, allowing the KIT gene to express the biologically active KIT protein normally. The preferred method of changing coat color is to change the pig's coat color from white to black. Typically, the KIT gene mRNA precursor in pigs containing redundant mutated copies of the KIT gene cannot be properly spliced ​​to form normal mRNA, thereby interfering with the expression of normal KIT protein, affecting the normal migration and survival of melanocytes, and resulting in a white coat trait. After gene editing, the KIT gene can express the biologically active KIT protein normally, making the pig's coat black, such as... Figure 1 As shown.

[0054] In some preferred embodiments, the pigs include Large White pigs, also known as Yorkshire pigs, which originated in the United Kingdom. Due to their high feed conversion ratio, slaughter rate, and adaptability, Large White pigs are raised in all countries with developed pig farming industries worldwide, making them the most famous and widely distributed dominant lean-type pig breed in the world. Gene editing methods are used to delete redundant mutated copies of the KIT gene in Large White pigs, causing the genome-edited Large White pigs to express only functional KIT mRNA and KIT protein, thereby changing the original white-haired trait of the Large White pig to the black-haired trait. The black-haired Large White pigs obtained through gene editing possess the characteristics of black hair and high lean meat percentage, providing excellent breeding material for the improvement of local black-haired pig breeds in my country.

[0055] According to one aspect of the present invention, the present invention also provides a method for changing the coat color of pigs, comprising introducing the sgRNA or the DNA molecule into porcine biological material.

[0056] In some alternative implementations, the coat color of pigs is altered by introducing the gene-editing element into pig fertilized eggs to obtain KIT gene-edited embryos.

[0057] In some optional embodiments, the coat color of pigs is altered by introducing the gene-editing element into pig somatic cells, screening for KIT gene-edited positive somatic cells, and then constructing reconstructed embryos through somatic cell nuclear transfer; wherein the somatic cells may be fibroblasts. The screening preferably includes PCR identification of somatic cells in which the gene-editing element has been introduced using a first primer pair and a second primer pair; the upstream and downstream primers of the first primer pair are located upstream and downstream of the 3-5 breakpoint, respectively; the second primer pair is used to amplify the 17 introns of the KIT gene; somatic cells with no amplification product from the first primer pair and with an amplification product from the second primer pair are KIT gene-edited positive somatic cells. Preferably, the sequences of the first primer pair are shown in SEQ ID NO. 9 and SEQ ID NO. 10. Preferably, the sequences of the second primer pair are shown in SEQ ID NO. 11 and SEQ ID NO. 12.

[0058] The gene editing element described in the two embodiments above includes the sgRNA or the DNA molecule encoding the sgRNA, and Cas9-related elements, including DNA encoding Cas9, mRNA encoding Cas9, or Cas9 protein molecules; the gene editing element may also include an expression vector that simultaneously encodes the sgRNA and Cas9.

[0059] The technical solution and beneficial effects of the present invention will be further described below with reference to preferred embodiments.

[0060] The porcine embryonic fibroblasts (PEFs) in this example were prepared as follows: 37-day-old Large White pig fetuses were decapitated, removing the head, tail, limbs, internal organs, and bones, and cleaning away any blood. The fetus was continuously minced for 30 minutes using curved ophthalmic scissors to ensure thorough fragmentation. The minced fetal tissue was aspirated into a 15mL centrifuge tube using the blue tip of the scissors, and 5mL of complete culture medium was added. After settling naturally for several minutes, the supernatant was removed, and a few drops of FBS were added to the lower tissue fragment. The tissue fragments were then aspirated using a 15cm glass Pasteur tube with a 1cm bend at the tip and spread evenly in two T75 culture flasks, bottom up. 15mL of complete culture medium was added to the opposite side. After 6-8 hours, the culture flasks were carefully inverted to immerse the tissue fragments in the culture medium. The medium was changed every two days. Once the cells had filled the T75 culture flasks, they were cryopreserved for later use. The Large White pigs used were from the Tianjin Wuqing Base of the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0061] Example 1

[0062] Construction of Cas9 / sgRNA expression vector specifically recognizing KIT gene target sites, and sgRNA activity detection.

[0063] (1) Construction of Cas9 / sgRNA expression vectors. First, a sequence downstream of the coding region of the porcine KIT gene was selected as the target sequence, as shown in SEQ ID NO.7. Based on the target sequence, sequences responsible for recognizing the target fragment region were designed, resulting in three sgRNAs responsible for recognizing the target fragment region, named sgRNA1, sgRNA2, and sgRNA3, respectively. The sgRNA1 sequence is shown in SEQ ID NO.1, the sgRNA2 sequence in SEQ ID NO.2, and the sgRNA3 sequence in SEQ ID NO.3. For detailed construction steps of the three Cas9 / sgRNA expression vectors, please refer to the instruction manual of the Cas9 / sgRNA Construction Kit (Catalog. No. VK001-01) from VST.

[0064] (2) Construction of sgRNA activity detection targeting vector. Based on the three sgRNA sequences in (1), an sgRNA activity detection targeting vector containing three sgRNA targeting sites was constructed. For details of the construction steps of the targeting vector, please refer to the instruction manual of the sgRNA activity fluorescence detection kit (Catalog. No. BG13701S) of Hesheng Gene Co., Ltd.

[0065] (3) sgRNA activity detection. HEK293 cells were co-transfected with 3 μg of the constructed Cas9 / sgRNA vector and 2 μg of the sgRNA activity detection targeting vector. A negative control group (cell lines without Cas9 / sgRNA plasmid) was set up. 48 h after transfection, the luminescence of the cells was observed under a fluorescence microscope, and the proportion of fluorescent cells was detected by flow cytometry. Compared with the negative control group: if a stronger fluorescence signal was detected in the experimental group, it indicated that the sgRNA activity was high. If a weaker fluorescence signal or no fluorescence signal was detected in the experimental group, it indicated that the sgRNA activity was weak or non-existent. The fluorescence chromatograms and flow cytometry results of the three sgRNA activity detection methods are as follows. Figure 2 and Figure 3 As shown in the figure. The results indicated that sgRNA2 activity was high, and subsequent experiments used gRNA2.

[0066] Example 2

[0067] Screening of large white pig fetal fibroblast cell lines with successful deletion of redundant mutated copies of the KIT gene.

[0068] (1) Cell transfection and single-clone culture. Primary large white pig embryonic fibroblasts were revived into 6 cm plates one day before transfection. Cell transfection was performed when the cells reached 70-80% confluence. The cell transfection method was electrotransfection based on a nuclear transfector. Electrotransfection was performed using the Basic Primary Fibroblasts Nucleofector Kit (Lonza) in the Amaxa Nucleofector (Lonza) single-well nuclear transfection system.

[0069] The specific operating procedure is as follows: a. Collect cells and adjust the cell count to 5×10⁶. 5 ~1×10 6 a. Centrifuge at 200g for 5 minutes to remove as much culture medium as possible. b. Resuspend cells in 100μL of electroporation reagent and add 5μg of Cas9 / sgRNA2 expression vector plasmid. Slowly add the electroporation system (including cells, transfection reagent, and plasmid) along the wall of the electroporation cuvette to avoid generating air bubbles that would reduce transfection efficiency. c. After transfection, add 500μL of complete culture medium (20% FBS + DMEM) to the electroporation cuvette, gently pipette the cells, and then transfer them to a 6cm culture dish. Incubate at 37.5℃ in a 5% CO2 incubator. After 48 hours of electroporation, when the cell confluence is approximately 90%, plate the cells and pick cell clones for positive cell identification.

[0070] (2) Identification of positive monoclonal cell lines. Primer pairs designed for detecting positive monoclonal cell lines were 3-5 breakpoint amplification primers (953F9 / 953R3), sequences shown in SEQ ID NO. 9 and SEQ ID NO. 10; and 17-intron amplification primers (K17F / K17R), sequences shown in SEQ ID NO. 11 and SEQ ID NO. 12. The genomes of the selected monoclonal cell lines were amplified using both primer pairs. Monoclonal cell lines that could not amplify a 152bp target band with the 953F9 / 953R3 primer pair but could amplify a 175bp target band with the K17F / K17R primer pair were selected as candidate positive cell lines. The 17-intron amplification sequences of the candidate positive cell lines were sequenced. Candidate positive clonal cell lines whose sequences matched the corresponding 17-intron amplification sequences of Duroc pigs were considered positive clonal cell lines. Electrophoresis diagrams of PCR amplification in Large White pigs and Duroc pigs and sequencing peak diagrams of PCR products are shown below. Figure 4 and Figure 5 As shown. Identification results: Positive clones (42#, 47#, 48#) were obtained from the selected cell clones. Electrophoresis images of the 3-5 breakpoint PCR amplification products and the 17-intron PCR amplification products, and sequencing results of the 17-intron PCR amplification products are shown below. Figure 6 and Figure 7 As shown.

[0071] Example 3

[0072] Somatic cell nuclear transfer was used to prepare lean black-haired pigs with deleted extra mutant copies of the KIT gene.

[0073] Using the cells obtained in Example 2 as donor cells for nuclear transfer, and young pig oocytes that had matured in vitro for 40 hours as recipient cells, the donor cells were transferred into enucleated oocytes. After electrofusion and activation, recombinant clonal embryos were constructed. Well-developed recombinant clonal embryos were selected and surgically transferred into the uterus of naturally estrous multiparous Large White sows for pregnancy. After embryo transfer, technicians closely monitored the return to estrus and regularly checked the pregnancy status of the recipient sows.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention. 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ggagatctcc cgatccgtcg acgtcaggtg 180 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 240 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 300 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 360 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 420 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 480 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 540 tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 600 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 660 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 720 cgatcggagg accgaaggag ctaaccgcttt tttgcacaa catgggggat catgtaactc 780 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 840 cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 900 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 960 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 1020 ggtcacgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 1080 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 1140 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 1200 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 1260 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 1320 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 1380 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 1440 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt 1500 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 1560 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 1620 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 1680 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 1740 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 1800 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 1860 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 1920 ggaaaaacgc cagcaacgcg gccttttac ggttcctggc ctttgctg ccttttgctc 1980 acatgttctt tattaccctg ttaccctaa ccggttcacc attgtatcc gatatcaact 2040 ttgtatagaa aagttggctc cgaatttctc gaggaatttcg gcaggaagg ggcctatttc 2100 ccatgattcc ttcatatttg catatacgat aaggctgt tagagagata attagaatta 2160 atttgactgt aacacaag atattac aaatacgtg acgtagaag taataatttc 2220 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa 2280 cttgaaagta tttcgatttc ttggcttat atatcttgtg gaaaggacga aacaccgaga 2340 gttcttgtcc atggatgtt tagagctaga atagcaagt taaatagg ctagtccgtt 2400 atcaacttga aaagtgca ccgagtcggt gctttttt aagcttggct ccggtgcccg 2460 tcagtgggca gagcgcacat cgcccacagt ccccgagaag tgtggggag gggtcggcaa 2520 ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg 2580 gctccgcctt ttcccgagg gtgggggaga accgtatata agtgcagtag tcgccgtgaa 2640 cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt gtggttcccg 2700 cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt ccacctggct 2760 gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc 2820 ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg 2880 ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct 2940 agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt 3000 aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg 3060 ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag 3120 aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc 3180 gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga 3240 aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg 3300 agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc 3360 ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt 3420 ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac 3480 tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt 3540 tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt 3600 ttttcttcca tttcaggtcc cgggtaactg atcataattc gacccaagtt tgtacaaaaa 3660 agcaggctga ttaccggaga attccaattg gcggccgcac cggtgccacc atgccaaaga 3720 agaagcggaa ggtcggtggc ggctcacccg ggatggacaa gaagtactcc attggcctcg 3780 acatcggaac aaatagcgtg ggctgggctg tcatcacaga tgagtacaag gtgcctagca 3840 agaaatttaa ggtgctggga aatacagaca gacatagcat caagaagaac ctcattggcg 3900 ctctcctgtt tgactccggc gaaacagccg aagctaccag actcaagaga accgctagga 3960 gaaggtacac cagaaggaaa aacaggattt gctacctgca ggaaattttt tccaacgaga 4020 tggccaaggt ggacgattcc ttcttccata ggctggaaga gagcttcctc gtggaggaag 4080 aaaaaaa cgagaggcat cctatttttg gcaatattgt ggatgaggtc gcctaccatg 4140 agaagttcc caaatctat catctgagaa aaaaactggt ggatagcacc gacaaggccg 4200 atctcaggct catttatctc gctctggctc acatgatcaa gtttaggggc cacttcctga 4260 tcgaaggcga cctgaatccc gacaactccg acgtggacaa actgttcatc cagctcgtcc 4320 agacctacaa tcaactcttc gaggaacc ccatcaatgc ttccggcgtg gatgccaagg 4380 ccatcctgag cgctaggctc tccaagtcca ggaggctgga aaatctgatc gcccaactcc 4440 ctggagaa gaaacggc ctgtttggca atctgattgc cctgagcctc ggactcaccc 4500 ccaacttcaa gatcaacttc gatctcgccg aagacgccaa actccaactg agaaggata 4560 cctacgacga cgatctcgat aatctctcg cccagatcgg cgatcaatat gccgacctct 4620 ttctggccgc caaaaaacctg agcgacgcta ttctgctcag cgacattctc agggtgaata 4680 cagaaatcac aaaagccccc ctgtccgcca gcatgatcaa aaggtacgat gaacaccatc 4740 aggacctcac cctgctgaag gctctggtca ggcagcaact ccccgaaaag 4800 ttttctttga tcagtccaag aatggatatg ctggctatat tgatggaggc gcctcccagg 4860 aggaatttta taaattcatc aagcccattc tcgaaagat ggacggacc gagagctgc 4920 tggtcaact caatagggag gatctgctga ggaagcaag gaccttcgac atggcagca 4980 tcccccacca gatccaccctc ggcgaactcc acgctatcct caggaggcag gaagacttct 5040 accctttcct gaaggataac agggagaaaa tcgagaaaat cctgaccttc agaatcccct 5100 actacgtcgg acctctcgcc aggggcaat ccagattcgc ctggatgaca aggagagcg 5160 aggaaacaat cacacatgg aacttcgaag aagtggtcga taagggcgcc agcgcccaga 5220 gcttcattga aaggatgacc aactttgata agaacctgcc caatgagaag gtgctgccta 5280 agcactccct gctgtatgag tattcaccg tgtataatga gctgaccaag gtcaagtacg 5340 tcaccgagggg atgagaag cctgctttc tctccggcga gcagaaaaaa gccatcgtgg 5400 acctgctgtt caaccac aggaggtga ccgtcaagca actcaggag gactacttta 5460 agaagattga gtgctttgat agcgtggaaa ttagcggagt cgaggacagg ttcaatgcct 5520 5580 agagaatga agacattctg gaggacattg tcctcaccct gaccctgttt gaggacagag 5640 agatgattga agagaggctg aaaacctatg cccacctgtt cgacgacaag gtgatgaagc 5700 agctcaaaag aaaggatat accggctggg gcagactgtc caggaagctg atcaacggca 5760 ttagggacaa gcagagcggc aagaccattc tcgactttct caagtccgac ggattcgcca 5820 5880 aggctcaggt cagcggccaa ggcgattccc tccatgagca catcgctaat ctggctggct 5940 cccctgctat caaaaagggc atcctccaga cagtcaaagt cgtcgatgag ctggtcaagg 6000 tgatgggcag gcataaaccc gagaacattg tgattgagat ggctagggag aaccagacca 6060 cccagaaagg ccagaaaaac agcagagaaa gaatgaag gatcgaggag ggcatcaaag 6120 aactgggcag ccaaatcctc aaggagcacc ccgtcgaaaa tacacaactc cagaacgaaa 6180 6240 tcacaggct ctccgattac gatgtggacc acatcgtccc tcagtcctttt ctgaagatg 6300 atagcatcga cacaggtg ctgaccaggt ccgacagaa taggggcag agcgataatg 6360 tgccctccga ggaggtcgtc aaaaaaatga aaactactg gagacaactc ctcaacgcta 6420 agctcatcac ccaagaag ttcgacaatc tgaccaagc cgagaggggc ggcctctccg 6480 aactggacaa ggccggcttc atcaaaggc aattggtgga aaccaggcag attackaagc 6540 atgtcgctca aattctcgat agcaggatga ataccaata tgacgagac gatagctga 6600 tcagagaggt caggtcatc acaccagt caagctcgt gagcgacttc agaaaagatt 6660 tccaatttta taaagtcagg gagatcaca attaccacca cgctcacgac gcttatctca 6720 acgctgtcgt gggaaccgcc ctgatcaaaa ataccccaa gctggaaagc gagttcgtgt 6780 atggcgatta taaagtgtac gacgtgagga agatgatcgc taaaagcgag caggaatcg 6840 gcaaggctac agccaagtac tttcttact ccacattat gaacttcttc agaccgaga 6900 ttaccctcgc caacggcgaa attaggaga ggccctgat tgaacaat ggagaacag 6960 gcgaaatcgt ctgggacaag ggcagggact tcgccacagt cagaaaagtg ctgtccatgc 7020 ctcaagtcaa catcgtcaa aagaccgagg tgcagaccgg cggctttagc aaagaaagca 7080 tcctgcccaa gagaactcc icaagctca tcgctaggaa gaaggactgg gacctaaga 7140 atacggagg atttgactcc cctaccgtcg cctattccgt cctcgtcgtc gctaaggtgg 7200 agaagggcaa gagcagaag ctcaagagcg tcaggagct gctgggaatc accatcatgg 7260 agaggagctc cttcgaaaaaaccctattg atttcctgga ggccaagggc tacaaggagg 7320 tcaagaagga cctcatcatc aagctgccca atacagcct cttcgaactg gaaaatggca 7380 ggaagagaat gctcgctagc gccggcgagc tccagaagg aaatgagctg gctctgccca 7440 gcaagtacgt caactccctc tatctcgcca gccactatga aaagctcaag ggcagccccg 7500 aagacaatga gcagaagcag ctcttcgtcg agcagcaca gcactacctc gatgaatca 7560 tcgagcaat cagcgagttt tccaaaggg tgatctcgc cgacgctaac ctcgataagg 7620 tcctctccgc ttacacaag catagagaca agcccatcag agacaggcc gagacatca 7680 7740. tccacctgtt tacactcaca aacctcggag cccctgccgc ttttaaatac ttcgatacaa ccattgatag gaagaggtac acctccacca aggaggtgct ggatgctacc ctgattcatc aatccatcac aggactctac gaaacaagga ttgacctgtc ccaactggga ggcgacacta gtggtggcgg ctcaaagcgt cctgctgcta ctaagaaagc tggtcaagct aagaaaaaga aatctagtga gggccgcggc agcctgctga cctgcggcga cgtggagga aacccggcc 7980. ccgattccat ggtgtctaag ggcgaagagc tgattaagga gaacatgcac atgaagctgt acatggaggg caccgtgaac aaccaccact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctacc agcttcatgt acggcagcaa aaccttcatc aaccacaccc 8220 agggcatccc cgacttcttt aagcagtcct tccctgaggg cttcacatgg gagagagtca 8340. ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag atcagagggg tgaacttccc atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggcctcca ccgagatgct gtaccccgct gacggcggcc 8460 tggaaggcag aagcgacatg gccctgaagc tcgtgggcgg gggccacctg atctgcaact 8520 tgaagaccac atacagatcc aagaaacccg ctaagaacct caagatgccc ggcgtctact 8580 atgtggacag aagactggaa agaatcaagg aggccgacaa agaaacctac gtcgagcagc 8640 acgaggtggc tgtggccaga tactgcgacc tccctagcaa actggggcac aaacttaatt 8700 aagtcgagac tagataactg atctacccag ctttcttgta caaagtggta cgcgtgaatt 8760 cactcctcag gtgcaggctg cctatcagaa ggtggtggct ggtgtggcca atgccctggc 8820 tcacaaatac cactgagatc tttttccctc tgccaaaaat tatggggaca tcatgaagcc 8880 ccttgagcat ctgacttctg gctaataaag gaaatttatt ttcattgcaa tagtgtgttg 8940 gaattttttg tgtctctcac tcggaaggac atatgggagg gcaaatcatt taaaacatca 9000 gaatgagtat ttggtttaga gtttggcaac atatgcccat atgctggctg ccatgaacaa 9060 aggttggcta taaagaggtc atcagtatat gaaacagccc cctgctgtcc attccttatt 9120 ccatagaaaa gccttgactt gaggttagat tttttttata ttttgttttg tgttattttt 9180 ttctttaaca tccctaaaat tttccttaca tgttttacta gccagatttt tcctcctctc 9240 ctgactactc ccagtcatag ctgtccctct tctcttatgg agatccctcg acctgcagcc 9300 caagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa 9360 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga 9420 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt 9480 gccagcggat cgacagtact aagcttggtg cgtttttatg cttgtagtat tgtataatgt 9540 ttttaagatc cttaattaat agggataa 9568 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <400> 9 taagtgaaag aagtcaatct gag 23 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <400> 10 ggcagtcatg taactatcac c 21 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <400> 11 gtattcacag agacttggcg gc 22 <210> 12 <211> 26 <212> DNA <213> Artificial sequence <400> 12 aaacctgcaa ggaaaatccttcacgg 26

Claims

1. A method for changing the color of pig hair, characterized in that, The method includes introducing a gene-editing element into a pig fertilized egg to obtain a KIT gene-edited embryo; or the method includes introducing a gene-editing element into a pig somatic cell, screening for KIT gene-edited positive somatic cells, and then constructing a reconstructed embryo by somatic cell nuclear transfer; the somatic cells include fibroblasts; The gene editing element consists of (a) or (b): (a) the sgRNA or the DNA molecule encoding the sgRNA; and Cas9-related elements, the Cas9-related elements including DNA encoding Cas9, mRNA encoding Cas9, or a Cas9 protein molecule; (b) An expression vector that simultaneously encodes the sgRNA and Cas9; The screening process includes PCR identification of somatic cells with introduced gene editing elements using a first primer pair and a second primer pair; the upstream and downstream primers of the first primer pair are located upstream and downstream of the 3-5 breakpoint, respectively; the second primer pair is used to amplify the 17 introns of the KIT gene; somatic cells with no amplification product from the first primer pair and with amplification product from the second primer pair are KIT gene-editing positive somatic cells. The nucleotide sequence in the sgRNA responsible for recognizing the target fragment region is the sequence shown in SEQ ID NO.2; The DNA molecule contains the sequence shown in SEQ ID NO.

5.

2. The method for changing pig hair color according to claim 1, characterized in that, The sequence of the vector that simultaneously encodes the sgRNA and Cas9 is shown in SEQ ID NO.

8.

3. The method for changing pig hair color according to claim 1, characterized in that, The gene editing element was introduced into porcine somatic cells using the calcium phosphate method, lipid transfection method, lentivirus transfection method, or electroporation method.

4. The method for changing pig hair color according to claim 1, characterized in that, The gene-editing element was introduced into pig somatic cells using electroporation.

5. The method for changing pig hair color according to claim 1, characterized in that, The gene-editing element was introduced into a pig zygote using a zygote microinjection method.

6. The method for changing pig hair color according to claim 1, characterized in that, The change of pig coat color includes changing the pig's coat color to black, where the pig's original coat color was white; the pig includes Large White pigs.

7. The method for changing pig hair color according to claim 1, characterized in that, The sequences of the first primer pair are shown in SEQ ID NO.9 and SEQ ID NO.10; The second primer pair sequence is shown in SEQ ID NO.11 and SEQ ID NO.12.

Images