Method for preparing pure hair and nail type ectodermal dysplasia model pig nuclear transfer donor cells and special gene editing system thereof

Abstract

The application discloses a method for preparing a pure hair and nail type ectodermal dysplasia model pig nuclear transfer donor cell and a special gene editing system thereof. The application provides a method for preparing a recombinant pig cell: HOXC13-gRNA1, HOXC13-gRNA4 and NCN protein are co-transfected into a pig cell to obtain the recombinant pig cell. The recombinant pig cell is a recombinant cell in which a HOXC13 gene is mutated. The application provides a kit comprising HOXC13-gRNA1 shown in SEQ ID NO: 16, HOXC13-gRNA4 shown in SEQ ID NO: 17 and NCN protein. The kit is used for: preparing the recombinant pig cell; preparing a pure hair and nail type ectodermal dysplasia model pig; preparing a pure hair and nail type ectodermal dysplasia cell model or a pure hair and nail type ectodermal dysplasia tissue model or a pure hair and nail type ectodermal dysplasia organ model. The application has great application value for research and development of a pure hair and nail type ectodermal dysplasia drug and revealing a pathogenesis of the disease.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, specifically to the field of gene editing, and more specifically relates to a method for preparing a pure hair and nail ectodermal dysplasia (pure hair and nail ectodermal dysplasia) model pig nuclear transfer donor cell and a special gene editing system therefor. BACKGROUND

[0002] Pure hair and nail ectodermal dysplasia (PHNED) is an autosomal recessive genetic disease, and the main clinical manifestations are complete loss or malnutrition of the patient's hair and nails from birth, which seriously affects the patient's appearance and fine motor function of the hands. The disease can be divided into four types, five types, six types, seven types and nine types, and the HOXC13 gene is the pathogenic gene of pure hair and nail ectodermal dysplasia type 9 (ECTD-9).

[0003] HOXC13 belongs to one of the Abd-B class members of the Hox (Homobox) gene family, and is closely related to hair follicle formation and hair growth. The expression of hair structure proteins KP (keratin) and KAP (keratin associated protein) is strictly regulated by HOXC13, and the expression level of HOXC13 gene is closely related to the formation of hair follicles and the growth of hair, and is essential for maintaining the normal morphology of hair follicles. Therefore, the study of HOXC13 helps to improve people's understanding of hair follicle development and hair growth mechanism, and further reveals the pathogenesis of ECTD-9 type.

[0004] The study of the occurrence and development mechanism of pure hair and nail ectodermal dysplasia and the development of corresponding drugs all need to be based on animal models, and the commonly used animal model is a mouse model. However, mice are very different from humans in terms of size, organ size, physiology, pathology, and other aspects, and cannot truly simulate the normal physiological and pathological state of humans. Pigs, as large animals, are similar in size and physiological function to humans, are easy to breed in large quantities, and have lower requirements in terms of ethics and animal protection, making them ideal animal models for human diseases.

[0005] Gene editing is a biological technology that has made significant progress in recent years, including gene editing based on homologous recombination, ZFN, TALEN, CRISPR / Cas9, and other editing technologies based on nucleases. Among them, CRISPR / Cas9 technology is the most advanced gene editing technology currently available. Currently, gene editing technology is being increasingly applied to the production of animal models. SUMMARY

[0006] The present application aims to provide a method for preparing pure hair and nail type ectodermal dysplasia model pig nuclear transfer donor cells and a special gene editing system thereof.

[0007] The present application provides a method for preparing recombinant pig cells, comprising the following steps: co-transfecting HOXC13-gRNA1, HOXC13-gRNA4 and NCN protein into pig cells to obtain recombinant pig cells.

[0008] The co-transfection is specifically in the form of electroporation.

[0009] The parameter setting of the electroporation is specifically 1450V, 10ms, 3pulse.

[0010] The co-transfection is specifically by using a mammalian nucleic acid transfection kit (Neon kit, Thermofisher) and a NeonTM transfection system electroporation instrument.

[0011] The present application also provides application of HOXC13-gRNA1, HOXC13-gRNA4 and NCN protein in preparation of a kit.

[0012] The present application also provides application of HOXC13-gRNA1, HOXC13-gRNA4 and PRONCN protein in preparation of a kit.

[0013] The present application also provides application of HOXC13-gRNA1, HOXC13-gRNA4 and specific plasmid in preparation of a kit.

[0014] The present application also provides a kit comprising HOXC13-gRNA1, HOXC13-gRNA4 and NCN protein.

[0015] The present application also provides a kit comprising HOXC13-gRNA1, HOXC13-gRNA4 and PRONCN protein.

[0016] The present application also provides a kit comprising HOXC13-gRNA1, HOXC13-gRNA4 and specific plasmid.

[0017] The kit according to any one of the above further comprises pig cells.

[0018] The use of any of the above-mentioned kits is as follows (a) or (b) or (c): (a) preparing a recombinant pig cell; (b) preparing a model pig of pure hair and nail type ectodermal dysplasia; (c) preparing a cell model or a tissue model or an organ model of pure hair and nail type ectodermal dysplasia of pure hair and nail type ectodermal dysplasia.

[0019] The ratio of HOXC13-gRNA1, HOXC13-gRNA4 and NCN protein is 0.8-1.2 μg HOXC13-gRNA1: 0.8-1.2 μg HOXC13-gRNA4: 3-5 μg NCN protein in turn.

[0020] The ratio of HOXC13-gRNA1, HOXC13-gRNA4 and NCN protein is 1 μg HOXC13-gRNA1: 1 μg HOXC13-gRNA4: 4 μg NCN protein in turn.

[0021] The ratio of pig cells, HOXC13-gRNA1, HOXC13-gRNA4 and NCN protein is 100,000 pig cells: 0.8-1.2 μg HOXC13-gRNA1: 0.8-1.2 μg HOXC13-gRNA4: 3-5 μg NCN protein in turn.

[0022] The ratio of pig cells, HOXC13-gRNA1, HOXC13-gRNA4 and NCN protein is 100,000 pig cells: 1 μg HOXC13-gRNA1: 1 μg HOXC13-gRNA4: 4 μg NCN protein in turn.

[0023] The HOXC13-gRNA1 of any of the above is an sgRNA, and the target sequence binding region is as shown in SEQ ID NO: 16.

[0024] Specifically, the HOXC13-gRNA1 is as shown in SEQ ID NO: 16.

[0025] Specifically, the HOXC13-gRNA1 is as shown in SEQ ID NO: 10.

[0026] The HOXC13-gRNA4 of any of the above is an sgRNA, and the target sequence binding region is as shown in SEQ ID NO: 17.

[0027] Specifically, the HOXC13-gRNA4 is as shown in SEQ ID NO: 17.

[0028] Specifically, the HOXC13-gRNA4 is shown as SEQ ID NO: 13.

[0029] The NCN protein is a Cas9 protein or a fusion protein having the Cas9 protein.

[0030] Specifically, the NCN protein is shown as SEQ ID NO: 3.

[0031] The pig cell is a pig fibroblast.

[0032] The pig cell is a primary pig fibroblast.

[0033] The pig cell is a primary pig fibroblast obtained from a newborn pig.

[0034] The method for preparing the NCN protein comprises the following steps:

[0035] (1) introducing the plasmid pKG-GE4 into E. coli BL21(DE3) to obtain a recombinant bacterium;

[0036] (2) culturing the recombinant bacterium in a liquid medium at 30°C, then adding IPTG and inducing the culture at 25°C, and then collecting the bacterial cells;

[0037] (3) subjecting the collected bacterial cells to cell disruption, and collecting a crude protein solution;

[0038] (4) purifying the fusion protein having His6 tag from the crude protein solution by affinity chromatography;

[0039] (5) subjecting the fusion protein having His6 tag to enterokinase digestion, and then removing the protein having His6 tag by Ni-NTA resin to obtain the purified NCN protein;

[0040] The fusion gene shown as nucleotides 5209-9852 in SEQ ID NO: 1 is in the plasmid pKG-GE4.

[0041] The method for preparing the NCN protein specifically comprises the following steps:

[0042] (1) introducing the plasmid pKG-GE4 into E. coli BL21(DE3) to obtain a recombinant bacterium.

[0043] (2) inoculating the recombinant bacterium obtained in step (1) into a liquid LB medium containing ampicillin, and subjecting to shaking culture;

[0044] (3) inoculating the bacterial solution obtained in step (2) into a liquid LB medium, and subjecting to shaking culture at 30°C and 230 rpm until the OD 600nmValue = 1.0, then IPTG is added to make its concentration in the system 0.5 mM, then 25℃, 230 rpm shaking culture for 12 hours, then centrifugal collection of bacterial bodies;

[0045] (4) The bacterial bodies obtained in step (3) are washed with PBS buffer;

[0046] (5) The bacterial bodies obtained in step (4) are suspended in crude extraction buffer, then the bacterial bodies are broken, then the supernatant is collected by centrifugation, and the filtrate is collected by using a filter membrane with a pore size of 0.22 μm;

[0047] (6) The fusion protein (the fusion protein shown in SEQ ID NO: 2) with His6 tag is purified from the filtrate obtained in step (5) by affinity chromatography;

[0048] (7) The post-column solution collected in step (6) is concentrated using an ultrafiltration tube, and then diluted with 25 mM Tris-HCl (pH 8.0);

[0049] (8) The recombinant bovine enterokinase with His6 tag is added to the solution obtained in step (7) for enzyme digestion;

[0050] (9) The solution after step (8) is mixed with Ni-NTA resin, incubated, and then the supernatant is collected by centrifugation;

[0051] (10) The supernatant obtained in step (9) is concentrated using an ultrafiltration tube, and then added to the enzyme storage solution, which is the NCN protein solution.

[0052] The specific method for purifying the fusion protein with His6 tag from the filtrate obtained in step (5) by affinity chromatography is as follows:

[0053] First, the Ni-NTA agarose column is equilibrated with 5 column volumes of equilibration buffer (flow rate of 1 ml / min); then 50 ml of the filtrate obtained in step (5) is loaded (flow rate of 0.5-1 ml / min); then the column is washed with 5 column volumes of equilibration buffer (flow rate of 1 ml / min); then the column is washed with 5 column volumes of buffer (flow rate of 1 ml / min) to remove impurities; then 10 column volumes of elution buffer are used to elute at a flow rate of 0.5-1 ml / min, and the post-column solution (90-100 ml) is collected.

[0054] Any of the above PRONCN proteins comprises the following elements from upstream to downstream: a signal peptide, a chaperone protein, a protein tag, a protease enzyme digestion site, a nuclear localization signal, a Cas9 protein, and a nuclear localization signal.

[0055] The signal peptide functions to promote protein secretion expression. The signal peptide can be selected from the group consisting of E. coli alkaline phosphatase (phoA) signal peptide, S. aureus protein A signal peptide, E. coli outer membrane protein (ompa) signal peptide, or any other prokaryotic gene signal peptide, preferably the alkaline phosphatase signal peptide (phoA signal peptide). The alkaline phosphatase signal peptide is used to guide the secretion expression of the target protein into the bacterial periplasmic cavity, so as to separate the intracellular protein from the bacteria, and the target protein secreted into the bacterial periplasmic cavity is soluble expression, which can be cleaved by the signal peptide enzyme in the bacterial periplasmic cavity.

[0056] The molecular chaperone functions to increase the solubility of the protein. The molecular chaperone can be any protein that helps form disulfide bonds, preferably thioredoxin (TrxA protein). Thioredoxin, which can act as a molecular chaperone to help the co-expressed target protein (such as Cas9 protein) form disulfide bonds, improve protein stability, folding accuracy, increase solubility and activity of the target protein.

[0057] The protein tag functions for protein purification. The tag can be a His tag (His-Tag, His6 protein tag), a GST tag, a Flag tag, an HA tag, a c-Myc tag, or any other protein tag, further preferably a His tag. The His tag can be combined with a Ni column, and the target protein can be purified by one-step Ni column affinity chromatography, which greatly simplifies the purification process of the target protein.

[0058] The protease cleavage site functions to remove the non-functional segment after purification to release the native form of the Cas9 protein. The protease can be selected from the group consisting of enterokinase, factor Xa, thrombin, TEV protease, HRV 3C protease, WELQut protease, or any other endoprotease, further preferably enterokinase. EK is the enterokinase cleavage site, which facilitates the removal of the fused TrxA-His segment using enterokinase to obtain the native form of the Cas9 protein. After the application uses commercial enterokinase with His tag to cleave the fusion protein, the TrxA-His segment and the enterokinase with His tag can be removed by one-step affinity chromatography to obtain the native form of the Cas9 protein, avoiding the damage and loss of the target protein caused by multiple purification and dialysis.

[0059] The nuclear localization signal can be any nuclear localization signal, preferably the SV40 nuclear localization signal and / or the nucleoplasmin nuclear localization signal. The NLS is the nuclear localization signal; an NLS site is designed at both the N-terminus and C-terminus of Cas9, enabling Cas9 to more effectively enter the cell nucleus for gene editing.

[0060] The Cas9 protein may be saCas9 or spCas9, preferably spCas9 protein.

[0061] The PRONCN protein is shown in SEQ ID NO: 2.

[0062] Each of the above-mentioned specific plasmids comprises the following elements from upstream to downstream: promoter, operon, ribosome binding site, gene encoding PRONCN protein, and terminator.

[0063] The promoter may specifically be the T7 promoter. The T7 promoter is a strong prokaryotic expression promoter that can efficiently drive the expression of exogenous genes.

[0064] The operon can specifically be the Lac operon. The Lac operon is a regulatory element for lactose-induced expression. After the bacteria have grown to a certain number, the expression of the target protein can be induced by IPTG at low temperature, which can avoid the impact of premature expression of the target protein on the growth of the host bacteria. Induction at low temperature also significantly improves the solubility of the expressed target protein.

[0065] The ribosome binding site is the ribosome binding site during protein translation, which is essential for protein translation.

[0066] The terminator can specifically be a T7 terminator. The T7 terminator can effectively terminate gene transcription at the end of the target gene, preventing other downstream sequences outside the target gene from being transcribed and translated.

[0067] For the codons of spCas9 protein, this application has optimized the codons to fully adapt to the codon preferences of the high-efficiency E. coli expression strain E. coli BL21(DE3) selected in this application, thereby improving the expression level of Cas9 protein.

[0068] The T7 promoter is shown as nucleotides 5121-5139 in SEQ ID NO: 1.

[0069] The Lac operon is shown as nucleotides 5140-5164 in SEQ ID NO: 1.

[0070] The ribosome binding site is shown as nucleotides 5178-5201 in SEQ ID NO: 1.

[0071] The coding sequence of the alkaline phosphatase signal peptide is shown as nucleotides 5209-5271 in SEQ ID NO: 1.

[0072] The coding sequence of the TrxA protein is shown as nucleotides 5272-5598 in SEQ ID NO: 1.

[0073] The coding sequence of His-Tag is shown as nucleotides 5620-5637 in SEQ ID NO: 1.

[0074] The coding sequence of the enterokinase cleavage site is shown as nucleotides 5638-5652 in SEQ ID NO: 1.

[0075] The coding sequence of the nuclear localization signal is shown as nucleotides 5656-5670 in SEQ ID NO: 1.

[0076] The coding sequence of the spCas9 protein is shown as nucleotides 5701-9801 in SEQ ID NO: 1.

[0077] The coding sequence of the nuclear localization signal is shown as nucleotides 9802-9849 in SEQ ID NO: 1.

[0078] The T7 terminator is nucleotides 9902-9949 in SEQ ID NO: 1.

[0079] Specifically, the specific plasmid is plasmid pKG-GE4.

[0080] The plasmid pKG-GE4 contains the DNA molecule represented by nucleotides 5121-9949 of SEQ ID NO: 1.

[0081] Specifically, any of the plasmids pKG-GE4 described above is shown in SEQ ID NO: 1.

[0082] This invention also protects recombinant porcine cells prepared by any of the methods described above.

[0083] The recombinant cells are recombinant pig cells with a mutation in the HOXC13 gene.

[0084] The recombinant pig cells can specifically be single-cell clones with genotypes of heterozygosity, identical biallelic mutants, or different biallelic mutants as shown in Table 1.

[0085] This invention also protects the application of the recombinant porcine cells in the preparation of pure trichome and nail-type ectodermal dysplasia model pigs. By using the recombinant porcine cells as nuclear transfer donor cells for somatic cell cloning, cloned pigs can be obtained, namely, pure trichome and nail-type ectodermal dysplasia model pigs.

[0086] This invention also protects porcine tissues from model pigs prepared using the recombinant porcine cells, specifically a tissue model of trichomes and toenail-type ectodermal dysplasia. This invention also protects porcine organs from model pigs prepared using the recombinant porcine cells, specifically an organ model of trichomes and toenail-type ectodermal dysplasia. This invention further protects porcine cells from model pigs prepared using the recombinant porcine cells, specifically a cell model of trichomes and toenail-type ectodermal dysplasia.

[0087] The present invention also protects the application of the recombinant porcine cells, the pure trichome and toenail ectodermal dysplasia tissue model, the pure trichome and toenail ectodermal dysplasia organ model, the pure trichome and toenail ectodermal dysplasia cell model, or the pure trichome and toenail ectodermal dysplasia model pig, as follows (d1) or (d2) or (d3) or (d4):

[0088] (d1) Screening for drugs to treat trichomes and nail ectodermal dysplasia;

[0089] (d2) Efficacy evaluation of drugs for pure trichomes and nail-type ectodermal dysplasia;

[0090] (d3) Evaluate the efficacy of gene therapy and / or cell therapy for pure trichomes and nail ectodermal dysplasia;

[0091] (d4) To study the pathogenesis of trichomes and nail-type ectodermal dysplasia.

[0092] The pig mentioned above can specifically be the Bama miniature pig.

[0093] The pigs mentioned above can specifically refer to newborn Bama miniature pigs.

[0094] The pig mentioned above can specifically refer to the Congjiang Xiang pig.

[0095] The pigs mentioned above can specifically refer to newborn Congjiang Xiang pigs.

[0096] All of the above-mentioned trichomeopathic and toenail-type ectodermal dysplasia is caused by mutations in the HOXC13 gene.

[0097] Pig HOXC13 gene information: encodes homeobox C13; located on chromosome 5; gene ID is 100518969, Susscrofa.

[0098] The protein encoded by the porcine HOXC13 gene is shown in SEQ ID NO: 8.

[0099] The porcine HOXC13 gene contains the DNA segment shown in SEQ ID NO: 9.

[0100] Any of the above-described mutations are deletions and / or insertions and / or substitutions of one or more nucleotides.

[0101] Any of the above-described mutations is the deletion of one or more nucleotides.

[0102] Any of the above mutations is an insertion of one or more nucleotides.

[0103] Any of the above-described mutations are deletions and insertions of one or more nucleotides.

[0104] The phenotype of pure trichomeopathic ectodermal dysplasia is: reduced hair growth and / or nail atrophy.

[0105] The phenotype of ectodermal dysplasia of pure trichomes and toenails is: hair loss and / or nail atrophy.

[0106] The phenotype of pure trichomes and toenail type ectodermal dysplasia is: reduced hair and / or hoof developmental abnormalities.

[0107] The phenotype of trichomeotypic ectodermal dysplasia is characterized by hair loss and / or hoof dysplasia.

[0108] The above-mentioned trichomeopathic ectodermal dysplasia is trichomeopathic ectodermal dysplasia type 9.

[0109] Compared with the prior art, the present invention has at least the following beneficial effects:

[0110] (1) The research object of this invention (pig) has better applicability than other animals (mice, mice, primates).

[0111] Rodents such as mice and rats differ greatly from humans in body size, organ size, physiology, and pathology, making it impossible to realistically simulate normal human physiological and pathological states. Studies have shown that over 95% of drugs proven effective in mice and rats are ineffective in human clinical trials. Among large animals, primates are the closest relatives to humans, but they are small, reach sexual maturity late (mating begins at 6-7 years old), and are single-birth animals, resulting in extremely slow population expansion and high rearing costs. Furthermore, primate cloning is inefficient, difficult, and costly.

[0112] Pigs, as model animals, do not have the aforementioned drawbacks. Pigs are the closest relatives to humans besides primates, and their body size, weight, and organ size are similar to humans. They are also remarkably similar to humans in anatomy, physiology, immunology, nutritional metabolism, and disease pathogenesis. Furthermore, pigs reach sexual maturity early (4-6 months), have high reproductive capacity, produce multiple offspring per litter, and can form a large herd within 2-3 years. In addition, pig cloning technology is very mature, and the costs of cloning and raising pigs are much lower than for primates. Therefore, pigs are very suitable animals to serve as human disease models.

[0113] (2) The vector constructed in this invention uses the strong promoter T7-lac, which can efficiently express the target protein, to express the target protein. The signal peptide of bacterial periplasmic protein alkaline phosphatase (phoA) guides the secretion of the target protein into the bacterial periplasmic lumen, thereby separating it from intracellular proteins. The target protein secreted into the bacterial periplasmic lumen is soluble. Simultaneously, the thioreduction protein TrxA is fused with the Cas9 protein for expression. TrxA helps the co-expressed target protein form disulfide bonds, improving protein stability, correct folding, and increasing the solubility and activity of the target protein. To facilitate the purification of the target protein, a His tag is designed, allowing for one-step Ni column affinity chromatography purification of the target protein, greatly simplifying the purification process. Furthermore, an enterokinase cleavage site is designed after the His tag to facilitate the removal of the fused TrxA-His polypeptide fragment, yielding the native form of the Cas9 protein. After cleaving the fusion protein with a His-tagged enterokinase, the TrxA-His polypeptide fragment and the His-tagged enterokinase can be removed by a single affinity chromatography step, yielding the native form of Cas9 protein. This avoids the damage and loss to the target protein caused by multiple purification dialysis steps. Furthermore, this invention also designs an NLS site at the N-terminus and C-terminus of Cas9, enabling Cas9 to more effectively enter the cell nucleus for gene editing. Additionally, this invention selects E. coli BL21(DE3) as the target protein expression strain, which can efficiently express exogenous genes cloned into expression vectors containing the phage T7 promoter (such as pET-32a). Moreover, this invention optimizes the codons for the Cas9 protein to perfectly suit the codon preferences of the expression strain, thereby improving the expression level of the target protein. Furthermore, this invention induces the expression of the target protein with IPTG at low temperature after the bacteria have grown to a certain quantity, avoiding the impact of premature expression on host bacterial growth. Low-temperature induction also significantly improves the solubility of the expressed target protein. After the above-mentioned optimization design and experimental implementation, the activity of the obtained Cas9 protein was significantly improved compared with that of the commercial Cas9 protein.

[0114] (3) Gene editing was performed using the Cas9 high-efficiency protein constructed and expressed in this invention in combination with in vitro transcribed gRNA, and the optimal ratio of Cas9 and gRNA was optimized. The final rate of gene-edited single-cell clones was as high as 96.8%, which is much higher than the conventional gene editing efficiency (10-30%).

[0115] (4) Using the target gene knockout single-cell clone obtained by the present invention to perform somatic cell nuclear transfer animal cloning, the target gene knockout clone pig can be directly obtained, and the gene mutation can be stably inherited.

[0116] The method of microinjecting gene-edited material into fertilized eggs followed by embryo transfer, used in mouse model creation, has a relatively low probability of directly obtaining gene-mutated offspring, requiring crossbreeding and selection of offspring. This method is not suitable for creating models of large animals (such as pigs) with long gestation periods. Therefore, this invention employs a technically challenging method of primary cell in vitro editing, Cas9 protein and double gRNA cleavage, and screening for positively edited single-cell clones. Subsequently, somatic cell nuclear transfer animal cloning technology is used to directly obtain pig models of the corresponding disease. This significantly shortens the pig model creation cycle and saves manpower, material resources, and financial resources.

[0117] This invention utilizes CRISPR / Cas9 technology combined with dual gRNA editing to knock out the HOXC13 gene, mimicking the genetic characteristics of trichomeotypic ectodermal dysplasia (THD). Single-cell clones with the HOXC13 gene knockout were obtained, laying the foundation for later development of a pig model of THD via somatic cell nuclear transfer animal cloning. This invention will contribute to the study and elucidation of the pathogenesis of THD caused by HOXC13 gene dysfunction. It can also be used for drug screening, efficacy evaluation, gene therapy, and cell therapy research, providing effective experimental data for further clinical applications and offering powerful experimental tools for the successful treatment of human THD. This invention has significant application value for the development of drugs for THD and elucidating the pathogenesis of this disease. Attached Figure Description

[0118] Figure 1 The results show the alignment of the forward sequencing of single-cell clone number 17 in Example 1 with the wild-type sequence.

[0119] Figure 2 The reverse sequencing results of the single-cell clone numbered 2 in Example 1 are compared with the wild-type sequence.

[0120] Figure 3 The reverse sequencing results of the single-cell clone numbered 22 in Example 1 are compared with the wild-type sequence.

[0121] Figure 4 The results show the alignment of the forward sequencing of the single-cell clone numbered 1 in Example 1 with the wild-type sequence.

[0122] Figure 5 This is an electrophoresis image of PCR amplification using different primer pairs with genome extracted from ear tissue of a pig named BX4 as a template in Example 2.

[0123] Figure 6 The image shows electrophoresis results of PCR amplification using primer pairs HOXC13-E1-JDF87 and HOXC13-E1-JDR605, respectively, with genomic DNA from 10 pigs as templates in Example 2.

[0124] Figure 7 This is a schematic diagram of the structure of plasmid pET-32a.

[0125] Figure 8 This is a schematic diagram of the structure of plasmid pKG-GE4.

[0126] Figure 9 This is an electrophoresis diagram showing the optimized ratio of gRNA to NCN protein in Example 4.

[0127] Figure 10 This is an electrophoresis diagram comparing the gene editing efficiency of NCN protein and commercial Cas9 protein in Example 4. Detailed Implementation

[0128] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0129] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. The recombinant plasmids constructed in the examples have all been sequenced and verified. The commercially available Cas9-A protein is a commercially available, effective Cas9 protein. The commercially available Cas9-B protein is a commercially available, effective Cas9 protein. Complete culture medium (% by volume): 15% fetal bovine serum (Gibco) + 83% DMEM medium (Gibco) + 1% Penicillin-Streptomycin (Gibco) + 1% HEPES (Solarbio). Cell culture conditions: 37°C, incubator with 5% CO2 and 5% O2.

[0130] The primary porcine fibroblasts used in Examples 1 and 2 were prepared from the ear tissue of newborn Bama miniature pigs. The primary porcine fibroblasts used in Example 4 were prepared from the ear tissue of newborn Jiangxiang pigs. Method for preparing porcine primary fibroblasts from ear tissue: ① Take 0.5g of porcine ear tissue, remove hair and bone tissue, then soak in 75% alcohol for 30-40s, wash 5 times with PBS buffer containing 5% (v / v) Penicillin-Streptomycin (Gibco), and then wash once with PBS buffer; ② Cut the tissue into small pieces with scissors, digest with 5mL of 0.1% collagenase solution (Sigma) at 37℃ for 1h, then centrifuge at 500g for 5min and discard the supernatant; ③ Resuspend the pellet in 1mL of complete culture medium, then plate it into a 10cm diameter cell culture dish containing 10mL of complete culture medium and sealed with 0.2% gelatin (VWR), and culture until the cells reach approximately 60% confluence with the bottom of the dish; ④ After completing step ③, digest with trypsin and collect the cells, then resuspend them in complete culture medium for subsequent electroporation experiments.

[0131] Example 1: Preparation of a single-cell clone of Bama miniature pig with HOXC13 gene knockout

[0132] Two highly efficient gRNA targets (HOXC13-E1-gRNA1 target and HOXC13-E1-gRNA4 target) were selected from the screening in Example 2.

[0133] The NCN protein used in Example 1 was provided by the NCN protein solution prepared in Example 3.

[0134] I. Preparation of gRNA

[0135] 1. Preparation of HOXC13-T7-gRNA1 and HOXC13-T7-gRNA4 transcription templates

[0136] The HOXC13-T7-gRNA1 transcription template is a double-stranded DNA molecule, as shown in SEQ ID NO: 14.

[0137] The HOXC13-T7-gRNA4 transcription template is a double-stranded DNA molecule, as shown in SEQ ID NO: 15.

[0138] 2. Obtain gRNA through in vitro transcription

[0139] Using the HOXC13-T7-gRNA1 transcription template, in vitro transcription was performed using the Transcript Aid T7 High Yield Transcription Kit (Fermentas, K0441), followed by MEGA clearing. TMThe HOXC13-gRNA1 was recovered and purified using a Transcription Clean-Up Kit (Thermo, AM1908). HOXC13-gRNA1 is a single-stranded RNA, as shown in SEQ ID NO: 16.

[0140] Using the HOXC13-T7-gRNA4 transcription template, in vitro transcription was performed using the Transcript Aid T7 High Yield Transcription Kit (Fermentas, K0441), followed by MEGA clearing. TM The HOXC13-gRNA4 was recovered and purified using a Transcription Clean-Up Kit (Thermo, AM1908). HOXC13-gRNA4 is a single-stranded RNA, as shown in SEQ ID NO: 17.

[0141] HOXC13-gRNA1 (SEQ ID NO: 16):

[0142] GGAGUGCUGAUACCCUUCGACGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAA

[0143] GUGGCACCGAGUCCGGUGCUUUU HOXC13-gRNA4 (SEQ ID NO: 17):

[0144] GGCAGUCAGGUGUACUGCUCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAA

[0145] GUGGCACCGAGUCGGUGCUUUU

[0146] II. Transfection of porcine primary fibroblasts

[0147] 1. Co-transfect porcine primary fibroblasts with HOXC13-gRNA1, HOXC13-gRNA4, and NCN protein. The ratio was approximately 100,000 porcine primary fibroblasts: 1 μg HOXC13-gRNA1 : 1 μg HOXC13-gRNA4 : 4 μg NCN protein. Co-transfection was performed using electroporation with a mammalian nuclear transfection kit (Neon kit, Thermofisher) and a Neon™ transfection system (parameters set to 1450V, 10ms, 3 pulses).

[0148] 2. After completing step 1, incubate in complete culture medium for 16-18 hours, then replace with fresh complete culture medium. The total incubation time after electroporation is 48 hours.

[0149] 3. After completing step 2, digest and collect cells with trypsin, wash with complete culture medium, resuspend in complete culture medium, and then pick each single clone and transfer it to a 96-well plate (1 cell per well, 100 μl of complete culture medium per well) and culture for 2 weeks (replace with fresh complete culture medium every 2-3 days).

[0150] 4. After completing step 3, digest the cells with trypsin and collect them (about 2 / 3 of the cells obtained from each well are seeded into a 6-well plate containing complete culture medium, and the remaining 1 / 3 are collected in a 1.5 mL centrifuge tube).

[0151] 5. Take the 6-well plate from step 4, culture until the cells reach 80% confluence, digest with trypsin and collect the cells, and freeze the cells using cell cryopreservation solution (90% complete culture medium + 10% DMSO, volume ratio).

[0152] 6. Take the centrifuge tube from step 4, collect the cells, lyse the cells and extract genomic DNA. Perform PCR amplification using primers consisting of HOXC13-E1-JDF87 and HOXC13-E1-JDR605, followed by electrophoresis. Use porcine primary fibroblasts as wild-type controls (WT).

[0153] 7. After completing step 6, recover the PCR amplification products and sequence them.

[0154] If a single-cell clone has only one sequencing result, its genotype is wild-type (also known as homozygous wild-type). If a single-cell clone has two sequencing results, one consistent with the sequencing result of a primary porcine fibroblast and the other showing a mutation (including deletion, insertion, or substitution of one or more nucleotides), the genotype of that single-cell clone is heterozygous. If a single-cell clone has two sequencing results, both showing mutations (including deletion, insertion, or substitution of one or more nucleotides) compared to the sequencing result of a primary porcine fibroblast, the genotype of that single-cell clone is biallelic mutant. If a single-cell clone has only one sequencing result and shows a mutation (including deletion, insertion, or substitution of one or more nucleotides) compared to the sequencing result of a primary porcine fibroblast, the genotype of that single-cell clone is biallelic mutant. If a single-cell clone has only one sequencing result and is consistent with the sequencing result of a primary porcine fibroblast, the genotype of that single-cell clone is wild-type (also known as homozygous wild-type).

[0155] The results are shown in Table 1. The genotype of single-cell clone numbered 17 was wild-type. The genotypes of single-cell clones numbered 2, 7, 15, 24, and 25 were heterozygous. The genotypes of single-cell clones numbered 3, 4, 5, 6, 8, 9, 11, 12, 13, 14, 16, 18, 19, 20, 21, 22, 23, 27, 28, 29, 30, and 31 were biallelic mutants. The genotypes of single-cell clones numbered 1, 10, and 26 were biallelic mutants. The success rate of obtaining HOXC13 gene-editing single-cell clones was 96.8%.

[0156] Example sequencing alignment results can be found in Figures 1 to 4 . Figure 1 The result is the alignment of the forward sequencing of single-cell clone number 17 with the wild-type sequence, which indicates that it is wild-type. Figure 2 The reverse sequencing results of single-cell clone number 2 were compared with the wild-type sequence, and it was determined to be heterozygous. Figure 3 The results are the reverse sequencing and wild-type sequence alignment of single-cell clone number 22, showing different biallelic mutants. Figure 4 This is the result of forward sequencing of single-cell clone number 1 and comparison with wild-type sequence, showing a biallelic mutant.

[0157] Table 1. Genotyping results of HOXC13 gene-edited single-cell clones.

[0158]

[0159]

[0160] The aforementioned heterozygous, biale-identical mutant, and biale-different mutant single-cell clones are all target single-cell clones. Using these cells as donor cells for nuclear transfer and performing somatic cell cloning, cloned pigs can be obtained, namely, pure trichomes and nail-type ectodermal dysplasia model pigs.

[0161] Example 2: Screening for highly efficient gRNA targets of the HOXC13 gene

[0162] Information on the porcine HOXC13 gene: Encoding the homeobox C13; located on chromosome 5; Gene ID: 100518969, Susscrofa. The amino acid sequence of the protein encoded by the porcine HOXC13 gene is shown in SEQ ID NO: 8. The porcine genomic DNA contains two exons; a portion of the first coding exon and its downstream sequence are shown in SEQ ID NO: 9.

[0163] I. Conservation analysis of the pre-defined deletion region of the HOXC13 gene and adjacent genomic sequences

[0164] Ten newborn Bama miniature pigs were selected, including six females (named BC1, BC2, BC3, BC4, BC5, and BC6) and four males (named BX1, BX2, BX3, and BX4).

[0165] HOXC13-E1-JDF113: CACCCGGGCGATAAGTACC;

[0166] HOXC13-E1-JDR604:GAACGTGTCGGAGCAGATTTC;

[0167] HOXC13-E1-JDF87: ACCTGCAGCAAAAACCTTGC;

[0168] HOXC13-E1-JDR605: GGAACGTGTCGGAGCAGATT.

[0169] Genomic DNA was extracted from ear tissue of a pig named BX4 and used as a template. PCR amplification was performed using different primer pairs, followed by 1% agarose gel electrophoresis. See the electrophoresis image below. Figure 5 . Figure 1Group 5: Primer pair consisting of HOXC13-E1-JDF87 and HOXC13-E1-JDR604; Group 2: Primer pair consisting of HOXC13-E1-JDF87 and HOXC13-E1-JDR605; Group 3: Primer pair consisting of HOXC13-E1-JDF113 and HOXC13-E1-JDR604; Group 4: Primer pair consisting of HOXC13-E1-JDF113 and HOXC13-E1-JDR605. The results indicate that the primer pair consisting of HOXC13-E1-JDF87 and HOXC13-E1-JDR605 is preferred for amplifying the target fragment.

[0170] Using genomic DNA from 10 pigs as templates, PCR amplification was performed using primer pairs consisting of HOXC13-E1-JDF87 and HOXC13-E1-JDR605, followed by 1% agarose gel electrophoresis. (See electrophoresis image below.) Figure 6 PCR amplification products were recovered and sequenced. The sequencing results were compared with HOXC13 gene sequences in public databases. Conserved regions common to 10 pigs were selected for gRNA target design.

[0171] II. Target Screening

[0172] Several targets were initially screened by NGG (avoiding possible mutation sites), and four targets were further screened out after preliminary experiments.

[0173] The four target points are as follows:

[0174] HOXC13-E1-gRNA1 target: AGTGCTGATACCCTTCGACG;

[0175] HOXC13-E1-gRNA2 target: CCAGTGCTGATACCCTTCGA;

[0176] HOXC13-E1-gRNA3 target: CAGCACTGGGCTCTCTCCAA;

[0177] HOXC13-E1-gRNA4 target: CAGTCAGGTGTACTGCTCCA.

[0178] III. Preparation of gRNA

[0179] The pKG-U6gRNA plasmid was digested with the restriction endonuclease BbsI, and the vector backbone (a large linear fragment of about 3kb) was recovered.

[0180] HOXC13-E1-gRNA1-S and HOXC13-E1-gRNA1-A were synthesized separately, then mixed and annealed to obtain a double-stranded DNA molecule with sticky ends. The sticky-ended double-stranded DNA molecule was ligated to a vector backbone to obtain the plasmid pKG-U6gRNA(HOXC13-E1-gRNA1). Plasmid pKG-U6gRNA(HOXC13-E1-gRNA1) expresses the sgRNA shown in SEQ ID NO: 10. HOXC13-E1-gRNA1 .

[0181] sgRNA HOXC13-E1-gRNA1 (SEQ ID NO: 10):

[0182] AGUGCUGAUACCCUUCGACGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

[0183] HOXC13-E1-gRNA2-S and HOXC13-E1-gRNA2-A were synthesized separately, then mixed and annealed to obtain a double-stranded DNA molecule with sticky ends. The sticky-ended double-stranded DNA molecule was ligated to a vector backbone to obtain plasmid pKG-U6gRNA(HOXC13-E1-gRNA2). Plasmid pKG-U6gRNA(HOXC13-E1-gRNA2) expresses the sgRNA shown in SEQ ID NO: 11. HOXC13-E1-gRNA2 .

[0184] sgRNA HOXC13-E1-gRNA2 (SEQ ID NO: 11):

[0185] CCAGUGCUGAUACCCUUCGAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

[0186] HOXC13-E1-gRNA3-S and HOXC13-E1-gRNA3-A were synthesized separately, then mixed and annealed to obtain a double-stranded DNA molecule with sticky ends. The sticky-ended double-stranded DNA molecule was ligated to a vector backbone to obtain plasmid pKG-U6gRNA(HOXC13-E1-gRNA3). Plasmid pKG-U6gRNA(HOXC13-E1-gRNA3) expresses the sgRNA shown in SEQ ID NO: 12. HOXC13-E1-gRNA3 .

[0187] sgRNA HOXC13-E1-gRNA3 (SEQ ID NO: 12):

[0188] CAGCACUGGGCUCUCUCCAAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

[0189] HOXC13-E1-gRNA4-S and HOXC13-E1-gRNA4-A were synthesized separately, then mixed and annealed to obtain a double-stranded DNA molecule with sticky ends. The sticky-ended double-stranded DNA molecule was ligated to a vector backbone to obtain the plasmid pKG-U6gRNA(HOXC13-E1-gRNA4). Plasmid pKG-U6gRNA(HOXC13-E1-gRNA4) expresses the sgRNA shown in SEQ ID NO: 13. HOXC13-E1-gRNA4 .

[0190] sgRNA HOXC13-E1-gRNA4 (SEQ ID NO: 13):

[0191] CAGUCAGGUGUACUGCUCCAguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

[0192] HOXC13-E1-gRNA1-S:caccgAGTGCTGATAACCCTTCGACG;

[0193] HOXC13-E1-gRNA1-A:aaacCGTCGAAGGGTATCAGCACTc;

[0194] HOXC13-E1-gRNA2-S:caccgCCAGTGCTGATAACCCTTCGA;

[0195] HOXC13-E1-gRNA2-A:aaacTCGAAGGGTATCAGCACTGGc;

[0196] HOXC13-E1-gRNA3-S: caccgCAGCACTGGGCTCTCTCCAA;

[0197] HOXC13-E1-gRNA3-A:aaacTTGGAGAGAGCCCAGTGCTGc;

[0198] HOXC13-E1-gRNA4-S: caccgCAGTCAGGTGTACTGCTCCA;

[0199] HOXC13-E1-gRNA4-A:aaacTGGAGCAGTACACCTGACTGc.

[0200] HOXC13-E1-gRNA1-S, HOXC13-E1-gRNA1-A, HOXC13-E1-gRNA2-S, HOXC13-E1-gRNA2-A, HOXC13-E1-gRNA3-S, HOXC13-E1-gRNA3-A, HOXC13-E1-gRNA4-S, and HOXC13-E1-gRNA4-A are all single-stranded DNA molecules.

[0201] IV. Editing efficiency testing for different target points

[0202] 1. Co-transfection

[0203] Group 1: Porcine primary fibroblasts were co-transfected with plasmid pKG-U6gRNA (HOXC13-E1-gRNA1) and plasmid pKG-GE3. The ratio was approximately 200,000 porcine primary fibroblasts: 0.92 μg of plasmid pKG-U6gRNA (HOXC13-E1-gRNA1): 1.08 μg of plasmid pKG-GE3.

[0204] Group 2: Porcine primary fibroblasts were co-transfected with plasmid pKG-U6gRNA (HOXC13-E1-gRNA2) and plasmid pKG-GE3. The ratio was approximately 200,000 porcine primary fibroblasts: 0.92 μg of plasmid pKG-U6gRNA (HOXC13-E1-gRNA2): 1.08 μg of plasmid pKG-GE3.

[0205] Group 3: Porcine primary fibroblasts were co-transfected with plasmid pKG-U6gRNA (HOXC13-E1-gRNA3) and plasmid pKG-GE3. The ratio was approximately 200,000 porcine primary fibroblasts: 0.92 μg of plasmid pKG-U6gRNA (HOXC13-E1-gRNA3): 1.08 μg of plasmid pKG-GE3.

[0206] Group 4: Porcine primary fibroblasts were co-transfected with plasmid pKG-U6gRNA (HOXC13-E1-gRNA4) and plasmid pKG-GE3. The ratio was approximately 200,000 porcine primary fibroblasts: 0.92 μg of plasmid pKG-U6gRNA (HOXC13-E1-gRNA4) : 1.08 μg of plasmid pKG-GE3.

[0207] Group 5: Primary porcine fibroblasts were electroporated without plasmids using the same electroporation parameters.

[0208] Co-transfection was performed using electroporation with a mammalian nuclear transfection kit (Neon kit, Thermofisher) and a Neon™ transfection system (parameters set to 1450V, 10ms, 3 pulses).

[0209] 2. After completing step 1, incubate in complete culture medium for 12-18 hours, then replace with fresh complete culture medium. The total incubation time after electroporation is 48 hours.

[0210] 3. After completing step 2, cells were digested and collected using trypsin, lysed, and genomic DNA was extracted. PCR amplification was performed using primer pairs consisting of HOXC13-E1-JDF87 and HOXC13-E1-JDR605, followed by 1% agarose gel electrophoresis to detect mutations in target genes.

[0211] After the target product was gel-extracted and recovered, it was sent to a sequencing company for sequencing. The sequencing results were then analyzed using the web-based Synthego ICE tool to determine the gene editing efficiency of different targets. The gene editing efficiencies of the first, second, third, and fourth groups were 84%, 48%, 43%, and 75%, respectively, while no gene editing occurred in the fifth group. The results indicate that the HOXC13-E1-gRNA1 and HOXC13-E1-gRNA4 targets have high editing efficiencies.

[0212] Example 3: Preparation and purification of NCN protein

[0213] I. Construction of a high-efficiency prokaryotic Cas9 expression vector

[0214] A schematic diagram of the structure of plasmid pET-32a is shown below. Figure 7 .

[0215] Plasmid pKG-GE4 was obtained by modifying plasmid pET-32a. Plasmid pET32a-T7lac-phoA:SP-TrxA-His-EK-NLS-spCas9-NLS-T7ter (abbreviated as plasmid pKG-GE4), as shown in SEQ ID NO: 1, is a circular plasmid; its structural diagram is shown below. Figure 8 .

[0216] In SEQ ID NO: 1, nucleotides 5121-5139 form the T7 promoter, nucleotides 5140-5164 encode the Lac operator, nucleotides 5178-5201 form the ribosome binding site (RBS), nucleotides 5209-5271 encode the alkaline phosphatase signal peptide (phoA signal peptide), nucleotides 5272-5598 encode the TrxA protein, nucleotides 5620-5637 encode the His-Tag (also known as the His6 tag), nucleotides 5638-5652 encode the enterokinase cleavage site (EK cleavage site), nucleotides 5656-5670 encode the nuclear localization signal, nucleotides 5701-9801 encode the spCas9 protein, nucleotides 9802-9849 encode the nuclear localization signal, and nucleotides 9902-9949 form the T7 terminator. The nucleotides encoding the spCas9 protein have been codon-optimized for Escherichia coli BL21(DE3) strain.

[0217] The main modifications to plasmid pKG-GE4 are as follows: ① The coding region of the TrxA protein was retained. The TrxA protein can help the expressed target protein form disulfide bonds, increasing the solubility and activity of the target protein. An alkaline phosphatase signal peptide coding sequence was added before the TrxA protein coding region. The alkaline phosphatase signal peptide can guide the expressed target protein to be secreted into the bacterial periplasmic lumen and can be cleaved by prokaryotic periplasmic signal peptidase. ② A His-Tag coding sequence was added after the TrxA protein coding sequence. The His-Tag can be used for... Enrichment of the target protein; ③ Add the coding sequence of the enterokinase cleavage site DDDDK (Asp-Asp-Asp-Asp-Lys) downstream of the His-Tag coding sequence. The purified protein will remove His-Tag and the upstream fused TrxA protein under the action of enterokinase; ④ Insert the Cas9 gene of suitable Escherichia coli BL21(DE3) strain with optimized codons, and add nuclear localization signal coding sequences upstream and downstream of this gene to increase the nuclear localization ability of the purified Cas9 protein in the later stage.

[0218] The fusion gene in plasmid pKG-GE4, as shown in nucleotides 5209-9852 of SEQ ID NO: 1, encodes the fusion protein shown in SEQ ID NO: 2 (fusion protein TrxA-His-EK-NLS-spCas9-NLS, abbreviated as PRONCN protein). Due to the presence of alkaline phosphatase signal peptide and enterokinase cleavage site, the fusion protein is cleaved by enterokinase to form the protein shown in SEQ ID NO: 3. The protein shown in SEQ ID NO: 3 is named NCN protein.

[0219] II. Induced Expression

[0220] 1. Plasmid pKG-GE4 was introduced into Escherichia coli BL21(DE3) to obtain recombinant bacteria.

[0221] 2. Inoculate the recombinant bacteria obtained in step 1 into liquid LB medium containing 100 μg / ml ampicillin and culture overnight at 37°C with shaking at 200 rpm.

[0222] 3. Inoculate the bacterial culture obtained in step 2 into liquid LB medium and incubate at 30°C with shaking at 230 rpm until OD reaches 100%. 600nm The concentration was set to 1.0, then isopropyl thiogalactoside (IPTG) was added to a concentration of 0.5 mM in the system. The mixture was then cultured at 25°C and 230 rpm for 12 hours with shaking. Finally, the cells were collected by centrifugation at 4°C and 10,000 g for 15 minutes.

[0223] 4. Take the bacterial cells obtained in step 3 and wash them with PBS buffer.

[0224] III. Purification of the fusion protein TrxA-His-EK-NLS-spCas9-NLS

[0225] 1. Take the bacterial cells obtained in step 2, add crude extraction buffer and suspend the cells. Then, homogenize the cells using a homogenizer (3 cycles at 1000 rpm). Centrifuge at 15000g for 30 min at 4℃, collect the supernatant, and filter the supernatant through a 0.22μm pore size filter membrane. Collect the filtrate. In this step, 10 ml of crude extraction buffer is prepared for every gram of wet weight of bacterial cells. Crude extraction buffer: contains 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 5 mM Imidazole, 1 mM PMSF, and the balance is ddH2O.

[0226] 2. Affinity chromatography was used to purify the fusion protein.

[0227] First, equilibrate the Ni-NTA agarose column with 5 column volumes of equilibration buffer (flow rate: 1 ml / min); then load 50 ml of the filtrate obtained in step 1 (flow rate: 0.5-1 ml / min); then wash the column with 5 column volumes of equilibration buffer (flow rate: 1 ml / min); then wash the column with 5 column volumes of buffer (flow rate: 1 ml / min) to remove contaminating proteins; finally, elute with 10 column volumes of elution buffer at a flow rate of 0.5-1 ml / min, and collect the post-column solution (90-100 ml).

[0228] Ni-NTA agarose column: GenScript, L00250 / L00250-C, 10ml packing material.

[0229] Equilibrium solution: contains 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 5 mM Imidazole, and the balance is ddH2O.

[0230] Buffer solution: containing 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 50 mM Imidazole, with the balance being ddH2O.

[0231] Eluent: Contains 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 500 mM Imidazole, and the balance is ddH2O.

[0232] IV. Enzymatic digestion of the fusion protein TrxA-His-EK-NLS-spCas9-NLS and purification of NCN protein

[0233] 1. Take 15 ml of the post-column solution collected in step 3, concentrate it to 200 μl using an Amicon ultrafiltration tube (Sigma, UFC9100, 15 ml capacity), and then dilute it to 1 ml with 25 mM Tris-HCl (pH 8.0). Use 6 ultrafiltration tubes to obtain a total of 6 ml.

[0234] 2. Add the commercially available His6-tagged recombinant bovine enterokinase (Sangon Biotech, C620031, Recombinant Bovine Enterokinase Light Chain, His6-tagged) to the solution obtained in step 1 (approximately 6 ml), and digest at 25°C for 16 hours. Add 2 units of enterokinase per 50 μg of protein.

[0235] 3. Take the solution from step 2 (about 6 ml), mix it with 480 μl of Ni-NTA resin (GenScript, L00250 / L00250-C), mix by rotation at room temperature for 15 min, then centrifuge at 7000 g for 3 min, and collect the supernatant (4-5.5 ml).

[0236] 4. Take the supernatant obtained in step 3 and concentrate it to 200 μl using an Amicon ultrafiltration tube (Sigma, UFC9100, 15 ml capacity). Then add it to the enzyme stock solution and adjust the protein concentration to 5 mg / ml to obtain the NCN protein solution. Enzyme stock solution (pH 7.4): contains 10 mM Tris, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% (v / v) glycerol, and the balance is ddH2O.

[0237] Sequencing revealed that the N-terminal 15 amino acid residues in the NCN protein solution are as shown in positions 1 to 15 of SEQ ID NO: 3, which is the NCN protein.

[0238] Example 4: Performance of NCN protein

[0239] The NCN protein used in Example 4 was provided by the NCN protein solution prepared in Example 3.

[0240] The following two gRNA targets targeting the TTN gene were selected:

[0241] TTN-gRNA1 target: AGAGCACAGTCAGCCTGGCG;

[0242] TTN-gRNA2 target: CTTCCAGAATTGGATCTCCG.

[0243] The primers used to identify target fragments containing gRNA from the TTN gene are as follows:

[0244] TTN-F55: TACGGAATTGGGGAGCCAGCGGA;

[0245] TTN-R560: CAAAGTTAACTCTCTGTGTCT.

[0246] I. Preparation of gRNA

[0247] 1. Preparation of TTN-T7-gRNA1 and TTN-T7-gRNA2 transcription templates

[0248] The TTN-T7-gRNA1 transcription template is a double-stranded DNA molecule, as shown in SEQ ID NO: 4.

[0249] The TTN-T7-gRNA2 transcription template is a double-stranded DNA molecule, as shown in SEQ ID NO: 5.

[0250] 2. Obtain gRNA through in vitro transcription

[0251] Using TTN-T7-gRNA1 as a transcription template, in vitro transcription was performed using the Transcript Aid T7 High Yield Transcription Kit (Fermentas, K0441), followed by MEGA clearing. TM The TTN-gRNA1 was recovered and purified using a Transcription Clean-Up Kit (Thermo, AM1908). TTN-gRNA1 is a single-stranded RNA, as shown in SEQ ID NO: 6.

[0252] Using TTN-T7-gRNA2 as a transcription template, in vitro transcription was performed using the Transcript Aid T7 High Yield Transcription Kit (Fermentas, K0441), followed by MEGA clearing. TM The TTN-gRNA2 was recovered and purified using a Transcription Clean-Up Kit (Thermo, AM1908). TTN-gRNA2 is a single-stranded RNA, as shown in SEQ ID NO: 7.

[0253] II. Optimization of the ratio of gRNA to NCN protein

[0254] 1. Co-transfection of porcine primary fibroblasts

[0255] Group 1: TTN-gRNA1, TTN-gRNA2, and NCN protein were co-transfected into porcine primary fibroblasts. The ratio was approximately 100,000 porcine primary fibroblasts: 0.5 μg TTN-gRNA1 : 0.5 μg TTN-gRNA2 : 4 μg NCN protein.

[0256] Group 2: TTN-gRNA1, TTN-gRNA2, and NCN protein were co-transfected into porcine primary fibroblasts. The ratio was approximately 100,000 porcine primary fibroblasts: 0.75 μg TTN-gRNA1 : 0.75 μg TTN-gRNA2 : 4 μg NCN protein.

[0257] Group 3: TTN-gRNA1, TTN-gRNA2, and NCN protein were co-transfected into porcine primary fibroblasts. The ratio was approximately 100,000 porcine primary fibroblasts: 1 μg TTN-gRNA1 : 1 μg TTN-gRNA2 : 4 μg NCN protein.

[0258] Group 4: TTN-gRNA1, TTN-gRNA2, and NCN protein were co-transfected into porcine primary fibroblasts. The ratio was approximately 100,000 porcine primary fibroblasts: 1.25 μg TTN-gRNA1 : 1.25 μg TTN-gRNA2 : 4 μg NCN protein.

[0259] Group 5: TTN-gRNA1 and TTN-gRNA2 were co-transfected into porcine primary fibroblasts. Ratio: approximately 100,000 porcine primary fibroblasts: 1 μg TTN-gRNA1: 1 μg TTN-gRNA2.

[0260] Co-transfection was performed using electroporation with a mammalian nuclear transfection kit (Neon kit, Thermofisher) and a Neon™ transfection system (parameters set to 1450V, 10ms, 3 pulses).

[0261] 2. After completing step 1, incubate in complete culture medium for 12-18 hours, then replace with fresh complete culture medium. The total incubation time after electroporation is 48 hours.

[0262] 3. After completing step 2, cells were digested and collected with trypsin, genomic DNA was extracted, and PCR amplification was performed using primers consisting of TTN-F55 and TTN-R560, followed by 1% agarose gel electrophoresis.

[0263] See electrophoresis image Figure 9 The 505bp band is the wild-type band (WT), and the band around 254bp (the wild-type band theoretically has a deletion of 251bp) is the deletion mutation band (MT).

[0264] Gene deletion mutation efficiency = (MT gray level / MT band bp) / (WT gray level / WT band bp + MT gray level / MT band bp) × 100%. The gene deletion mutation efficiency of the first group is 19.9%, the gene deletion mutation efficiency of the second group is 39.9%, the gene deletion mutation efficiency of the third group is 79.9%, and the gene deletion mutation efficiency of the fourth group is 44.3%. No mutation occurred in the fifth group.

[0265] The results showed that the gene editing efficiency was highest when the mass ratio of the two gRNAs to the NCN protein was 1:1:4, and the actual dosage was 1 μg:1 μg:4 μg. Therefore, the optimal dosage of the two gRNAs to the NCN protein was determined to be 1 μg:1 μg:4 μg.

[0266] III. Comparison of gene editing efficiency between NCN protein and commercial Cas9 protein

[0267] 1. Co-transfection of porcine primary fibroblasts

[0268] Cas9-A group: TTN-gRNA1, TTN-gRNA2, and commercial Cas9-A protein were co-transfected into porcine primary fibroblasts. Ratio: approximately 100,000 porcine primary fibroblasts: 1 μg TTN-gRNA1 : 1 μg TTN-gRNA2 : 4 μg Cas9-A protein.

[0269] pKG-GE4 group: TTN-gRNA1, TTN-gRNA2, and NCN protein were co-transfected into porcine primary fibroblasts. Ratio: approximately 100,000 porcine primary fibroblasts: 1 μg TTN-gRNA1 : 1 μg TTN-gRNA2 : 4 μg NCN protein.

[0270] Cas9-B group: TTN-gRNA1, TTN-gRNA2, and commercial Cas9-B protein were co-transfected into porcine primary fibroblasts. Ratio: approximately 100,000 porcine primary fibroblasts : 1 μg TTN-gRNA1 : 1 μg TTN-gRNA2 : 4 μg Cas9-B protein.

[0271] Control group: porcine primary fibroblasts were co-transfected with TTN-gRNA1 and TTN-gRNA2. Ratio: approximately 100,000 porcine primary fibroblasts: 1 μg TTN-gRNA1 : 1 μg TTN-gRNA2.

[0272] Co-transfection was performed using electroporation with a mammalian nuclear transfection kit (Neon kit, Thermofisher) and a Neon™ transfection system (parameters set to 1450V, 10ms, 3 pulses).

[0273] 2. After completing step 1, incubate in complete culture medium for 12-18 hours, then replace with fresh complete culture medium. The total incubation time after electroporation is 48 hours.

[0274] 3. After completing step 2, cells were digested and collected with trypsin, genomic DNA was extracted, and PCR amplification was performed using primers consisting of TTN-F55 and TTN-R560, followed by 1% agarose gel electrophoresis.

[0275] See electrophoresis image Figure 10 The gene deletion mutation efficiency using commercial Cas9-A protein was 28.5%, that using NCN protein was 85.6%, and that using commercial Cas9-B protein was 16.6%.

[0276] The results showed that, compared with commercially available Cas9 protein, the NCN protein prepared using this invention significantly improved gene editing efficiency.

[0277] The plasmid pKG-GE3 in Example 2 is a circular plasmid, as shown in SEQ ID NO: 2 of patent application 202010084343.6. In SEQ ID NO: 2 of patent application 202010084343.6, nucleotides 395-680 form the CMV enhancer, nucleotides 682-890 form the EF1a promoter, nucleotides 986-1006 encode the nuclear localization signal (NLS), nucleotides 1016-1036 encode the nuclear localization signal (NLS), nucleotides 1037-5161 encode the Cas9 protein, nucleotides 5162-5209 encode the nuclear localization signal (NLS), nucleotides 5219-5266 encode the nuclear localization signal (NLS), and nucleotides 5276-5332 encode polypeptide P2A (the amino acid sequence of polypeptide P2A is “ATNFSLLKQAGDVEENPGP”, with the break position being…). Nucleotides 5333-6046 (between the first and second amino acid residues at the C-terminus) encode the EGFP protein, nucleotides 6056-6109 encode polypeptide T2A (the amino acid sequence of polypeptide T2A is “EGRGSLLTCGDVEENPGP”, with the break point between the first and second amino acid residues at the C-terminus), nucleotides 6110-6703 encode the Puromycin protein (abbreviated as Puro protein), nucleotides 6722-7310 form the WPRE sequence element, nucleotides 7382-7615 form the 3'LTR sequence element, and nucleotides 7647-7871 form the bGH poly(A)signal sequence element. In SEQ ID NO: 2 of patent application 202010084343.6, nucleotides 911-6706 form a fusion gene, expressing a fusion protein. Due to the presence of the self-cleaving peptide P2A and the self-cleaving peptide T2A, the fusion protein spontaneously forms the following three proteins: a protein with Cas9 protein, a protein with EGFP protein, and a protein with Puro protein.

[0278] The pKG-U6gRNA vector, or plasmid pKG-U6gRNA, in Example 2 is a circular plasmid, as shown in SEQ ID NO: 3 of patent application 202010084343.6. In SEQ ID NO: 3 of patent application 202010084343.6, nucleotides 2280-2539 form the hU6 promoter, and nucleotides 2558-2637 are used for transcription to form the gRNA backbone. In use, a DNA molecule of approximately 20 bp (the target sequence binding region for transcription to form gRNA) is inserted into the plasmid pKG-U6gRNA to form a recombinant plasmid. The recombinant plasmid is then transcribed into gRNA in cells.

[0279] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for preparing recombinant porcine cells, comprising the following steps: co-transfecting porcine cells with HOXC13-gRNA1, HOXC13-gRNA4 and NCN protein to obtain recombinant porcine cells; The HOXC13-gRNA1 is an sgRNA, and its target sequence binding region is shown as nucleotides 3-22 in SEQ ID NO: 16; the HOXC13-gRNA4 is an sgRNA, and its target sequence binding region is shown as nucleotides 3-22 in SEQ ID NO: 17; the NCN protein is shown as in SEQ ID NO: 3; The method for preparing the NCN protein includes the following steps: (1) Plasmid pKG-GE4 was introduced into Escherichia coli BL21(DE3) to obtain recombinant bacteria; (2) The recombinant bacteria were cultured in liquid culture medium at 30°C, then IPTG was added and the culture was induced at 25°C, and then the bacterial cells were collected; (3) The collected bacterial cells were broken down to collect the crude protein solution; (4) The His6-tagged fusion protein was purified from the crude protein solution by affinity chromatography; (5) The His6-tagged fusion protein was digested with His6-tagged enterokinase, and then the His6-tagged protein was removed with Ni-NTA resin to obtain purified NCN protein. The plasmid pKG-GE4 is shown in SEQ ID NO: 1; The mass ratio of HOXC13-gRNA1, HOXC13-gRNA4, and NCN protein is 1:1:

4.

2. Application of HOXC13-gRNA1, HOXC13-gRNA4, and NCN proteins in the preparation of the kit; HOXC13-gRNA1 is the HOXC13-gRNA1 described in claim 1; HOXC13-gRNA4 is the HOXC13-gRNA4 described in claim 1; NCN protein is the NCN protein described in claim 1; The mass ratio of HOXC13-gRNA1, HOXC13-gRNA4 and NCN protein is 1:1:4; The kit is intended for use as follows (a) or (b) or (c): (a) to prepare recombinant porcine cells; (b) to prepare pure trichomeal and toenail ectodermal dysplasia model pigs; (c) to prepare pure trichomeal and toenail ectodermal dysplasia cell models, pure trichomeal and toenail ectodermal dysplasia tissue models, or pure trichomeal and toenail ectodermal dysplasia organ models.

3. A kit comprising HOXC13-gRNA1, HOXC13-gRNA4 and NCN protein; HOXC13-gRNA1 is the HOXC13-gRNA1 described in claim 1; HOXC13-gRNA4 is the HOXC13-gRNA4 described in claim 1; NCN protein is the NCN protein described in claim 1; The mass ratio of HOXC13-gRNA1, HOXC13-gRNA4 and NCN protein is 1:1:4; The kit is intended for use as follows (a) or (b) or (c): (a) to prepare recombinant porcine cells; (b) to prepare pure trichomeal and toenail ectodermal dysplasia model pigs; (c) to prepare pure trichomeal and toenail ectodermal dysplasia cell models, pure trichomeal and toenail ectodermal dysplasia tissue models, or pure trichomeal and toenail ectodermal dysplasia organ models.

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