CN116103339A - Kit and application thereof in construction of pig nuclear transfer donor cells of COL2A1 gene mutation type II collagen disease model - Google Patents

Abstract

The invention discloses a kit and application thereof in constructing a model pig nuclear transfer donor cell of a COL2A1 gene mutation type II collagen disease. The present invention provides SEQ ID NO:16, COL2A1-gRNA3, SEQ ID NO:17 and NCN protein of COL2A1-gRNA4 in the preparation of the kit. The invention also provides a method for preparing the recombinant pig cell: and co-transfecting the COL2A1-gRNA3, the COL2A1-gRNA4 and the NCN protein into the pig cells to obtain the recombinant pig cells. The recombinant porcine cell is a recombinant porcine cell with mutation of COL2A1 gene. The kit has the following purposes: preparing recombinant pig cells; preparing a model pig of type II collagen diseases; preparing a type II collagenosis cell model, a type II collagenosis tissue model or a type II collagenosis organ model. The invention has great application value for researching and developing type II collagen diseases and revealing pathogenesis of the diseases.

Description

Kit and its application in constructing a pig model of type II collagen disease with COL2A1 gene mutation Nuclear transplantation donor cells

Technical Field

The present invention belongs to the field of biotechnology, specifically to the field of gene editing technology, and more specifically to a kit and its application in constructing a pig nuclear transplant donor cell with a COL2A1 gene mutation type II collagen disease model.

Background Art

Collagen is the largest type of protein in the human body, and type II collagen is an important protein that constitutes articular cartilage and hyaline cartilage. The COL2A1 gene can encode the precursor of type II collagen, so mutations in the COL2A1 gene will cause changes in the structure of type II collagen, leading to type II collagen disease (also known as type II collagen disease). Type II collagen disease is an autosomal dominant genetic disease, and the main symptoms of patients include various cartilage dysplasias and osteoarthritis.

The COL2A1 gene encodes the α chain of type II collagen precursor protein. Three identical α chains can form precursor protein homotrimers after folding each other, and the homotrimers then form mature type II collagen through cross-linking. There is a special triple helix region in the α chain, and the characteristic sequence of this region is a repeated Gly-X-Y sequence (Gly is glycine, X and Y are other amino acids). The triple helix structure plays an important role in the stability of collagen molecules.

The study of the occurrence and development mechanism of type II collagen disease and the development of corresponding drugs need to be based on animal models. The commonly used animal model is the mouse model. However, mice are very different from humans in terms of body shape, organ size, physiology, pathology, etc., and cannot truly simulate the normal physiological and pathological state of humans. Pigs are large animals with similar body size and physiological functions to humans. They are easy to breed and raise on a large scale, and have lower requirements in ethics and animal protection. They are ideal human disease model animals.

Gene editing is a biotechnology that has made significant progress in recent years. It includes gene editing based on homologous recombination to nuclease-based ZFN, TALEN, CRISPR/Cas9 and other editing technologies. Among them, CRISPR/Cas9 technology is the most advanced gene editing technology. At present, gene editing technology is increasingly being used in the preparation of animal models.

Summary of the invention

The purpose of the present invention is to provide a kit and its application in constructing a pig nuclear transplant donor cell with a COL2A1 gene mutation type II collagen disease model.

The present invention provides use of COL2A1-gRNA3, COL2A1-gRNA4 and NCN protein in preparing a kit.

The present invention also provides the use of COL2A1-gRNA3, COL2A1-gRNA4 and PRONCN protein in preparing a kit.

The present invention also provides use of COL2A1-gRNA3, COL2A1-gRNA4 and a specific plasmid in preparing a kit.

The present invention provides a kit comprising COL2A1-gRNA3, COL2A1-gRNA4 and NCN protein.

The present invention also provides a kit, comprising COL2A1-gRNA3, COL2A1-gRNA4 and PRONCN protein.

The present invention also provides a kit, comprising COL2A1-gRNA3, COL2A1-gRNA4 and a specific plasmid.

Any of the above kits further comprises pig cells.

The use of any of the above kits is as follows (a) or (b) or (c): (a) preparing recombinant pig cells; (b) preparing pigs with type II collagen disease model; (c) preparing cell model of type II collagen disease or tissue model of type II collagen disease or organ model of type II collagen disease.

The present invention provides a method for preparing recombinant pig cells, comprising the following steps: co-transfecting COL2A1-gRNA3, COL2A1-gRNA4 and NCN protein into pig cells to obtain recombinant pig cells.

The co-transfection specifically adopts the method of electroporation transfection.

The specific parameters for electroporation transfection can be set as: 1450V, 10ms, 3pulse.

The co-transfection can specifically be performed using a mammalian nuclear transfection kit (Neon kit, Thermofisher) and a NeonTM transfection system electroporator.

The ratios of COL2A1-gRNA3, COL2A1-gRNA4 and NCN protein are: 0.8-1.2μg COL2A1-gRNA3: 0.8-1.2μg COL2A1-gRNA4: 3-5μg NCN protein.

The ratios of COL2A1-gRNA3, COL2A1-gRNA4 and NCN protein were: 1 μg COL2A1-gRNA3: 1 μg COL2A1-gRNA4: 4 μg NCN protein.

The ratios of pig cells, COL2A1-gRNA3, COL2A1-gRNA4 and NCN protein are: 100,000 pig cells: 0.8-1.2μg COL2A1-gRNA3: 0.8-1.2μg COL2A1-gRNA4: 3-5μg NCN protein.

The ratios of pig cells, COL2A1-gRNA3, COL2A1-gRNA4 and NCN protein are: 100,000 pig cells: 1μg COL2A1-gRNA3: 1μg COL2A1-gRNA4: 4μg NCN protein.

Any of the above COL2A1-gRNA3 is a sgRNA, and its target sequence binding region is shown in nucleotides 3 to 22 in SEQ ID NO: 16.

Specifically, the COL2A1-gRNA3 is shown in SEQ ID NO: 16.

Specifically, the COL2A1-gRNA3 is shown in SEQ ID NO: 12.

Any of the above COL2A1-gRNA4 is a sgRNA, and its target sequence binding region is shown in nucleotides 3 to 22 in SEQ ID NO: 17.

Specifically, the COL2A1-gRNA4 is shown in SEQ ID NO: 17.

Specifically, the COL2A1-gRNA4 is shown in SEQ ID NO:13.

Any of the above-mentioned NCN proteins is a Cas9 protein or a fusion protein with a Cas9 protein.

Specifically, the NCN protein is shown in SEQ ID NO:3.

Any of the above pig cells is a pig fibroblast.

Any of the above pig cells is a primary pig fibroblast.

Any of the above pig cells is a primary pig fibroblast obtained from a newborn pig.

The preparation method of the NCN protein comprises the following steps:

(1) Introducing plasmid pKG-GE4 into Escherichia coli BL21 (DE3) to obtain recombinant bacteria;

(2) culturing the recombinant bacteria in a liquid medium at 30° C., then adding IPTG and inducing the culture at 25° C., and then collecting the bacteria;

(3) crushing the collected bacterial cells and collecting the crude protein solution;

(4) purifying the fusion protein with the His ₆ tag from the crude protein solution using affinity chromatography;

(5) using enterokinase with a His ₆ tag to digest the fusion protein with a His ₆ tag, and then using Ni-NTA resin to remove the protein with the His ₆ tag to obtain a purified NCN protein;

Plasmid pKG-GE4 contains the fusion gene shown by nucleotides 5209 to 9852 in SEQ ID NO:1.

The preparation method of the NCN protein specifically comprises the following steps:

(1) The plasmid pKG-GE4 was introduced into Escherichia coli BL21 (DE3) to obtain recombinant bacteria.

(2) inoculating the recombinant bacteria obtained in step (1) into a liquid LB medium containing ampicillin and culturing with shaking;

(3) The bacterial solution obtained in step (2) was inoculated into liquid LB medium, and cultured at 30° C. and 230 rpm with shaking until the OD _{600 nm} value was 1.0, and then IPTG was added to a concentration of 0.5 mM in the system, and then cultured at 25° C. and 230 rpm with shaking for 12 hours, and then the bacteria were collected by centrifugation;

(4) Take the bacterial cells obtained in step (3) and wash them with PBS buffer;

(5) taking the bacterial cells obtained in step (4), adding a crude extraction buffer to suspend the bacterial cells, and then breaking the bacterial cells, and then centrifuging to collect the supernatant, filtering with a 0.22 μm pore size filter membrane, and collecting the filtrate;

(6) Purifying the fusion protein with the His ₆ tag (the fusion protein shown in SEQ ID NO: 2) from the filtrate obtained in step (5) by affinity chromatography;

(7) taking the column solution collected in step (6), concentrating it using an ultrafiltration tube, and then diluting it with 25 mM Tris-HCl (pH 8.0);

(8) adding recombinant bovine enterokinase with a His ₆ tag to the solution obtained in step (7) and performing enzyme digestion;

(9) mixing the solution obtained in step (8) with Ni-NTA resin, incubating, and then centrifuging to collect the supernatant;

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

The specific method of purifying the fusion protein with the His ₆ tag from the filtrate obtained in step (5) by affinity chromatography is as follows:

First, use 5 column volumes of equilibration solution to equilibrate the Ni-NTA agarose column (flow rate is 1 ml/min); then load 50 ml of the filtrate obtained in step (5) (flow rate is 0.5-1 ml/min); then wash the column with 5 column volumes of equilibration solution (flow rate is 1 ml/min); then wash the column with 5 column volumes of buffer (flow rate is 1 ml/min) to remove impurities; then elute with 10 column volumes of eluent at a flow rate of 0.5-1 ml/min, and collect the post-column solution (90-100 ml).

Any of the above PRONCN proteins includes the following elements from upstream to downstream: a signal peptide, a molecular chaperone protein, a protein tag, a protease cleavage site, a nuclear localization signal, a Cas9 protein, and a nuclear localization signal.

The function of the signal peptide is to promote protein secretion expression. The signal peptide can be selected from the Escherichia coli alkaline phosphatase (phoA) signal peptide, the Staphylococcus aureus protein A signal peptide, the Escherichia coli outer membrane protein (ompa) signal peptide or any other prokaryotic gene signal peptide, preferably an 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, thereby separating it from the bacterial intracellular protein, and the target protein secreted into the bacterial periplasmic cavity is soluble and can be cleaved by the signal peptidase in the bacterial periplasmic cavity.

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

The function of the protein tag is to purify the protein. The tag may be a His tag (His-Tag, His ₆ protein tag), a GST tag, a Flag tag, an HA tag, a c-Myc tag or any other protein tag, more preferably a His tag. The His tag can bind to a Ni column, and the target protein can be purified by one-step Ni column affinity chromatography, which can greatly simplify the purification process of the target protein.

The function of the protease cleavage site is to be used to cut off the non-functional segment after purification to release the natural form of Cas9 protein. The protease can be selected from enterokinase, factor Xa, thrombin, TEV protease, HRV 3C protease, WELQut protease or any other endoprotease, and enterokinase is further preferred. EK is an enterokinase cleavage site, which is convenient for using enterokinase to remove the fused TrxA-His segment to obtain a natural form of Cas9 protein. After the commercial enterokinase with a His tag is used to cut the fusion protein, the TrxA-His segment and the enterokinase with a His tag can be removed by one affinity chromatography to obtain a natural form of Cas9 protein, avoiding damage and loss to the target protein by multiple purification dialysis.

The nuclear localization signal can be any nuclear localization signal, preferably an SV40 nuclear localization signal and/or a nucleoplasmin nuclear localization signal. NLS is a nuclear localization signal, and an NLS site is designed at the N-terminus and the C-terminus of Cas9, respectively, so that Cas9 can more effectively enter the cell nucleus for gene editing.

The Cas9 protein may be saCas9 or spCas9, preferably spCas9 protein.

The PRONCN protein is specifically shown in SEQ ID NO:2.

Any of the above-mentioned specific plasmids includes the following elements from upstream to downstream: a promoter, an operator, a ribosome binding site, a gene encoding PRONCN protein, and a terminator.

The promoter may specifically be a T7 promoter, which is a strong promoter for prokaryotic expression and can efficiently drive the expression of exogenous genes.

The operon may 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, IPTG can be used to induce the expression of the target protein at low temperature, which can avoid the effect of premature expression of the target protein on the growth of the host bacteria. Inducing expression at low temperature also significantly improves the solubility of the expressed target protein.

The ribosome binding site is a ribosome binding site during protein translation and is necessary for protein translation.

The terminator may specifically be a T7 terminator. The T7 terminator can effectively terminate gene transcription at the end of the target gene to prevent other downstream sequences other than the target gene from being transcribed and translated.

For the codons of the spCas9 protein, the present application optimizes the codons to make them fully adapt to the codon preference of the Escherichia coli high-efficiency expression strain E. coli BL21 (DE3) selected in the present application, thereby improving the expression level of the Cas9 protein.

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

The lac operon is shown in nucleotides 5140-5164 of SEQ ID NO:1.

The ribosome binding site is shown in nucleotides 5178-5201 of SEQ ID NO:1.

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

The coding sequence of TrxA protein is shown in nucleotides 5272-5598 in SEQ ID NO:1.

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

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

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

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

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

The T7 terminator is shown as nucleotides 9902-9949 in SEQ ID NO:1.

Specifically, the specific plasmid is plasmid pKG-GE4.

Plasmid pKG-GE4 contains the DNA molecule represented by nucleotides 5121 to 9949 in SEQ ID NO:1.

Specifically, any of the above plasmids pKG-GE4 is shown as SEQ ID NO:1.

The present invention also protects the recombinant pig cells prepared by any of the above methods.

The recombinant pig cell is a recombinant pig cell in which the COL2A1 gene is mutated.

The recombinant pig cell can specifically be a single cell clone having a genotype of heterozygous, bi-allelic identical mutation or bi-allelic different mutation in Table 1.

The present invention also protects the use of the recombinant pig cells in preparing type II collagen disease model pigs.

The recombinant pig cells are used as nuclear transplant donor cells for somatic cell cloning to obtain cloned pigs, namely, type II collagen disease model pigs.

The present invention also protects the pig tissue of the model pig prepared by using the recombinant pig cells, that is, the type II collagen disease tissue model.

The present invention also protects the pig organs of the model pigs prepared by using the recombinant pig cells, namely the organ model of type II collagen disease.

The present invention also protects pig cells of a model pig prepared by using the recombinant pig cells, namely a type II collagen disease cell model.

The present invention also protects the use of the recombinant pig cell, the type II collagen disease tissue model, the type II collagen disease organ model, the type II collagen disease cell model or the type II collagen disease model pig, which is as follows (d1) or (d2) or (d3) or (d4):

(d1) Screening for drugs to treat type II collagen diseases;

(d2) Evaluate the efficacy of drugs for type II collagen diseases;

(d3) Evaluate the efficacy of gene therapy and/or cell therapy for type II collagen diseases;

(d4) To study the pathogenesis of type II collagen disease.

Any of the above-mentioned pigs can specifically be Congjiang Xiang pigs.

Any of the above-mentioned pigs can specifically be newborn Congjiang Xiang pigs.

Any of the above-mentioned pigs can specifically be Bama Xiang pigs.

Any of the above-mentioned pigs can specifically be newborn Bama Xiang pigs.

Any of the above mentioned type II collagen diseases is caused by mutation of COL2A1 gene.

Pig COL2A1 gene information: encoding type II collagen α1 chain; located on chromosome 5; Gene ID is 397323, Sus scrofa.

The protein encoded by the porcine COL2A1 gene is shown in NCBI as XP_020948270.1 (13-MAY-2017).

The protein encoded by the porcine COL2A1 gene has a protein segment shown in SEQ ID NO:8.

The porcine COL2A1 gene has a DNA segment shown in SEQ ID NO:9.

Any of the above mutations is a deletion and/or insertion and/or substitution of one or more nucleotides.

Any of the above mutations is a deletion of one or more nucleotides.

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

Any of the above mutations is a deletion or insertion of one or more nucleotides.

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

(1) The research object of the present invention (pig) has better applicability than other animals (rat, mouse, and primate).

Rodents such as mice and rats are very different from humans in terms of body shape, organ size, physiology, and pathology, and cannot truly simulate the normal physiological and pathological conditions of humans. Studies have shown that more than 95% of drugs that have been proven effective in mice and rats are ineffective in human clinical trials. As far as large animals are concerned, primates are the animals that are most closely related to humans, but they are small in size, mature late (mating begins at 6-7 years old), and are single-birth animals. The population expansion rate is extremely slow, and the breeding cost is very high. In addition, primate cloning is inefficient, difficult, and costly.

As a model animal, pigs do not have the above disadvantages. They are the animals that are most closely related to humans except primates. Their body shape, weight, organ size, etc. are similar to humans. They are very similar to humans in anatomy, physiology, immunology, nutritional metabolism, and disease pathogenesis. At the same time, pigs mature early (4-6 months), have high fertility, and can produce many offspring per litter. A large group can be formed within 2-3 years. In addition, pig cloning technology is very mature, and the cost of cloning and raising is much lower than that of primates. Therefore, pigs are very suitable animals as models of human diseases.

(2) The vector constructed by the present invention uses a strong promoter T7-lac capable of efficiently expressing the target protein to express the target protein, and uses the signal peptide of bacterial periplasmic protein alkaline phosphatase (phoA) to guide the secretion and expression of the target protein into the bacterial periplasmic cavity, thereby separating it from the bacterial intracellular protein, and the target protein secreted into the bacterial periplasmic cavity is soluble. At the same time, the thioredoxin TrxA and Cas9 protein fusion expression are also used. TrxA can help the co-expressed target protein to form a disulfide bond, improve the stability of the protein, the correctness of folding, and increase the solubility and activity of the target protein. In order to facilitate the purification of the target protein, a His tag is designed, and the target protein can be purified by one-step Ni column affinity chromatography, which greatly simplifies the purification process of the target protein. At the same time, an enterokinase cleavage site is designed after the His tag to facilitate the excision of the fused TrxA-His polypeptide fragment to obtain a natural form of the Cas9 protein. After the fusion protein is cleaved by enterokinase with a His tag, the TrxA-His polypeptide fragment and the enterokinase with a His tag can be removed by an affinity chromatography to obtain a natural form of Cas9 protein, avoiding the damage and loss of the target protein by multiple purification dialysis. At the same time, the present invention also designs an NLS site at the N-terminus and C-terminus of Cas9, so that Cas9 can more effectively enter the nucleus for gene editing. In addition, the present invention selects E.coli BL21 (DE3) strain as the target protein expression strain, which can efficiently express exogenous genes cloned in an expression vector (such as pET-32a) containing a bacteriophage T7 promoter. At the same time, for the codons of the Cas9 protein, the present invention performs codon optimization to fully adapt to the codon preference of the expression strain, thereby improving the expression level of the target protein. In addition, after the bacteria grow to a certain number, the present invention uses IPTG to induce the expression of the target protein at low temperature, which can avoid the influence of premature expression of the target protein on the growth of the host bacteria, and the induced expression at low temperature also significantly improves the solubility of the expressed target protein. After the above-mentioned optimization designs and experimental implementation, the activity of the obtained Cas9 protein was significantly improved compared with the commercial Cas9 protein.

(3) The Cas9 high-efficiency protein constructed and expressed by the present invention was combined with in vitro transcribed gRNA for gene editing, and the optimal dosage ratio of Cas9 and gRNA was optimized. The final rate of gene-edited single-cell clones was as high as 97.1%, which is much higher than the conventional gene editing efficiency (10-30%).

(4) Using the target gene knockout single cell clone obtained by the present invention to carry out somatic cell nuclear transplantation animal cloning can directly obtain cloned pigs with target gene knockout, and the gene mutation can be stably inherited.

The method of embryo transplantation after microinjection of gene editing materials into fertilized eggs used in mouse model making is not suitable for large animal (such as pig) model making with a long gestation period because of its low probability of directly obtaining offspring with gene mutations, and requires hybrid breeding of offspring. Therefore, the present invention adopts a method of in vitro editing of primary cells with high technical difficulty and challenge, and cutting and screening of positive edited single cell clones with Cas9 protein and double gRNA, and then directly obtains the corresponding disease model pigs through somatic cell nuclear transplantation animal cloning technology in the later stage, which can greatly shorten the model pig production cycle and save manpower, material resources and financial resources.

The present invention uses CRISPR/Cas9 technology combined with dual gRNA editing to knock out the COL2A1 gene, simulate the genetic characteristics of type II collagen disease, and obtain single-cell clones of COL2A1 gene knockout, laying the foundation for the later breeding of type II collagen disease model pigs through somatic cell nuclear transfer animal cloning technology. The present invention will help to study and reveal the pathogenesis of type II collagen disease caused by abnormal function of the COL2A1 gene, and can also be used for drug screening, drug efficacy evaluation, gene therapy and cell therapy research, which can provide effective experimental data for further clinical applications, and thus provide a powerful experimental means for the successful treatment of human type II collagen disease. The present invention has great application value for the research and development of drugs for type II collagen disease and revealing the pathogenesis of the disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is the comparison result of the forward sequencing of the single cell clone numbered 33 and the wild-type sequence.

FIG2 is the comparison result of the reverse sequencing of the single cell clone numbered 14 and the wild-type sequence.

FIG3 is the comparison result of the reverse sequencing of the single cell clone numbered 24 and the wild-type sequence.

FIG. 4 is the comparison result of the forward sequencing of the single cell clone numbered 2 and the wild-type sequence.

FIG. 5 is an electrophoretic diagram of PCR amplification using different primer pairs using the genome extracted from the ear tissue of a pig named BX4 as a template in Example 2.

FIG6 is an electrophoretic diagram of PCR amplification performed using the primer pair consisting of COL2A1-E27-JDF219 and COL2A1-E27-JDR660 with the genomic DNA of 10 pigs as templates in Example 2.

FIG. 7 is a schematic diagram of the structure of plasmid pET-32a.

FIG. 8 is a schematic diagram of the structure of plasmid pKG-GE4.

Figure 9 is an electrophoresis diagram of the optimized dosage ratio of gRNA and NCN protein in Example 4.

Figure 10 is an electrophoretic diagram comparing the gene editing efficiency of NCN protein and commercial Cas9 protein in Example 4.

DETAILED DESCRIPTION

The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources. The recombinant plasmids constructed in the examples have all been sequenced and verified. The commercial Cas9-A protein is a commercially available Cas9 protein with good effects. The commercial Cas9-B protein is a commercially available Cas9 protein with good effects. Complete culture medium (% is volume ratio): 15% fetal bovine serum (Gibco) + 83% DMEM medium (Gibco) + 1% Penicillin-Streptomycin (Gibco) + 1% HEPES (Solarbio). Cell culture conditions: 37°C, 5% CO ₂ , 5% O ₂ constant temperature incubator.

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

Plasmid pKG-GE3 is a circular plasmid, as shown in SEQ ID NO: 2 in patent application 202010084343.6. In SEQ ID NO: 2 in patent application 202010084343.6, nucleotides 395-680 constitute the CMV enhancer, nucleotides 682-890 constitute 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 the polypeptide P2A (the amino acid sequence of polypeptide P2A is "ATNFSLLKQAGDVEENPGP", and the break position is The nucleotides at positions 5333-6046 encode EGFP protein, the nucleotides at positions 6056-6109 encode polypeptide T2A (the amino acid sequence of polypeptide T2A is "EGRGSLLTCGDVEENPGP", and the break position is between the first amino acid residue and the second amino acid residue at the C-terminus), the nucleotides at positions 6110-6703 encode Puromycin protein (abbreviated as Puro protein), the nucleotides at positions 6722-7310 constitute the WPRE sequence element, the nucleotides at positions 7382-7615 constitute the 3'LTR sequence element, and the nucleotides at positions 7647-7871 constitute the bGH poly (A) signal sequence element. In SEQ ID NO: 2 in patent application 202010084343.6, the nucleotides at positions 911-6706 form a fusion gene to express a fusion protein. Due to the presence of the self-cleaving polypeptide P2A and the self-cleaving polypeptide 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.

The pKG-U6gRNA vector, i.e., the plasmid pKG-U6gRNA, is a circular plasmid, as shown in SEQ ID NO: 3 in patent application 202010084343.6. In SEQ ID NO: 3 in patent application 202010084343.6, nucleotides 2280-2539 constitute the hU6 promoter, and nucleotides 2558-2637 are used to transcribe to form the gRNA backbone. When used, a DNA molecule of about 20 bp (used to transcribe the target sequence binding region of the gRNA) is inserted into the plasmid pKG-U6gRNA to form a recombinant plasmid, and the recombinant plasmid is transcribed in the cell to obtain the gRNA.

Example 1: Preparation of COL2A1 gene knockout Bama Xiang pig single cell clone

Two highly efficient gRNA target sites (COL2A1-E27-gRNA3 target site and COL2A1-E27-gRNA4 target site) were screened and obtained in Example 2.

The NCN protein is provided by the NCN protein solution prepared in Example 3.

1. Preparation of gRNA

1. Preparation of COL2A1-T7-gRNA3 and COL2A1-T7-gRNA4 transcription templates

The COL2A1-T7-gRNA3 transcription template is a double-stranded DNA molecule, as shown in SEQ ID NO:14.

The COL2A1-T7-gRNA4 transcription template is a double-stranded DNA molecule, as shown in SEQ ID NO:15.

2. Obtain gRNA by in vitro transcription

The COL2A1-T7-gRNA3 transcription template was taken and in vitro transcription was performed using Transcript Aid T7 High Yield Transcription Kit (Fermentas, K0441), and then recovered and purified using MEGA clear ^TM Transcription Clean-Up Kit (Thermo, AM1908) to obtain COL2A1-gRNA3. COL2A1-gRNA3 is a single-stranded RNA, as shown in SEQ ID NO: 16.

The COL2A1-T7-gRNA4 transcription template was taken and in vitro transcription was performed using Transcript Aid T7 High Yield Transcription Kit (Fermentas, K0441), and then recovered and purified using MEGA clear ^TM Transcription Clean-Up Kit (Thermo, AM1908) to obtain COL2A1-gRNA4. COL2A1-gRNA4 is a single-stranded RNA, as shown in SEQ ID NO: 17.

COL2A1-gRNA3 (SEQ ID NO: 16):

GGCCAGGGAAACCCAUGACACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAGUGGCACCGAGUCCGGUGCUUUUCOL2A1-gRNA4 (SEQ ID NO: 17):

GGCCAGGGCGACCAUCUUCACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUU

2. Transfection of primary porcine fibroblasts

1. Co-transfect COL2A1-gRNA3, COL2A1-gRNA4 and NCN protein into porcine primary fibroblasts. Ratio: about 100,000 porcine primary fibroblasts: 1μg COL2A1-gRNA3: 1μg COL2A1-gRNA4: 4μg NCN protein. Co-transfection was performed by electroporation using a mammalian nuclear transfection kit (Neon kit, Thermofisher) and a Neon TM transfection system electroporator (parameter settings: 1450V, 10ms, 3pulse).

2. After completing step 1, culture in complete culture medium for 16-18 hours, then replace with new complete culture medium for culture. The total culture time after electroporation is 48 hours.

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

4. After completing step 3, use trypsin to digest and collect the cells (about 2/3 of the cells obtained in each well are inoculated into a 6-well plate filled with complete culture medium, and the remaining 1/3 are collected in a 1.5 mL centrifuge tube).

5. Take the 6-well plate from step 4 and culture the cells until they grow to 80% confluence, digest and collect the cells using trypsin, and freeze the cells using cell freezing solution (90% complete culture medium + 10% DMSO, volume ratio).

6. Take the centrifuge tube from step 4, take the cells, lyse the cells and extract genomic DNA, perform PCR amplification using the primer pair consisting of COL2A1-E27-JDF219 and COL2A1-E27-JDR660, and then perform electrophoresis. Use porcine primary fibroblasts as wild-type control (WT).

7. After completing step 6, recover the PCR amplification product and sequence it.

There is only one sequencing result of primary porcine fibroblasts, and its genotype is wild type (also called homozygous wild type). If there are two sequencing results for a single cell clone, one is consistent with the sequencing result of primary porcine fibroblasts, and the other is mutated compared with the sequencing result of primary porcine fibroblasts (mutation includes deletion, insertion or substitution of one or more nucleotides), the genotype of the single cell clone is heterozygous; if there are two sequencing results for a single cell clone, both of which are mutated compared with the sequencing result of primary porcine fibroblasts (mutation includes deletion, insertion or substitution of one or more nucleotides), the genotype of the single cell clone is biallelic different mutation type; if there is one sequencing result for a single cell clone, and it is mutated compared with the sequencing result of primary porcine fibroblasts (mutation includes deletion, insertion or substitution of one or more nucleotides), the genotype of the single cell clone is biallelic identical mutation type; if there is one sequencing result for a single cell clone, and it is consistent with the sequencing result of primary porcine fibroblasts, the genotype of the single cell clone is wild type (also called homozygous wild type).

The results are shown in Table 1. The genotype of the single cell clone numbered 33 is wild type. The genotype of the single cell clones numbered 4, 10, 14, 20, 21, and 22 is heterozygous. The genotype of the single cell clones numbered 1, 3, 5, 6, 7, 9, 11, 12, 13, 15, 16, 18, 19, 24, 25, 26, 28, 29, 30, 31, 32, 34, and 35 is a double allele different mutation type. The genotype of the single cell clones numbered 2, 8, 17, 23, and 27 is a double allele identical mutation type. The ratio of COL2A1 gene-edited single cell clones was 97.1%.

Exemplary sequencing comparison results are shown in Figures 1 to 4. Figure 1 is the comparison result of the forward sequencing of the single cell clone numbered 33 with the wild-type sequence, which is determined to be wild-type. Figure 2 is the comparison result of the reverse sequencing of the single cell clone numbered 14 with the wild-type sequence, which is determined to be heterozygous. Figure 3 is the comparison result of the reverse sequencing of the single cell clone numbered 24 with the wild-type sequence, which is a double allele different mutation type. Figure 4 is the comparison result of the forward sequencing of the single cell clone numbered 2 with the wild-type sequence, which is a double allele identical mutation type.

Table 1 Genotyping results of COL2A1 gene-edited single-cell clones

The above-mentioned single cell clones of heterozygous type, double allele identical mutation type and double allele different mutation type are all target single cell clones. Using the cells as nuclear transplant donor cells for somatic cell cloning can obtain cloned pigs, which are type II collagen disease model pigs.

Example 2: Screening of efficient gRNA targets for COL2A1 gene

Pig COL2A1 gene information: Encodes type II collagen α1 chain; located on chromosome 5; Gene ID is 397323, Sus scrofa. The protein encoded by the pig COL2A1 gene is shown in NCBI as XP_020948270.1 (13-MAY-2017). Part of the protein encoded by the pig COL2A1 gene is shown in SEQ ID NO: 8. In the pig genomic DNA, the COL2A1 gene has 54 exons, and the 27th coding exon and its upstream and downstream 300bp are shown in SEQ ID NO: 9.

1. Conservative analysis of the presumed deletion region of the COL2A1 gene and adjacent genomic sequences

There were 10 newborn Bama Xiang pigs, including 6 females (named BC1, BC2, BC3, BC4, BC5, and BC6) and 4 males (named BX1, BX2, BX3, and BX4).

COL2A1-E27-JDF220:CTGTGCCCTTCCACAGTAGG;

COL2A1-E27-JDR660: GGCTAAAGCCGAGTATCCCC;

COL2A1-E27-JDF219: CCTGTGCCCTTCCACAGTAG;

COL2A1-E27-JDR633: CTGCCCCATGGAAGACTGTT.

The genomic DNA extracted from the ear tissue of the pig named BX4 was used as a template, and PCR amplification was performed using different primer pairs, and then 1% agarose gel electrophoresis was performed. The electrophoresis diagram is shown in Figure 5. In Figure 5: Group 1: using a primer pair consisting of COL2A1-E27-JDF219 and COL2A1-E27-JDR633; Group 2: using a primer pair consisting of COL2A1-E27-JDF219 and COL2A1-E27-JDR660; Group 3: using a primer pair consisting of COL2A1-E27-JDF220 and COL2A1-E27-JDR633; Group 4: using a primer pair consisting of COL2A1-E27-JDF220 and COL2A1-E27-JDR660. The results showed that the primer pair consisting of COL2A1-E27-JDF219 and COL2A1-E27-JDR660 was preferably used for amplification of the target fragment.

The genomic DNA of 10 pigs was used as a template, and the primer pair consisting of COL2A1-E27-JDF219 and COL2A1-E27-JDR660 was used for PCR amplification, followed by 1% agarose gel electrophoresis. The electrophoresis diagram is shown in Figure 6. The PCR amplification product was recovered and sequenced, and the sequencing results were compared with the COL2A1 gene sequence in the public database. The conserved region common to the 10 pigs was selected for the design of gRNA target sites.

2. Target Screening

By screening NGG (avoiding possible mutation sites), several targets were initially screened, and 4 targets were further screened after preliminary experiments.

The four targets are as follows:

COL2A1-E27-gRNA1 target: TGGACCTCCAGGTCCCCAAG;

COL2A1-E27-gRNA2 target: CGAGCCCCTTGGGGACCTGG;

COL2A1-E27-gRNA3 target: CCAGGGAAACCCATGACACC;

COL2A1-E27-gRNA4 target: CCAGGGCGACCATCTTCACC.

3. Preparation of gRNA

The plasmid pKG-U6gRNA was taken and digested with restriction endonuclease BbsI to recover the vector backbone (a large linear fragment of about 3 kb).

COL2A1-E27-gRNA1-S and COL2A1-E27-gRNA1-A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with sticky ends. The double-stranded DNA molecules with sticky ends were connected to the vector backbone to obtain plasmid pKG-U6gRNA (COL2A1-E27-gRNA1). Plasmid pKG-U6gRNA (COL2A1-E27-gRNA1) expresses sgRNA _{COL2A1-E27-gRNA1} shown in SEQ ID NO: 10.

sgRNA _{COL2A1-E27-gRNA1} (SEQ ID NO: 10):

UGGACCUCCAGGUCCCCAAGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

COL2A1-E27-gRNA2-S and COL2A1-E27-gRNA2-A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with sticky ends. The double-stranded DNA molecules with sticky ends were connected to the vector backbone to obtain plasmid pKG-U6gRNA (COL2A1-E27-gRNA2). Plasmid pKG-U6gRNA (COL2A1-E27-gRNA2) expressed sgRNA _{COL2A1-E27-gRNA2} shown in SEQ ID NO: 11.

sgRNA _{COL2A1-E27-gRNA2} (SEQ ID NO: 11):

CGAGCCCCUUGGGGACCUGGguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

COL2A1-E27-gRNA3-S and COL2A1-E27-gRNA3-A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with sticky ends. The double-stranded DNA molecules with sticky ends were connected to the vector backbone to obtain plasmid pKG-U6gRNA (COL2A1-E27-gRNA3). Plasmid pKG-U6gRNA (COL2A1-E27-gRNA3) expressed sgRNA _{COL2A1-E27-gRNA3} shown in SEQ ID NO: 12.

sgRNA _{COL2A1-E27-gRNA3} (SEQ ID NO: 12):

CCAGGGAAACCCAUGACACCguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

COL2A1-E27-gRNA4-S and COL2A1-E27-gRNA4-A were synthesized separately, then mixed and annealed to obtain double-stranded DNA molecules with sticky ends. The double-stranded DNA molecules with sticky ends were connected to the vector backbone to obtain plasmid pKG-U6gRNA (COL2A1-E27-gRNA4). Plasmid pKG-U6gRNA (COL2A1-E27-gRNA4) expressed sgRNA _{COL2A1-E27-gRNA4} shown in SEQ ID NO: 13.

sgRNA _{COL2A1-E27-gRNA4} (SEQ ID NO: 13):

CCAGGGCGACCAUCUUCACCguuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu

COL2A1-E27-gRNA1-S:caccgTGGACCTCCAGGTCCCCAAG;

COL2A1-E27-gRNA1-A:aaacCTTGGGGACCTGGAGGTCCAc;

COL2A1-E27-gRNA2-S: caccgCGAGCCCCTTGGGGACCTGG;

COL2A1-E27-gRNA2-A:aaacCCAGGTCCCCAAGGGGCTCGc;

COL2A1-E27-gRNA3-S: caccgCCAGGGAAACCCCATGACACC;

COL2A1-E27-gRNA3-A:aaacGGTGTCATGGGTTTCCCTGGc;

COL2A1-E27-gRNA4-S: caccgCCAGGGCGACCATCTTCACC;

COL2A1-E27-gRNA4-A:aaacGGTGAAGATGGTCGCCCTGGc.

COL2A1-E27-gRNA1-S, COL2A1-E27-gRNA1-A, COL2A1-E27-gRNA2-S, COL2A1-E27-gRNA2-A, COL2A1-E27-gRNA3-S, COL2A1-E27-gRNA3-A, COL2A1-E27-gRNA4-S, and COL2A1-E27-gRNA4-A are all single-stranded DNA molecules.

4. Comparison of editing efficiency of different target combinations

1. Co-transfection

Group 1: Co-transfect porcine primary fibroblasts with plasmid pKG-U6gRNA (COL2A1-E27-gRNA1) and plasmid pKG-GE3. Ratio: about 200,000 porcine primary fibroblasts: 0.92μg plasmid pKG-U6gRNA (COL2A1-E27-gRNA1): 1.08μg plasmid pKG-GE3.

Group 2: Plasmid pKG-U6gRNA (COL2A1-E27-gRNA2) and plasmid pKG-GE3 were co-transfected into primary porcine fibroblasts. Ratio: about 200,000 primary porcine fibroblasts: 0.92μg plasmid pKG-U6gRNA (COL2A1-E27-gRNA2): 1.08μg plasmid pKG-GE3.

Group 3: Plasmid pKG-U6gRNA (COL2A1-E27-gRNA3) and plasmid pKG-GE3 were co-transfected into primary porcine fibroblasts. Ratio: about 200,000 primary porcine fibroblasts: 0.92μg plasmid pKG-U6gRNA (COL2A1-E27-gRNA3): 1.08μg plasmid pKG-GE3.

Group 4: Plasmid pKG-U6gRNA (COL2A1-E27-gRNA4) and plasmid pKG-GE3 were co-transfected into porcine primary fibroblasts. Ratio: about 200,000 porcine primary fibroblasts: 0.92μg plasmid pKG-U6gRNA (COL2A1-E27-gRNA4): 1.08μg plasmid pKG-GE3.

Group 5: Porcine primary fibroblasts, electroporated with the same electroporation parameters but without adding plasmids.

Co-transfection was performed by electroporation using a mammalian nuclear transfection kit (Neon kit, Thermofisher) and a Neon TM transfection system electroporator (parameter settings: 1450 V, 10 ms, 3 pulses).

2. After completing step 1, culture in complete culture medium for 12-18 hours, then replace with new complete culture medium for culture. The total culture time after electroporation is 48 hours.

3. After completing step 2, trypsin was used to digest and collect the cells, the cells were lysed, genomic DNA was extracted, PCR amplification was performed using a primer pair consisting of COL2A1-E27-JDF219 and COL2A1-E27-JDR660, and then 1% agarose gel electrophoresis was performed to detect the mutation of the cell target gene.

After the target product was excised and recovered, it was sent to a sequencing company for sequencing. Then the sequencing results were analyzed using the web version of the Synthego ICE tool to analyze the sequencing peak graph to obtain the gene editing efficiency of different targets. The gene editing efficiency of the first, second, third, and fourth groups was 5%, 63%, 81%, and 68%, respectively, and no gene editing occurred in the fifth group. The results showed that the editing efficiency of the COL2A1-E27-gRNA3 target and the COL2A1-E27-gRNA4 target was high.

Example 3. Preparation and purification of NCN protein

1. Construction of prokaryotic Cas9 efficient expression vector

The schematic diagram of the structure of plasmid pET-32a is shown in FIG7 .

Plasmid pKG-GE4 is obtained by transforming plasmid pET-32a as the starting plasmid. Plasmid pET32a-T7lac-phoA:SP-TrxA-His-EK-NLS-spCas9-NLS-T7ter (referred to as plasmid pKG-GE4), as shown in SEQ ID NO: 1, is a circular plasmid, and the schematic diagram of the structure is shown in Figure 8.

In SEQ ID NO: 1, nucleotides 5121-5139 constitute a T7 promoter, nucleotides 5140-5164 encode a Lac operator, nucleotides 5178-5201 constitute a ribosome binding site (RBS), nucleotides 5209-5271 encode an alkaline phosphatase signal peptide (phoA signal peptide), nucleotides 5272-5598 encode a TrxA protein, nucleotides 5620-5637 encode a His-Tag (also known as a His ₆ tag), nucleotides 5638-5652 encode an enterokinase cleavage site (EK cleavage site), nucleotides 5656-5670 encode a nuclear localization signal, nucleotides 5701-9801 encode a spCas9 protein, nucleotides 9802-9849 encode a nuclear localization signal, and nucleotides 9902-9949 constitute a T7 terminator. The nucleotide encoding the spCas9 protein has been codon-optimized for the Escherichia coli BL21(DE3) strain.

The main modifications of plasmid pKG-GE4 are as follows: ① The coding region of TrxA protein is retained. TrxA protein can help the expressed target protein to form disulfide bonds and increase the solubility and activity of the target protein; the coding sequence of alkaline phosphatase signal peptide is added before the coding region of TrxA protein. The alkaline phosphatase signal peptide can guide the expressed target protein to be secreted into the membrane periplasmic cavity of bacteria and can be cleaved by prokaryotic periplasmic signal peptidase; ② The coding sequence of His-Tag is added after the coding sequence of TrxA protein. His-Tag can be used for 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 the His-Tag and the TrxA protein fused upstream under the action of enterokinase; ④ Insert the codon-optimized Cas9 gene suitable for expression in the Escherichia coli BL21 (DE3) strain, and add the nuclear localization signal coding sequence upstream and downstream of the gene to increase the nuclear localization ability of the Cas9 protein purified later.

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

2. Inducible Expression

1. Introduce plasmid pKG-GE4 into Escherichia coli BL21 (DE3) to obtain recombinant bacteria.

2. The recombinant bacteria obtained in step 1 were inoculated into liquid LB medium containing 100 μg/ml ampicillin and cultured overnight at 37°C and 200 rpm with shaking.

3. The bacterial solution obtained in step 2 was inoculated into liquid LB medium, and cultured at 30°C and 230 rpm with shaking until the OD _600nm value = 1.0, and then isopropylthiogalactoside (IPTG) was added to a concentration of 0.5 mM in the system, and then cultured at 25°C and 230 rpm with shaking for 12 hours, and then centrifuged at 4°C and 10000 g for 15 minutes to collect the bacteria.

4. Take the bacteria obtained in step 3 and wash them with PBS buffer.

3. Purification of fusion protein TrxA-His-EK-NLS-spCas9-NLS

1. Take the bacteria obtained in step 2, add crude extraction buffer and suspend the bacteria, then use a homogenizer to break the bacteria (1000par cycle three times), then centrifuge at 4°C and 15000g for 30min, collect the supernatant, filter the supernatant with a 0.22μm pore size filter membrane, and collect the filtrate. In this step, 10ml crude extraction buffer is added for every gram of wet weight of bacteria.

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 ddH ₂ O.

2. Purify the fusion protein using affinity chromatography.

First, use 5 column volumes of equilibration solution to equilibrate the Ni-NTA agarose column (flow rate is 1 ml/min); then load 50 ml of the filtrate obtained in step 1 (flow rate is 0.5-1 ml/min); then wash the column with 5 column volumes of equilibration solution (flow rate is 1 ml/min); then wash the column with 5 column volumes of buffer (flow rate is 1 ml/min) to remove impurities; then elute with 10 column volumes of eluent at a flow rate of 0.5-1 ml/min, and collect the post-column solution (90-100 ml).

Ni-NTA agarose column: GenScript, L00250/L00250-C, filler volume 10 ml.

Balance solution: contains 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 5 mM Imidazole, and the balance is ddH ₂ O.

Buffer: Contains 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 50 mM Imidazole, and the balance is ddH ₂ O.

The eluent contained 20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, 500 mM Imidazole, and the balance was ddH ₂ O.

IV. Enzyme cleavage of fusion protein TrxA-His-EK-NLS-spCas9-NLS and purification of NCN protein

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, capacity 15 ml), 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.

2. Add commercially available recombinant bovine enterokinase with a His ₆ tag (Sangon Biotech, C620031, Recombinant Bovine Enterokinase Light Chain, His) to the solution (about ₆ ml) obtained in step 1 and digest at 25°C for 16 hours. Add 2 units of enterokinase for every 50 μg of protein.

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

4. Take the supernatant obtained in step 3, concentrate it to 200 μl using an Amicon ultrafiltration tube (Sigma, UFC9100, capacity 15 ml), then add it to the enzyme storage solution and adjust the protein concentration to 5 mg/ml to obtain the NCN protein solution.

After sequencing, the protein in the NCN protein solution has 15 amino acid residues at the N-terminus as shown in SEQ ID NO: 3, positions 1 to 15, namely, the NCN protein.

Enzyme storage solution (pH 7.4): contains 10 mM Tris, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% (volume ratio) glycerol, and the balance is ddH ₂ O.

Example 4. Performance of NCN protein

The NCN protein is provided by the NCN protein solution prepared in Example 3.

The two gRNA targets targeting the TTN gene were selected as follows:

TTN-gRNA1 target: AGAGCACAGTCAGCCTGGCG;

TTN-gRNA2 target: CTTCCAGAATTGGATCTCCG.

The primers used to identify the target fragment containing the gRNA in the TTN gene are as follows:

TTN-F55: TACGGAATTGGGGAGCCAGCGGA;

TTN-R560: CAAAGTTAACTCTCTGTGTCT.

1. Preparation of gRNA

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

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

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

2. Obtain gRNA by in vitro transcription

The TTN-T7-gRNA1 transcription template was taken and in vitro transcription was performed using Transcript Aid T7 High Yield Transcription Kit (Fermentas, K0441), and then recovered and purified using MEGA clear ^TM Transcription Clean-Up Kit (Thermo, AM1908) to obtain TTN-gRNA1. TTN-gRNA1 is a single-stranded RNA, as shown in SEQ ID NO:6.

The TTN-T7-gRNA2 transcription template was taken, and in vitro transcription was performed using Transcript Aid T7 High Yield Transcription Kit (Fermentas, K0441), and then recovered and purified using MEGA clear ^TM Transcription Clean-Up Kit (Thermo, AM1908) to obtain TTN-gRNA2. TTN-gRNA2 is a single-stranded RNA, as shown in SEQ ID NO:7.

2. Optimization of the ratio of gRNA to NCN protein

1. Co-transfection of primary porcine fibroblasts

Group 1: TTN-gRNA1, TTN-gRNA2 and NCN protein were co-transfected into porcine primary fibroblasts. Ratio: about 100,000 porcine primary fibroblasts: 0.5μg TTN-gRNA1: 0.5μg TTN-gRNA2: 4μg NCN protein.

Group 2: TTN-gRNA1, TTN-gRNA2 and NCN protein were co-transfected into porcine primary fibroblasts. Ratio: about 100,000 porcine primary fibroblasts: 0.75μg TTN-gRNA1: 0.75μg TTN-gRNA2: 4μg NCN protein.

Group 3: TTN-gRNA1, TTN-gRNA2 and NCN protein were co-transfected into porcine primary fibroblasts. Ratio: about 100,000 porcine primary fibroblasts: 1μg TTN-gRNA1: 1μg TTN-gRNA2: 4μg NCN protein.

Group 4: TTN-gRNA1, TTN-gRNA2 and NCN protein were co-transfected into porcine primary fibroblasts. Ratio: about 100,000 porcine primary fibroblasts: 1.25μg TTN-gRNA1: 1.25μg TTN-gRNA2: 4μg NCN protein.

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

Co-transfection was performed by electroporation using a mammalian nuclear transfection kit (Neon kit, Thermofisher) and a Neon TM transfection system electroporator (parameter settings: 1450 V, 10 ms, 3 pulses).

2. After completing step 1, culture in complete culture medium for 12-18 hours, then replace with new complete culture medium for culture. The total culture time after electroporation is 48 hours.

3. After completing step 2, digest and collect the cells with trypsin, extract genomic DNA, perform PCR amplification using a primer pair consisting of TTN-F55 and TTN-R560, and then perform 1% agarose gel electrophoresis.

The electrophoresis pattern is shown in Figure 9. The 505 bp band is the wild-type band (WT), and the 254 bp band (the wild-type band is 505 bp and theoretically lacks 251 bp) is the deletion mutation band (MT).

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

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 1μg:1μg:4μg. Therefore, the optimal dosage of the two gRNAs to the NCN protein was determined to be 1μg:1μg:4μg.

3. Comparison of gene editing efficiency between NCN protein and commercial Cas9 protein

1. Co-transfection of primary porcine fibroblasts

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

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

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

Control group: TTN-gRNA1 and TTN-gRNA2 were co-transfected into porcine primary fibroblasts. Ratio: about 100,000 porcine primary fibroblasts: 1μg TTN-gRNA1: 1μg TTN-gRNA2.

Co-transfection was performed by electroporation using a mammalian nuclear transfection kit (Neon kit, Thermofisher) and a Neon TM transfection system electroporator (parameter settings: 1450 V, 10 ms, 3 pulses).

2. After completing step 1, culture in complete culture medium for 12-18 hours, then replace with new complete culture medium for culture. The total culture time after electroporation is 48 hours.

3. After completing step 2, digest and collect the cells with trypsin, extract genomic DNA, perform PCR amplification using a primer pair consisting of TTN-F55 and TTN-R560, and then perform 1% agarose gel electrophoresis.

The electrophoresis diagram is shown in Figure 10. The gene deletion mutation efficiency using the commercial Cas9-A protein was 28.5%, the gene deletion mutation efficiency using the NCN protein was 85.6%, and the gene deletion mutation efficiency using the commercial Cas9-B protein was 16.6%.

The results show that compared with commercial Cas9 protein, the NCN protein prepared by the present invention significantly improves the gene editing efficiency.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims (13)

1. Application of COL2A1-gRNA3, COL2A1-gRNA4 and NCN protein in the preparation of the kit; The COL2A1-gRNA3 is an sgRNA, and its target sequence binding region is shown in nucleotides 3-22 of SEQ ID NO: 16; the COL2A1-gRNA4 is an sgRNA, and its target sequence binding region is shown in nucleotides 3-22 of SEQ ID NO: 17; the NCN protein is a Cas9 protein or a fusion protein with a Cas9 protein; The purpose of the kit is as follows (a) or (b) or (c): (a) preparing recombinant pig cells; (b) preparing pigs with type II collagen disease model; (c) preparing cell model of type II collagen disease or tissue model of type II collagen disease or organ model of type II collagen disease. 2. Application of COL2A1-gRNA3, COL2A1-gRNA4 and PRONCN protein in the preparation of the kit; COL2A1-gRNA3 is the COL2A1-gRNA3 described in claim 1; COL2A1-gRNA4 is the COL2A1-gRNA4 described in claim 1; The PRONCN protein includes the following elements from upstream to downstream: a signal peptide, a molecular chaperone protein, a protein tag, a protease cleavage site, a nuclear localization signal, a Cas9 protein, and a nuclear localization signal; The purpose of the kit is as follows (a) or (b) or (c): (a) preparing recombinant pig cells; (b) preparing pigs with type II collagen disease model; (c) preparing cell model of type II collagen disease or tissue model of type II collagen disease or organ model of type II collagen disease. 3. Application of COL2A1-gRNA3, COL2A1-gRNA4 and specific plasmids in preparing kits; COL2A1-gRNA3 is the COL2A1-gRNA3 described in claim 1; COL2A1-gRNA4 is the COL2A1-gRNA4 described in claim 1; The specific plasmid includes the following elements from upstream to downstream: a promoter, an operator, a ribosome binding site, a gene encoding a PRONCN protein, and a terminator; the PRONCN protein includes the following elements from upstream to downstream: a signal peptide, a molecular chaperone protein, a protein tag, a protease cleavage site, a nuclear localization signal, a Cas9 protein, and a nuclear localization signal; The purpose of the kit is as follows (a) or (b) or (c): (a) preparing recombinant pig cells; (b) preparing pigs with type II collagen disease model; (c) preparing cell model of type II collagen disease or tissue model of type II collagen disease or organ model of type II collagen disease. 4. A kit comprising COL2A1-gRNA3, COL2A1-gRNA4 and NCN protein; COL2A1-gRNA3 is the COL2A1-gRNA3 described in claim 1; COL2A1-gRNA4 is the COL2A1-gRNA4 described in claim 1; NCN protein is the NCN protein described in claim 1; The purpose of the kit is as follows (a) or (b) or (c): (a) preparing recombinant pig cells; (b) preparing pigs with type II collagen disease model; (c) preparing cell model of type II collagen disease or tissue model of type II collagen disease or organ model of type II collagen disease. 5. A kit comprising COL2A1-gRNA3, COL2A1-gRNA4 and PRONCN protein; COL2A1-gRNA3 is the COL2A1-gRNA3 described in claim 1; COL2A1-gRNA4 is the COL2A1-gRNA4 described in claim 1; PRONCN protein is the PRONCN protein described in claim 2; The purpose of the kit is as follows (a) or (b) or (c): (a) preparing recombinant pig cells; (b) preparing pigs with type II collagen disease model; (c) preparing cell model of type II collagen disease or tissue model of type II collagen disease or organ model of type II collagen disease. 6. A kit comprising COL2A1-gRNA3, COL2A1-gRNA4 and a specific plasmid; COL2A1-gRNA3 is the COL2A1-gRNA3 described in claim 1; COL2A1-gRNA4 is the COL2A1-gRNA4 described in claim 1; the specific plasmid is the specific plasmid described in claim 3; The purpose of the kit is as follows (a) or (b) or (c): (a) preparing recombinant pig cells; (b) preparing pigs with type II collagen disease model; (c) preparing cell model of type II collagen disease or tissue model of type II collagen disease or organ model of type II collagen disease. 7. A method for preparing recombinant pig cells, comprising the following steps: co-transfecting COL2A1-gRNA3, COL2A1-gRNA4 and NCN protein into pig cells to obtain recombinant pig cells; COL2A1-gRNA3 is the COL2A1-gRNA3 described in claim 1; COL2A1-gRNA4 is the COL2A1-gRNA4 described in claim 1; and NCN protein is the NCN protein described in claim 1. 8. The use according to claim 1 or the kit according to claim 4 or the method according to claim 7, characterized in that: the NCN protein is shown in SEQ ID NO: 3. 9. The use or kit or method according to claim 8, characterized in that: The preparation method of the NCN protein comprises the following steps: (1) Introducing plasmid pKG-GE4 into Escherichia coli BL21 (DE3) to obtain recombinant bacteria; (2) culturing the recombinant bacteria in a liquid medium at 30° C., then adding IPTG and inducing the culture at 25° C., and then collecting the bacteria; (3) crushing the collected bacterial cells and collecting the crude protein solution; (4) purifying the fusion protein with the His ₆ tag from the crude protein solution using affinity chromatography; (5) using enterokinase with a His ₆ tag to digest the fusion protein with a His ₆ tag, and then using Ni-NTA resin to remove the protein with the His ₆ tag to obtain a purified NCN protein; Plasmid pKG-GE4 contains the fusion gene shown by nucleotides 5209 to 9852 in SEQ ID NO:1. 10. The recombinant pig cell prepared by the method of claim 7, 8 or 9. 11. Use of the recombinant pig cell according to claim 10 in preparing a pig model of type II collagen disease. 12. Pig tissue, pig organ or pig cell of a type II collagen disease model pig prepared using the recombinant pig cell according to claim 10. 13. Use of the recombinant pig cell according to claim 10, the pig tissue according to claim 12, the pig organ according to claim 12, the pig cell according to claim 12, or the type II collagen disease model pig prepared using the recombinant pig cell according to claim 10, as follows (d1) or (d2) or (d3) or (d4): (d1) Screening for drugs to treat type II collagen diseases; (d2) Evaluate the efficacy of drugs for type II collagen diseases; (d3) Evaluate the efficacy of gene therapy and/or cell therapy for type II collagen diseases; (d4) To study the pathogenesis of type II collagen disease.

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