CN118667818B

CN118667818B - Method for targeting knocking-in human blood coagulation factor VII and VIII coding genes at pig blood coagulation factor VII and VIII gene loci and application

Info

Publication number: CN118667818B
Application number: CN202410995788.8A
Authority: CN
Inventors: 陆芳; 汪金玲
Original assignee: Guangzhou Zhongkefei Dolphin Biotechnology Co ltd
Current assignee: Guangzhou Zhongkefei Dolphin Biotechnology Co ltd
Priority date: 2024-07-24
Filing date: 2024-07-24
Publication date: 2025-04-22
Anticipated expiration: 2044-07-24
Also published as: CN118667818A

Abstract

The present invention relates to a method and application for targeted knock-in of human coagulation factor VII and VIII coding genes at pig coagulation factor VII and VIII gene loci, and belongs to the field of gene editing technology. The present invention provides a group of sgRNAs for targeted knock-in of human coagulation factor coding genes at pig coagulation factor gene loci. Targeting the specific sites of the first intron of pig coagulation factor VII coding gene F7 and pig coagulation factor VIII coding gene F8, sgRNA and targeting vectors are designed respectively, and human coagulation factor FVII and FVIII protein coding genes are site-specifically knocked in, and human coagulation factor FVII protein expression is initiated by the endogenous promoter of pig F7 gene, and human coagulation factor FVIII protein expression is initiated by the endogenous promoter of pig F8 gene. The expression level is equivalent to the physiological level, and will not have a significant impact on the health status of the prepared gene-edited pigs, and is used for large-scale production of human coagulation factor FVII and FVIII protein products.

Description

Method for targeting knocking-in human blood coagulation factor VII and VIII coding genes at pig blood coagulation factor VII and VIII gene loci and application

Technical Field

The invention relates to the technical field of gene editing, in particular to a method for targeting knocking-in human blood coagulation factor VII and VIII coding genes at pig blood coagulation factor VII and VIII gene loci and application thereof.

Background

Coagulation factors are a general term for blood plasma and a large class of substances in tissues directly involved in coagulation, most of which are proteins synthesized by the liver and secreted into the blood plasma, and are critical for coagulation after bleeding in the body. In the coagulation process, different coagulation factors are activated sequentially in a certain order, and finally the coagulation function is realized through the coagulation cascade reaction of the intrinsic and extrinsic coagulation pathways (the theory of coagulation cascade).

The deficiency of blood coagulation factors leads to coagulation dysfunction, leading to various hemorrhagic diseases such as hemophilia. Clinically, replacement therapy with exogenously infused missing clotting factors is an effective means of current treatment of hemophilia. The blood coagulation factor VIII (Coagulation factorVIII, FVIII) is a key factor of an endogenous blood coagulation pathway, and hemophilia A caused by defects accounts for 80% -85% of human hemophilia patients, so that the demand of the treatment FVIII is large. In addition, for patients partially receiving exogenous infusion of coagulation factors, the production of antibodies to the corresponding coagulation factors in vivo reduces the therapeutic effect and increases the risk of treatment. For such patients, factor VII (FVII), a key factor involved in the extrinsic pathway of coagulation, is the first alternative drug. FVII is also widely used in the treatment of traumatic bleeding, and there is also a greater clinical demand for FVII.

The existing human blood coagulation factor products for treatment mainly originate from human blood plasma, the sources of raw materials are limited, pig blood plasma blood coagulation factor products are directly used in early stage, but pig blood coagulation factors are not completely the same as human blood coagulation factors, recombinant human FVII and FVIII proteins expressed by Chinese hamster ovary cells (CHO cells) cultured in vitro and recombinant pig FVIII protein products are available abroad by means of genetic engineering technology, but due to low protein expression level and incomplete post-translational modification of proteins, secretion, protein activity, half-life and the like are influenced, and the technology barriers are high, the production process is complex, the yield is low, the cost is high, and the existing domestic products are also lacking. Thus, there is a need to develop new methods for efficiently preparing human FVII and FVIII. In recent decades, the technology of producing human functional proteins by using genetically modified animals or plants as bioreactors is expected to provide a new source for human coagulation factor production. For example, human FVII or FVIII proteins are expressed in the mammary glands of mammals such as sheep or rabbits by transgenic techniques using mammary gland specific expression vectors. The mammary gland bioreactor has the limitations that, on one hand, the integration of target genes in animal genome has larger randomness, unstable result and larger individual difference of target protein expression level, and on the other hand, the protein processing and modifying system has larger difference between species and tissues, and influences the activity, immunogenicity, pharmacokinetics and other coagulation factor characteristics of target proteins. Meanwhile, the target protein produced by the mammary gland bioreactor only occupies a small proportion of total protein secreted by mammary glands, so that the purification difficulty of the target protein is increased. The functional protein is knocked into an endogenous site of an animal genome, so that the functional protein is expressed in situ, and the target protein with more complete post-translational modification can be obtained. Sheep and cattle can also be used to develop blood bioreactors, but the risk of zoonosis is higher (e.g., brucellosis, prion infection, tuberculosis, etc.). Compared with human beings, the pig has more similar physiological, metabolic and protein post-translational modification systems, is convenient for large-scale breeding and raising, and has low zoonosis risk. Currently, human albumin production by knocking human serum albumin coding sequences into pig albumin gene loci at fixed points and using pigs as blood bioreactors has been reported. The use of pigs as blood bioreactors for the production of human coagulation factors has yet to be developed.

In addition, pigs are expected to solve the problem of donor organ shortage in human organ transplantation as an ideal xenogeneic organ transplantation donor. Solving the incompatibility between human and pig organs is a long-term critical person in this field, where the incompatibility of the coagulation system is an important aspect. In the past, studies have focused mainly on the problem of immune rejection, and the coagulation aspect mainly expresses thrombomodulin. With the intensive research, the humanization of functional proteins between pigs and humans is becoming more and more important, and the humanization of blood coagulation factors is an important part. The construction of a humanized pig model expressing only human coagulation factors but not porcine coagulation factors is of great value for improving the xenograft effect, in particular liver xenograft (most of the coagulation factors are synthesized by the liver). For example, it has been reported that human FVII and human albumin expression sequences are knocked in at the porcine FVII gene (F7) site simultaneously, humanized modifications (Li L,Meng H,Zou Q,et al.Establishment of gene-edited pigs expressing human blood-coagulation factor VII and albumin for bioartificial liver use[J].J Gastroenterol Hepatol,2019,34(10):1851-1859.). are made but due to the additional knockin of human albumin expression sequences, human FVII humanized modifications are not fully mimicked and porcine FVII protein may be expressed.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for effectively cutting target sites and targeting knocking-in human coagulation factor VII and VIII coding genes at pig coagulation factor VII and VIII gene sites and application thereof.

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:

In a first aspect, a set of sgRNAs targeting a gene encoding a knockin human clotting factor at a porcine clotting factor gene locus,

The group of sgrnas comprises a first sgRNA targeted to knock-in a human factor VII encoding gene at a porcine factor VII gene locus and a second sgRNA targeted to knock-in a human factor VIII encoding gene at a porcine factor VIII gene locus;

The nucleotide sequence of the first sgRNA is shown as SEQ ID NO. 2, and the nucleotide sequence of the second sgRNA is shown as SEQ ID NO. 5.

The accession number of the pig blood coagulation factor VII gene on NCBI is NC_010453, and the accession number of the pig blood coagulation factor VIII gene on NCBI is NC_010461. The sgRNA provided by the invention can effectively cut the porcine blood coagulation factor VII gene locus and the porcine blood coagulation factor VIII gene locus at fixed points.

In a second aspect, the invention also provides an expression vector for expressing the sgRNA.

Further, the nucleotide sequence of the expression vector for expressing the first sgRNA is shown as SEQ ID NO. 18, and the nucleotide sequence of the expression vector for expressing the second sgRNA is shown as SEQ ID NO. 19.

In the third aspect, a CRISPR/Cas9 gene editing technology is used for targeting a targeting vector knocking in human blood coagulation factor coding genes at pig blood coagulation factor gene loci, the nucleotide sequence of the targeting vector knocking in human blood coagulation factor FVII coding genes at pig blood coagulation factor VII gene loci is shown as SEQ ID NO. 16, and the nucleotide sequence of the targeting vector knocking in human blood coagulation factor VIII coding genes at pig blood coagulation factor VIII gene loci is shown as SEQ ID NO. 17.

In a fourth aspect, the invention provides the use of the sgRNA, the expression vector and the targeting vector in the preparation of porcine fetal fibroblasts targeted to knock-in human coagulation factor encoding genes.

In a fifth aspect, the invention provides a method for preparing a pig fetal fibroblast targeted to knock-in a human coagulation factor coding gene, wherein the sgRNA expression vector and the targeting vector are co-transfected in the pig fetal fibroblast, and then the pig fetal fibroblast is cultured.

Further, the sgRNA targeting recognition site is in the 1 st intron of the pig blood coagulation factor encoding gene, the nucleotide sequence of the sense strand of the sgRNA targeting recognition site of the pig blood coagulation factor VII gene site is shown as SEQ ID NO. 8, the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 9, the nucleotide sequence of the sense strand of the sgRNA targeting recognition site of the pig blood coagulation factor VIII gene site is shown as SEQ ID NO. 14, and the nucleotide sequence of the antisense strand is shown as SEQ ID NO. 15.

In a specific embodiment of the invention, the preparation method comprises the following steps:

s1, taking 5-15 mug, preferably 10 mug of each of the sgRNA expression vector and the targeting vector, and mixing to obtain a mixed plasmid;

S2, transfecting the mixed plasmid in the step S1 into a swine fetal fibroblast, and electrotransferring to obtain an electrotransferred cell;

And S3, culturing the cells subjected to electrotransformation in the step S2 into monoclonal cells to obtain the pig fetal fibroblasts targeted to knock-in the human coagulation factor coding genes.

Further, in step S1, 10. Mu.g of the sgRNA expression vector with the nucleotide sequence shown in SEQ ID NO. 18 and 10. Mu.g of the targeting vector with the nucleotide sequence shown in SEQ ID NO. 16 are mixed to obtain a mixed plasmid 1, and 10. Mu.g of the sgRNA expression vector with the nucleotide sequence shown in SEQ ID NO. 19 and 10. Mu.g of the targeting vector with the nucleotide sequence shown in SEQ ID NO. 17 are mixed to obtain a mixed plasmid 2.

The pig fetal fibroblasts obtained by culturing the mixed plasmid 1 and targeted knocking in the human blood coagulation factor coding genes are pig fetal fibroblasts targeted and knocked in the human blood coagulation factor VII coding genes at pig blood coagulation factor VII gene loci;

the pig fetal fibroblasts obtained by culturing the mixed plasmid 2 and targeting the knockin human blood coagulation factor encoding genes are the pig fetal fibroblasts targeting the knockin human blood coagulation factor VIII encoding genes at pig blood coagulation factor VIII gene loci.

Further, the parameters of the step S2 electric rotation are 1350V, 30ms and 1pulse.

Further, in the step S3, the cells are spread in the culture dish with the density of 50-5000 cells/dish. Preferably, the cell density of the mixed plasmid 1 after electrotransformation in the step S2 is 3000 to 5000 cells/dish, and the cell density of the mixed plasmid 2 after electrotransformation in the step S2 is 50 to 100 cells/dish.

Further, step S3 cultures the cells in high-sugar DMEM medium containing fetal bovine serum.

Further, the concentration of the fetal bovine serum in step S3 was 15% (v/v).

Further, the culture conditions in the step S3 are 38.5 ℃ and 5% CO ₂ incubator.

Further, the concentration of puromycin in the medium of step S3 is 0.3 to 1. Mu.g/mL, preferably 1. Mu.g/mL.

Further, the culture time in the step S3 is 8-12 days.

In step S3, the cells obtained by electrotransformation and prepared from the mixed plasmid 1 are screened by adding puromycin into the culture medium.

In a sixth aspect, the invention provides a pig fetal fibroblast targeted to knock-in a human coagulation factor encoding gene, which is prepared by the preparation method.

In a seventh aspect, the invention provides an application of the pig fetal fibroblasts in preparing a gene editing pig targeted to knock-in human coagulation factor encoding genes.

In an eighth aspect, the invention provides a method for preparing a gene editing pig targeted to knock-in human coagulation factor coding genes, wherein the nuclei of the pig fetal fibroblasts are transplanted into enucleated oocytes, the enucleated oocytes are fused to form recombinant embryos, the recombinant embryos are transplanted into a sow body, and the gene editing pig is obtained after gestation and farrowing.

Compared with the prior art, the invention has the beneficial effects that:

the invention designs sgRNA and targeting vectors respectively aiming at specific sites of 1 st introns of a pig blood coagulation factor VII coding gene F7 and a pig blood coagulation factor VIII coding gene F8, knocks in human blood coagulation factor FVII and FVIII protein coding genes at fixed points, starts human blood coagulation factor FVII protein expression by a pig F7 gene endogenous promoter, starts human blood coagulation factor FVII protein expression by a pig F8 gene endogenous promoter, has the expression level equivalent to the physiological level, does not have obvious influence on the health condition of the prepared gene editing pig, is used for producing human blood coagulation factor FVII and FVIII protein products on a large scale, and simultaneously ensures that the pig endogenous blood coagulation factor coding genes are blocked and cannot be expressed due to the knocked-in human blood coagulation factor coding genes. The humanized single protein and the humanized single protein of human FVII and FVIII can be realized, and the humanized single protein can be mated with pigs humanized by other functional proteins (such as serum albumin), so that a pig model with more functional proteins and humanized simultaneously is obtained, and the humanized modified human FVII and FVIII can be used for perfecting the modification of xenograft related genes.

Drawings

FIG. 1 shows the target site sequences and sgRNAs of the F7 and F8 genes of pigs. Wherein A is F7 gene target site sequence and sgRNA, and B is F8 gene target site sequence and sgRNA.

FIG. 2 shows the sgRNA mediated cleavage effect at the target site. Wherein A is pF7-sgRNA-1, B is pF7-sgRNA-2, C is pF8-sgRNA-0;D is pF8-sgRNA-1, E is pF8-sgRNA-2;F is the base difference of the pF8-sgRNA-0 target site sequence (pig F8) and the corresponding human FVIII protein coding sequence (human F8).

FIG. 3 is a schematic diagram of the accurate targeting principle of the pig F7 gene locus.

Fig. 4 is a schematic diagram of the accurate targeting principle of the swine F8 gene locus.

FIG. 5 is a graph showing the genotyping results of cloned pigs. Wherein, A is F7 locus genotype identification, and B is F8 locus genotype identification.

FIG. 6 shows Sanger sequencing confirming that the pig F7 site knocks in the gene encoding human FVII protein. Wherein A is Sanger sequencing peak diagram of cloned pig target site knock-in human FVII protein coding gene, and B is protein sequence corresponding to knock-in sequence of sequencing result and human FVII protein sequence comparison result.

FIG. 7 shows Sanger sequencing confirming that the pig F8 site knocks in the gene encoding human FVIII protein. Wherein A is Sanger sequencing peak diagram of cloned pig target site knock-in human FVIII protein coding gene, and B is protein sequence corresponding to knock-in sequence of sequencing result and human FVIII protein sequence comparison result.

Detailed Description

For a better description of the objects, technical solutions and advantages of the present invention, the present invention will be further described with reference to the following specific examples. Other materials, reagents, etc. used in the examples are commercially available unless otherwise specified.

Example 1 design of pig F7 and F8 Gene target site specific sgRNA

Sequence information of a porcine factor FVII protein-encoding gene F7 (NCBI accession number: NC_ 010453) and a factor FVIII protein-encoding gene F8 (NCBI accession number: NC_ 010461) was obtained from the porcine reference genome (Sscofa 11.1) in NCBI, specific sgRNAs were designed with the vicinity of the initiation codon ATG as target sites, and the designed sgRNAs were tested for efficacy. And (3) sequencing to verify whether the target site sequence is consistent with the reference sequence, and controlling the sequencing result.

1. Design of sgRNA

As shown in FIG. 1A, the sgRNA sequences of 2 porcine FVII protein-encoding genes F7, pF7-sgRNA-1 and pF7-sgRNA-2, respectively, were designed. As shown in FIG. 1B, 3 sgRNA sequences of the F8 gene encoding porcine FVIII protein were designed, pF8-sgRNA-0, pF8-sgRNA-1 and pF8-sgRNA-2, respectively.

The sgRNA sequences and their corresponding target sequences are shown in tables 1 and 2.

TABLE 1

TABLE 2

2. Construction of sgRNA expression vectors

(1) PX330 plasmid (brand: addgene, cat# 42230, nucleic acid sequence containing SpCas9 effector protein and U6 promoter) is used as blank vector, and cut with restriction enzyme BpiI to obtain enzyme-digested vector skeleton. Preparing 50 mu L of enzyme digestion reaction system, namely 10X FAST DIGEST buffer (brand: thermo Scientific, product number: B64) 5 mu L, bpiI (brand: thermo Scientific, product number: FD 1014) 2 mu L, PX330 plasmid 5 mu g, adding double distilled water (ddH ₂ O) to complement to 50 mu L, incubating for enzyme digestion for 2h at 37 ℃, purifying and recovering the digested fragments, and obtaining the PX330 enzyme digestion carrier skeleton.

(2) The two complementary primers specific to the target site are synthesized, 4 additional bases are respectively added at the 5' end of the sequence shown in the table 2 so as to complementarily pair with the sticky end of the PX330 enzyme-cut carrier skeleton, the 5' -additional base of the sense strand in the table 2 is 5' -CACC-3', which is used as a forward primer, and the 5' -additional base of the antisense strand in the table 2 is 5' -AAAC-3', which is used as a reverse primer. The forward primer and the reverse primer were each 5. Mu.L in a concentration of 4. Mu.M, mixed, and annealed. The annealing procedure was 98℃for 10min and naturally cooled to room temperature (25 ℃) to give an annealed product (double-stranded DNA fragment containing cohesive ends).

(3) Cloning the annealed product of the step (2) onto the PX330 enzyme-cleaved vector backbone of the step (1).

PX330 enzyme-cleaved vector backbone of step (1) was mixed with 0.5. Mu.L of the annealed product of step (2) and 2.5. Mu.L of Solution I (Takara Co.) and incubated at 16℃for 2 hours to give a ligation product.

(4) And (3) converting each connecting product (5 mu L) in the step (3) into 100 mu L of escherichia coli DH5 alpha competent cells, incubating for 30min on ice, and then performing heat shock conversion at 42 ℃ for 45s to obtain the converted escherichia coli DH5 alpha cells. The transformed E.coli DH 5. Alpha. Cells were plated on LB agar plates supplemented with ampicillin (100. Mu.g/mL), cultured at 37℃for 12 hours, single colonies were picked up, and cultured in liquid LB medium supplemented with ampicillin (100. Mu.g/mL) at 37℃for 8 hours, to obtain E.coli DH 5. Alpha. Cells after screening culture. E.coli DH 5. Alpha. Cells after screening culture were lysed, extracted and purified to obtain 5 PX330-sgRNA expression vectors (PX 330-sgRNA expression vectors expressing the pF7-sgRNA-1, pF7-sgRNA-2, pF8-sgRNA-0, pF8-sgRNA-1 and pF8-sgRNA-2 sequences in Table 1 were inserted into PX330 plasmids, respectively, i.e., the sequences GGGTCTTCGAGAAGACCT in the original PX330 vectors were replaced with the corresponding sense strand sequences in Table 2 to obtain PX330-sgRNA expression vectors expressing the pF7-sgRNA-1, pF7-sgRNA-2, pF8-sgRNA-0, pF8-sgRNA-1 and pF8-sgRNA-2 sequences, respectively).

(5) And (3) verifying that the sequences of the 5 PX330-sgRNA expression vectors prepared in the step (4) are correct through sequencing.

3. Testing sgrnas

The cleavage effect of the sgrnas designed in step 1 was tested by transfection.

(1) Fetal swine fibroblasts (Porcine fetal fibroblast, PFF) were cultured in PFF medium (high sugar modified eagle medium DMEM medium supplemented with 15% v/v fetal bovine serum) and PFF cells were harvested by digestion when they were grown to 80% confluency in a 38.5 ℃ 5% CO ₂ incubator.

(2) And (2) respectively transfecting the 5 PX330-sgRNA expression vectors constructed in the step (2) into the PFF cells treated in the step (1) by using an Invitrogen ^TMNeon^TM transfection system to obtain transfected PFF cells. Each group of cells had 2.5X10 ⁵ cells, each group of PX330-sgRNA expression vectors had a mass of 5. Mu.g, and the electrotransformation parameters were 1350V, 30ms and 1pulse. The transfected cells were plated in 24-well plates, fresh PFF medium was added and cultured in a 38.5℃5% CO ₂ incubator for 48h.

(3) Collecting the PFF cells cultured in the step (2), extracting genome, and carrying out PCR amplification by taking the extracted genome as a template to obtain fragments (PCR products) of the sgRNA target site range.

The PCR reaction was performed using a 2X RAPID TAQ MASTER Mix (cat No. P222-02, brand Vazyme) with a 2X RAPID TAQ MASTER Mix of 10. Mu.L upstream primer concentration of 10. Mu.M, 0.2. Mu.L downstream primer concentration of 10. Mu.M, 1.5. Mu.L extracted genomic template, 8.1. Mu.L ultrapure water, and a total volume of 20. Mu.L. The PCR reaction conditions were 95℃for 3min, [95℃for 15s,55℃for 15s,72℃for 10s ] for 36 cycles, 72℃for 5min, and 12℃for incubation.

The forward primer of the F7 site is CCGACCGGGAAAGTCAACAGAC, the reverse primer of the F7 site is CACTTGGTACCGGAGTCAGGA, the forward primer of the F8 site is TCGTGCTAATGCTGCTGTCA, and the reverse primer of the F8 site is AGACCCTCTAGACACGCCTT.

(4) And (3) carrying out Sanger sequencing (first generation gene sequencing) on the fragment (PCR product) of the target site range of the sgRNA obtained in the step (3), judging whether the target site is cut according to a sequencing peak diagram, and determining the sgRNA for effectively cutting the target site DNA double strand.

As shown in FIGS. 2A and 2B, pF7-sgRNA-1 was not cleaved to produce a double peak, and there was no cleavage activity, whereas pF7-sgRNA-2 had a distinct cleavage double peak, effectively mediating target site cleavage. As shown in FIGS. 2C, 2D and 2E, pF8-sgRNA-1 did not have a distinct cleavage double peak, whereas pF8-sgRNA-0 and pF8-sgRNA-2 had a distinct cleavage double peak, effectively mediating cleavage of the target site, wherein the pF8-sgRNA-0 target site is located within the first exon and the pF8-sgRNA-2 target site is located within the first intron.

Example 2 design of targeting vectors for porcine F7 and F8 loci

When the human coagulation factor coding sequence expression element is knocked in to the pig endogenous site at fixed points, a targeting vector is required to be provided, and a repair template is provided for homologous recombination after CRISPR/Cas9 (a gene editing technology) cuts double-stranded DNA.

1. As shown in FIG. 3, the nucleotide sequence of the targeting vector of the F7 locus of the porcine FVII protein-encoding gene is shown as SEQ ID NO:16, and consists of a left homology arm of the F7 locus, a human full-length FVII protein-encoding gene (comprising a start codon and an end stop codon, CCDS database accession number: CCDS 9528.1), a tailing signal of transcription termination (PolyA, PA in FIG. 3), an independently expressed puromycin (Puro) selection tag (loxP-PGK-Puro-PA-loxP), a right homology arm of the F7 locus and a diphtheria toxin A chain (DTA) selection tag (PGK-DTA-pA) for negatively selecting and killing transgenic cells to prevent transgene but not homologous recombination.

2. As shown in FIG. 4, the nucleotide sequence of the targeting vector of the F8 locus of the pig FVIII protein coding gene is shown as SEQ ID NO:17, and consists of a left homology arm of the F8 locus, a human full-length FVIII protein coding gene (comprising a start codon and a tail stop codon, CCDS database accession number: CCDS 35457.1), a transcription termination tailing signal (PolyA, PA in FIG. 4), a right homology arm of the F8 locus and an expression DTA screening tag.

The Puro screening tag was removed because the FVIII protein coding sequence itself was longer (> 7 kb), resulting in a larger targeting vector. On the one hand, the oversized vector affects plasmid copy number, yield and transfection efficiency, and on the other hand, the exogenous puro tag needs to be removed in the subsequent pig model application.

EXAMPLE 3 pig somatic Gene modification, site-directed knock-in of human FVII or FVIII expression elements

1. Experimental method

1. Human FVII expression element site-directed knock-in:

(1) The PX330-sgRNA expression vector (nucleotide sequence shown as SEQ ID NO: 18) prepared in example 1 and the targeting vector of the F7 site of the pig prepared in example 2 were mixed to obtain F7 mixed plasmids, each 10. Mu.g.

(2) The F7-mixed plasmid of step (1) was transfected into PFF cells (5X 10 ⁵) and electrotransformed by the same method as in example 1 to obtain electrotransformed cells.

(3) The cells after the electrotransformation in the step (2) are spread in a culture dish with the density of 10cm (3000-5000 pieces per dish), and are cultured in a culture box with high sugar DMEM medium added with 15% (v/v) fetal bovine serum at 38.5 ℃ and 5% CO ₂, and 1 mug/mL puromycin is added in the culture medium for screening. Culturing for 8-12 days, and growing single cells into clone samples to obtain single-cell-derived clone cells.

(4) And (3) selecting the single-cell-derived cloned cells in the step (3), and continuously culturing the single-cell-derived cloned cells in a 24-well plate until the cells grow into culture holes, wherein the culture conditions are the same as those in the step (3).

(5) And (3) spreading three-quarter cells on a 24-pore plate for continuous culture until the culture holes are full, wherein the culture conditions are the same as those of the step (3), performing cell lysis on the remaining one-quarter cells, extracting the genome of the cells as a template, and using PCR amplification to identify whether target sequences (human full-length FVII protein coding genes+PolyA+Puro screening tag fragments) are accurately integrated on target sites of pig genomes, so as to obtain human FVII knocked-in cells, and freezing for later use.

The PCR reaction was performed in the same manner as in step 3 of example 1, wherein the PCR reaction conditions were 95℃for 3min, [95℃for 15s,55℃for 15s,72℃for 2min ] for 36 cycles, 72℃for 5min and 12℃for heat preservation. The PCR primer sequences are shown in Table 3. The cells are diploid and have a pair of homologous chromosomes. Single allele knock-ins represent heterozygotes and double allele knock-ins represent homozygotes. In tables 3 and 4F 7-homozygous/heterozygous means to identify whether one of the two homologous chromosomes is knocked in (heterozygous/homoallelic knock-in) or both chromosomes are knocked in (homoallelic/homoallelic knock-in)

TABLE 3 Table 3

2. Human FVIII expression element site-directed knock-in:

(1) 10. Mu.g each of PX330-sgRNA expression vector for pF8-sgRNA-0 prepared in example 1 and targeting vector for pig F8 site prepared in example 2 was mixed to obtain F8 mixed plasmid 0. pF8-sgRNA-0 was replaced with pF8-sgRNA-2 (nucleotide sequence shown in SEQ ID NO: 19), and the other conditions were unchanged, to obtain F8-mixed plasmid 1.

(2) According to the method of steps (2) - (5) in the fixed-point knocking-in method of human FVII expression element of the present embodiment, human FVII-knocked-in cells are prepared. Because the targeting vector of the F8 locus of the pig does not contain Puro screening tags, puromycin is not added into the culture medium for screening, and cells after electrotransformation are paved in a 10cm culture dish at the density of 50-100 cells per dish. The PCR primer sequences are shown in Table 4.

TABLE 4 Table 4

2. Experimental results

Cell clone statistics for F7 and F8 gene locus edits are shown in Table 5. Since the F8 site is located on the X chromosome, the screen cell uses male cells, and only a single X chromosome is used, so that only single-allele knocked-in cells can be finally obtained, and double-allele knocked-in cells are not used.

TABLE 5

When human FVIII knockin cells were prepared with PX330-sgRNA expression vector containing pF8-sgRNA-0, expected human FVIII knockin cells were not successfully obtained. Further analysis found that the sequence of the designed target site for pF8-sgRNA-0 was only 2 bases different from the corresponding human FVIII protein coding sequence (FIG. 2F), so that pF8-sgRNA-0 was able to cleave not only the porcine F8 gene target site, but also the targeting vector, resulting in the eventual failure to obtain the target modified cell clone. Thus, human FVIII knockin cells were prepared with PX330-sgRNA expression vector containing pF8-sgRNA-2 and frozen for use.

Example 4 obtaining of a porcine model of FVII or FVIII protein humanization by somatic cell nuclear transfer techniques

1. Experimental method

Human FVII knock-in cells obtained in example 3 (numbered #1, #2 and #3, respectively) and human FVIII knock-in cells (numbered #77, #106 and #165, respectively) were used as donor cells for somatic cell nuclear transfer.

Donor cells were injected into perivitelline spaces of enucleated porcine mature oocytes under a micromanipulator and fused and activated (parameters: 120V/mm, 30 μsec, 2 pulses) by means of an ECM2001 (BTX) cell fusion electroporator by electro-activation to obtain recombinant embryos.

The recombinant embryos were transferred into the oviduct of sow recipients (about 200 recombinant embryos per sow recipient) and 767 recombinant embryos were transferred in total, wherein 3 sow recipients successfully matured with gestation and 11 cloned piglets were born (table 6).

Ear tissues of small cloned piglets are taken, genomic DNA is extracted as a template for PCR amplification, and the PCR amplification steps are the same as those of the embodiment 3, so that genotypes of F7 and F8 target sites are identified. The resulting PCR amplified product was further sequenced by Sanger to confirm that the target site was knocked into the human FVII or FVIII protein encoding gene.

2. Experimental results

As shown in FIGS. 5A and 5B, the total 3 cloned piglets numbered 451-5, 306-3 and 306-7 were positive model pigs knocked in by human FVII, the total 7 cloned piglets numbered 451-1, 451-3, 451-7, 451-9, 306-1, 306-5 and 306-9 were positive model pigs knocked in by human FVIII, and the 010-1 cloned pigs were wild-type pigs. As shown in fig. 6A, 6B, 7A and 7B, sanger sequencing results confirmed that the target site was exactly knocked in to human FVII or FVIII protein coding sequence.

TABLE 6 statistics of cloning pig status obtained by somatic cell nuclear transplantation

Finally, it should be noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted equally without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A group of sgRNAs for knocking in human coagulation factor coding genes at the porcine coagulation factor gene locus, characterized in that:

The set of sgRNAs includes a first sgRNA that targets and knocks in a human coagulation factor VII encoding gene at a porcine coagulation factor VII gene site, and a second sgRNA that targets and knocks in a human coagulation factor VIII encoding gene at a porcine coagulation factor VIII gene site;

The nucleotide sequence of the first sgRNA is shown in SEQ ID NO: 2, and the nucleotide sequence of the second sgRNA is shown in SEQ ID NO: 5.

2. An expression vector for expressing the sgRNA according to claim 1, characterized in that the expression vector is an expression vector for expressing the first sgRNA or an expression vector for expressing the second sgRNA.

3. The expression vector according to claim 2, characterized in that the nucleotide sequence of the expression vector expressing the first sgRNA is shown as SEQ ID NO: 18, and the nucleotide sequence of the expression vector expressing the second sgRNA is shown as SEQ ID NO: 19.

4. A targeting vector for targeted knock-in of a human coagulation factor encoding gene at a porcine coagulation factor gene locus using CRISPR/Cas9 gene editing technology, characterized in that the nucleotide sequence of the targeting vector for targeted knock-in of a human coagulation factor FVII encoding gene at a porcine coagulation factor VII gene locus is as shown in SEQ ID NO: 16; the nucleotide sequence of the targeting vector for targeted knock-in of a human coagulation factor VIII encoding gene at a porcine coagulation factor VIII gene locus is as shown in SEQ ID NO: 17.

5. Use of the sgRNA according to claim 1, the expression vector according to claim 2 or 3, and the targeting vector according to claim 4 in preparing porcine fetal fibroblasts for targeted knock-in of a gene encoding a human coagulation factor.

6. A method for preparing porcine fetal fibroblasts with targeted knock-in of a gene encoding a human coagulation factor, characterized in that the expression vector according to claim 2 or 3 and the targeting vector according to claim 4 are co-transfected into porcine fetal fibroblasts and cultured.

7. The preparation method according to claim 6, characterized in that the sgRNA targeting recognition site is in the first intron of the porcine coagulation factor encoding gene, the sense chain nucleotide sequence of the sgRNA targeting recognition site of the porcine coagulation factor VII gene site is shown in SEQ ID NO: 8, and the antisense chain nucleotide sequence is shown in SEQ ID NO: 9; the sense chain nucleotide sequence of the sgRNA targeting recognition site of the porcine coagulation factor VIII gene site is shown in SEQ ID NO: 14, and the antisense chain nucleotide sequence is shown in SEQ ID NO: 15.

8. A porcine fetal fibroblast with a targeted knock-in of a gene encoding a human coagulation factor, characterized in that it is prepared by the preparation method described in claim 6 or 7.

9. Use of the porcine fetal fibroblasts according to claim 8 in the preparation of gene-edited pigs with targeted knock-in of human coagulation factor encoding genes.

10. A method for preparing a gene-edited pig with targeted knock-in of a gene encoding a human coagulation factor, characterized in that the nucleus of the pig fetal fibroblast described in claim 8 is transplanted into an enucleated oocyte, fused to form a recombinant embryo, and the recombinant embryo is transplanted into a sow, and the gene-edited pig is obtained after pregnancy and birth.