CN112442515A

CN112442515A - Application of gRNA target combination in construction of hemophilia model pig cell line

Info

Publication number: CN112442515A
Application number: CN201910823017.XA
Authority: CN
Inventors: 牛冬; 汪滔; 王德华; 王磊; 程锐; 曾为俊; 马翔
Original assignee: Nanjing Qizhen Genetic Engineering Co Ltd
Current assignee: Nanjing Qizhen Genetic Engineering Co Ltd
Priority date: 2019-09-02
Filing date: 2019-09-02
Publication date: 2021-03-05
Anticipated expiration: 2039-09-02
Also published as: CN112442515B; WO2021042470A1

Abstract

The invention discloses application of a gRNA target combination in construction of hemophilia model pig cells, which comprises three gRNA target combinations, specifically, a pair of target sequences are designed for pig F8 and F9 genes respectively, three CRISPR/Cas9 systems are constructed, the three CRISPR/Cas9 systems are transferred into pig fibroblasts respectively, and gene mutant strains are screened to obtain a A, B, A & B hemophilia model pig cell line. According to the invention, by modifying the Cas9 expression vector, the gene editing efficiency is obviously improved. By adjusting the molar ratio of the gRNA expression vector to the Cas9 expression vector, the editing efficiency was also significantly improved. The invention can obtain A, B, A & B hemophilia disease model pig by using somatic cell nuclear transplantation cloning technology, is used for research on drug screening, drug effect evaluation, pharmacology and toxicology, disease pathology, gene therapy, cell therapy and the like, provides effective experimental data for further clinical application, and also provides powerful experimental means for successfully treating human hemophilia.

Description

Application of gRNA target combination in construction of hemophilia model pig cell line

Technical Field

The invention belongs to the technical field of gene editing, and particularly relates to application of a gRNA target combination in construction of A, B and A & B hemophilia model pig cell lines.

Background

Hemophilia (heamophilia) generally refers to a group of X-linked recessive inherited bleeding diseases caused by deficiency of blood coagulation factors due to gene mutation, and no effective cure method exists at present. According to epidemiological investigation results, the number of hemophilia patients in China is about 13 thousands as of 2018 years, and the number of patients who can really perform preventive treatment is only two. The coagulation process is a series of limited hydrolysis processes of proteins, generally divided into three stages: thromboplastin formation, thrombin formation and fibrin formation. While hemophilia is generally a disorder of thromboplastin in the first stage of coagulation, it is generally classified into three categories according to the different coagulation factors that are deficient: hemophilia a (factor viii deficiency), hemophilia B (factor ix deficiency) and hemophilia C (factor xi deficiency). The incidence rate is more than that of the hemophilia A, and is about 85 percent; hemophilia B accounts for about 15%; hemophilia C is less common. Hemophilia a is caused by deficiency of factor viii, an antihemophilic globulin (AHG), and the associated gene is located at Xq28, spanning more than 186kb, and more than 46 mutations have been found. Hemophilia B is caused by deficiency of factor IX, a Plasma Thromboplastin Component (PTC), and the related gene is located at Xq27.1, the total length of the gene is about 34kb, and there are over 100 identified mutations. Hemophilia C is factor XI, plasma antithrombin deficiency (PTA), and the related gene is located at 15q11, has a 23kb gene length, and has 3 mutations. Research shows that about 1 of 5000 male live infants is hemophilia A patients, about 1 of B patients is suffered from hemophilia B patients, about 1 of 25000 male patients is suffered from hemophilia C ethnicity is obvious, most of the hemophilia C ethnicity is descendants of jews in the south of the Turkey, and the inheritance pattern is autosomal recessive inheritance.

Hemophilia is generally treated by means of blood infusion, prothrombin complex, coagulation factor and the like, but blood products risk to infect infectious diseases of patients, and protein preparations such as the prothrombin complex, the coagulation factor and the like cause the patients to generate antibodies against the proteins, so that the treatment effect is reduced, and even more serious bleeding symptoms are caused. Therefore, the construction of an animal model of hemophilia will have a very important role in therapeutic studies. Currently, the animal model for hemophilia is mainly a mouse model. In the early 90s of the 20 th century, researchers have successfully constructed hemophilia a models of mice based on the gene targeting principle. Thereafter, a mouse model of hemophilia B was also successfully constructed. However, rodents such as mice are very different from humans in body type, physiology, pathology and the like, and cannot well simulate human diseases, and in fact, more than 95% of drugs validated by mice are ineffective in human clinical trials. Therefore, the development of a large animal hemophilia model closer to the human body shape and physiological function is the key to the study of hemophilia treatment. However, no artificially constructed large animal hemophilia model has been reported at present. In large animals, primates are animals having a close relationship with humans, but primates are significantly different from humans in body type and organ size, and primates are late in sexual maturity (macaques are 6 to 7 years old in first mating age), and are monofetuses, and the propagation rate is extremely slow. Meanwhile, the cloning efficiency of the primates is low, the difficulty is high, the cost is high, the economic benefit is poor, and the large-scale scientific research and commercial requirements cannot be met. The pig is the animal which is most suitable as a disease model, the size, the physiological function and the like of the pig are very close to those of the human, and the physiological and pathological characteristics of the human can be well simulated. Meanwhile, the pig has early sexual maturity, short breeding period, one litter and multiple fetuses, mature cloning technology and lower cost, and can form a large-scale colony in a short period. The long-term close breeding ensures that the pig has high gene purity and genetic stability, definite genetic background, relatively stable phenotype and good repeatability in biomedical research, and meets the requirements of scientific research; meanwhile, the miniature pig is easy to feed and low in cost, and the pig serving as a carnivorous animal for a long time and the pig serving as a disease model animal have no ethical problems and the like.

Therefore, the pig serving as the model animal can overcome the defects of large rodent and human species difference, such as large rodent, mouse and the like, and the defects of high cost and long breeding period of primates, can completely replace large, small and primate model animals, provides a model animal with very high cost performance for wide pharmaceutical enterprises, universities and scientific research institutions, and greatly promotes the rapid development of biological medicines.

The gene editing technology is used for respectively causing independent mutation and combined mutation on genes of factor VIII (F8) and factor IX (F9) in primary pig fibroblasts to obtain F8, F9, F8 and F9 mutant cells, and the cloning technology is used for the next step to obtain model pigs with A, B, A and B hemophilia diseases, so that the research on drug screening, drug effect detection, disease pathology, gene therapy, cell therapy and the like can be carried out, effective experimental data can be provided for further clinical application, and powerful experimental means can be provided for successfully treating human hemophilia.

Disclosure of Invention

The invention designs a pair of target sequences aiming at pig F8 and F9 genes respectively, constructs three CRISPR/Cas9 systems by utilizing the two pairs of target sequences, transfers the three CRISPR/Cas9 systems into pig fibroblasts respectively, and obtains a pig cell line of A, B and A & B hemophilia models by screening.

The invention provides application of a gRNA target combination in constructing a hemophilia model pig cell line, wherein the gRNA target combination consists of a first gRNA target and a second gRNA target, and/or a third gRNA target and a fourth gRNA target,

wherein the base sequence of the first gRNA target point is TATAGTTGTGACAGGGACAT, and the base sequence of the second gRNA target point is CACAAGTCCAGAAGATGACG;

the base sequence of the third gRNA target is ATGCCACCAAAATTCTGCAT; the fourth gRNA target has a nucleotide sequence of AAACTGGAAGAGTTTGTTCG.

The invention also provides a double-stranded DNA molecule comprising a cohesive end and a target fragment, wherein the base sequences of the target fragment are respectively as follows:

TATAGTTGTGACAGGGACAT；

or CACAAGTCCAGAAGATGACG;

or ATGCCACCAAAATTCTGCAT;

or AAACTGGAAGAGTTTGTTCG.

The invention also provides an expression cassette or gRNA expression vector comprising the double-stranded DNA molecule as described.

The invention also provides a CRISPR/Cas9 system, which comprises a first gRNA expression vector, a second gRNA expression vector and a Cas9 expression vector, wherein the target sequence of the first gRNA expression vector is TATAGTTGTGACAGGGACAT, and the target sequence of the second gRNA is CACAAGTCCAGAAGATGACG.

Optionally, the molar ratio of the first gRNA expression vector, the second gRNA expression vector and the Cas9 expression vector is 1.5-2: 1. Further optionally, the molar ratio of the first gRNA expression vector, the second gRNA expression vector, and the Cas9 expression vector is 1.5:1.5: 1.

The invention also provides another CRISPR/Cas9 system, which comprises a third gRNA expression vector, a fourth gRNA expression vector and a Cas9 expression vector, wherein the target sequence of the third gRNA expression vector is ATGCCACCAAAATTCTGCAT, and the target sequence of the fourth gRNA expression vector is AAACTGGAAGAGTTTGTTCG.

Optionally, the molar ratio of the third gRNA expression vector, the fourth gRNA expression vector and the Cas9 expression vector is 1.5-2: 1. Further optionally, the molar ratio of the first gRNA expression vector, the second gRNA expression vector, and the Cas9 expression vector is 1.5:1.5: 1.

The invention also provides a third CRISPR/Cas9 system, which comprises a first gRNA expression vector, a second gRNA expression vector, a third gRNA expression vector, a fourth gRNA expression vector and a Cas9 expression vector,

the target sequence of the first gRNA expression vector is TATAGTTGTGACAGGGACAT, and the target sequence of the second gRNA is CACAAGTCCAGAAGATGACG;

the target sequence of the third gRNA expression vector is ATGCCACCAAAATTCTGCAT, and the target sequence of the fourth gRNA expression vector is AAACTGGAAGAGTTTGTTCG.

Optionally, the molar ratio of the first gRNA expression vector, the second gRNA expression vector, the third gRNA expression vector, the fourth gRNA expression vector and the Cas9 expression vector is 0.75-1: 1. Further optionally, a molar ratio of the first gRNA expression vector, the second gRNA expression vector, the third gRNA expression vector, the fourth gRNA expression vector, and the Cas9 expression vector is 0.75:0.75:0.75: 1. Alternatively, the base sequence of the Cas9 expression vector is shown as SEQ ID NO. 70.

Optionally, the original vector of the gRNA expression vector is pKG-U6gRNA, and the complete sequence of the pKG-U6gRNA is shown in SEQ ID NO: 71.

The invention also provides a method for constructing the hemophilia model pig cell line, which comprises the following steps: any one of the CRISPR/Cas9 systems is transferred into a pig ear primary fibroblast, and a gene mutation monoclonal cell is screened.

The hemophilia includes types a, B and a & B.

The invention also provides a hemophilia model pig cell line constructed according to the method.

Optionally, the constructing step of the Cas9 expression vector comprises:

the vector pX330-U6-Chimeric _ BB-CBh-hSpCas9 is used as an original vector, and the original vector has a gRNA framework sequence, a CMV enhancer and a Cas9 gene; replacing the chicken β -actin promoter downstream of the CMV enhancer with the EF1a promoter; adding at least one nuclear localization coding sequence NLS at the N end and the C end of the Cas9 gene respectively;

the method can also comprise the following steps: the gRNA backbone sequence was engineered to be as shown in SEQ ID NO:70 to remove invalid redundant sequences.

The method can also comprise the following steps: a P2A-EGFP-T2A-PURO sequence, a WPRE gene, a 3' LTR gene and a bGH polyA sequence are sequentially inserted into the downstream of a C-end NLS of a Cas9 gene, so that a fluorescence and resistance screening marker is increased, and the expression efficiency of Cas9 is enhanced.

According to the invention, through conservative analysis of F8 and F9 gene knockout preset target spot exons and adjacent genome sequences, a high-efficiency knockout target spot sequence is designed, a gRNA expression vector is constructed by using the target spot sequence, and the gRNA expression vector and an expression vector for high-efficiency expression of Cas9 are transferred into a host cell, so that the gene editing efficiency is obviously improved.

The invention mutates F8, F9, F8 and F9 in primary pig fibroblasts by a gene editing technology to obtain various gene mutant cells, and then a somatic cell nuclear transfer cloning technology is used for obtaining model pigs with the disease of the haemophilia type A, B and A & B, so that the model pigs are used for researching drug screening, drug effect evaluation, pharmacological toxicology, disease pathology, gene therapy, cell therapy and the like, effective experimental data are provided for further clinical application, and powerful experimental means are provided for successfully treating human haemophilia.

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

(1) the subject of the invention (pig) has better applicability than other animals (rats, mice, primates). No large animal hemophilia model has been successfully developed. The selected research object is the pig, a hemophilia model cell line of the pig is constructed, and then the hemophilia disease model pig is cloned and produced for research on drug screening, pharmacology, pathology, toxicology and the like.

To date, only mouse hemophilia models have been constructed, and no large animal hemophilia model has been successfully developed. Rodents such as rats and mice have great differences from humans in body types, organ sizes, physiology, pathology and the like, and cannot truly simulate normal physiological and pathological states of humans. Studies have shown that over 95% of drugs validated to be effective in large mice are not effective in human clinical trials.

The large animals, primates, which are the animals most closely related to humans, are small in size, late in sexual maturity (mating starts at age 6-7), and are single-birth animals, and the population propagation rate is extremely slow, and the raising cost is high. In addition, primate cloning efficiency is low, difficulty is high, and cost is high.

However, pigs, which are animals that have a close relationship with humans except primates, do not have the above-mentioned disadvantages as model animals, and have body types, body weights, organ sizes, and the like close to those of humans, and are very similar to those of humans in terms of anatomy, physiology, nutritional metabolism, disease pathogenesis, and the like. Meanwhile, the pigs have early sexual maturity (4-6 months), high reproductive capacity and multiple births, and can form a large group within 2-3 years. In addition, the cloning technology of the pig is very mature, and the cloning and feeding cost is much lower than that of a primate; and the pig is taken as a carnivorous animal of human for a long time, and the pig is taken as a disease model animal, so that the problems of animal protection, ethics and the like do not exist.

(2) The improved cas9 high-efficiency expression vector is adopted for gene editing, and the editing efficiency is improved by about 300-400% compared with that of the original vector.

(3) gRNA vector and cas9 vector were not routinely 1:1, but in a ratio of 3:1 in terms of mole ratio.

For grnas that ultimately function: cas9 protein complex, the gRNA vector transcribes gRNA earlier than cas9 protein forms, and the transcribed gRNA degrades rapidly, so if at the DNA vector level, the molar ratio is 1:1, due to early transcription and degradation of grnas, there is eventually more moles of cas9 protein than undegraded grnas. Through experimental comparison, the molar ratio editing efficiency of 3: 1or 4:1 is higher than that of 1:1 gRNA: cas9 vector. Thus, a carrier molar ratio of gRNA to cas9 of 3:1 is preferably used in the present invention.

(4) For each target gene, the invention adopts double gRNA combination to carry out mutation, compared with the method adopting single gRNA, the invention can effectively reduce the generation of non-frame-shift mutation, and can directly detect the gene editing efficiency by PCR.

If a single gRNA is used to mutate the target gene, there is a probability of 1/3 resulting in a non-frameshift mutation of the base in random repair of non-homologous end joining (NHEJ) of the DNA, and the non-frameshift mutation is likely to fail to disrupt the function of the target gene and fail to achieve the intended goal of inactivating the target gene. When the double gRNA is used for cutting and mutating the target gene, a fragment can be removed from the target gene, and the fragment deletion frame shift mutation of the target gene can be effectively generated by designing a base fragment with non-3 times removed. Meanwhile, the gene editing product of the deletion fragment can be directly detected by a PCR means, and the efficiency of gene editing can be directly estimated by the ratio of the gene editing product to a wild-type product (i.e. unedited product).

Drawings

FIG. 1 is a map of pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO vector (pKG-GE 3 for short).

FIG. 2 is a structural map of original vector pX330-U6-Chimeric _ BB-CBh-hSpCas 9.

FIG. 3 shows the result of the electrophoresis of the original vector pX330 by BbsI and XbaI.

FIG. 4 is an electrophoretogram of a gRNAsc1-6 insert synthesized from the whole gene.

FIG. 5 is a structural map of recombinant vector pU6gRNAcas 9.

FIG. 6 is a structural map of the pU6gRNA-eEF1a Cas9 vector.

FIG. 7 shows the result of cutting the pU6gRNAcas9 vector with XbaI and AgeI enzymes.

FIG. 8 shows the electrophoresis results of eEF1a1-14 synthesized from the whole gene.

FIG. 9 is a pU6gRNA-eEF1a Cas9+ nNLS vector map.

FIG. 10 is a graph showing the result of AgeI and BglII enzymatic cleavage of Cas9 with pU6gRNA-eEF1 a.

FIG. 11 is a diagram showing the results of electrophoresis of N-NLS 1-12 in whole gene synthesis.

FIG. 12 shows FseI and SbfI cleavage maps of vector pU6gRNA-eEF1a Cas9+ nNLS.

FIG. 13 is the result of a gel map of the spliced 2727bp fragment.

FIG. 14 depicts a map of the constructed pKG-U6gRNA vector.

FIG. 15 is a schematic diagram showing the ligation of the pKG-U6gRNA vector after annealing of the DNA oligo.

FIG. 16 is a graph showing the results of the optimal molar ratio screening assay of gRNA and cas9 vector.

FIG. 17 is a graph showing comparison of the editing efficiency of MSTN gene.

FIG. 18 is a graph showing the comparison of the editing efficiency of the FNDC5 gene.

FIG. 19 is a porcine F8 gene transcription profile.

FIG. 20 is a diagram showing the results of optimal primer screening PCR for F8 gene amplification.

FIG. 21 is a graph showing the results of PCR amplification of 8 porcine F8 genes using the selected primer set.

FIG. 22 is an alignment analysis chart of the amplified F8 gene sequence and the published F8 gene sequence.

FIG. 23 is a B1-gRNA1 insertion sequence.

FIG. 24 is a B1-gRNA2 insertion sequence.

FIG. 25 is a B1-gRNA3 insertion sequence.

FIG. 26 is a B1-gRNA4 insertion sequence.

FIG. 27 is a diagram showing the results of PCR of F8 high-efficiency target gRNA combined screening.

FIG. 28 is a graph showing the results of F8 PCR (primers B1-F843/B1-R1258) assay of group 1F8 mutant monoclonal.

FIG. 29 is a graph of the alignment of the forward and reverse sequencing results of monoclonal F8 without nested peaks to the published F8 sequence.

FIG. 30 is a graph comparing the sequencing results of clone No. 4B 1-F843 with that of WT B1-F843.

FIG. 31 is a deletion map theoretically created by F8-gRNA1 and F8-gRNA 4.

FIG. 32 is a graph that plots deletion sequences resulting from F8-gRNA1 and F8-gRNA 4.

FIG. 33 is a graph of the alignment of the Miseq sequencing results of F8 monoclonal 1-23 with the wild type sequence.

FIG. 34 is a porcine F9 gene transcription profile.

FIG. 35 is a diagram showing the results of optimal primer screening PCR for F9 gene amplification.

FIG. 36 is a graph showing the results of PCR amplification of 8 porcine F9 genes using the selected primer set.

FIG. 37 is an alignment analysis chart of the amplified F9 gene sequence and the published F9 gene sequence.

FIG. 38 is a B2-gRNA1 insertion sequence.

FIG. 39 is a B2-gRNA2 insertion sequence.

FIG. 40 is a B2-gRNA3 insertion sequence.

FIG. 41 is a B2-gRNA4 insertion sequence.

FIG. 42 is a diagram showing the results of PCR of F9 high-efficiency target gRNA combined screening.

FIG. 43 is a graph showing the results of F9 PCR (primers B2-F539/B2-R853) of group 2F9 mutant monoclonals.

FIG. 44 is a graph of the alignment of the sequencing results of monoclonal F9 without nested peaks to the published F9 sequence.

FIG. 45 is an alignment of the F9 gene editing monoclonal 24-50Miseq sequencing results with the wild type sequence.

FIG. 46F8+ F9 mutant monoclonal F8 (primers B1-F843/B1-R1258) PCR assay results.

FIG. 47F8+ F9 mutant monoclonal F9 (primers B2-F539/B2-R853) PCR assay results.

FIG. 48 is a PCR product sequencing analysis diagram of the F8+ F9 monoclonal F8 gene.

FIG. 49F8+ F9 PCR product sequencing analysis chart of monoclonal F9 gene.

FIG. 50 analysis of the F8 Miseq sequencing results for different monoclonals.

FIG. 51 analysis profile of F9 Miseq sequencing results for different monoclonals.

Detailed Description

Construction and application effect detection of Cas9 high-efficiency expression vector

1.1 construction of Cas9 high-efficiency expression vector

The pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO vector (pKG-GE 3 for short) is modified from addge (Plasmid #42230, from Zhang Feng lab) pX330-U6-Chimeric _ BB-CBh-hSpCas9 vector, a PURO gene is screened by modifying a promoter, increasing a nuclear localization signal, adding a WPRE sequence for enhancing translation and adding green fluorescence and resistance, the map is shown in figure 1, and the base sequence is shown in SEQ ID NO: 1 is shown.

The original vector used, pX330-U6-Chimeric _ BB-CBh-hSpCas9, the structure of which is shown in FIG. 2, was purchased from addge (Plasmid #42230, from Zhang Feng lab).

The construction steps are as follows:

(1) removal of excess short gRNA backbone

pX330-U6-Chimeric _ BB-CBh-hSpCas9 (FIG. 2) is digested with BbsI and XbaI, the vector fragment (about 8313bp left and right) is recovered, the insert 175bp (SEQ I D NO: 1) is synthesized from the whole gene, and the insert is recombined with the recovered vector fragment to obtain the pU6gRNAcas9 vector (FIG. 2).

The construction method comprises the following specific steps:

1) the plasmid pX330-U6-Chimeric _ BB-CBh-hSpCas9 was digested with restriction enzymes BbsI and XbaI (the digestion system is shown in Table 1, and the reaction is carried out at 37 ℃ for 2 h.

TABLE 1

Composition of	Measurement of
		ddH₂O	To 50ul
pX330 plasmid	2ug
		10XFD buffer	5ul
FD BbsI	1.5ul
		FD XbaI	1.5ul
Total amount of	50

2) The digested pX330-U6-Chimeric _ BB-CBh-hSpCas9 plasmid was isolated by agarose Gel separation, and as a result, as shown in FIG. 3, the large fragment of the vector was recovered by Gel recovery Kit (Novonop Fastpur Gel DNA Extraction Mini Kit # DC301), and the desired fragment was dissolved in 50ul ddH₂O at-20 ℃ for later use.

3) Using DNAworks design, 175bp inserts were synthesized in the whole gene, and the primers for whole gene synthesis are shown in Table 2:

TABLE 2

Whole Gene Synthesis PCR Using Phanta Max (Novozam P505), the mixture was mixed according to the system shown in Table 3:

TABLE 3

Composition of	Volume ul
		ddH₂O	To 50ul
2×Phanta Max Buffer	25ul
		dNTP(10mM)	1ul
DMSO	2ul
		Primer mix (10uM 0.1ul per Primer)	0.6ul
F primer gRNAsc-1(10uM)	1ul
		R primer gRNAsc-6(10uM)	1ul
Phanta Max	1ul
		Total amount of	50ul

PCR conditions were as follows: circulating at 95 deg.C for 3min (95 deg.C for 15s and 58 deg.C for 15s and 72 deg.C for 20s) for 32 times and 5min at 72 deg.C; storing at 4 ℃. A175 bp insert (SEQ ID NO: 1) was obtained by whole gene synthesis, and after completion of PCR, the PCR product was subjected to 1% agarose electrophoresis and separated, and as a result, as shown in FIG. 4, the target fragment was recovered with a Gel recovery Kit (Novonopause FastPuDNA Extraction Mini Kit # DC301), and dissolved in 50ul ddH₂O at-20 ℃ for later use.

4) Recombination of the vector and the 175bp insert was performed using a Cloning recombination Kit (Novozam Clonexpress II One Step Cloning Kit # C112). The components were added according to the system shown in Table 4, mixed and reacted at 37 ℃ for 30min, immediately after completion of the reaction on ice and used for the conversion.

TABLE 4

Composition of	Volume ul
		Linearized vector	150ng
Insert fragment	0.04Xbp number ng
		5x CE II Buffer	2ul
Exnase II	1ul
		ddH₂O	To 10ul

5) Transformation, cloning, detection and plasmid miniextraction

a) 100 μ L of DH5 α chemically competent cells (Vazyme # C502) were placed in an ice bath;

b) adding 10 mu L of the recombinant reaction product obtained in the step (4) into a centrifuge tube filled with competent cells, uniformly mixing, and standing in an ice bath for 30 min;

c) placing the competent cells in ice bath for 30min in water bath at 42 deg.C for 90s, and rapidly transferring to ice bath to cool the cells for 3 min;

d) adding 300 μ L sterile LB culture medium (without antibiotic) into the centrifuge tube, mixing, and shake culturing at 37 deg.C with 220rpm shaking table for 60 min;

f) adding 100uL of competent cells to an LB solid agar medium containing corresponding antibiotics, and uniformly coating the competent cells by using a sterile coating rod; and (3) inverting the LB solid agar culture medium coated with the competent cells into an incubator at 37 ℃ for culturing for 12-16 h.

6) Selecting clone, culturing, testing bacterial liquid, correctly cloning and extracting.

4 clones are picked from the constructed plate, respectively placed in 300ul of LB culture medium containing Amp resistance, cultured overnight at 37 ℃, 100ul of the clones separated in the next day are respectively sequenced by using a universal primer LKO1_5, the clone with the correct sequencing result is obtained, respectively 20ul of bacterial liquid is taken and cultured overnight in a test tube containing 3ml of Amp LB, plasmid extraction is carried out by using a plasmid miniprep kit in the next day, and the plasmid is stored for standby at-20 ℃. The resulting recombinant vector pU6gRNAcas9 is shown in FIG. 5.

(2) Engineering promoters and enhancers

The constructed pU6gRNAcas9 vector is treated with XbaI and AgeI endonuclease to eliminate promoter (CMV enhancer) and enhancer sequence (chicken beta-actin), linear vector sequence of about 7650bp is recovered and 554bp sequence containing CMV enhancer and EF1a promoter (SEQ ID NO: 8) is synthesized, and the DNA is recombined with the enzyme digestion vector pU6gRNAcas9 to obtain pU6gRNA-eEF1a Cas9 vector (figure 6).

The construction method comprises the following specific steps:

1) the modified pU6gRNAcas9 plasmid was digested with restriction enzymes XbaI and AgeI

See the section of the pU6gRNAcas9 vector linearized during the engineering process as described previously.

The result of cutting the XbaI and AgeI enzyme gel map of the pU6gRNAcas9 vector is shown in FIG. 7, and the large fragment of the vector is recovered.

2) The 554bp insert was synthesized from the whole gene, and the primers for whole gene synthesis are shown in Table 5:

TABLE 5

eEF1a-1	TCTGCAGACAAATGGCTCTAGAGGTACCCG(SEQ ID NO：9)
		eEF1a-2	GGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGGGTACCTCTAGAGCCAT(SEQ ID NO：10)
eEF1a-3	GCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAGTAACGCCAATAGGGA(SEQ ID NO：11)
		eEF1a-4	TACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATTGA(SEQ ID NO：12)
eEF1a-5	AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATG(SEQ ID NO：13)
		eEF1a-6	TACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCC(SEQ ID NO：14)
eEF1a-7	CCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTGTGCCCAGTACATGACCTT(SEQ ID NO：15)
		eEF1a-8	GACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCAC(SEQ ID NO：16)
eEF1a-9	TGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGGGGCAGAGCGCACATCGCC(SEQ ID NO：17)
		eEF1a-10	GGATCAATTGCCGACCCCTCCCCCCAACTTCTCGGGGACTGTGGGCGATGTGCGCTCTG(SEQ ID NO：18)
eEF1a-11	GGGGTCGGCAATTGATCCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGA(SEQ ID NO：19)
		eEF1a-12	CCCCCACCCTCGGGAAAAAGGCGGAGCCAGTACACGACATCACTTTCCCAGTTTACCCC(SEQ ID NO：20)
eEF1a-13	TCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTC(SEQ ID NO：21)
		eEF1a-14	GTTGCGAAAAAGAACGTTCACGGCG(SEQ ID NO：22)

See above for the whole gene synthesis part of the pU6gRNAcas9 vector engineering process.

3) Recombination of linearized vector and synthetic insert see above for cloning of the recombinant portion in the engineering of the pU6gRNAcas9 vector. The electrophoresis result of the eEF1a1-14 whole gene synthesis is shown in FIG. 8, and the target fragment 554bp is recovered by gel.

4) Selecting clone, culturing, transferring bacteria liquid, correctly cloning and sampling

See above for the pU6gRNAcas9 vector engineering procedure picking clones, culture, bacterial transfer (again using the universal primer LKO1_5 for sequencing) and correct clones spot. The resulting pU6gRNA-eEF1a Cas9 vector is shown in FIG. 6.

(3) Cas9 gene N-terminal increasing NLS sequence

The constructed vector pU6gRNA-eEF1a Cas9 is cut by AgeI and BglII, a 7786bp vector sequence is recovered, the sequence with the increased NLS is supplemented to the enzyme cutting site, the following sequence 447bp including 2 nuclear localization signals and a partially cut Cas9 coding sequence (SEQ ID NO: 23) is synthesized, and the pU6gRNA-eEF1a Cas9+ nNLS vector (figure 9) is obtained through recombination.

The construction method comprises the following specific steps:

1) pU6gRNA-eEF1a Cas9 plasmid cut and modified by restriction enzymes AgeI and BglII

See above for the linear portion of the pU6gRNAcas9 vector during the engineering process.

The AgeI and BglII enzyme cleavage map of pU6gRNA-eEF1a Cas9 shows that the result is shown in FIG. 10, and the large fragment of the vector is recovered.

2) The 447bp insert was synthesized from the whole gene, and the primers for whole gene synthesis are shown in Table 6:

TABLE 6

Referring to the whole gene synthesis part in the process of modifying the pU6gRNAcas9 vector, the electrophoresis result of N-NLS 1-12 whole gene synthesis is shown in FIG. 11, and 447bp target fragment is recovered from the gel.

3) Linearization vectors and recombination of synthetic inserts

See above for cloning of recombinant portions during engineering of the pU6gRNAcas9 vector.

See above the pU6gRNAcas9 vector was engineered by picking clones, culturing, bacterial transfer (sequencing using synthetic primer gRNA-F: ttttagagctaGAAAtagcaag) and cloning of correct spot. The vector map of the obtained pU6gRNA-eEF1a Cas9+ nNLS is shown in FIG. 9.

(4) NLS, P2A-EGFP-T2A-PURO and WPRE-3' LTR-bGH polyA signals are added to the C end of Cas9 gene

The constructed vector is named as pU6gRNA-eEF1a Cas9+ nNLS, FseI and SbfI are used for enzyme digestion, a vector sequence 7781bp is recovered, a synthetic sequence 2727bp comprises a sequence of NLS-P2A-EGFP-T2A-PURO-WPRE-3' LTR-bGH polyA signals (SEQ ID NO: 36), and the vector is recombined with a vector fragment to obtain the pU6gRNA eEF1a-mNLS-hSpCas 9-EGFP-PURO.

The construction method comprises the following specific steps:

1) the pU6gRNA-eEF1a Cas9+ nNLS plasmid which is enzyme-cut and transformed by restriction endonucleases FseI and SbfI is used for recovering a 7781bp linear vector fragment:

see the above section of the pU6gRNAcas9 vector linearization during the engineering process. The FseI and SbfI enzymatic cleavage gel images of the vector pU6gRNA-eEF1a Cas9+ nNLS are shown in FIG. 12, and the large vector fragment is recovered.

2) 2727bp insert fragment for whole gene synthesis

See above for the whole gene synthesis part of the pU6gRNAcas9 vector engineering process. The 2727bp synthetic fragment is derived from 3 fragment overlap extension PCR, and specifically comprises the following steps:

fragment one: the sequence containing the nuclear localization signal coding sequence and P2A, EGFP overlapping sequence 192bp (SEQ ID NO: 37) were synthesized, the primer sequences were synthesized as shown in Table 7, and the primers were obtained by whole gene synthesis (see the whole gene synthesis part in the process of engineering pU6gRNAcas9 vector).

TABLE 7

C-NLS-1	CGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAG(SEQ ID NO：38)
		C-NLS-2	AGGCCGCTTGGAGCCGCCCTTTTTCTTTTTTGCCTGGCCGGCCTTTTTCGTGGCCGCCG(SEQ ID NO：39)
C-NLS-3	GGCTCCAAGCGGCCTGCCGCGACGAAGAAAGCGGGACAGGCCAAGAAAAAGAAAGGATC(SEQ ID NO：40)
		C-NLS-4	TCCGGCTTGTTTCAGCAGAGAGAAGTTTGTTGCGCCGGATCCTTTCTTTTTCTTGGCCT(SEQ ID NO：41)
C-NLS-5	CTGCTGAAACAAGCCGGAGATGTCGAAGAGAATCCTGGACCGGTGAGCAAGGGCGAGGA(SEQ ID NO：42)
		C-NLS-6	CGGTGAACAGCTCCTCGCCCTTGCTCAC(SEQ ID NO：43)

Fragment two: the EGFP fragment 744bp (SEQ ID NO: 68), the template was the commercial vector EGFP-N1, and the primers are shown in Table 8, obtained by conventional PCR.

TABLE 8

EGFP-F	GTGAGCAAGGGCGAGGAGCTGTTCACCGG(SEQ ID NO：44)
		EGFP-R	TAGAAGACTTCCCCTGCCCTCGCCGGAGCCCTTGTACAGCTCGTCCATGCCGAGAGTG(SEQ ID NO：45)

Fragment three: T2A-PURO-WPRE-3' LTR-polyA signals sequence 1840bp (SEQ ID NO: 69), the template is LentiCRISPERV 2 (adddge Plasmid #52961), the primers are shown in Table 9, the primer PCR is carried out by using LentiCRISPERV 2 as the template V2-F/V2-R primer, the PCR product in the previous step is used as the template, and the target fragment 1840bp is obtained by T2A-F and V2-R PCR.

TABLE 9

T2A-F	GAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCA(SEQ ID NO：46)
		V2-F	TGCGGGGACGTGGAGGAAAATCCCGGCCCAACCGAGTACAAGCCCACGGTGCGCCTCG(SEQ ID NO：47)
V2-R	taccgcatcaggcgcccctgcaggccatagagcccaccgcatccccagcatgcctg(SEQ ID NO：48)

The three fragments are used as templates, a primer C-NLS-1/V2-R is used for PCR to obtain a 2727bp target fragment (SEQ ID NO: 36) with the full length, and the final assembled 2727bp fragment gel diagram result is shown in FIG. 13.

3) Linearization vectors and recombination of synthetic inserts

The insert fragments of the linearized vector pU6gRNA-eEF1a Cas9+ nNLS 7781bp and 2727bp are recombined, and the method refers to the cloning and recombination part in the process of modifying the pU6gRNAcas9 vector.

See above for the pU6gRNAcas9 vector engineering procedure clones were picked, cultured, tested in broth (using synthetic primers Cas 9-5-F: CCACCAGAGCATCACCGGCCTG (SEQ ID NO: 49) and F1ori-R: cacacccgccgcgcttaatgcg (SEQ ID NO: 50)) and miniaspirates were correctly cloned. The map of the obtained final modified vector pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO is shown in figure 1, and the base sequence (SEQ ID NO: 70) is shown.

The main elements of the modified vector pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO are as follows:

1) gRNA expression elements: U6-gRNA scaffold.

2) A promoter: CMV enhancer and EF1a hybrid promoter.

3) Cas9 gene containing multiple NLS: cas9 gene containing N-and C-terminal multinuclear localization signals (NLS).

4) Screening for marker genes: the fluorescent and resistant double selection marker original P2A-EGFP-T2A-PURO.

5) Elements that enhance translation: WPRE and 3' LTR, enhance translation of cas9 and the selectable marker gene.

6) Transcription termination signal: bGH polyA signal.

7) Carrier skeleton: including Amp resistance elements and ori replicons.

1.2 construction of MSTN and FNDC5 Gene gRNA target vectors to detect the efficiency of the modified cas9 vector

pKG-U6gRNA vector: a pUC57 vector is used as a source, a pKG-U6gRNA insertion sequence (a DNA fragment containing a U6 promoter, a BbsI enzyme cutting site and a sgRNA framework sequence, namely SEQ ID NO: 67) is connected through an EcoRV enzyme cutting site, the pKG-U6gRNA insertion sequence is reversely inserted into a pUC57 vector, and a positive clone is obtained after bacteria are transformed. The complete sequence of pKG-U6gRNA vector (SEQ ID NO: 71).

pKG-U6gRNA insertion sequence (the first underlined part is U6 promoter sequence, the capital base letter segment is the sequence of two BbsI enzyme cutting sites, the second underlined part is sgRNA framework sequence):

gataaacatgtgagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttaga gagataattggaattaatttgactgtaaacacaaagatattagtacaaaatacgtgacgtagaaagtaataatttc ttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcga tttcttggctttatatatcttgtggaaaggacgaaacaccGGGTCTTCGAGAAGACCTgttttagagctagaaata gcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttttctagcgcgtgcgccaattctgcagacaaatggctctagaggtacccatag

the constructed pKG-U6gRNA vector map is shown in FIG. 14:

synthesizing 2 pairs of complementary DNA oligos for each target, and annealing to form a DNA double strand complementary to the digested sticky end of the BbsI vector pKG-U6gRNA, as shown in FIG. 15, when synthesizing a sense strand of a target insertion sequence, since the U6 promoter starts to transcribe from the first g after the promoter, when the target is not the g start, one g is added in front of the target, and cacc is added in front of the g to be complementary to the digested U6 end of the BbsI vector; when synthesizing a complementary strand of the target insertion sequence, a complementary sequence of g and the target needs to be synthesized, and then an aaac sequence is added to the 5' end to be complementary with the sequence of the gRNA framework end digested by BbsI. When the target is g, g can be not added before the target of the sense strand, and c which is complementary to the added g is added to the 3 end of the complementary strand.

Designing two gRNA targets of MSTN:

MSTN-gRNA1: GCTGATTGTTGCTGGTCCCG (SEQ ID NO: 51) and

MSTN-gRNA2:TTTCCAGGCGAAGTTTACTG(SEQ ID NO：52)。

two gRNA targets were designed for FNDC 5:

FNDC5-gRNA 1: TGTACTCAGTGTCCTCCTCC (SEQ ID NO: 53) and

FNDC5-gRNA2：GCTCTTCAAGACGCCTCGCG(SEQ ID NO：54)。

and primers for detecting later gene editing efficiency are respectively designed on two sides of the target spot:

MSTN detection primer:

MSTN-F896 TCTCTCAGACAGTGCAGGCATTA(SEQ ID NO：55)

MSTN-R1351 CGTTTCCGTCGTAGCGTGATAAT(SEQ ID NO：56)

FNDC5 detection primers:

FNDC5-F209 CAGTTCTCACTTGATGGCCTTGG(SEQ ID NO：57)

FNDC5-R718 AGGGGTCTGGGGAGGAATGG(SEQ ID NO：58)

the following double strands were synthesized according to the four targets:

cloning a gRNA target sequence to a pKG-U6gRNA vector framework, and specifically comprising the following steps:

(1) digesting 1ug pKG-U6gRNA plasmid with restriction enzyme BbsI;

(2) carrying out agarose gel separation on the digested pKG-U6gRNA plasmid, and purifying and recovering a digested product by using a gel recovery kit;

(3) the sequences of the oligonucleotide strands gRNA-S and gRNA-A were annealed according to the following procedure:

95 ℃ for 5min and then reduced to 25 ℃ at a rate of 5 ℃/min.

(4) The ligation reaction was carried out according to the following system at 37 ℃ for 60 min:

(5) transformation of

1) 100. mu.L of competent cells (Vazyme) were placed in an ice bath;

2) adding 20 mu L of the ligation plasmid solution obtained in the step (4) into a centrifuge tube filled with competent cells, uniformly mixing, and standing in an ice bath for 30 min;

3) placing the competent cells in ice bath for 30min in water bath at 42 deg.C for 90s, and rapidly transferring to ice bath to cool the cells for 3 min;

4) adding 300 μ L sterile LB culture medium (without antibiotic) into the centrifuge tube, mixing, and shake culturing at 37 deg.C with 220rpm shaking table for 60 min;

5) adding 100uL of competent cells to an LB solid agar medium containing corresponding antibiotics, and uniformly coating the competent cells by using a sterile coating rod; inverting the LB solid agar culture medium coated with the competent cells into an incubator at 37 ℃ for culturing for 12-16 h;

6) selecting, cloning, culturing, sending to a company for sequencing, and performing small plasmid extraction after determining that the vector containing the target gRNA is constructed correctly.

2 clones, 16 clones in total, are picked from each constructed plate, and are respectively placed in LB culture medium containing 300ul of Amp resistance, and are cultured overnight at 37 ℃, 100ul of the clones separated in the next day are respectively sequenced by using universal primers M13F or M13R (sequencing is carried out by a general biological company), the clones with correct sequencing results are respectively picked, 20ul of bacterial liquid is respectively taken and is cultured in a test tube containing 3ml of Amp LB overnight, plasmid extraction is carried out by using a plasmid miniprep kit in the next day, and the plasmids are respectively named as pKG-U6gRNA (MSTN-1), pKG-U6gRNA (MSTN-2), pKG-U6gRNA (FNDC5-1) and pKG-U6gRNA (FNDC5-2) for standby. According to the same method, 10 tubes of pKG-GE3 plasmid were drawn in small tubes for use.

1.3gRNA vector and Cas9 vector optimal molar ratio screening

In order to determine the optimal proportion of three plasmids added during common electrotransformation when two gRNA vectors pKG-U6gRNA (about 3.0kb) containing target spots and a Cas9 vector pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO (abbreviated as pKG-GE3 about 10.5kb) are used for causing genome fragment deletion by using double gRNAs, the application performs electrotransformation after the gRNA vectors and the Cas9 vectors are mixed according to different proportions, and detects the gene editing efficiency of the electrotransfer cells after the gRNA and the Cas9 plasmids are mixed according to different proportions, so as to determine the optimal proportion.

The method comprises the following specific steps:

(1) preparation of primary pig fibroblasts

1) Taking 0.5g of pig ear tissues of 4 female pigs (1234) and 4 male pigs (AB C D) which are born just before the jungle river fragrant pigs respectively, removing external tissues, and soaking for 30-40s with 75% of alcohol.

2) 5 washes with PBS containing 5% P/S (Gibco Penicillin-Streptomyces) and one wash with PBS without P/S:

wherein the PBS formulation of 5% P/S is: 5% P/S (Gibco Penicillin-Streptomyces) + 95% PBS, 5%, 95% by volume.

3) The tissue was minced with scissors, 5mL of 1% collagenase (Sigma) was added and digested in a shaker at 37 ℃ for 1 h.

4)500g was centrifuged for 5min, the supernatant removed, and the pellet resuspended in 1mL complete medium and plated into a 9cm cell culture dish containing 10mL complete medium and sealed with 0.2% gelatin (VWR).

Wherein, the formula of the complete cell culture medium is as follows: 15% fetal bovine serum (Gibco) + 83% DMEM medium (Gibco) + 1% P/S (Gibco Penicillin-Streptomyces) + 1% HEPES (solarbio), 15%, 83%, 1% in volume percentage.

5) Standing at 37 deg.C for 5% CO₂(volume percent) 5% O₂The culture was carried out in a constant temperature incubator (volume percent).

6) When the cells are cultured to be about 60% of the bottom of the dish, 0.25% (Gibco) trypsin is used for digesting the cells, then complete culture medium is added to stop digestion, the cell suspension is transferred into a 15mL centrifuge tube, 400g of centrifuge is carried out for 4min, the supernatant is discarded, and the cells are frozen and stored for later use by using cell freezing medium (90% complete culture medium + 10% DMSO, volume ratio).

(2) gRNA and Cas9 expression vector mixed electric transfer pig primary cell with different molar ratios

The Cas9 expression vector pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO and the gRNA expression vector pKG-U6gRNA (MSTN-1) and pKG-U6gRNA (MSTN-2) constructed above are co-transfected into porcine primary fibroblasts respectively.

Electrotransformation experiments were carried out using a mammalian nuclear transfection kit (Neon kit, Thermofeisher) with a Neon TM transfection system electrotransfer apparatus.

The grouping is as follows:

group B (control): pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO, the actual dosage of the plasmid is 2ug

Group 1: pKG-U6gRNA (MSTN-1) + pKG-U6gRNA (MSTN-2) + pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO, in a molar ratio of 0.5: 0.5: 1, the actual dosage of the plasmid is 0.22ug +0.22ug +1.56ug, and the total amount is 2ug

Group 2: pKG-U6gRNA (MSTN-1) + pKG-U6gRNA (MSTN-2) + pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO, in a molar ratio of 1: 1:1, the actual dosage of the plasmid is 0.36ug +0.36ug +1.27ug, and the total amount is 2ug

Group 3: pKG-U6gRNA (MSTN-1) + pKG-U6gRNA (MSTN-2) + pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO, in a molar ratio of 1.5:1.5:1, the actual dosage of the plasmid is 0.46ug +0.46ug +1.08ug, and the total amount is 2ug

Group 4: pKG-U6gRNA (MSTN-1) + pKG-U6gRNA (MSTN-2) + pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO, in a molar ratio of 2: 2:1, the actual dosage of the plasmid is 0.53ug +0.53ug +0.93ug, and the total amount is 2ug

Preparing an electrotransformation reaction liquid, wherein the system is as follows:

the bubbles are not generated by carelessness in the process of uniformly mixing;

carrying out electrotransformation on primary pig cells and collecting the cells:

1) digesting the cells with pancreatin, washing the obtained cell suspension once with PBS phosphate buffer (Solarbio), centrifuging for 6min at 600g, discarding the supernatant, and resuspending the cells by using 7uL of electrotransfer basic solution R, wherein bubbles are prevented from being generated in the process of resuspension;

2) sucking 7uL of cell suspension, adding the cell suspension into the electrotransformation reaction liquid obtained in the step 1), and uniformly mixing, wherein no air bubble is generated in the uniformly mixing process;

3) placing the electric rotating cup with the reagent cassette in a cup groove of a Neon (TM) transformation system electric rotating instrument, and adding 3mL of E Buffer;

4) sucking 10uL of the mixed solution obtained in the step 3) by using an electric rotating gun, inserting the mixed solution into a click cup, selecting an electric rotating program (1450V 10ms3pulse), immediately transferring the mixed solution in the electric rotating gun into a 6-hole plate in a super clean bench after electric shock transfection, wherein each hole contains 2mL of complete culture solution of 15% fetal calf serum (Gibco) + 83% DMEM medium (Gibco) + 1% P/S (Gibco penillin-Streptomycin) + 1% HEPES (Solarbio);

5) mixing, and standing at 37 deg.C for 5% CO₂、5％O₂Culturing in a constant-temperature incubator;

6) and (3) performing electrotransformation for 6-12h for liquid exchange, performing electrotransformation for 48h by using trypsin for digestion, collecting cells into a 1.5ml EP tube, and performing mutation efficiency PCR detection at the later stage.

(3) PCR detection of MSTN gene deletion mutation efficiency

1) To the cells collected in the 1.5mL centrifuge tube in the previous step (depending on the cell amount, the cells are too much to be diluted appropriately and then a part is lysed), 10uL of a lysis solution prepared (Kapa biosystems: Kapa hotspot mouse mutagenesis kit, cat # KK7352) is added to lyse the cells and crude extract the genomic DNA of the cells.

The system for preparing KAPA2G lysate is as follows:

10X extract Buffer 1uL

Kapa Express extract enzyme 0.2uL

ddH₂O 8.8uL

and (3) cracking: 5 min-4 ℃ at 75 ℃ to 95 ℃, and storing the genome DNA at-20 ℃ after the reaction is finished;

2) the MSTN group uses MSTN-F896/MSTN-R1351 primer for PCR detection, and the PCR reaction system is as follows:

the reaction conditions were as follows

The results of the electrophoretic analysis are shown in FIG. 16: the molar ratio of gRNA1 to gRNA2 to cas9 is lane1 (0.5: 0.5: 1); lane2 (1.0: 1.0: 1); lane3 (1.5: 1.5: 1); lane4 (2.0: 2.0: 1); lane5 No gRNA control. The result shows that the 456bp band is a wild-type band (WT), and the band around 329bp (456 bp-127 bp theoretically deleted) is a deletion mutant band (MT).

According to the formula: the gene deletion mutation efficiency is 100 × (MT gray level/MT band bp number)/(WT gray level/WT band bp number + MT gray level/MT band bp number)%, and the gene deletion mutation efficiency of MSTN group 1 is 28.6%, the gene deletion mutation efficiency of MSTN group 2 is 77.8%, the gene deletion mutation efficiency of MSTN group 3 is 86.8%, and the gene deletion mutation efficiency of MSTN group 4 is 81.5%, wherein the group 3 gene editing efficiency is the highest, and the optimal usage of two gRNA plasmids and Cas9 plasmids is determined as a molar ratio of 1.5:1.5:1, the actual amount of plasmid is 0.46ug +0.46ug +1.08 ug.

1.4 Cas9 expression vector Gene editing Effect test

In order to detect the gene editing efficiency of the modified pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO vector relative to the pX330-U6-Chimeric _ BB-CBh-hSpCas9 vector before modification. The invention utilizes two gRNA target point vectors of the constructed pig MSTN gene and FNDC5 gene and the modified cas9 vector, and determines the gene editing efficiency of the vector by electrotransfering the pig primary fibroblast and detecting the deletion mutation efficiency of each gene through PCR.

The method comprises the following specific steps:

(1) electroporation of porcine primary cells

Cas9 expression vector pX330 or modified pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO and gRNA expression vector pKG-U6gRNA (MSTN-1) and pKG-U6gRNA (MSTN-2) or pKG-U6gRNA (FNDC5-1) and pKG-U6gRNA (FNDC5-2) are co-transfected into pig primary fibroblasts respectively.

Electrotransfer experiments were performed using a mammalian nuclear transfection kit (Neon) with a Neon TM transfection system electrotransfer instrument.

MSTN group B: pKG-U6gRNA (MSTN-1) and pKG-U6gRNA (MSTN-2)

MSTN set 330: pX330+ pKG-U6gRNA (MSTN-1) and pKG-U6gRNA (MSTN-2)

MSTN group KG: pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO + pKG-U6gRNA (MSTN-1) and pKG-U6gRNA (MSTN-2)

FNDC5 group B: pKG-U6gRNA (FNDC5-1) and pKG-U6gRNA (FNDC5-2)

FNDC5 group 330: pX330+ pKG-U6gRNA (FNDC5-1) and pKG-U6gRNA (FNDC5-2)

FNDC5 group KG: pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO + pKG-U6gRNA (FNDC5-1) and pKG-U6gRNA (FNDC5-2)

see section above (1.3gRNA vector and Cas9 vector for optimal molar ratio screening) for the method of electroporation and cell collection of porcine primary cells.

(2) PCR detection of MSTN and FNDC5 gene mutation efficiency

Cell lysis, PCR detection and electrophoresis were performed according to the procedure described above (1.3gRNA vector and Cas9 vector optimal molar ratio screening).

3ul of PCR product was collected and analyzed by agarose gel electrophoresis, and the results are shown in FIGS. 17 and 18. FIG. 17 is a comparison of the editing efficiency of MSTN genes, and the ratio of KG to 330 mutant/wild-type bands was higher, indicating that the editing efficiency of KG genes was higher than 330. FIG. 18 is a comparison of the editing efficiency of FNDC5 gene, and the ratio of KG gene to mutant/wild-type gene was higher than that of group 330, indicating that the editing efficiency of KG gene was higher than that of group 330.

According to the formula: the gene deletion mutation efficiency is 100 × (MT gray level/MT band bp number)/(WT gray level/WT band bp number + MT gray level/MT band bp number)%, and the gene deletion mutation efficiency of the MSTN-330 group and the gene deletion mutation efficiency of the MSTN-KG group are respectively calculated to be 27.6% and 86.5%. The FNDC5-330 group gene deletion mutation efficiency is 18.6%, the FNDC5-KG group gene deletion mutation efficiency is 81.7%, and the editing efficiency of the modified vector pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO gene is obviously improved (about 3-4 times).

Knock-out of F8 Gene

2.1F 8 gene knockout target gRNA design and construction

(1) Extraction of genomic DNA from porcine ear tissue

Column extraction of genomic DNA from the ear Tissue of 8 piglets (male A B C D female 1234) was performed using the Fastpure Cell/Tissue DNA Isolation Mini Kit (Vazyme Cat. DC102-01) from Vazyme, respectively, and finally the genomic DNA was dissolved in sterilized deionized water and quantified using NanoDrop and stored at-20 ℃ for future use.

(2) Conservation analysis of F8 gene knockout preset target point exon and adjacent genome sequence

1) The pig F8 gene information is searched as follows

F8 coagulation factor VIII[Sus scrofa(pig)]Gene ID:397339

Location:chromosome:X Exon count:26

The F8 gene transcript pattern is as in FIG. 19 (dark wide lines for coding exons and light wide lines for non-coding exons). Including 26 exons, with the 15 th exon being the largest of 2454bp (mutations in the hemophilian F8 gene also frequently occur in the largest exon), the present invention contemplates targeting at an early position within the 15 th exon of the F8 gene.

2) F8 gene knockout preset target point exon and adjacent genome sequence PCR amplification primer design

According to the found porcine F8 genome sequence

(https://www.ncbi.nlm.nih.gov/nuccore/NC_010461.5report＝genbank& from＝125265330&to＝125350853&strand＝true) Primers were designed to amplify the 8 porcine genomic samples F8 gene exon 15 forward site.

Primer design was performed using Primer3, and the design results were as follows:

3) f8 genome PCR amplification primer screening

PCR was performed using the genome extracted from ear tissue of swine (female 2) as a template using the designed two upstream and two downstream combinations, Max enzyme (Vazyme: P505), and electrophoresed to select the amplification primers, the result is shown in FIG. 20, in which 47 is an amplification band of F8-GT-F491/F8-GT-R1463 primer; 48 is an amplified band of the F8-GT-F491/F8-GT-R1490 primer; 49 is an amplification band of a F8-GT-F572/F8-GT-R1463 primer; the 50 is F8-GT-F572/F8-GT-R1490 primer, and 48, namely F8-GT-F491/F8-GT-R1490 primer is used for amplification of the band.

4) PCR amplification of F8 gene fragment of 8 pigs

Amplification of the F8 genomic fragment was performed with 8 genomic templates (male AB C D female 1234), primers F8-GT-F491/F8-GT-R1490, Max enzyme, respectively, and the results are shown in FIG. 21.

5) F8 gene sequence conservation analysis

The PCR amplification products were sequenced using the amplification primers (sequencing by general Bio Inc.). The sequencing result is analyzed by comparing the Snapgene with the sequence of the F8 gene published on the Internet, the result is shown in FIG. 22, the white vertical line represents mutation or deletion mutation, the black vertical line represents insertion mutation, and the design of target point and detection primer needs to be avoided on the mutation sequence.

(3) Double gRNA targets are designed in a conserved region on a preset target exon, so that a code shift mutation of a coding exon can be caused

(1) Targeted gRNA design using synthego

Designing a target to avoid possible mutation sites, and designing the gRNA of the target by using synthgo:

https://www.synthego.com/products/bioinformatics/crispr-design-tool

the F8 gene knockout target is designed as follows:

B1-sgRNA1：TATAGTTGTGACAGGGACAT(SEQ ID NO：76)

B1-sgRNA2：ATTCCAGGCTTCTTGCTGAG(SEQ ID NO：77)

B1-sgRNA3：GTCATCTTCTGGACTTGTGA(SEQ ID NO：78)

B1-sgRNA4：CACAAGTCCAGAAGATGACG(SEQ ID NO：79)

the combinations of targets and the resulting theoretical deletions are shown in table 10.

TABLE 10 combination of targets and resulting theoretical deletions

Based on the alignment, possible mutation sites were avoided and primers for later detection of mutations were designed as shown in table 11:

TABLE 11F 8 detection primers

B1-F843	ctgggctgtgagtaaccagagtt(SEQ ID NO：80)
		B1-R1258	GGCTTCTTGAAGATCAGATGAGG(SEQ ID NO：81)

Synthetic insert complementary DNA oligos for 4 targets of F8 are shown in Table 12:

TABLE 12

B1-1S	caccgTATAGTTGTGACAGGGACAT(SEQ ID NO：82)
		B1-1A	aaacATGTCCCTGTCACAACTATAc(SEQ ID NO：83)
B1-2S	caccgATTCCAGGCTTCTTGCTGAG(SEQ ID NO：84)
		B1-2A	aaacCTCAGCAAGAAGCCTGGAATc(SEQ ID NO：85)
B1-3S	caccGTCATCTTCTGGACTTGTGA(SEQ ID NO：86)
		B1-3A	aaacTCACAAGTCCAGAAGATGAC(SEQ ID NO：87)
B1-4S	caccgCACAAGTCCAGAAGATGACG(SEQ ID NO：88)
		B1-4A	aaacCGTCATCTTCTGGACTTGTGc(SEQ ID NO：89)

Each target synthesized 2 pairs of complementary DNA oligos, which upon annealing formed a DNA duplex complementary to the cleaved sticky ends of pKG-U6gRNA vector BbsI (FIG. 15).

The synthetic F8 gene target insert sequence is shown in FIGS. 23-26, and the double-stranded insert sequence formed after annealing complementary DNA oligo:

the B1-gRNA1 insertion sequence is shown in FIG. 23; the B1-gRNA2 insertion sequence is shown in FIG. 24; the B1-gRNA3 insertion sequence is shown in FIG. 25; the B1-gRNA4 insertion sequence is shown in FIG. 26.

F8 gRNA expression vector construction was performed by referring to the gRNA expression vector construction process described above (1.2 construction of MSTN and FNDC5 gene gRNA target vectors to examine the efficiency of the modified cas9 vector).

The correct clones were designated pKG-U6gRNA (B1-1), pKG-U6gRNA (B1-2), pKG-U6gRNA (B1-3), pKG-U6gRNA (B1-4), plasmid miniaspirate and stored at-20 ℃ until use.

2.2 different target gRNA vector combination and Cas9 vector electrotransfer pig ear primary fibroblast

Porcine primary fibroblasts were co-transfected with the 4 gRNA expression vectors pKG-U6gRNA (B1) constructed and the Cas9 expression vector pKG-GE 3. Electrotransformation experiments were performed using a mammalian fibroblast cell nuclear transfection kit (Neon) with a Neon TM transfection system electrotransfer instrument.

Group 5: the non-electrotransfer cell is prepared into an electrotransfer reaction solution according to the molar ratio of gRNA1, gRNA2 and cas9 of 1.5:1.5:1, and the system is as follows:

the porcine primary cells were electroporated and cell harvested according to the electroporation method mentioned above (optimal molar ratio screening of 1.3gRNA vector and Cas9 vector).

2.3 PCR detection of the cells after electroporation to determine highly efficient editing target combinations

Cell lysis, PCR detection (detection primers: B1-F843/B1-R1258) and electrophoretic analysis were performed according to the procedures described above (screening of optimal molar ratio of 1.3gRNA vector and Cas9 vector), and the results were as follows.

The PCR detection result of F8 gRNA combined mutation efficiency is shown in FIG. 27, B1-51 is combination 1(gRNA 1, 3); b1-52 is combination 2(gRNA 1, 4); b1-53 is combination 3(gRNA 2, 3); b1-54 is combination 4(gRNA 2, 4).

The larger band is the wild-type band, the smaller band is the band after deletion mutation, and the brighter the smaller band (mutated band) is relative to the larger band (wild-type band), the higher the mutation efficiency. The experimental result shows that the combination 2(pKG-U6gRNA (B1-1) + pKG-U6gRNA (B1-4) + pKG-GE3) theoretically deletes 151bp, and the mutation efficiency is highest.

2.4 preparation of pig ear Primary fibroblast F8 Gene editing monoclonal cells

(1) Porcine ear primary fibroblast cell electrotransformed by using high-efficiency target gRNA combination and Cas9 vector

Cell electrotransformation is carried out according to a method for electrotransfering pig ear primary fibroblasts by combining the target gRNA vector and the Cas9 vector (see the optimal molar ratio screening of the 1.3gRNA vector and the Cas9 vector), and number 2 cells (female and blood type AO) are used and are divided into three groups for electrotransformation:

f8 mutation: pKG-U6gRNA (B1-1) (0.46ug) + pKG-U6gRNA (B1-4) (0.46ug) + pKG-GE3(1.08 ug). The molar ratio of each component is as follows: pKG-U6gRNA (B1-1): pKG-U6gRNA (B1-4): pKG-GE3 ═ 1.5:1.5: 1.

(2) isolating single clone, amplifying culture

1) Culturing the cells after electrotransfer for 36-48 hr, performing monoclonal sorting, digesting with trypsin, neutralizing with complete culture medium, centrifuging for 5min at 500g, removing supernatant, resuspending the precipitate with 1mL of complete culture medium, diluting appropriately, picking monoclonal with oral pipette, transferring into 96-well plate containing 200ul of complete culture medium, picking 96 monoclonal per cell group, placing at 37 deg.C and 5% CO₂、5％O₂Culturing in a constant-temperature incubator, changing a cell culture medium every 2-3 days, observing the growth condition of cells in each hole by using a microscope during the culture, and removing holes without cells and non-monoclonal cells;

2) after the cells grew to the bottom of the wells of the 96-well plate (approximately 2 weeks or so), cells were digested with trypsin and collected, wherein 2/3 cells were seeded into a 6-well plate containing complete medium and the remaining 1/3 cells were collected in a 1.5mL centrifuge tube;

3) when the 6-well plate cells were grown to 50% confluency, they were digested with 0.25% (Gibco) trypsin and harvested, and frozen using cell cryopreservation (90% complete medium + 10% DMSO by volume).

(3) PCR detection is carried out after monoclonal cell culture, and sequencing is carried out to determine the mutation type

1) PCR detection

Cells in a 1.5mL centrifuge tube are collected, and monoclonal PCR detection is carried out according to the method (2 pairs of cells after electrotransformation are subjected to PCR detection in four high-efficiency target gRNA combined screening to determine high-efficiency editing target combination). The detection results are shown in FIG. 28. FIG. 28 shows the results of F8 PCR (primers B1-F843/B1-R1258) tests of group-1F 8 mutant monoclonals, from which it was determined that 3,4,10,11,12,14,15 were homozygous deletion mutant monoclonals, 5,6,7,16,19,22 were heterozygous deletion mutant monoclonals, and that 1,2,8,9,13,17,18,20,21 had not undergone large-fragment deletion.

2) Sequencing further confirmed each monoclonal mutation type

The PCR products were sequenced by gel recovery (all bands were recovered) using PCR primers (general purpose biology Co., Ltd.), and the sequencing results were analyzed to determine the mutation of the F8 gene of each monoclonal.

The sequencing result is divided into two types, namely no nested peak and nested peak, the non-nested peak can be directly compared with the corresponding genome sequence of F8 through the sequencing result, the front side has no nested peak, the back side has 2 nested peaks, two sequences contained in the nested peaks need to be determined according to a wild type sequence or a sequence after theoretical deletion, and if the front side has no nested peak and the back side has random nested peaks, the clone can be judged to be a non-monoclonal cell. FIG. 29 shows the alignment of the sequencing results of monoclonal F8 with no nested peaks (1,2,3,10,12,13,14,15,18,20,21, WT) with the wild-type sequence of F8, white blank spaces represent deletion mutations, insert bars represent insertion mutations, it can be seen that 3,10,12,14,15,18 have large fragment deletions, 1,20,21 have small fragment insertions or deletion mutations at two targets, and 2,13 are wild-type.

The sequencing result containing the set peak needs to combine a glue picture, a wild type sequence, a theoretical deletion sequence and the like to carry out sequence speculation, and the genotype can be determined by cloning a large number of sequences through a cloning vector. Taking the monoclonal antibody No. 4 as an example, a comparison graph of the sequencing result of the clone No. 4B 1-F843 and the sequencing result of WT B1-F843 is shown in FIG. 30, and the result shows that no wild-type sequence exists in the double peaks of the clone No. 4 (the result is consistent with the judgment that the homozygous deletion mutation occurs in No. 4 in a glue picture), and the deletion theoretically caused by F8-gRNA1 and F8-gRNA4 is combined with the results of FIG. 31 and FIG. 32, so that the sequence after the theoretical deletion occurs (the blank spaces are deletion points):

TTGTGACAGGGA ACGTGGAGCTTGACCCGCAGTCTGGAGAGA(SEQ ID NO：90)

one of the sequences is: TTGTGACAGGGANNACGTGGAGCTTGACCC (SEQ ID NO: 91)

Presumably, the other sequence was: TTGTGACAGGGANNTTGACCCGCAGTCT (SEQ ID NO: 92) NN is GC, presumed from the sequence;

these two sequences are: TTGTGACAGGGATGACGTGGAGCTTGACCC (SEQ ID NO: 93) and TTGTGACAGGGAGCTTGACCCGCAGTCT (SEQ ID NO: 94) with a deletion of the 149bp fragment and a deletion of the 158bp fragment.

The coding sequence of the clone B1 gene 4 has-149 bp and-158 bp mutation.

All sets of peak clones were subjected to sequence analysis according to such methods.

(4) Miseq sequencing further determines the type of monoclonal cell mutation

1) High throughput sequencing library construction and sequencing

The genotype of each monoclonal was further accurately determined by high throughput sequencing (Miseq sequencing). The construction, library quality inspection and on-machine sequencing of the monoclonal Miseq sequencing library are all finished by a third-party sequencing service company (Jinzhi company).

2) High throughput sequencing results and analysis

FIG. 33 is a graph showing the alignment of Miseq sequencing results of F8 monoclones 1-23 with wild-type sequences (each clone has 1-4 sequences), and from the results, it can be determined that most of the clones have large fragment deletion, some sequences have deletion or insertion mutation at one of the two targets, and some sequences are wild-type sequences.

The genotype of each clone was shown in table 13 by analysis of specific sequences:

watch 13

The Miseq analysis result is consistent with the PCR product sequencing analysis result. Among F8 gene editing cell monoclonals 1-23, homozygous mutant cell clones had: 1,3,4,10,11,12,14,15,16,18,19,20,21, 22; wild type cells were cloned with: 2, 13; heterozygous mutant clones had: 5,6, 7; non-monoclonal antibodies are: 8,9,17.

Knock-out of F9 Gene

3.1F 9 Gene knockout target gRNA design and construction

(1) Extraction of genomic DNA from porcine ear tissue

The method is shown in 2.1F 8 gene knockout target gRNA design and construction.

(2) Conservation analysis of F9 gene knockout preset target point exon and adjacent genome sequence

1) The pig F9 gene information is searched as follows:

F9 coagulation factor IX[Sus scrofa(pig)]Gene ID:397518

Location:X Exon count:8

the F9 gene transcription map is shown in FIG. 34 (the dark wide line in the map is coding exon, the light wide line is non-coding exon), and comprises 8 exons, the No. 2 exon is 164bp, and the invention plans to design the target point in the No. 2 exon of the F9 gene.

2) F9 gene knockout preset target point exon and adjacent genome sequence PCR amplification primer design

According to the found porcine F9 genome sequence

(https://www.ncbi.nlm.nih.gov/nuccore/NC_010461.5report＝genbank& from＝114218704&to 114250436), primers are designed to amplify 8 porcine genome samples F9 gene exon 2And flanking sequences.

3) f9 genome PCR amplification primer screening

The genome extracted from ear tissue of pig (female 2) was used as a template, PCR was carried out using the designed combinations of two upstream and two downstream, Max enzyme (Vazyme: P505), and the PCR was electrophoresed to select a good amplification primer. The electrophoresis result is shown in FIG. 35, and the amplified band of the primer 51 is F9-GT-F333/F9-GT-R1074; 52 is an F9-GT-F333/F9-GT-R1211 primer amplification band; 53 is an amplified band of the F9-GT-F421/F9-GT-R1074 primer; 54 is F9-GT-F421/F9-GT-R1211 primer amplification band, and the screening 52, namely F9-GT-F333/F9-GT-R1211 is used as an amplification primer.

4) PCR amplification of 8 pig genome templates F9 genome fragments

The F9 genome fragment was amplified with 8 genome templates (male A B C D female 1234), primers F9-GT-F333/F9-GT-R1211, Max enzyme, respectively, and the results are shown in FIG. 36.

5) F9 gene sequence conservation analysis

The PCR amplification products were sequenced using the amplification primers (sequencing by general Bio Inc.). The sequencing result is analyzed by comparing the Snapgene with the sequence of the F9 gene published on the Internet, the result is shown in FIG. 37, the white vertical line represents mutation or deletion mutation, the black vertical line represents insertion mutation, and the design of target point and detection primer needs to be avoided on the mutation sequence.

(1) Targeted gRNA design using synthego

Designing target to avoid possible mutation sites, and designing target gRNA by using synthgo

https://www.synthego.com/products/bioinformatics/crispr-design-tool

The F9 gene knockout target is designed as follows:

F9-sgRNA1：TGGTGGCATTTTCACGATCA(SEQ ID NO：99)

F9-sgRNA2：ATGCCACCAAAATTCTGCAT(SEQ ID NO：100)

F9-sgRNA3：AAACTGGAAGAGTTTGTTCG(SEQ ID NO：101)

F9-sgRNA4：AACTGGAAGAGTTTGTTCGA(SEQ ID NO：102)

the target combinations and resulting theoretical deletions are shown in table 14.

TABLE 14 combination of targets and resulting theoretical deletions

5 terminal target sequence

Direction

Numbering

3 terminal target sequence

Direction

Numbering

Deletion length bp

TGGTGGCATTTTCACGATCA

-

1

AACTGGAAGAGTTTGTTCGA

+

4

70

ATGCCACCAAAATTCTGCAT

+

2

AAACTGGAAGAGTTTGTTCG

+

3

44

Based on the alignment results, possible mutation sites were avoided, and primers for mutation detection at the later stage were designed, see table 15.

TABLE 15F 9 detection primers

B2-F539	gggattaccgggattaaaaacaa(SEQ ID NO：103)
		B2-R853	tttttattctttgctccgcatgt(SEQ ID NO：104)

Synthetic insert complementary DNA oligos for 4 targets of F9 (named B2) are shown in Table 16:

complementary DNA oligo for insertion sequence of 4 targets of Table 16F 9

B2-1S	caccgTGGTGGCATTTTCACGATCA(SEQ ID NO：105)
		B2-1A	aaacTGATCGTGAAAATGCCACCAc(SEQ ID NO：106)
B2-2S	caccgATGCCACCAAAATTCTGCAT(SEQ ID NO：107)
		B2-2A	aaacATGCAGAATTTTGGTGGCATc(SEQ ID NO：108)
B2-3S	caccgAAACTGGAAGAGTTTGTTCG(SEQ ID NO：109)
		B2-3A	aaacCGAACAAACTCTTCCAGTTTc(SEQ ID NO：110)
B2-4S	caccgAACTGGAAGAGTTTGTTCGA(SEQ ID NO：111)
		B2-4A	aaacTCGAACAAACTCTTCCAGTTc(SEQ ID NO：112)

Each target synthesized 2 pairs of complementary DNA oligos, which upon annealing formed a DNA duplex complementary to the cleaved sticky ends of pKG-U6gRNA vector BbsI (FIG. 18).

The double-stranded insert sequence obtained by annealing the synthetic F9 gene target insert sequence-complementary DNA oligo is shown in FIGS. 38-41:

the B2-gRNA1 insertion sequence is shown in FIG. 38; the B2-gRNA2 insertion sequence is shown in figure 39; the B2-gRNA3 insertion sequence is shown in figure 40; the B2-gRNA4 insertion sequence is shown in FIG. 41.

3.2 different target gRNA vector combination and Cas9 vector electrotransfer pig ear primary fibroblast

Porcine primary fibroblasts were co-transfected with the 4 gRNA expression vectors pKG-U6gRNA (B2) constructed and the Cas9 expression vector pKG-GE 3. Electrotransformation experiments were carried out using a mammalian fibroblast cell nuclear transfection kit (Neon kit, Thermofeisher) with a Neon TM transfection system electrotransfer instrument.

Combination 1: pKG-U6gRNA (B2-1) + pKG-U6gRNA (B2-4) + pKG-GE3 theoretical B2 deletion of 70bp

And (3) combination 2: pKG-U6gRNA (B2-2) + pKG-U6gRNA (B2-3) + pKG-GE3 theory B2 deletion of 44bp

Group 3: non-electrotransferred cells

Preparing an electrotransformation reaction solution according to a molar ratio of gRNA1, gRNA2 and cas9 of 1.5:1.5:1, wherein the system is as follows:

3.3 PCR detection of the cells after electroporation to determine highly efficient editing target combinations

Cell lysis, PCR detection (detection primers: B2-F539/B2-R853) and electrophoretic analysis were performed according to the procedures described above (screening of optimal molar ratio of 1.3gRNA vector and Cas9 vector), and the results were as follows.

The PCR detection result of the mutation efficiency of the F9 combined gRNA is shown in figure 42; b21, 4 is combination 1; b22, 3 is combination 2; b2WT is group 3.

The larger band is the wild-type band, the smaller band is the band after deletion mutation, and the brighter the smaller band (mutated band) is relative to the larger band (wild-type band), the higher the mutation efficiency. According to the experimental results, the combination of 2pKG-U6gRNA (B2-2) + pKG-U6gRNA (B2-3) + pKG-GE3 in B2 (namely F9) has the highest mutation efficiency of deleting 44bp in B2 theoretically.

3.4 preparation of pig ear Primary fibroblast F9 Gene editing monoclonal cells

group 2F9 mutations: pKG-U6gRNA (B2-2) (0.46ug) + pKG-U6gRNA (B2-3) (0.46ug) + pKG-GE3(1.08 ug). The molar ratio of each component is as follows: pKG-U6gRNA (B2-2): pKG-U6gRNA (B2-3): pKG-GE3 ═ 1.5:1.5: 1.

(2) isolating single clone, amplifying culture

The method steps are referred to the preparation of pig ear primary fibroblast F8 gene editing monoclonal cells in 2.4.

1) PCR detection

Cells collected in 1.5mL centrifuge tubes were subjected to monoclonal cell lysis, PCR detection and electrophoretic analysis according to the methods above (1.3gRNA vector and Cas9 vector optimal molar ratio selection). The results of F9 PCR (primers B2-F539/B2-R853) of group 2F9 mutant monoclonals are shown in FIG. 43, and it is judged from the results that 24,26,29,31,37,38,40,49,50 are homozygous deletion mutant monoclonals, 25,28,39,45,46,48 are heterozygous deletion mutant monoclonals, and 27,30,32,33,34,35,36,41,42,43,44,47 have not undergone large-scale deletion.

2) Sequencing further confirmed each monoclonal mutation type

The PCR products were sequenced by gel recovery (all bands were recovered) using PCR primers (general purpose biology Co., Ltd.), and the sequencing results were analyzed to determine the mutation of the F9 gene of each monoclonal.

The sequencing result is divided into two types, namely no nested peak and nested peak, the non-nested peak can be directly compared with the corresponding genome sequence of F9 through the sequencing result, the front side has no nested peak, the back side has 2 nested peaks, two sequences contained in the nested peaks need to be determined according to a wild type sequence or a sequence after theoretical deletion, and if the front side has no nested peak and the back side has random nested peaks, the clone can be judged to be a non-monoclonal cell.

FIG. 44 is a graph showing the alignment of the sequencing results (24,26,27,28,29, 3031, 33,34,35,36,38,40,41,42,43,44,47, WT) of monoclonal F9 without overlapping peaks with the wild-type sequence of F9, and from the alignment results, it was determined that 24,26,28,29,31,38,40 had homozygous deletion mutations, and the remaining monoclonal 27,30,33,34,35,36,41,42,43,44,47 were wild-type, and no clones with small fragment insertion or deletion mutations at either target were found.

The sequencing result containing the set peak needs to combine a gel map, a wild type sequence, a theoretical deletion sequence and the like to carry out sequence speculation, and the genotype can also be determined by cloning and sequencing a PCR product.

(4) Miseq sequencing further determines the type of monoclonal cell mutation

1) High throughput sequencing library construction and sequencing

2) High throughput sequencing results and analysis

FIG. 45 shows the alignment of the F9 gene editing monoclonal 24-50Miseq sequencing results with wild-type sequences (each clone has 1-2 sequences), from which it can be determined that most of the clones have large fragment deletion, some have deletion or insertion mutation at one of the two targets, and some have wild-type sequences.

The genotype of each clone was shown in table 17 by analysis of specific sequences:

TABLE 17

From the above analysis results, successful construction resulted in F9 gene mutant cell line, in which F9 homozygous mutant cell clone had: 24,25,26,28,29,31,37,38,40,48,49, 50; heterozygous mutant clones had: 32,39,45,46.

4. Preparation of monoclonal cells with combined knockout of F8 and F9 genes

(1) Porcine ear primary fibroblast transfected by high-efficiency target gRNA combinations of B1 and B2 and Cas9 vector

The optimum B1 and B2 target gRNA combinations screened by the experiment are used for cell electrotransformation of gRNA combinations and Cas9 vectors according to the method for electrotransformation of pig ear primary fibroblasts (see 1.3gRNA vector and Cas9 vector optimum molar ratio screening), and No. 2 cells (female, blood type AO) are used and are divided into three groups for electrotransformation:

f8 mutation: pKG-U6gRNA (B1-1) (0.23ug) + pKG-U6gRNA (B1-4) (0.23ug) + pKG-GE3(0.54 ug);

f9 mutation: pKG-U6gRNA (B2-2) (0.23ug) + pKG-U6gRNA (B2-3) (0.23ug) + pKG-GE3(0.54 ug).

The F8 mutation and the F9 mutation are mixed together to electrically transform the porcine primary fibroblast.

The molar ratio of the components is as follows: pKG-U6gRNA (B1-1): pKG-U6gRNA (B1-4): pKG-U6gRNA (B2-2): pKG-U6gRNA (B2-3): pKG-GE3 ═ 0.75:0.75:0.75:0.75: 1.

(2) Isolating single clone, amplifying culture

1) PCR detection

Cells collected in 1.5mL centrifuge tubes were subjected to monoclonal cell lysis, PCR detection and electrophoretic analysis according to the methods above (1.3gRNA vector and Cas9 vector optimal molar ratio selection). The detection results are shown in fig. 46 and 47.

From the PCR detection results of F8 (primers B1-F843/B1-R1258) and F9 (primers B2-F539/B2-R853) of the group 3F8+ F9 mutant monoclonals, 51,53,57 and 70 are F8F 9 double-gene deletion mutant monoclonals, 52,55,59,67 and 75 are F8 and F9 are not deletion mutant monoclonals, 54,56,61,66,69 and 73 are F8 homozygous deletion mutant F9 and are not deletion mutant monoclonals, 62,63,64 and 68 are F8 heterozygous deletion mutant F9 and are not deletion mutant monoclonals, 65 is F8 and is heterozygous deletion mutant F9 and is homozygous deletion mutant monoclonals, 71 and 72 are F8 deletion mutant F9 deletion heterozygous mutant monoclonals.

2) Sequencing further confirmed each monoclonal mutation type

The PCR products were sequenced by gel recovery (all bands were recovered) using PCR primers (general purpose biosciences), and the sequencing results were analyzed to further determine the mutation status of each of the monoclonal F8 and F9 genes.

An analysis chart of a PCR product sequencing result of the F8 gene of the F8+ F9 monoclonal is shown in a figure 48, the sequencing result is divided into two types, namely, a non-nested peak and a nested peak, the non-nested peak can be directly compared with genome sequences corresponding to F8 and F9 through the sequencing result, the front side of the non-nested peak is not nested, the back side of the non-nested peak is provided with 2 nested peaks, two sequences contained in the nested peaks are determined according to a wild type sequence or a sequence after theoretical deletion, and if the front side of the non-nested peak is not nested, the back side of the nested peak is disordered, the monoclonal cell can be judged to be a non-monoclonal cell.

FIG. 48 shows the result of F8 sequencing without overlapping peaks and the result of forward and reverse sequencing of single clone (51,52,53,56,57,58,66,67,69,70,71,72,75, WT) with the F8 wild-type sequence alignment, white blank spaces represent deletion mutations, insert lines represent insertion mutations, the result shows that

clones

51,53,56,57,58,66,69,70,71,72 have large-fragment deletion homozygous mutation of F8 gene,

clone

52,67 have homozygous segment deletion at target 1or target 4 of F8 gene, and 75 is the F8 gene wild-type.

The sequencing result of F9 gene product of F8+ F9 monoclonal is analyzed as shown in FIG. 49. FIG. 49 shows the alignment of F9 monoclonal positive and negative sequencing results (52,53,54,55,56,59,62,64,65,66,68,69,75, WT) with F9 wild-type sequence, white blank space represents deletion mutation, insert line represents insertion mutation,

clone

53,65 shows that F9 gene large fragment deletion homozygous mutation occurs,

clone

52,54,55,56,59,62,64,66,68,69,75 is F9 gene wild-type. The sequencing result containing the set peak needs to combine a gel map, a wild type sequence, a theoretical deletion sequence and the like to carry out sequence speculation, and the genotype can also be determined by cloning and sequencing a PCR product.

(4) Miseq sequencing further determines the type of monoclonal cell mutation

1) High throughput sequencing library construction and sequencing

2) High throughput sequencing results and analysis

The analysis map of F8 Miseq sequencing results of different monoclonals (51-75) is shown in FIG. 50, the Miseq sequencing result of F8 is compared with the F8 wild-type sequence except 60 (no library product is obtained), and the result can judge that most of the clones have F8 genome fragment deletion, partial sequences respectively have insertion or deletion mutation at one of two targets, and partial clones contain the wild-type sequence.

The results of the F9 Miseq sequencing of different monoclonals (51,53,57,60,61,63,65,67,70,71,72,73,74) are aligned with the F9 wild-type sequence in figure 51. From the results, it was determined that most of the clones had F9 genome fragment deletion, partial sequences had insertion or deletion mutations at one of 2 targets, respectively, and some clones had wild-type sequences.

The genotypes of the clones were shown in Table 18 by analysis of the specific sequences.

TABLE 18 analysis of monoclonal miseq sequencing results F8+ F9

Among the monoclonal cells (51-75) with simultaneous editing of the F8 and F9 genes, the cell clones successfully constructed for simultaneous homozygous mutation of F8 and F9 were: 51,53,57,61,65,70,73.

SEQUENCE LISTING

<110> Nanjing King Gene engineering Co., Ltd

<120> Cas9 overexpression vector and application thereof

<130>

<160> 112

<170> PatentIn version 3.3

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tcgctattac catgggggca gagcgcacat cgcccacagt ccccgagaag ttggggggag 360

gggtcggcaa ttgatccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg 420

tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag 480

tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggtt ggaccggtgc 540

caccatggac tata 554

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<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 17

tggcagtaca tctacgtatt agtcatcgct attaccatgg gggcagagcg cacatcgcc 59

<210> 18

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 18

ggatcaattg ccgacccctc cccccaactt ctcggggact gtgggcgatg tgcgctctg 59

<210> 19

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 19

ggggtcggca attgatccgg tgcctagaga aggtggcgcg gggtaaactg ggaaagtga 59

<210> 20

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 20

cccccaccct cgggaaaaag gcggagccag tacacgacat cactttccca gtttacccc 59

<210> 21

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 21

tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttc 59

<210> 22

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 22

gttgcgaaaa agaacgttca cggcg 25

<210> 23

<211> 447

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 23

ccagaacaca ggttggaccg gtgccaccat ggactataag gaccacgacg gagactacaa 60

ggatcatgat attgattaca aagacgatga cgataagatg gcccccaaaa agaaacgaaa 120

ggtgggtggg tccccaaaga agaagcggaa ggtcggtatc cacggagtcc cagcagccga 180

caagaagtac agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac 240

cgacgagtac aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag 300

catcaagaag aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac 360

ccggctgaag agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct 420

gcaagagatc ttcagcaacg agatggc 447

<210> 24

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 24

ccagaacaca ggttggaccg gtgc 24

<210> 25

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 25

gatccttgta gtctccgtcg tggtccttat agtccatggt ggcaccggtc caacctgtg 59

<210> 26

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 26

cgacggagac tacaaggatc atgatattga ttacaaagac gatgacgata agatggccc 59

<210> 27

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 27

tcttctttgg ggacccaccc acctttcgtt tctttttggg ggccatctta tcgtcatcg 59

<210> 28

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 28

ggtgggtccc caaagaagaa gcggaaggtc ggtatccacg gagtcccagc agccgacaa 59

<210> 29

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 29

cccacagagt tggtgccgat gtccaggccg atgctgtact tcttgtcggc tgctgggac 59

<210> 30

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 30

cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaaga 59

<210> 31

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 31

cttgatgctg tgccggtcgg tgttgcccag caccttgaat ttcttgctgg gcaccttgt 59

<210> 32

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 32

gaccggcaca gcatcaagaa gaacctgatc ggagccctgc tgttcgacag cggcgaaac 59

<210> 33

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 33

tatcttcttc tggcggttct cttcagccgg gtggcctcgg ctgtttcgcc gctgtcgaa 59

<210> 34

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 34

gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagaga 59

<210> 35

<211> 40

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 35

gccatctcgt tgctgaagat ctcttgcaga tagcagatcc 40

<210> 36

<211> 2727

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 36

cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gggcggctcc aagcggcctg 60

ccgcgacgaa gaaagcggga caggccaaga aaaagaaagg atccggcgca acaaacttct 120

ctctgctgaa acaagccgga gatgtcgaag agaatcctgg accggtgagc aagggcgagg 180

agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca 240

agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt 300

tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct 360

acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt 420

ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact 480

acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga 540

agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca 600

acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca 660

agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca 720

cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg 780

ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg 840

ccgccgggat cactctcggc atggacgagc tgtacaaggg ctccggcgag ggcaggggaa 900

gtcttctaac atgcggggac gtggaggaaa atcccggccc aaccgagtac aagcccacgg 960

tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg caccctcgcc gccgcgttcg 1020

ccgactaccc cgccacgcgc cacaccgtcg atccggaccg ccacatcgag cgggtcaccg 1080

agctgcaaga actcttcctc acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg 1140

acgacggcgc cgcggtggcg gtctggacca cgccggagag cgtcgaagcg ggggcggtgt 1200

tcgccgagat cggcccgcgc atggccgagt tgagcggttc ccggctggcc gcgcagcaac 1260

agatggaagg cctcctggcg ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg 1320

tcggagtctc gcccgaccac cagggcaagg gtctgggcag cgccgtcgtg ctccccggag 1380

tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc 1440

tccccttcta cgagcggctc ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac 1500

cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaac gcgttaagtc gacaatcaac 1560

ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta 1620

cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt 1680

tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg 1740

ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg 1800

gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca 1860

cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca 1920

ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg 1980

ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag 2040

cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc 2100

gccctcagac gagtcggatc tccctttggg ccgcctcccc gcgtcgactt taagaccaat 2160

gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg 2220

gctaattcac tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt 2280

agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca 2340

ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa 2400

ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcaggg cccgtttaaa 2460

cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc 2520

ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg 2580

aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg 2640

acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta 2700

tggcctgcag gggcgcctga tgcggta 2727

<210> 37

<211> 192

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 37

cggcggccac gaaaaaggcc ggccaggcaa aaaagaaaaa gggcggctcc aagcggcctg 60

ccgcgacgaa gaaagcggga caggccaaga aaaagaaagg atccggcgca acaaacttct 120

ctctgctgaa acaagccgga gatgtcgaag agaatcctgg accggtgagc aagggcgagg 180

agctgttcac cg 192

<210> 38

<211> 35

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 38

cggcggccac gaaaaaggcc ggccaggcaa aaaag 35

<210> 39

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 39

aggccgcttg gagccgccct ttttcttttt tgcctggccg gcctttttcg tggccgccg 59

<210> 40

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 40

ggctccaagc ggcctgccgc gacgaagaaa gcgggacagg ccaagaaaaa gaaaggatc 59

<210> 41

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 41

tccggcttgt ttcagcagag agaagtttgt tgcgccggat cctttctttt tcttggcct 59

<210> 42

<211> 59

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 42

ctgctgaaac aagccggaga tgtcgaagag aatcctggac cggtgagcaa gggcgagga 59

<210> 43

<211> 28

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 43

cggtgaacag ctcctcgccc ttgctcac 28

<210> 44

<211> 29

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 44

gtgagcaagg gcgaggagct gttcaccgg 29

<210> 45

<211> 58

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 45

tagaagactt cccctgccct cgccggagcc cttgtacagc tcgtccatgc cgagagtg 58

<210> 46

<211> 54

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 46

gagggcaggg gaagtcttct aacatgcggg gacgtggagg aaaatcccgg ccca 54

<210> 47

<211> 58

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 47

tgcggggacg tggaggaaaa tcccggccca accgagtaca agcccacggt gcgcctcg 58

<210> 48

<211> 56

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 48

taccgcatca ggcgcccctg caggccatag agcccaccgc atccccagca tgcctg 56

<210> 49

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 49

ccaccagagc atcaccggcc tg 22

<210> 50

<211> 22

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 50

cacacccgcc gcgcttaatg cg 22

<210> 51

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 51

gctgattgtt gctggtcccg 20

<210> 52

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 52

tttccaggcg aagtttactg 20

<210> 53

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 53

tgtactcagt gtcctcctcc 20

<210> 54

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 54

gctcttcaag acgcctcgcg 20

<210> 55

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 55

tctctcagac agtgcaggca tta 23

<210> 56

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 56

cgtttccgtc gtagcgtgat aat 23

<210> 57

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 57

cagttctcac ttgatggcct tgg 23

<210> 58

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 58

aggggtctgg ggaggaatgg 20

<210> 59

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 59

caccgctgat tgttgctggt cccg 24

<210> 60

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 60

aaaccgggac cagcaacaat cagc 24

<210> 61

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 61

caccgtttcc aggcgaagtt tactg 25

<210> 62

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 62

aaaccagtaa acttcgcctg gaaac 25

<210> 63

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 63

caccgtgtac tcagtgtcct cctcc 25

<210> 64

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 64

aaacggagga ggacactgag tacac 25

<210> 65

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 65

caccgctctt caagacgcct cgcg 24

<210> 66

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 66

aaaccgcgag gcgtcttgaa gagc 24

<210> 67

<211> 410

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 67

gataaacatg tgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg 60

ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata 120

cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa 180

tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct 240

tgtggaaagg acgaaacacc gggtcttcga gaagacctgt tttagagcta gaaatagcaa 300

gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt 360

ctagcgcgtg cgccaattct gcagacaaat ggctctagag gtacccatag 410

<210> 68

<211> 744

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 68

gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 60

gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 120

aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 180

gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 240

cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 300

aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 360

aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 420

ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 480

atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 540

cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 600

ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 660

ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagggctcc 720

ggcgagggca ggggaagtct tcta 744

<210> 69

<211> 1840

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 69

gagggcaggg gaagtcttct aacatgcggg gacgtggagg aaaatcccgg cccaaccgag 60

tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc 120

gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc 180

gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag 240

gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa 300

gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg 360

gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg 420

ttcctggcca ccgtcggagt ctcgcccgac caccagggca agggtctggg cagcgccgtc 480

gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc 540

gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag 600

gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa 660

gtcgacaatc aacctctgga ttacaaaatt tgtgaaagat tgactggtat tcttaactat 720

gttgctcctt ttacgctatg tggatacgct gctttaatgc ctttgtatca tgctattgct 780

tcccgtatgg ctttcatttt ctcctccttg tataaatcct ggttgctgtc tctttatgag 840

gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc 900

cccactggtt ggggcattgc caccacctgt cagctccttt ccgggacttt cgctttcccc 960

ctccctattg ccacggcgga actcatcgcc gcctgccttg cccgctgctg gacaggggct 1020

cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg 1080

ctgctcgcct gtgttgccac ctggattctg cgcgggacgt ccttctgcta cgtcccttcg 1140

gccctcaatc cagcggacct tccttcccgc ggcctgctgc cggctctgcg gcctcttccg 1200

cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt gggccgcctc cccgcgtcga 1260

ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta aaagaaaagg 1320

ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt gcttgtactg 1380

ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac 1440

tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt 1500

gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca 1560

gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt 1620

gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc 1680

taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt 1740

ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat 1800

gcggtgggct ctatggcctg caggggcgcc tgatgcggta 1840

<210> 70

<211> 10476

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 70

gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60

ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120

aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180

atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240

cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 300

gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttc tagcgcgtgc 360

gccaattctg cagacaaatg gctctagagg tacccgttac ataacttacg gtaaatggcc 420

cgcctggctg accgcccaac gacccccgcc cattgacgtc aatagtaacg ccaataggga 480

ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 540

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 600

ggcattgtgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 660

tagtcatcgc tattaccatg ggggcagagc gcacatcgcc cacagtcccc gagaagttgg 720

ggggaggggt cggcaattga tccggtgcct agagaaggtg gcgcggggta aactgggaaa 780

gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg 840

cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggttggac 900

cggtgccacc atggactata aggaccacga cggagactac aaggatcatg atattgatta 960

caaagacgat gacgataaga tggcccccaa aaagaaacga aaggtgggtg ggtccccaaa 1020

gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt acagcatcgg 1080

cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt acaaggtgcc 1140

cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga agaacctgat 1200

cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga agagaaccgc 1260

cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga tcttcagcaa 1320

cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct tcctggtgga 1380

agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg aggtggccta 1440

ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca gcaccgacaa 1500

ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc ggggccactt 1560

cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt tcatccagct 1620

ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg gcgtggacgc 1680

caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc tgatcgccca 1740

gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga gcctgggcct 1800

gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc agctgagcaa 1860

ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc agtacgccga 1920

cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca tcctgagagt 1980

gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat acgacgagca 2040

ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg agaagtacaa 2100

agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg gcggagccag 2160

ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg gcaccgagga 2220

actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct tcgacaacgg 2280

cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc ggcaggaaga 2340

tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga ccttccgcat 2400

cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga tgaccagaaa 2460

gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg gcgcttccgc 2520

ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg agaaggtgct 2580

gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga ccaaagtgaa 2640

atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga aaaaggccat 2700

cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga aagaggacta 2760

cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag atcggttcaa 2820

cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg acttcctgga 2880

caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac tgtttgagga 2940

cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg acaaagtgat 3000

gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga agctgatcaa 3060

cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt ccgacggctt 3120

cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta aagaggacat 3180

ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg ccaatctggc 3240

cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg acgagctcgt 3300

gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca gagagaacca 3360

gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg aagagggcat 3420

caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc agctgcagaa 3480

cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg accaggaact 3540

ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga gctttctgaa 3600

ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg gcaagagcga 3660

caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc agctgctgaa 3720

cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga gaggcggcct 3780

gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc ggcagatcac 3840

aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg agaatgacaa 3900

gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg atttccggaa 3960

ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc acgacgccta 4020

cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg aaagcgagtt 4080

cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga gcgagcagga 4140

aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact ttttcaagac 4200

cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga caaacggcga 4260

aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga aagtgctgag 4320

catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct tcagcaaaga 4380

gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg actgggaccc 4440

taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg tggtggccaa 4500

agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg ggatcaccat 4560

catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca agggctacaa 4620

agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg agctggaaaa 4680

cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg aactggccct 4740

gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc tgaagggctc 4800

ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact acctggacga 4860

gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg ctaatctgga 4920

caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc aggccgagaa 4980

tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca agtactttga 5040

caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg ccaccctgat 5100

ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc tgggaggcga 5160

caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagg gcggctccaa 5220

gcggcctgcc gcgacgaaga aagcgggaca ggccaagaaa aagaaaggat ccggcgcaac 5280

aaacttctct ctgctgaaac aagccggaga tgtcgaagag aatcctggac cggtgagcaa 5340

gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa 5400

cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac 5460

cctgaagttc atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac 5520

cctgacctac ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt 5580

cttcaagtcc gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga 5640

cggcaactac aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat 5700

cgagctgaag ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta 5760

caactacaac agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt 5820

gaacttcaag atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca 5880

gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac 5940

ccagtccgcc ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt 6000

cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagggct ccggcgaggg 6060

caggggaagt cttctaacat gcggggacgt ggaggaaaat cccggcccaa ccgagtacaa 6120

gcccacggtg cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc 6180

cgcgttcgcc gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg 6240

ggtcaccgag ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg 6300

ggtcgcggac gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg 6360

ggcggtgttc gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc 6420

gcagcaacag atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct 6480

ggccaccgtc ggagtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct 6540

ccccggagtg gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc 6600

ccgcaacctc cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc 6660

cgaaggaccg cgcacctggt gcatgacccg caagcccggt gcctgaacgc gttaagtcga 6720

caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc 6780

tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg 6840

tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt 6900

gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac 6960

tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc 7020

tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct 7080

gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct 7140

cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct 7200

caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct 7260

tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta 7320

agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga 7380

ctggaagggc taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct 7440

ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt 7500

aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac 7560

tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagggcc 7620

cgtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg 7680

cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata 7740

aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt 7800

ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt 7860

gggctctatg gcctgcaggg gcgcctgatg cggtattttc tccttacgca tctgtgcggt 7920

atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg 7980

cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 8040

ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 8100

taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 8160

aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 8220

ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 8280

tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt tcggcctatt 8340

ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt 8400

ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc 8460

cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 8520

cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat 8580

caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca 8640

tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc 8700

ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct 8760

gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg 8820

cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg 8880

tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc 8940

tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca 9000

cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac 9060

tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa 9120

agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg 9180

ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt 9240

ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg 9300

aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc 9360

gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga 9420

tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta 9480

ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca gcactggggc 9540

cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg 9600

atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt 9660

cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa 9720

ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt 9780

cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 9840

ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 9900

tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 9960

taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 10020

caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 10080

agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 10140

gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 10200

gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 10260

ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 10320

acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 10380

tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 10440

ggttcctggc cttttgctgg ccttttgctc acatgt 10476

<210> 71

<211> 3120

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 71

gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60

cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120

tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180

aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240

ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300

ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360

tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420

tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480

actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540

gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600

acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660

gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720

acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780

gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840

ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900

gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960

cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020

agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080

catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140

tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200

cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260

gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320

taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc 1380

ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440

tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500

ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560

cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620

agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680

gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740

atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800

gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860

gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920

ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980

cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040

cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100

acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160

cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2220

accatgatta cgccaagctt gcatgcaggc ctctgcagtc gacgggcccg ggatccgatg 2280

ataaacatgt gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc 2340

tgttagagag ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac 2400

gtgacgtaga aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat 2460

ggactatcat atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt 2520

gtggaaagga cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag 2580

ttaaaataag gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttc 2640

tagcgcgtgc gccaattctg cagacaaatg gctctagagg tacccataga tctagatgca 2700

ttcgcgaggt accgagctcg aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa 2760

accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta 2820

atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat 2880

ggcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcatatggt 2940

gcactctcag tacaatctgc tctgatgccg catagttaag ccagccccga cacccgccaa 3000

cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg 3060

tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga 3120

<210> 72

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 72

cgtactctag aacccatgcc act 23

<210> 73

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 73

atcatcacca aagagtccgt gtg 23

<210> 74

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 74

acgtgtctga tggaattgtt ggc 23

<210> 75

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 75

aatgtgtcct ttcagtctcc gct 23

<210> 76

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 76

tatagttgtg acagggacat 20

<210> 77

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 77

attccaggct tcttgctgag 20

<210> 78

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 78

gtcatcttct ggacttgtga 20

<210> 79

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 79

cacaagtcca gaagatgacg 20

<210> 80

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 80

ctgggctgtg agtaaccaga gtt 23

<210> 81

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 81

ggcttcttga agatcagatg agg 23

<210> 82

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 82

caccgtatag ttgtgacagg gacat 25

<210> 83

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 83

aaacatgtcc ctgtcacaac tatac 25

<210> 84

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 84

caccgattcc aggcttcttg ctgag 25

<210> 85

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 85

aaacctcagc aagaagcctg gaatc 25

<210> 86

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 86

caccgtcatc ttctggactt gtga 24

<210> 87

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 87

aaactcacaa gtccagaaga tgac 24

<210> 88

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 88

caccgcacaa gtccagaaga tgacg 25

<210> 89

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 89

aaaccgtcat cttctggact tgtgc 25

<210> 90

<211> 42

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 90

ttgtgacagg gaacgtggag cttgacccgc agtctggaga ga 42

<210> 91

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> misc_feature

<222> (13)..(14)

<223> n is a, c, g, or t

<400> 91

ttgtgacagg gannacgtgg agcttgaccc 30

<210> 92

<211> 28

<212> DNA

<213> Artificial sequence (Artificial sequence)

<220>

<221> misc_feature

<222> (13)..(14)

<223> n is a, c, g, or t

<400> 92

ttgtgacagg gannttgacc cgcagtct 28

<210> 93

<211> 30

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 93

ttgtgacagg gatgacgtgg agcttgaccc 30

<210> 94

<211> 28

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 94

ttgtgacagg gagcttgacc cgcagtct 28

<210> 95

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 95

gccaaagtgt gacgttaact gct 23

<210> 96

<211> 24

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 96

accaaagctc tcctgactca aagt 24

<210> 97

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 97

tgtttcgtat accagctaga gga 23

<210> 98

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 98

atctctcatg ctggcgtaaa cct 23

<210> 99

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 99

tggtggcatt ttcacgatca 20

<210> 100

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 100

atgccaccaa aattctgcat 20

<210> 101

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 101

aaactggaag agtttgttcg 20

<210> 102

<211> 20

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 102

aactggaaga gtttgttcga 20

<210> 103

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 103

gggattaccg ggattaaaaa caa 23

<210> 104

<211> 23

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 104

tttttattct ttgctccgca tgt 23

<210> 105

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 105

caccgtggtg gcattttcac gatca 25

<210> 106

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 106

aaactgatcg tgaaaatgcc accac 25

<210> 107

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 107

caccgatgcc accaaaattc tgcat 25

<210> 108

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 108

aaacatgcag aattttggtg gcatc 25

<210> 109

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 109

caccgaaact ggaagagttt gttcg 25

<210> 110

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 110

aaaccgaaca aactcttcca gtttc 25

<210> 111

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 111

caccgaactg gaagagtttg ttcga 25

<210> 112

<211> 25

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 112

aaactcgaac aaactcttcc agttc 25

Claims

Use of a gRNA target combination for the construction of a hemophilia model pig cell line, the gRNA target combination consisting of a first gRNA target and a second gRNA target, and/or a third gRNA target and a fourth gRNA target, characterized in that,

wherein the base sequence of the first gRNA target point is TATAGTTGTGACAGGGACAT, and the base sequence of the second gRNA target point is CACAAGTCCAGAAGATGACG;

the base sequence of the third gRNA target is ATGCCACCAAAATTCTGCAT; the fourth gRNA target has a nucleotide sequence of AAACTGGAAGAGTTTGTTCG.
2. A double stranded DNA molecule comprising a cohesive end and a target fragment, wherein the base sequences of the target fragment are:

TATAGTTGTGACAGGGACAT；

or CACAAGTCCAGAAGATGACG;

or ATGCCACCAAAATTCTGCAT;

or AAACTGGAAGAGTTTGTTCG.
3. An expression cassette or gRNA expression vector comprising the double stranded DNA molecule of claim 2.
A CRISPR/Cas9 system comprising a first gRNA expression vector, a second gRNA expression vector, and a Cas9 expression vector,

the target sequence of the first gRNA expression vector is TATAGTTGTGACAGGGACAT, and the target sequence of the second gRNA expression vector is CACAAGTCCAGAAGATGACG.
5. The CRISPR/Cas9 system according to claim 4, wherein the molar ratio of the first gRNA expression vector, the second gRNA expression vector and the Cas9 expression vector is 1.5-2: 1.
A CRISPR/Cas9 system including a third gRNA expression vector, a fourth gRNA expression vector, and a Cas9 expression vector,

the target sequence of the third gRNA expression vector is ATGCCACCAAAATTCTGCAT, and the target sequence of the fourth gRNA expression vector is AAACTGGAAGAGTTTGTTCG.
7. The CRISPR/Cas9 system according to claim 6, wherein the molar ratio of the third gRNA expression vector, the fourth gRNA expression vector and the Cas9 expression vector is 1.5-2: 1.
A CRISPR/Cas9 system, comprising a first gRNA expression vector, a second gRNA expression vector, a third gRNA expression vector, a fourth gRNA expression vector and a Cas9 expression vector,

the target sequence of the first gRNA expression vector is TATAGTTGTGACAGGGACAT, and the target sequence of the second gRNA expression vector is CACAAGTCCAGAAGATGACG;

the target sequence of the third gRNA expression vector is ATGCCACCAAAATTCTGCAT, and the target sequence of the fourth gRNA expression vector is AAACTGGAAGAGTTTGTTCG.
9. The CRISPR/Cas9 system according to claim 8, wherein the molar ratio of the first gRNA expression vector, the second gRNA expression vector, the third gRNA expression vector, the fourth gRNA expression vector and the Cas9 expression vector is 0.75-1: 1.
10. The CRISPR/Cas9 system according to any one of claims 4-9, wherein the base sequence of the Cas9 expression vector is shown as SEQ ID NO. 70.
11. The CRISPR/Cas9 system according to any of claims 4-9, characterized in that the original vector of the gRNA expression vector is pKG-U6 gRNA.
12. A method of constructing a hemophilia model porcine cell line, comprising the steps of: transferring the CRISPR/Cas9 system of any one of claims 4-9 into porcine ear primary fibroblasts, and screening for gene mutation monoclonal cells.
13. An A, B and hemophilia a & B model pig cell line constructed according to the method of claim 12.