WO2021042470A1 - gRNA靶点组合在构建A型、B型和A＆B型血友病模型猪细胞系中的应用 - Google Patents

gRNA靶点组合在构建A型、B型和A＆B型血友病模型猪细胞系中的应用 Download PDF

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WO2021042470A1
WO2021042470A1 PCT/CN2019/114157 CN2019114157W WO2021042470A1 WO 2021042470 A1 WO2021042470 A1 WO 2021042470A1 CN 2019114157 W CN2019114157 W CN 2019114157W WO 2021042470 A1 WO2021042470 A1 WO 2021042470A1
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grna
expression vector
target
cas9
gene
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牛冬
汪滔
王德华
王磊
程锐
曾为俊
马翔
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南京启真基因工程有限公司
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Definitions

  • the invention belongs to the technical field of gene editing, and specifically relates to the application of gRNA target combination in the construction of A, B and A&B hemophilia model pig cell lines.
  • Heamophilia usually refers to a group of X-chromosome-linked recessive hereditary bleeding disorders that are caused by gene mutations that lack coagulation factors. There is currently no effective cure. According to the results of an epidemiological survey, as of 2018, the number of hemophilia patients in my country is about 130,000, and only 20% of patients can really do preventive treatment.
  • the coagulation process is a series of limited hydrolysis of proteins, generally divided into three stages: thromboplastin formation, thrombin formation and fibrin formation. Hemophilia is generally the first stage of coagulation, that is, thromboplastin disorder.
  • hemophilia A deficiency of factor VIII
  • hemophilia B Factor IX deficiency
  • hemophilia C factor IX deficiency
  • the incidence of hemophilia A is more common, accounting for about 85%; hemophilia B accounts for about 15%; hemophilia C is less common.
  • Hemophilia A is caused by factor VIII-antihemophilic globulin (AHG) deficiency.
  • AHG factor VIII-antihemophilic globulin
  • the related gene is located at Xq28, the gene span is over 186kb, and more than 46 mutations have been found.
  • Hemophilia B is caused by the lack of factor IX-plasma thrombin component (PTC).
  • hemophilia The treatment of hemophilia generally adopts blood transfusion, prothrombin complex, clotting factor and other means, but blood products have the risk of patients with infectious diseases. Protein preparations such as prothrombin complex and clotting factors can cause patients to have targeted Antibodies to these proteins reduce their therapeutic effects and even cause more severe bleeding symptoms. Therefore, the construction of animal models of hemophilia will play a very important role in treatment research. At present, the animal models of hemophilia are mainly mouse models. As early as the early 1990s, researchers had successfully constructed a mouse model of hemophilia A based on the principle of gene targeting. Since then, a mouse model of hemophilia B has also been successfully constructed.
  • mice are very different from humans in terms of body size, physiology, pathology, etc., and cannot simulate human diseases well. In fact, more than 95% of drugs that have been validated by mice are ineffective in human clinical trials. . Therefore, the development of a large animal model of hemophilia that is closer to human body size and physiological function is the key to the study of hemophilia treatment. However, there have not been any reports of artificially constructed large animal hemophilia models.
  • pigs have early sexual maturity, short reproductive cycles, multiple litters, mature cloning technology and low cost, and large-scale groups can be formed in a short period of time.
  • Long-term inbreeding makes pigs have high genetic homozygosity and genetic stability, clear genetic background, relatively stable phenotype, good reproducibility in biomedical research, and meets the requirements of scientific research; at the same time, small pigs are easy to raise and cost. It is low, and pigs have long been carnivorous animals for humans, and there are no ethical issues in using pigs as disease model animals.
  • Rats and primate model animals provide the most cost-effective model animals for pharmaceutical companies, universities and research institutes, which will greatly promote the rapid development of biomedicine.
  • the present invention designs a pair of target sequences for each of the pig F8 and F9 genes, uses the two pairs of target sequences to construct three CRISPR/Cas9 systems, and transfers the three CRISPR/Cas9 systems into pig fibroblasts respectively, and screens Obtain A, B and A&B hemophilia model pig cell lines.
  • the base sequence of the first gRNA target is TATAGTTGTGACAGGGACAT
  • the base sequence of the second gRNA target is CACAAGTCCAGAAGATGACG
  • the base sequence of the third gRNA target is ATGCCACCAAAATTCTGCAT; the base sequence of the fourth gRNA target is AAACTGGAAGAGTTTGTTCG.
  • the present invention also provides double-stranded DNA molecules, including sticky ends and target fragments, the base sequences of the target fragments are:
  • the molar ratio of the first gRNA expression vector, the second gRNA expression vector and the Cas9 expression vector is 1.5-2:1.5-2:1. 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 present invention also provides another 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 fourth gRNA expression vector The target sequence is AAACTGGAAGAGTTTGTTCG.
  • the molar ratio of the third gRNA expression vector, the fourth gRNA expression vector and the Cas9 expression vector is 1.5-2:1.5-2:1.
  • 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 present invention also provides a third CRISPR/Cas9 system, including 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 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 to 1:0.75 to 1:0.75 to 1:0.75 ⁇ 1:1. Further 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 0.75:0.75:0.75:0.75:1.
  • the base sequence of the Cas9 expression vector is shown in SEQ ID NO: 70.
  • the original vector of the gRNA expression vector is pKG-U6gRNA, and the full sequence of pKG-U6gRNA is shown in SEQ ID NO: 71.
  • the present invention also provides a method for constructing a pig cell line of a hemophilia model, which includes the following steps: transferring any of the CRISPR/Cas9 systems into pig ear primary fibroblasts, and screening gene mutation monoclonal cells.
  • the original vector has gRNA backbone sequence, CMV enhancer and Cas9 gene; replace the chicken ⁇ -actin promoter downstream of the CMV enhancer with the EF1a promoter; At least one nuclear localization coding sequence NLS is added to the N-terminus and C-terminus of the Cas9 gene;
  • It may also include: transforming the gRNA backbone sequence into a sequence as shown in SEQ ID NO: 70 to remove invalid redundant sequences.
  • It may also include: sequentially inserting P2A-EGFP-T2A-PURO sequence, WPRE gene, 3'LTR gene and bGH polyA sequence downstream of the C-terminal NLS of Cas9 gene to increase fluorescence and resistance selection markers and enhance the expression efficiency of Cas9.
  • the present invention designs an efficient knockout target sequence by analyzing the conservative analysis of the F8 and F9 gene knockout pre-determined target exons and adjacent genomic sequences, uses the target sequence to construct a gRNA expression vector, and efficiently express the expression of Cas9
  • the vectors are transferred into host cells together, which significantly improves the efficiency of gene editing.
  • the invention uses gene editing technology to cause mutations in F8, F9, F8 & F9 in pig primary fibroblasts to obtain mutant cells of each gene.
  • the next step is to use somatic cell nuclear transfer cloning technology to obtain A, B and A&B hemophilia disease model pigs It is used for drug screening, drug efficacy evaluation, pharmacology and toxicology, disease pathology, gene therapy and cell therapy, etc., providing effective experimental data for further clinical applications, and also providing powerful experimental means for the successful treatment of human hemophilia .
  • the present invention has at least the following beneficial effects:
  • primates are the animals closest to humans, but they are small in size, late in sexual maturity (mating at the age of 6-7), and are singleton animals, their population expansion speed is extremely slow, and the cost of breeding Also high. In addition, cloning of primates is inefficient, difficult, and costly.
  • Pigs do not have the above shortcomings. Pigs are the closest relatives to humans except primates. Their body size, weight, organ size, etc. are similar to humans, and they have anatomy, physiology, nutritional metabolism, and disease pathogenesis. It is very similar to human beings in other aspects. At the same time, pigs have early sexual maturity (4-6 months), high fecundity, multiple litters, and a larger group can be formed within 2-3 years. In addition, pig cloning technology is very mature, and the cost of cloning and feeding is much lower than that of primates. Moreover, pigs have long been carnivorous animals for humans, and there are no animal protection and ethical issues in using pigs as disease model animals.
  • the gRNA vector and the cas9 vector are not based on the conventional 1:1 molar ratio, but based on the 3:1 molar ratio.
  • Figure 1 is a map of pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO vector (referred to as pKG-GE3).
  • Figure 8 is a graph showing the electrophoresis results of eEF1a1-14 for full gene synthesis.
  • Figure 9 is a vector map of pU6gRNA-eEF1a Cas9+nNLS.
  • Fig. 10 is a diagram showing the results of the digestion gel map of pU6gRNA-eEF1a Cas9 with AgeI and BglII.
  • Figure 11 shows the results of N-NLS 1-12 electrophoresis of the whole gene synthesis.
  • Figure 14 The constructed pKG-U6gRNA vector map.
  • Figure 15 is a schematic diagram of DNA oligo connection with pKG-U6gRNA vector after annealing.
  • Figure 17 is a comparison diagram of MSTN gene editing efficiency.
  • Figure 18 is a comparison diagram of FNDC5 gene editing efficiency.
  • Figure 19 is a transcription map of pig F8 gene.
  • Figure 21 is a graph showing the results of PCR amplification of the F8 gene of 8 pigs using the selected primers.
  • Figure 23 shows the B1-gRNA1 insert sequence.
  • Figure 25 shows the B1-gRNA3 insert sequence.
  • Figure 27 is a graph showing the results of PCR for F8 high-efficiency target gRNA combination screening.
  • Figure 30 is a comparison diagram of the sequencing result of clone No. 4 B1-F843 and the sequencing result of WT B1-F843.
  • Figure 31 is a theoretical deletion map of F8-gRNA1 and F8-gRNA4.
  • Figure 32 is a sequence diagram of deletions caused by F8-gRNA1 and F8-gRNA4.
  • Figure 34 is a transcription map of pig F9 gene.
  • Figure 35 is a graph showing the PCR results of screening the best primers for F9 gene amplification.
  • Figure 36 is a graph showing the results of PCR amplification of the F9 gene of 8 pigs using the selected primers.
  • Fig. 37 is a comparison analysis diagram of the amplified F9 gene sequence and the published F9 gene sequence.
  • Figure 41 shows the B2-gRNA4 insert sequence.
  • Figure 42 is a graph showing the PCR results of F9 high-efficiency target gRNA combinatorial screening.
  • Figure 43 is a graph showing the results of F9 PCR (primers B2-F539/B2-R853) of the F9 mutant monoclonal in group 2.
  • Figure 44 is a diagram showing the alignment results of the monoclonal F9 sequencing results without the set peaks and the published F9 sequences.
  • Figure 45 shows the sequencing result of F9 gene editing monoclonal 24-50 Miseq and the comparison result of the wild-type sequence.
  • Figure 48 Analysis diagram of sequencing results of F8+F9 monoclonal F8 gene PCR products.
  • FIG. 49 Analysis diagram of sequencing results of F8+F9 monoclonal F9 gene PCR products.
  • the pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO vector (pKG-GE3 for short) is modified from the addgene (Plasmid#42230, from Zhang Feng lab) pX330-U6-Chimeric_BB-CBh-hSpCas9 vector.
  • the promoter is modified to increase For nuclear localization signal, add WPRE sequence to enhance translation, add green fluorescence and resistance screening puro gene, the map is shown in Figure 1, and the base sequence is shown in SEQ ID NO:1.
  • the build steps are as follows:
  • gRNAsc-1 TGTGGAAAGGACGAAACACC
  • gRNAsc-2 TGCTATTTCTAGCTCTAAAACAGGTCTTCTCGAAGACCCGGTGTTTCGTCCTTTCCACA
  • gRNAsc-3 CCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA
  • gRNAsc-4 CACGCTAGAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGC (SEQ ID NO: 5)
  • gRNAsc-5 GTGCTTTTTTCTAGCGTGCGCCAATTCTGCAGACAAATGGCTCTAGAGGTACCCGTT (SEQ ID NO: 6)
  • gRNAsc-6 TTATGTAACGGGTACCTCTAGAGCC (SEQ ID NO: 7)
  • Phanta Max (Novizan P505) was used for total gene synthesis PCR, and mixed according to the system shown in Table 3:
  • PCR conditions 95°C 3min (95°C 15s 58°C 15s 72°C 20s) cycle 32 times 72°C 5min; store at 4°C.
  • the 175bp insert (SEQ ID NO: 1) was obtained by whole gene synthesis. After PCR, the PCR products were separated by 1% agarose electrophoresis. Gel DNA Extraction Mini Kit#DC301) recover the target fragment, and dissolve the target fragment in 50ul ddH 2 O at -20°C for use.
  • step (4) Add 10 ⁇ L of the recombination reaction product obtained in step (4) to the centrifuge tube containing competent cells, mix well and let stand in an ice bath for 30 minutes;
  • pU6gRNACas9 vector For the constructed pU6gRNACas9 vector, use XbaI and AgeI endonuclease to remove the promoter (CMV enhancer) and enhancer sequence (chicken ⁇ -actin), recover the linear vector sequence of about 7650bp, and synthesize 554bp including CMV enhancer and EF1a promoter
  • the sequence (SEQ ID NO: 8) is recombined with the digestion vector pU6gRNACas9 to obtain the pU6gRNA-eEF1a Cas9 vector ( Figure 6).
  • eEF1a-1 TCTGCAGACAAATGGCTCTAGAGGTACCCG (SEQ ID NO: 9)
  • eEF1a-2 GGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGGGTACCTCTAGAGCCAT (SEQ ID NO: 10)
  • eEF1a-3 GCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAGTAACGCCAATAGGGA
  • eEF1a-4 TACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATTGA
  • eEF1a-5 AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATG
  • eEF1a-6 TACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCC (SEQ ID NO: 14)
  • eEF1a-7 CCTATTGACGTCAATGACGGTAAA
  • the constructed vector pU6gRNA-eEF1a Cas9 was digested with AgeI and BglII, and the 7786bp vector sequence was recovered, and the NLS-added sequence was added to the restriction site.
  • the following sequence 447bp was synthesized including 2 nuclear localization signals and partially excised Cas9 code Sequence (SEQ ID NO: 23, recombined to obtain pU6gRNA-eEF1a Cas9+nNLS vector ( Figure 9).
  • N-NLS-1 CCAGAACACAGGTTGGACCGGTGC
  • N-NLS-2 GATCCTTGTAGTCTCCGTCGTGGTCCTTATAGTCCATGGTGGCACCGGTCCAACCTGTG
  • N-NLS-3 CGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCC
  • N-NLS-4 TCTTCTTTGGGGACCCACCCACCTTTCGTTTCTTTTTTTGGGGGCCATCTTATCGTCATCG
  • N-NLS-5 GGTGGGTCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAA
  • N-NLS-6 CCCACAGAGTTGGTGCCGATGTCCAGGCCGATGCTGTACTTCTTGTCGGCTGCTGGGAC
  • N-NLS-7 CGGCACCAACTCTGTGGGCTGGGCCGT
  • N-NLS-9 GACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAAC
  • N-NLS-10 TATCTTCTTCTGGCGGTTCTCTTCAGCCGGGTGGCCTCGGCTGTTTCGCCGCTGTCGAA
  • N-NLS-11 GAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGA
  • N-NLS-12 GCCATCTCGTTGCTGAAGATCTCTTGCAGATAGCAGATCC (SEQ ID NO: 35)
  • the vector constructed above was named pU6gRNA-eEF1a Cas9+nNLS, and it was digested with FseI and SbfI.
  • the vector sequence was recovered 7781bp, and the synthetic sequence 2727bp included NLS-P2A-EGFP-T2A-PURO-WPRE-3'LTR-bGH polyA signals( SEQ ID NO: 36) is recombined with the vector fragment to obtain the vector pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO.
  • the 2727bp synthetic fragment is derived from 3-segment overlap extension PCR, which specifically includes:
  • Fragment 1 Synthesize 192bp (SEQ ID NO: 37) containing the nuclear localization signal coding sequence and P2A, EGFP overlapping sequence, the synthetic primer sequence is shown in Table 7, and it is obtained by whole gene synthesis (for the method, please refer to the whole gene synthesis part in the transformation process of pU6gRNACas9 vector ).
  • Fragment 2 EGFP fragment 744bp (SEQ ID NO: 68), template is commercial vector EGFP-N1, primers are shown in Table 8, obtained by conventional PCR.
  • T2A-PURO-WPRE-3'LTR-polyA signals sequence 1840bp (SEQ ID NO: 69), template is LentiCRISPRV2 (addgene Plasmid#52961), primers are shown in Table 9, use LentiCRISPRV2 as template V2-F/V2- R primer PCR, and the previous PCR product as a template, T2A-F and V2-R PCR to obtain the target fragment of 1840bp.
  • the linearized vector pU6gRNA-eEF1a Cas9+nNLS 7781bp and 2727bp insert fragments are recombined.
  • Promoter CMV enhancer and EF1a hybrid promoter.
  • Cas9 gene containing multiple NLS Cas9 gene containing N-terminal and C-terminal multinuclear localization signal (NLS).
  • Screening marker gene the original P2A-EGFP-T2A-PURO of fluorescence and resistance double screening markers.
  • Vector backbone including Amp resistant original and ori replicon.
  • pKG-U6gRNA vector source pUC57 vector, connect pKG-U6gRNA insert sequence (DNA fragment containing U6 promoter, BbsI restriction site and sgRNA backbone sequence, namely SEQ ID NO: 67) through EcoRV restriction site, pKG -U6gRNA insert sequence is reversely inserted into pUC57 vector, and positive clones are obtained after transformation of bacteria.
  • the full sequence of pKG-U6gRNA vector SEQ ID NO: 71).
  • pKG-U6gRNA insert sequence (the first underlined part is the U6 promoter sequence, the uppercase base letter is the sequence where the two BbsI restriction sites are located, and the second underlined part is the sgRNA backbone sequence):
  • Each target site synthesizes 2 pairs of complementary DNA oligo, which can be annealed to form a DNA double-strand complementary to the sticky end of the pKG-U6gRNA vector BbsI after digestion, as shown in Figure 15.
  • synthesizing the target site s sense strand, it is activated by U6
  • the promoter starts to be transcribed from the first g after the promoter, so when the target is not g, you need to add a g before the target, and add cacc before the g to complement the U6 end of the vector after BbsI digestion; synthetic target When inserting the sequence complementary strand, it is necessary to synthesize the complementary sequence of g and the target, and then add the aaac sequence at the 5'end, which is complementary to the gRNA backbone end sequence cut by BbsI.
  • the target point starts with g, you can not add g before the target point of the sense strand, and at the same time add one less c that is complementary to the added g
  • MSTN-gRNA1 GCTGATTGTTGCTGGTCCCG (SEQ ID NO: 51) and
  • MSTN-gRNA2 TTTCCAGGCGAAGTTTACTG (SEQ ID NO: 52).
  • FNDC5-gRNA1 TGTACTCAGTGTCCTCCTCC (SEQ ID NO: 53) and
  • FNDC5-gRNA2 GCTCTTCAAGACGCCTCGCG (SEQ ID NO: 54).
  • step (4) 1) Add 20 ⁇ L of the ligated plasmid solution obtained in step (4) to the centrifuge tube containing competent cells, mix well and let stand in an ice bath for 30 minutes;
  • plasmid small extraction kit for plasmid extraction, and name them as pKG-U6gRNA (MSTN-1), pKG-U6gRNA (MSTN-2), pKG-U6gRNA (FNDC5-1), pKG-U6gRNA (FNDC5-2) plasmids are available for use. According to the same method, a small sample of 10 tubes of pKG-GE3 plasmid for use.
  • two target-containing gRNA vectors pKG-U6gRNA about 3.0kb
  • Cas9 vector pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO referred to as pKG-GE3 about 10.5kb
  • the PBS formula of 5% P/S is: 5% P/S (Gibco Penicillin-Streptomycin) + 95% PBS, and 5% and 95% are volume percentages.
  • the formula of complete cell culture medium is: 15% fetal bovine serum (Gibco) + 83% DMEM medium (Gibco) + 1% P/S (Gibco Penicillin-Streptomycin) + 1% HEPES (Solarbio), 15%, 83%, 1%, and 1% are volume percentages.
  • 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 were co-transfected into porcine primary fibroblasts.
  • the grouping is as follows:
  • Group 1 pKG-U6gRNA(MSTN-1)+pKG-U6gRNA(MSTN-2)+pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO, the molar ratio is 0.5:0.5:1, the actual amount of plasmid is 0.22ug+0.22ug +1.56ug, total 2ug
  • Group 2 pKG-U6gRNA(MSTN-1)+pKG-U6gRNA(MSTN-2)+pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO, the molar ratio is 1:1:1, the actual amount of plasmid is 0.36ug+0.36ug +1.27ug, total 2ug
  • Group 3 pKG-U6gRNA(MSTN-1)+pKG-U6gRNA(MSTN-2)+pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO, the molar ratio is 1.5:1.5:1, the actual amount of plasmid is 0.46ug+0.46ug +1.08ug, total 2ug
  • Group 4 pKG-U6gRNA(MSTN-1)+pKG-U6gRNA(MSTN-2)+pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO, the molar ratio is 2:2:1, the actual amount of plasmid is 0.53ug+0.53ug +0.93ug, total 2ug
  • each well contains 2 mL of 15% fetal bovine serum (Gibco) + 83% DMEM medium (Gibco) + 1% P/S (Gibco Penicillin-Streptomycin) + 1% HEPES (Solarbio) complete culture medium;
  • the electroporation is 6-12h to change the medium, and the electroporation is 48h and trypsinization is used to collect the cells in a 1.5ml EP tube, and the mutation efficiency PCR detection is performed in the later period.
  • the KAPA2G lysate preparation system is as follows:
  • Lysis process 75°C for 15min—95°C for 5min—4°C, after the reaction, the genomic DNA is stored at -20°C;
  • the MSTN group uses MSTN-F896/MSTN-R1351 primers for PCR detection, and the PCR reaction system is as follows:
  • the results of electrophoresis analysis are shown in Figure 16:
  • the molar ratio of gRNA1:gRNA2: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. From the results, it is judged that the 456bp band is a wild-type band (WT), and the band around 329bp (456bp-theoretical deletion 127bp) is a deletion mutation band (MT).
  • WT wild-type band
  • MT deletion mutation band
  • gene deletion mutation efficiency 100*(MT gray scale/MT band bp number)/(WT gray scale/WT band bp number+MT gray scale/MT band bp number)%
  • MSTN group was calculated separately 1 gene deletion mutation efficiency was 28.6%
  • MSTN group 2 gene deletion mutation efficiency was 77.8%
  • MSTN group 3 gene deletion mutation efficiency was 86.8%
  • MSTN group 4 gene deletion mutation efficiency was 81.5%
  • group 3 gene editing efficiency was the highest. It is determined that the most suitable amount of the two gRNA plasmids and the Cas9 plasmid is the molar ratio of 1.5:1.5:1, and the actual amount of plasmid is 0.46ug+0.46ug+1.08ug.
  • the present invention uses the constructed pig MSTN gene and FNDC5 gene each of two gRNA target vectors and the modified cas9 vector, and determines the gene editing efficiency of the vector by electrotransforming pig primary fibroblasts and detecting the deletion mutation efficiency of each gene by PCR .
  • the Cas9 expression vector pX330 or the modified pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO and the gRNA expression vector pKG-U6gRNA (MSTN-1) and pKG-U6gRNA (MSTN-2) or pKG-U6gRNA (FNDC5-1) and pKG-U6gRNA (FNDC5-2) was co-transfected into porcine primary fibroblasts.
  • Neon mammalian nuclear transfection kit
  • NeonTM transfection system electroporation instrument for electroporation experiments.
  • MSTN group B pKG-U6gRNA (MSTN-1) and pKG-U6gRNA (MSTN-2)
  • MSTN group 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)
  • Figure 17 is a comparison of MSTN gene editing efficiency.
  • the KG group accounts for more mutation bands/wild-type bands than the 330 group, indicating that the KG gene editing efficiency is higher than that of the 330 group.
  • Figure 18 is a comparison of FNDC5 gene editing efficiency.
  • the KG group accounts for more mutation bands/wild-type bands than the 330 group, indicating that the KG gene editing efficiency is higher than that of the 330 group.
  • gene deletion mutation efficiency 100*(MT grayscale/MT band bp number)/(WT grayscale/WT band bp number+MT grayscale/MT band bp number)%, respectively calculate MSTN-
  • the 330 group gene deletion mutation efficiency was 27.6%, and the MSTN-KG group gene deletion mutation efficiency was 86.5%.
  • the efficiency of gene deletion and mutation in the FNDC5-330 group is 18.6%, and the efficiency of gene deletion and mutation in the FNDC5-KG group is 81.7%.
  • the modified vector pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO gene editing efficiency is significantly improved (about 3-4 times) .
  • the transcription form of the F8 gene is shown in Figure 19 (the dark broad lines are coding exons, and the light broad lines are non-coding exons). Including 26 exons, the 15th exon is the largest at 2454bp, (mutations of the F8 gene in hemophilia patients often occur on the largest exon), so the present invention plans to design the target on the 15th F8 gene The front position within the exon.
  • Primer3 is used for primer design, and the design results are as follows:
  • 47 is the amplified band of F8-GT-F491/F8-GT-R1463 primer
  • 48 is the amplified band of F8-GT-F491/F8-GT-R1490
  • 49 is F8-GT-F572 /F8-GT-R1463 primer amplified band
  • 50 is the F8-GT-F572/F8-GT-R1490 primer amplified band, and it is determined that 48, F8-GT-F491/F8-GT-R1490 is used as the primer.
  • the above PCR amplification products were sequenced using amplification primers (General Biologicals Sequencing).
  • the sequencing results were compared and analyzed using Snapgene and the F8 gene sequence published on the Internet. The results are shown in Figure 22.
  • the white vertical lines represent mutations or deletion mutations, and the black vertical lines represent insertion mutations. Design the target and detection primers to avoid designing the mutation sequence. .
  • F8 gene knockout target is 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)
  • 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 site synthesizes 2 pairs of complementary DNA oligo, which can be annealed to form a DNA double strand complementary to the sticky end of the pKG-U6gRNA vector BbsI after digestion ( Figure 15).
  • the insert sequence of the synthetic F8 gene target is complementary to the DNA oligo annealing and the double-stranded insert sequence formed after annealing is shown in Figure 23 to Figure 26:
  • the B1-gRNA1 insertion sequence is shown in Figure 23; the B1-gRNA2 insertion sequence is shown in Figure 24; the B1-gRNA3 insertion sequence is shown in Figure 25; and the B1-gRNA4 insertion sequence is shown in Figure 26.
  • the correct clones were named pKG-U6gRNA(B1-1), pKG-U6gRNA(B1-2), pKG-U6gRNA(B1-3), pKG-U6gRNA(B1-4), plasmid mini-pump, stored at -20°C for later use .
  • gRNA expression vectors pKG-U6gRNA (B1) and Cas9 expression vector pKG-GE3 were used to co-transfect porcine primary fibroblasts.
  • Mammalian fibroblast nuclear transfection kit (Neon) and NeonTM transfection system electroporator were used for electroporation experiments.
  • Combination 1 pKG-U6gRNA(B1-1)+pKG-U6gRNA(B1-3)+pKG-GE3 theoretical deletion of 136bp
  • Combination 2 pKG-U6gRNA(B1-1)+pKG-U6gRNA(B1-4)+pKG-GE3 theoretically deleted 151bp
  • Combination 3 pKG-U6gRNA(B1-2)+pKG-U6gRNA(B1-3)+pKG-GE3 theoretical deletion 86bp
  • the electroporation reaction solution was prepared, and the system was as follows:
  • the pig primary cell electrotransformation and cell collection were carried out.
  • 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
  • the brighter the smaller band (mutant band) is relative to the larger band (wild-type band), the higher the mutation efficiency.
  • combination 2 pKG-U6gRNA(B1-1)+pKG-U6gRNA(B1-4)+pKG-GE3 has a theoretical deletion of 151bp and has the highest mutation efficiency.
  • F8 mutation pKG-U6gRNA(B1-1)(0.46ug)+pKG-U6gRNA(B1-4)(0.46ug)+pKG-GE3(1.08ug).
  • Figure 28 is the F8 PCR (primer B1-F843/B1-R1258) test results of the group-1 F8 mutant monoclonal. From the results, it is judged that 3, 4, 10, 11, 12, 14, 15 are homozygous deletion mutant monoclonals. 5, 6, 7, 16, 19, 22 are single clones of heterozygous deletion mutations, and 1, 2, 8, 9, 13, 17, 18, 20, and 21 have no large fragment deletions.
  • PCR products are recovered by gel (multiple bands need to be recovered) and sequenced with PCR primers (General Biotech), and the sequencing results are analyzed to further determine the mutations of each monoclonal F8 gene.
  • the sequencing results are divided into two types, unset peaks and set peaks.
  • the unset peaks can be directly compared with the corresponding genome sequence of F8 through the sequencing results. There are no sets of peaks in the front and two sets of peaks in the back, which need to be based on the wild-type sequence or After theoretical deletion, the sequence is performed to determine the two sequences contained in the set of peaks. If there is no set of peaks in the front and the set of peaks in the back are messy, the clone can be judged as a non-monoclonal cell.
  • Figure 29 shows the results of monoclonal F8 positive and negative sequencing without jacket peaks (1,2,3,10,12,13,14,15,18,20,21,WT) and F8 wild-type sequence alignment results
  • white space represents deletion mutations
  • the insertion line represents insertion mutations. From the results, we can see that large fragment deletions occurred at 3, 10, 12, 14, 15, 18, and small fragment insertions or deletions occurred at two target sites at 1, 20, 21. Mutations, 2,13 are wild-type.
  • Sequencing results with nested peaks need to be combined with gel maps, wild-type sequences and theoretical deletion sequences for sequence estimation, and genotypes can also be determined by cloning vectors and cloning a large number of sequencing.
  • cloning vectors and cloning a large number of sequencing Taking monoclonal No. 4 as an example, the comparison between the sequencing results of clone No. 4 B1-F843 and the sequencing results of WT B1-F843 is shown in Figure 30. From the results, it can be seen that there is no wild-type sequence in the double peak of clone No. 4 (and The homozygous deletion mutation of No. 4 in the gel picture is judged the same), combined with Fig. 31 and Fig. 32, the deletion caused by the theory of F8-gRNA1 and F8-gRNA4, the sequence after the theoretical deletion (the space is the deletion point):
  • TTGTGACAGGGATGACGTGGAGCTTGACCC (SEQ ID NO: 93) with a 149 bp fragment deletion
  • TTGTGACAGGGAGCTTGACCCGCAGTCT (SEQ ID NO: 94) with a 158 bp fragment deletion.
  • the B1 gene coding sequence of clone 4 has mutations of -149bp and -158bp.
  • Miseq sequencing was used to further accurately determine the genotype of each single clone.
  • the construction of the monoclonal Miseq sequencing library, library quality inspection and on-machine sequencing were all commissioned to a third-party sequencing service company (Jinweizhi Company).
  • Figure 33 is the result of Miseq sequencing result of F8 monoclonal 1-23 and wild-type sequence comparison (each clone has 1 to 4 sequences). From the results, it can be judged that most of the clones have large fragment deletions, and some sequences are different A deletion or insertion mutation occurred in one of the two targets, and some sequences were wild-type sequences.
  • the F9 gene transcription map is shown in Figure 34 (the dark and wide lines in the figure are coding exons, and the light and wide lines are non-coding exons), including 8 exons and the second exon 164bp, the present invention It is planned to design the target in the second exon of F9 gene.
  • Primer3 is used for primer design, and the design results are as follows:
  • the Max enzyme (Vazyme's catalog number: P505) was used for PCR and electrophoresis to screen good amplification primers.
  • FIG. 35 The results of electrophoresis are shown in Figure 35, and 51 is the amplified band with F9-GT-F333/F9-GT-R1074 primer; 52 is the amplified band with F9-GT-F333/F9-GT-R1211 primer; 53 is F9-GT-F421/F9-GT-R1074 primer amplified band; 54 is F9-GT-F421/F9-GT-R1211 primer amplified band, screening 52 is F9-GT-F333/F9-GT-R1211 As an amplification primer.
  • the above PCR amplification products were sequenced using amplification primers (General Biologicals Sequencing).
  • the sequencing results were compared and analyzed using Snapgene and the F9 gene sequence published on the Internet. The results are shown in Figure 37.
  • the white vertical lines represent mutations or deletion mutations, and the black vertical lines represent insertion mutations. Design the target and detection primers to avoid designing the mutation sequence. .
  • F9 gene knockout target is 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 synthesized F9 (named B2) has a total of 4 target insertion sequence complementary DNA oligo as shown in Table 16:
  • 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 site synthesizes 2 pairs of complementary DNA oligo, which can be annealed to form a DNA double strand complementary to the sticky end of the pKG-U6gRNA vector BbsI after digestion ( Figure 18).
  • the B2-gRNA1 insertion sequence is shown in Figure 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 Figure 41.
  • the correct clones were named pKG-U6gRNA(B2-1), pKG-U6gRNA(B2-2), pKG-U6gRNA(B2-3), pKG-U6gRNA(B2-4), plasmid mini-pump, stored at -20°C for later use .
  • gRNA expression vectors pKG-U6gRNA (B2) and Cas9 expression vector pKG-GE3 were used to co-transfect porcine primary fibroblasts.
  • Mammalian fibroblast nuclear transfection kit (Neon kit, Thermofisher) and NeonTM transfection system electroporator were used for electroporation experiments.
  • Combination 1 pKG-U6gRNA(B2-1)+pKG-U6gRNA(B2-4)+pKG-GE3 theoretical B2 deletion 70bp
  • the electroporation reaction solution was prepared, and the system was as follows:
  • the pig primary cell electrotransformation and cell collection were carried out.
  • the larger band is the wild-type band
  • the smaller band is the band after deletion mutation
  • the brighter the smaller band (mutant band) is relative to the larger band (wild-type band), the higher the mutation efficiency.
  • B2 ie F9
  • the theoretical B2 deletion 44bp mutation efficiency is the highest.
  • Group 2 F9 mutation pKG-U6gRNA(B2-2)(0.46ug)+pKG-U6gRNA(B2-3)(0.46ug)+pKG-GE3(1.08ug).
  • the sequencing results are divided into two types, unset peaks and set peaks.
  • the unset peaks can be directly compared with the corresponding genome sequence of F9 through the sequencing results.
  • the sequence is performed to determine the two sequences contained in the set of peaks. If there is no set of peaks in the front and the set of peaks in the back are messy, the clone can be judged to be a non-monoclonal cell.
  • Figure 44 shows the sequencing results of monoclonal F9 without a set of peaks (24, 26, 27, 28, 29, 30 31, 33, 34, 35, 36, 38, 40, 41, 42, 43, 44, 47, WT)
  • the result of the comparison with the F9 wild-type sequence shows that 24,26,28,29,31,38,40 have homozygous deletion mutations, and the remaining monoclonal 27,30,33,34,35,36, 41,42,43,44,47 are wild-type, and there are no clones with small insertion or deletion mutations at one of the two targets.
  • Sequencing results with sets of peaks need to be combined with gel maps, wild-type sequences and theoretical deletion sequences for sequence estimation, and the genotype can also be determined by PCR product cloning and sequencing.
  • Miseq sequencing was used to further accurately determine the genotype of each single clone.
  • the construction of the monoclonal Miseq sequencing library, library quality inspection and on-machine sequencing were all commissioned to a third-party sequencing service company (Jinweizhi Company).
  • Figure 45 is the result of F9 gene editing monoclonal 24-50 Miseq sequencing result compared with the wild-type sequence (each clone has 1-2 kinds of sequences). From the results, it can be judged that most of the clones have large fragment deletions, and some of the sequences are in One of the two targets has a deletion or insertion mutation, and some sequences are wild-type sequences.
  • F8 mutation pKG-U6gRNA(B1-1)(0.23ug)+pKG-U6gRNA(B1-4)(0.23ug)+pKG-GE3(0.54ug);
  • F9 mutation pKG-U6gRNA(B2-2)(0.23ug)+pKG-U6gRNA(B2-3)(0.23ug)+pKG-GE3(0.54ug).
  • F8 mutation and F9 mutation gene editing components are mixed together to electrotransform pig primary fibroblasts.
  • the analysis diagram of the sequencing results of F8 gene PCR products of F8+F9 monoclonal F8 gene is shown in Figure 48.
  • the sequencing results are divided into two categories, unset peaks and set peaks.
  • the unset peaks can be directly compared with the corresponding genome sequences of F8 and F9 through the sequencing results. Yes, there is no set of peaks in the front and two sets of peaks in the back.
  • the two sequences contained in the set of peaks should be determined based on the wild-type sequence or the sequence after the theoretical deletion. If there is no set of peaks in the front and the set of peaks in the back is messy, you can judge this Clones are non-monoclonal cells.
  • Figure 48 shows the F8 sequencing results of non-set peak monoclonal forward and reverse sequencing results (51, 52, 53, 56, 57, 58, 66, 67, 69, 70, 71, 72, 75, WT) and F8 wild-type sequence
  • white spaces represent deletion mutations
  • insertion lines represent insertion mutations. From the results, we can see that clones 51,53,56,57,58,66,69,70,71,72 have homozygous mutations in large fragments of the F8 gene.
  • Clone 52, 67 had F8 gene target 1 or target 4 homozygous deletion of small fragments, 75 was F8 gene wild-type.
  • Figure 49 shows the results of F9 unset peak monoclonal forward and reverse sequencing results (52,53,54,55,56,59,62,64,65,66,68,69,75,WT) and F9 wild-type sequence alignment results , White spaces represent deletion mutations, and insertion lines represent insertion mutations. From the results, we can see that clones 53,65 have large fragment deletion homozygous mutations in the F9 gene, clones 52,54,55,56,59,62,64,66,68, 69 and 75 are wild-type F9 genes. Sequencing results with sets of peaks need to be combined with gel maps, wild-type sequences and theoretical deletion sequences for sequence estimation, and the genotype can also be determined by PCR product cloning and sequencing.
  • Miseq sequencing was used to further accurately determine the genotype of each single clone.
  • the construction of the monoclonal Miseq sequencing library, library quality inspection and on-machine sequencing were all commissioned to a third-party sequencing service company (Jinweizhi Company).

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Abstract

gRNA靶点组合在构建血友病模型猪细胞中的应用，包括三种gRNA靶点组合，具体是针对猪F8和F9基因各设计了一对靶点序列，构建了三种CRISPR/Cas9系统，将三种CRISPR/Cas9系统分别转入猪成纤维细胞，筛选基因突变株，获得A、B、A&B型血友病模型猪细胞系。通过改造Cas9表达载体，显著提高了基因编辑的效率。通过调整gRNA表达载体和Cas9表达载体的摩尔比，也显著提高编辑效率。运用体细胞核移植克隆技术可得到A、B、A&B型血友病疾病模型猪，用于进行药物筛选、药效评价、药理毒理、疾病病理、基因治疗及细胞治疗等研究，为进一步的临床应用提供有效的实验数据，也为成功治疗人类血友病提供有力的实验手段。

Description

gRNA靶点组合在构建A型、B型和A＆B型血友病模型猪细胞系中的应用 技术领域
本发明属于基因编辑技术领域，具体涉及gRNA靶点组合在构建A、B和A&B型血友病模型猪细胞系中的应用。
背景技术
血友病(heamophilia)通常是指一组由基因突变造成凝血因子缺乏的、X染色体连锁隐性遗传性出血性疾病，目前尚无有效的治愈方法。据流行病学调查结果显示，截至2018年，我国血友病患者数量约为13万，而真正能够做好预防性治疗的患者仅两成。凝血过程是一系列蛋白质的有限水解过程，一般分为三个阶段：凝血活酶形成、凝血酶形成和纤维蛋白形成。而血友病一般是在凝血的第一阶段即凝血活酶发生障碍，按照所缺乏的不同凝血因子通常可分为三类：A型血友病(因子Ⅷ缺乏)、B型血友病(因子Ⅸ缺乏)和C型血友病(因子Ⅺ缺乏)。发病率以A型血友病较多，约占85％；B型血友病约占15％；C型血友病则较少见。A型血友病为因子Ⅷ——抗血友病球蛋白(antihemophilic globulin,AHG)缺乏导致，相关基因定位于Xq28，基因跨度超过186kb，已发现有46种以上突变。B型血友病为因子Ⅸ——血浆凝血活酶成分(plasma thrombin component,PTC)缺乏导致，相关基因定位于Xq27.1，基因总长度为34kb左右，已鉴定出的突变有100种之多。C型血友病为因子Ⅺ——血浆凝血活酶前质(plasma thromboplastic antecedent deficiency,PTA)缺乏，相关基因定位于15q11，基因长度为23kb，现已发现3种突变。研究表明，在世界范围内，每5000名男性活婴中就有大约1人为A型血友病患者，B型血友病在每25000名男性中约有1人患病，而C型血友病种族倾向明显，多见于土耳其南部犹太人后裔，且遗传方式属常染色体隐性遗传。
血友病的治疗一般采用血液输注、凝血酶原复合物、凝血因子等手段，但血液制品存在着患者感染传染病的风险，凝血酶原复合物、凝血因子等蛋白制剂会导致患者产生针对这些蛋白的抗体，从而降低其治疗效果，甚至引起更严重的出血症状。因此，血友病动物模型的构建将在治疗研究中具有非常重要的作用。目前，关于血友病的动物模型以小鼠模型为主。早在20世纪90年代初，研究者就已依据基因打靶原理成功构建了小鼠的A型血友病模型。此后，B型血友病小鼠模型也被成功构建。但小鼠等啮齿类动物从体型、生理、病理等方面与人相差巨大，不能很好地模拟人类疾病，事实上，95％以上经小鼠验证有效的药物在人的临床试验中是无效的。因此，研发与人类体型及生理功能更近的大动物血友病模型是血友病治疗研究的关键。但目前尚未有任何人工构建的大动物血友病模型的报道。就大动物而言，灵长类是和人类亲缘关系最近的动物，但灵长类体型和器官大小与人相差较大，而且灵长类性成熟晚(猕猴初次交配年龄为6-7岁)，且为单胎动物，扩繁速度极慢。同时，灵长类动物克隆效率低、难度大、成本高，经济效益差，无法满足大规模的科研及商业需求。而猪是最适合作为疾病模型的动物，其体型大小、生理功能等与人非常接近，能很好地模拟人类的生理、病理特征。同时，猪性成熟早、繁殖周期短，一窝多胎、克隆技术成熟且成本较低，短时期内即可形成较大规模的群体。长期的近亲培育使猪的基因纯合度和遗传稳定性很高，遗传背景明确，表型相对稳定，在生物医学研究中的重复性好，符合科学研究的要求；同时，小型猪易饲养，成本低，而且猪作为人类长期以来的肉食性动物，用猪作为疾病模型动物不存在伦理等问题。
因此，用猪作为模型动物既可以克服大、小鼠等啮齿类动物与人种属差异较大的缺点，又可以克服灵长类动物成本高、繁殖周期长的缺陷，可以完全替代大、小鼠及灵长类模型动物，为广大药企、高校及科研院所提供性价比非常高的模式动物，将极大地促进生物医药的快速发展。
在此，我们通过基因编辑技术在猪原代成纤维细胞中对因子Ⅷ(F8)、因子Ⅸ(F9)基因分别造成单独突变和联合突变，得到F8、F9、F8&F9基因突变细胞，下一步运用克隆技术可 得到A、B、A&B型三种血友病疾病模型猪，用于进行药物筛选、药效检测、疾病病理、基因治疗及细胞治疗等研究，为进一步的临床应用提供有效的实验数据，也为成功治疗人类血友病提供有力的实验手段。
发明内容
本发明针对猪F8和F9基因各设计了一对靶点序列，利用该两对靶点序列构建了三种CRISPR/Cas9系统，将该三种CRISPR/Cas9系统分别转入猪成纤维细胞，筛选得到A、B及A&B型血友病模型猪细胞系。
本发明提供了gRNA靶点组合在构建血友病模型猪细胞系中的应用，所述gRNA靶点组合由第一gRNA靶点和第二gRNA靶点，和/或，第三gRNA靶点和第四gRNA靶点组成，
其中，所述第一gRNA靶点的碱基序列为TATAGTTGTGACAGGGACAT，第二gRNA靶点的碱基序列为CACAAGTCCAGAAGATGACG；
第三gRNA靶点的碱基序列为ATGCCACCAAAATTCTGCAT；第四gRNA靶点的碱基序列为AAACTGGAAGAGTTTGTTCG。
本发明还提供了双链DNA分子，包括粘性末端和靶点片段，所述靶点片段的碱基序列分别为：
TATAGTTGTGACAGGGACAT；
或CACAAGTCCAGAAGATGACG；
或ATGCCACCAAAATTCTGCAT；
或AAACTGGAAGAGTTTGTTCG。
本发明还提供了包含如所述双链DNA分子的表达盒或gRNA表达载体。
本发明还提供了一种CRISPR/Cas9系统，包括第一gRNA表达载体、第二gRNA表达载体和Cas9表达载体，所述第一gRNA表达载体的靶点序列为TATAGTTGTGACAGGGACAT，所述第二gRNA的靶点序列为CACAAGTCCAGAAGATGACG。
可选的，所述第一gRNA表达载体、第二gRNA表达载体和Cas9表达载体的摩尔比为1.5～2:1.5～2:1。进一步可选的，所述第一gRNA表达载体、第二gRNA表达载体和Cas9表达载体的摩尔比为1.5:1.5:1。
本发明还提供另一种CRISPR/Cas9系统，包括第三gRNA表达载体、第四gRNA表达载体和Cas9表达载体，所述第三gRNA表达载体的靶点序列为ATGCCACCAAAATTCTGCAT，所述第四gRNA表达载体的靶点序列为AAACTGGAAGAGTTTGTTCG。
可选的，所述第三gRNA表达载体、第四gRNA表达载体和Cas9表达载体的摩尔比为1.5～2:1.5～2:1。进一步可选的，所述第一gRNA表达载体、第二gRNA表达载体和Cas9表达载体的摩尔比为1.5:1.5:1。
本发明还提供第三种CRISPR/Cas9系统，包括第一gRNA表达载体、第二gRNA表达载体、第三gRNA表达载体、第四gRNA表达载体和Cas9表达载体，
所述第一gRNA表达载体的靶点序列为TATAGTTGTGACAGGGACAT，所述第二gRNA的靶点序列为CACAAGTCCAGAAGATGACG；
所述第三gRNA表达载体的靶点序列为ATGCCACCAAAATTCTGCAT，所述第四gRNA表达载体的靶点序列为AAACTGGAAGAGTTTGTTCG。
可选的，所述第一gRNA表达载体、第二gRNA表达载体、第三gRNA表达载体、第四gRNA表达载体和Cas9表达载体的摩尔比为0.75～1:0.75～1:0.75～1:0.75～1:1。进一步可选的，所述第一gRNA表达载体、第二gRNA表达载体、第三gRNA表达载体、第四gRNA表达载体和Cas9表达载体的摩尔比为0.75:0.75:0.75:0.75:1。可选的，所述Cas9表达载体的碱基序列如SEQ ID NO:70所示。
可选的，所述gRNA表达载体的原始载体为pKG-U6gRNA，pKG-U6gRNA的全序列如SEQ ID NO:71。
本发明还提供了构建血友病模型猪细胞系的方法，包括以下步骤：将任一种所述的CRISPR/Cas9系统转入猪耳原代成纤维细胞中，筛选基因突变单克隆细胞。
所述血友病包括A型、B型和A&B型。
本发明还提供了根据所述的方法构建的血友病模型猪细胞系。
可选的，所述Cas9表达载体的构建步骤包括：
以载体pX330-U6-Chimeric_BB-CBh-hSpCas9为原始载体，原始载体具有gRNA骨架序列、CMV增强子和Cas9基因；将所述CMV增强子下游的chickenβ-actin启动子替换为EF1a启动子；在所述Cas9基因的N端和C端各增加至少一个核定位编码序列NLS；
还可以包括：将所述gRNA骨架序列改造为如SEQ ID NO：70所示序列，以去除无效冗余序列。
还可以包括：在Cas9基因的C端NLS下游依次插入P2A-EGFP-T2A-PURO序列、WPRE基因、3’LTR基因和bGH polyA序列，增加荧光和抗性筛选标记及增强Cas9的表达效率。
本发明通过对F8、F9基因敲除预设靶点外显子及邻近基因组序列保守性分析，设计高效敲除的靶点序列，用该靶点序列构建gRNA表达载体，与高效表达Cas9的表达载体一起转入宿主细胞，显著提高了基因编辑效率。
本发明通过基因编辑技术在猪原代成纤维细胞中对F8、F9、F8&F9造成突变，得到各基因突变细胞，下一步运用体细胞核移植克隆技术可得到A、B及A&B型血友病疾病模型猪，用于进行药物筛选、药效评价、药理毒理、疾病病理、基因治疗及细胞治疗等研究，为进一步的临床应用提供有效的实验数据，也为成功治疗人类血友病提供有力的实验手段。
与现有技术相比，本发明至少具有如下有益效果：
(1)本发明研究对象(猪)比其他动物(大小鼠、灵长类)具有更好的应用性。目前未有任何大动物血友病模型被成功研发。本发明选取的研究对象为猪，构建其血友病模型细胞系，进而克隆生产血友病疾病模型猪，用来进行药物筛选及药理病理毒理等研究。
目前为止，仅有小鼠血友病模型被构建出，未有任何大动物血友病模型被成功研发。大小鼠等啮齿类动物不论从体型、器官大小、生理、病理等方面都与人相差巨大，无法真实地模拟人类正常的生理、病理状态。研究表明，95％以上在大小鼠中验证有效的药物在人类临床试验中是无效的。
就大动物而言，灵长类是与人亲缘关系最近的动物，但其体型小、性成熟晚(6-7岁开始交配)，且为单胎动物，群体扩繁速度极慢，饲养成本也高。另外，灵长类动物克隆效率低、难度大、成本高。
而猪作为模型动物就没有上述缺点，猪是除灵长类外与人亲缘关系最近的动物，其体型、体重、器官大小等与人相近，在解剖学、生理学、营养代谢、疾病发病机制方面等方面与人类极为相似。同时，猪的性成熟早(4-6个月)，繁殖力高，一窝多胎，在2-3年内即可形成一个较大群体。另外，猪的克隆技术非常成熟，克隆及饲养成本较灵长类低得多；而且猪作为人类长期以来的肉食性动物，用猪作为疾病模型动物不存在动物保护和伦理等问题。
(2)采用本发明改造的cas9高效表达载体进行基因编辑,编辑效率比原载体提高300～400％左右。
(3)gRNA载体和cas9载体不是按常规的1：1摩尔数比，而是按3：1的摩尔数比。
针对最终起作用的gRNA：cas9蛋白复合物来讲，gRNA载体转录出gRNA的时间要比cas9蛋白形成的时间早，而且转录出来的gRNA降解速度很快，因此如果在DNA载体水平，摩尔数比按1：1的话，由于gRNA的早转录及降解，最终会使cas9蛋白的摩尔数多于未降解的gRNA。经过实验比对，发现3:1或4:1要比1:1的gRNA:cas9载体摩尔比编辑效率更高。因此，本发明优选采用了3:1的gRNA:cas9的载体摩尔比。
(4)对每个靶基因，本发明采用双gRNA组合进行突变，与采用单gRNA相比，可有效降低非移码突变的产生，并可直接用PCR来检测基因编辑效率。
如果用单gRNA对靶基因进行突变，在DNA的非同源末端连接(NHEJ)随机修复中， 会有1/3的概率产生碱基的非移码突变，而非移码突变很可能不能破坏靶基因的功能，达不到使靶基因失活的预期目标。而用双gRNA对靶基因进行切割突变时，可使靶基因去除掉一个片段，通过设计去除非3倍数的碱基片段，可有效产生靶基因的片段缺失移码突变。同时也可以直接通过PCR手段来检测缺失片段的基因编辑产物，通过基因编辑产物与野生型产物(即未编辑产物)的比率可直接估算基因编辑的效率。
附图说明
图1为pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO载体(简称pKG-GE3)的图谱。
图2为原始载体pX330-U6-Chimeric_BB-CBh-hSpCas9的结构图谱。
图3为原始载体pX330经BbsI、XbaI酶切电泳结果图。
图4为全基因合成的gRNAsc1-6插入片段电泳图谱。
图5为重组载体pU6gRNACas9载体的结构图谱。
图6为pU6gRNA-eEF1a Cas9载体的结构图谱。
图7为pU6gRNACas9载体经XbaI、AgeI酶切胶图结果图。
图8为全基因合成的eEF1a1-14电泳结果图。
图9为pU6gRNA-eEF1a Cas9+nNLS载体图谱。
图10为pU6gRNA-eEF1a Cas9的AgeI、BglII酶切胶图结果图。
图11为全基因合成的N-NLS 1-12电泳结果图。
图12为载体pU6gRNA-eEF1a Cas9+nNLS的FseI、SbfI酶切胶图。
图13为拼接的2727bp片段的胶图结果。
图14所构建的pKG-U6gRNA载体图谱。
图15为DNA oligo退火后与pKG-U6gRNA载体连接示意图。
图16为gRNA和cas9载体最佳摩尔比筛选检测结果图。
图17为MSTN基因编辑效率的对比图。
图18为FNDC5基因编辑效率的对比图。
图19为猪F8基因转录图谱。
图20为F8基因扩增最佳引物筛选PCR结果图。
图21为采用经筛选的引物对8只猪F8基因的PCR扩增结果图。
图22为扩增得到的F8基因序列与发表的F8基因序列比对分析图。
图23为B1-gRNA1插入序列。
图24为B1-gRNA2插入序列。
图25为B1-gRNA3插入序列。
图26为B1-gRNA4插入序列。
图27为F8高效靶点gRNA组合筛选PCR结果图。
图28为组1 F8突变单克隆的F8 PCR(引物B1-F843/B1-R1258)检测结果图。
图29为无套峰的单克隆F8正反测序结果与发表的F8序列比对结果图。
图30为4号克隆B1-F843测序结果与WT的B1-F843测序结果对比图。
图31为F8-gRNA1和F8-gRNA4理论造成的缺失图谱。
图32为F8-gRNA1和F8-gRNA4造成的缺失测序图。
图33为F8单克隆1-23的Miseq测序结果与野生型序列比对结果图。
图34为猪F9基因转录图谱。
图35为F9基因扩增最佳引物筛选PCR结果图。
图36为采用经筛选的引物对8只猪F9基因的PCR扩增结果图。
图37为扩增得到的F9基因序列与发表的F9基因序列比对分析图。
图38为B2-gRNA1插入序列。
图39为B2-gRNA2插入序列。
图40为B2-gRNA3插入序列。
图41为B2-gRNA4插入序列。
图42为F9高效靶点gRNA组合筛选PCR结果图。
图43为组2 F9突变单克隆的F9 PCR(引物B2-F539/B2-R853)结果图。
图44为无套峰的单克隆F9测序结果与发表的F9序列比对结果图。
图45为F9基因编辑单克隆24-50Miseq测序结果与野生型序列比对结果。
图46 F8+F9突变单克隆的F8(引物B1-F843/B1-R1258)PCR检测结果。
图47 F8+F9突变单克隆的F9(引物B2-F539/B2-R853)PCR检测结果。
图48 F8+F9单克隆F8基因PCR产物测序结果分析图。
图49 F8+F9单克隆F9基因PCR产物测序结果分析图。
图50不同单克隆的F8 Miseq测序结果分析图谱。
图51不同单克隆的F9 Miseq测序结果分析图谱。
具体实施方式
1.Cas9高效表达载体的构建及应用效果检测
1.1 Cas9高效表达载体的构建
pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO载体(简称pKG-GE3)，从addgene(Plasmid#42230，from Zhang Feng lab)pX330-U6-Chimeric_BB-CBh-hSpCas9载体改造而来，通过改造启动子，增加核定位信号，加入增强翻译的WPRE序列，加入绿色荧光及抗性筛选puro基因，图谱如图1，碱基序列如SEQ ID NO：1所示。
所用原始载体pX330-U6-Chimeric_BB-CBh-hSpCas9的结构如图2所示，购自addgene(Plasmid#42230，from Zhang Feng lab)。
构建步骤如下：
(1)去除多余的短gRNA骨架
pX330-U6-Chimeric_BB-CBh-hSpCas9(图2)用BbsI、XbaI酶切，回收载体片段(约8313bp左右)，全基因合成插入片段175bp(SEQ I D NO：1)，与回收后载体片段重组得到pU6gRNACas9载体(图2)。
构建具体步骤如下：
1)用限制性内切酶BbsI、XbaI酶切pX330-U6-Chimeric_BB-CBh-hSpCas9质粒(酶切体系如表1，37℃反应2h。
表1
组份 量
ddH 2O To 50ul
pX330质粒 2ug
10XFD buffer 5ul
FD BbsI 1.5ul
FD XbaI 1.5ul
总量 50
2)酶切的pX330-U6-Chimeric_BB-CBh-hSpCas9质粒跑琼脂糖胶分离，结果如图3所示，用胶回收试剂盒(诺唯赞FastPure Gel DNA Extraction Mini Kit#DC301)纯化回收载体大片段，目的片段溶解在50ul ddH 2O中，-20℃备用。
3)使用DNAworks设计，全基因合成175bp插入片段，全基因合成引物如表2所示：
表2
gRNAsc-1 TGTGGAAAGGACGAAACACC(SEQ ID NO：2)
gRNAsc-2 TGCTATTTCTAGCTCTAAAACAGGTCTTCTCGAAGACCCGGTGTTTCGTCCTTTCCACA(SEQ ID NO：3)
gRNAsc-3 CCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAA(SEQ ID NO：4)
gRNAsc-4 CACGCGCTAGAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGC(SEQ ID NO：5)
gRNAsc-5 GTGCTTTTTTCTAGCGCGTGCGCCAATTCTGCAGACAAATGGCTCTAGAGGTACCCGTT(SEQ ID NO：6)
gRNAsc-6 TTATGTAACGGGTACCTCTAGAGCC(SEQ ID NO：7)
全基因合成PCR使用Phanta Max(诺唯赞P505)，按表3所示体系混合：
表3
组份 体积ul
ddH 2O To 50ul
2×Phanta Max Buffer 25ul
dNTP(10mM) 1ul
DMSO 2ul
Primer mix(10uM每条引物0.1ul) 0.6ul
F引物gRNAsc-1(10uM) 1ul
R引物gRNAsc-6(10uM) 1ul
Phanta Max 1ul
总量 50ul
PCR条件：95℃3min(95℃ 15s 58℃ 15s 72℃ 20s)循环32次72℃ 5min；4℃保存。通过全基因合成得到175bp插入片段(SEQ ID NO：1)，完成PCR后，PCR产物跑1％的琼脂糖电泳，进行分离，结果如图4所示，用胶回收试剂盒(诺唯赞FastPure Gel DNA Extraction Mini Kit#DC301)回收目的片段，目的片段溶解在50ul ddH 2O中，-20℃备用。
4)使用克隆重组试剂盒(诺唯赞ClonExpress II One Step Cloning Kit#C112)进行载体和175bp插入片段的重组。按表4所示体系加入各组份，混合，37℃反应30min，反应完成立即至于冰上，并用于转化。
表4
组份 体积ul
线性化载体 150ng
插入片段 0.04Xbp数ng
5x CE II Buffer 2ul
Exnase II 1ul
ddH 2O To 10ul
5)转化，克隆送测并质粒小抽
a)取100μL DH5α化学感受态细胞(Vazyme#C502)置于冰浴中；
b)向装有感受态细胞的离心管中加入10μL步骤(4)中得到的重组反应产物，混匀后在冰浴中静置30min；
c)将冰浴30min的感受态细胞置于42℃水浴中90s，然后迅速转移到冰浴中，使细胞冷却3min；
d)向离心管中加入300μL无菌的LB培养基(不含抗生素)，混匀后置于37℃摇床220rpm振荡培养60min；
f)将100uL的感受态细胞加到含相应抗生素的LB固体琼脂培养基上，用无菌的涂布棒将感受态细胞涂布均匀；将涂布有感受态细胞的LB固体琼脂培养基倒置于37℃培养箱中培养12～16h。
6)挑取克隆，培养，菌液送测，正确克隆小抽。
构建的平板挑取4个克隆，分别置于含300ul的含Amp抗性的LB培养基中，37℃培养过夜，第二天分出100ul分别用通用引物LKO1_5进行测序，测序结果正确的克隆，分别取菌液20ul到含3ml Amp LB的试管中过夜培养，第二天使用质粒小抽试剂盒进行质粒抽提，质粒-20℃保存备用。得到的重组载体pU6gRNACas9载体如图5。
(2)改造启动子及增强子
对构建好的pU6gRNACas9载体，用XbaI和AgeI内切酶去除启动子(CMV增强子)及增强子序列(chickenβ-actin)，回收线性载体序列约7650bp，并合成554bp包含CMV增强子及EF1a启动子的序列(SEQ ID NO：8)，与酶切载体pU6gRNACas9重组得到pU6gRNA-eEF1a Cas9载体(图6)。
构建具体步骤如下：
1)用限制性内切酶XbaI、AgeI酶切改造的pU6gRNACas9质粒
具体实验方法，参看如前所述pU6gRNACas9载体的改造过程中载体线性化部分。
pU6gRNACas9载体XbaI、AgeI酶切胶图结果如图7所示，回收载体大片段。
2)全基因合成554bp插入片段，全基因合成引物如表5所示：
表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)
全基因合成方法参看以上pU6gRNACas9载体的改造过程中全基因合成部分。
3)线性化载体和合成插入片段的重组参看以上pU6gRNACas9载体的改造过程中克隆重组部分。eEF1a1-14全基因合成电泳结果如图8所示，胶回收目的片段554bp。
4)挑取克隆、培养、菌液送测、正确克隆小抽
参看以上pU6gRNACas9载体的改造过程中挑取克隆、培养、菌液送测(同样使用通用引物LKO1_5进行测序)及正确克隆小抽部分。得到pU6gRNA-eEF1a Cas9载体如图6所示。
(3)Cas9基因N端增加NLS序列
构建好的载体pU6gRNA-eEF1a Cas9使用AgeI、BglII酶切，回收7786bp载体序列，并将增加了NLS的序列补充到酶切位点，合成以下序列447bp包括2个核定位信号及部分切除的Cas9编码序列(SEQ ID NO：23，重组得到pU6gRNA-eEF1a Cas9+nNLS载体(图9)。
构建具体步骤如下：
1)用限制性内切酶AgeI、BglII酶切改造的pU6gRNA-eEF1a Cas9质粒
具体实验方法，参看以上pU6gRNACas9载体的改造过程中载体线性化部分。pU6gRNA-eEF1a Cas9的AgeI、BglII酶切胶图结果如图10所示，回收载体大片段。
2)全基因合成447bp插入片段，全基因合成引物如表6所示：
表6
N-NLS-1 CCAGAACACAGGTTGGACCGGTGC(SEQ ID NO：24)
N-NLS-2 GATCCTTGTAGTCTCCGTCGTGGTCCTTATAGTCCATGGTGGCACCGGTCCAACCTGTG(SEQ ID NO：25)
N-NLS-3 CGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAGATGGCCC(SEQ ID NO：26)
N-NLS-4 TCTTCTTTGGGGACCCACCCACCTTTCGTTTCTTTTTGGGGGCCATCTTATCGTCATCG(SEQ ID NO：27)
N-NLS-5 GGTGGGTCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAA(SEQ ID NO：28)
N-NLS-6 CCCACAGAGTTGGTGCCGATGTCCAGGCCGATGCTGTACTTCTTGTCGGCTGCTGGGAC(SEQ ID NO：29)
N-NLS-7 CGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGA(SEQ ID NO：30)
N-NLS-8 CTTGATGCTGTGCCGGTCGGTGTTGCCCAGCACCTTGAATTTCTTGCTGGGCACCTTGT(SEQ ID NO：31)
N-NLS-9 GACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAAC(SEQ ID NO：32)
N-NLS-10 TATCTTCTTCTGGCGGTTCTCTTCAGCCGGGTGGCCTCGGCTGTTTCGCCGCTGTCGAA(SEQ ID NO：33)
N-NLS-11 GAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGA(SEQ ID NO：34)
N-NLS-12 GCCATCTCGTTGCTGAAGATCTCTTGCAGATAGCAGATCC(SEQ ID NO：35)
全基因合成方法参看以上pU6gRNACas9载体的改造过程中全基因合成部分，N-NLS 1-12全基因合成电泳结果如图11所示，胶回收447bp目的片段。
3)线性化载体和合成插入片段的重组
参看以上pU6gRNACas9载体的改造过程中克隆重组部分。
4)挑取克隆、培养、菌液送测、正确克隆小抽
参看以上pU6gRNACas9载体的改造过程中挑取克隆、培养、菌液送测(使用合成引物gRNA-F：ttttagagctaGAAAtagcaag进行测序)以及正确克隆小抽部分。得到pU6gRNA-eEF1a Cas9+nNLS载体图谱如图9所示。
(4)Cas9基因C端加入NLS、P2A-EGFP-T2A-PURO、WPRE-3’LTR-bGH polyA signals
以上构建好的载体命名为pU6gRNA-eEF1a Cas9+nNLS，使用FseI、SbfI酶切，回收载体序列7781bp，合成序列2727bp包括NLS-P2A-EGFP-T2A-PURO-WPRE-3’LTR-bGH polyA signals(SEQ ID NO：36)的序列，与载体片段重组得到载体pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO。
构建具体步骤如下：
1)用限制性内切酶FseI、SbfI酶切改造的pU6gRNA-eEF1a Cas9+nNLS质粒，回收7781bp线性载体片段：
具体实验方法参看以上pU6gRNACas9载体的改造过程中载体线性化部分。载体pU6gRNA-eEF1a Cas9+nNLS的FseI、SbfI酶切胶图如图12所示，回收载体大片段。
2)全基因合成2727bp插入片段
全基因合成方法参看以上pU6gRNACas9载体的改造过程中全基因合成部分。2727bp合成片段来源于3段片段重叠延伸PCR，具体包括：
片段一：合成含核定位信号编码序列及P2A，EGFP重叠序列192bp(SEQ ID NO：37)，合成引物序列见表7，通过全基因合成得到(方法参看pU6gRNACas9载体的改造过程中全基因合成部分)。
表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)
片段二：EGFP片段744bp(SEQ ID NO：68)，模板为商业化载体EGFP-N1，引物为表8，常规PCR得到。
表8
EGFP-F GTGAGCAAGGGCGAGGAGCTGTTCACCGG(SEQ ID NO：44)
EGFP-R TAGAAGACTTCCCCTGCCCTCGCCGGAGCCCTTGTACAGCTCGTCCATGCCGAGAGTG(SEQ ID NO：45)
片段三：T2A-PURO-WPRE-3’LTR-polyA signals序列1840bp(SEQ ID NO：69)，模板为LentiCRISPRV2(addgene Plasmid#52961)，引物如表9，用LentiCRISPRV2为模板V2-F/V2-R引物PCR，再以前步PCR产物为模板，T2A-F和V2-R PCR得到目的片段1840bp。
表9
T2A-F GAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGCCCA(SEQ ID NO：46)
V2-F TGCGGGGACGTGGAGGAAAATCCCGGCCCAACCGAGTACAAGCCCACGGTGCGCCTCG(SEQ ID NO：47)
V2-R taccgcatcaggcgcccctgcaggccatagagcccaccgcatccccagcatgcctg(SEQ ID NO：48)
上述三个片段为模板、使用引物C-NLS-1/V2-R PCR得到全长2727bp目的片段(SEQ ID NO：36)，最终拼接的2727bp片段胶图结果如图13所示。
3)线性化载体和合成插入片段的重组
线性化载体pU6gRNA-eEF1a Cas9+nNLS 7781bp和2727bp插入片段重组，方法参看以上pU6gRNACas9载体的改造过程中克隆重组部分。
4)挑取克隆、培养、菌液送测、正确克隆小抽
参看以上pU6gRNACas9载体的改造过程中挑取克隆，培养，菌液送测(使用合成引物Cas9-5-F：CCACCAGAGCATCACCGGCCTG(SEQ ID NO：49)和f1ori-R:cacacccgccgcgcttaatgcg(SEQ ID NO：50)进行测通)，正确克隆小抽部分。得到的最终改造载体pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO图谱如图1，碱基序列(SEQ ID NO：70)。
改造后载体pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO主要原件：
1)gRNA表达原件：U6-gRNA scaffold。
2)启动子：CMV增强子和EF1a杂合启动子。
3)含多个NLS的cas9基因：含N端和C端多核定位信号(NLS)的Cas9基因。
4)筛选标记基因：荧光和抗性双筛选标记原件P2A-EGFP-T2A-PURO。
5)增强翻译的元件：WPRE和3‘LTR，增强cas9及筛选标记基因的翻译。
6)转录终止信号：bGH polyA signal。
7)载体骨架：包括Amp抗性原件和ori复制子等。
1.2构建MSTN和FNDC5基因gRNA靶点载体来检测所改造的cas9载体的效率
pKG-U6gRNA载体：来源pUC57载体，通过EcoRV酶切位点，连接pKG-U6gRNA插入序列(含U6启动子、BbsI酶切位点和sgRNA骨架序列的DNA片段，即SEQ ID NO：67)，pKG-U6gRNA插入序列反向插入到pUC57载体，转化细菌后得到阳性克隆。pKG-U6gRNA载体全序列(SEQ ID NO：71)。
pKG-U6gRNA插入序列(第一段下划线部分为U6启动子序列，大写碱基字母段为两个BbsI酶切位点所在序列，第二段下划线部分为sgRNA骨架序列)：
Figure PCTCN2019114157-appb-000001
所构建的pKG-U6gRNA载体图谱如图14：
每个靶点合成2对互补的DNA oligo，通过退火可以形成与pKG-U6gRNA载体BbsI酶切后粘性末端互补的DNA双链，如图15，在合成靶点插入序列正义链时，由于U6启动子从启动子后第一个g开始转录，所以当靶点不为g开始的需要在靶点前加入一个g，同时在g前加入cacc与BbsI酶切后的载体U6端互补；合成靶点插入序列互补链时，需要合成g与靶点的互补序列，然后在5’端加入aaac序列，与BbsI酶切的gRNA骨架端序列互补。当靶点为g开始的，可以在正义链靶点前不加入g，同时在互补链的3端少加入一个与加入g互补的c。
设计MSTN两个gRNA靶点：
MSTN-gRNA1:GCTGATTGTTGCTGGTCCCG(SEQ ID NO：51)和
MSTN-gRNA2:TTTCCAGGCGAAGTTTACTG(SEQ ID NO：52)。
设计FNDC5两个gRNA靶点：
FNDC5-gRNA1：TGTACTCAGTGTCCTCCTCC(SEQ ID NO：53)和
FNDC5-gRNA2：GCTCTTCAAGACGCCTCGCG(SEQ ID NO：54)。
并分别在靶点两侧设计用于后期基因编辑效率检测的引物：
MSTN检测引物：
MSTN-F896 TCTCTCAGACAGTGCAGGCATTA(SEQ ID NO：55)
MSTN-R1351 CGTTTCCGTCGTAGCGTGATAAT(SEQ ID NO：56)
FNDC5检测引物：
FNDC5-F209 CAGTTCTCACTTGATGGCCTTGG(SEQ ID NO：57)
FNDC5-R718 AGGGGTCTGGGGAGGAATGG(SEQ ID NO：58)
根据四个靶点分别合成以下双链：
Figure PCTCN2019114157-appb-000002
将gRNA靶点序列克隆到pKG-U6gRNA载体骨架上，具体步骤如下：
(1)用限制性内切酶BbsI消化1ug pKG-U6gRNA质粒；
(2)酶切的pKG-U6gRNA质粒跑琼脂糖胶分离，用胶回收试剂盒纯化回收酶切产物；
(3)将上述寡核苷酸链gRNA-S和gRNA-A序列按照以下程序退火：
Figure PCTCN2019114157-appb-000003
95℃，5min然后以5℃/min的速率降至25℃。
(4)按照以下体系进行连接反应，37℃反应60min：
Figure PCTCN2019114157-appb-000004
(5)转化
1)取100μL感受态细胞(Vazyme)置于冰浴中；
2)向装有感受态细胞的离心管中加入20μL步骤(4)得到的连接质粒溶液，混匀后在冰浴中静置30min；
3)将冰浴30min的感受态细胞置于42℃水浴中90s，然后迅速转移到冰浴中，使细胞冷却3min；
4)向离心管中加入300μL无菌的LB培养基(不含抗生素)，混匀后置于37℃摇床220rpm 振荡培养60min；
5)将100uL的感受态细胞加到含相应抗生素的LB固体琼脂培养基上，用无菌的涂布棒将感受态细胞涂布均匀；将涂布有感受态细胞的LB固体琼脂培养基倒置于37℃培养箱中培养12～16h；
6)挑取克隆、培养、送公司测序，确定含靶点gRNA载体构建正确后，进行质粒小抽。
每个构建的平板挑取2个克隆，一共16个克隆，分别置于含300ul的含Amp抗性的LB培养基中，37℃培养过夜，第二天分出100ul分别用通用引物M13F或M13R进行测序(通用生物公司进行测序)，测序结果正确的克隆，分别取菌液20ul到含3ml Amp LB的试管中过夜培养，第二天使用质粒小抽试剂盒进行质粒抽提，并分别命名为pKG-U6gRNA(MSTN-1)，pKG-U6gRNA(MSTN-2)，pKG-U6gRNA(FNDC5-1)，pKG-U6gRNA(FNDC5-2)质粒备用。按照同样方法，小抽10管pKG-GE3质粒备用。
1.3 gRNA载体及Cas9载体最佳摩尔比筛选
为了确定使用双gRNA造成基因组片段缺失时，两个含靶点gRNA载体pKG-U6gRNA(约3.0kb)与Cas9载体pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO(简称pKG-GE3约10.5kb)，共同电转时三种质粒加入的最佳比例，本申请进行了gRNA载体和Cas9载体按照不同比例混合后电转，检测gRNA和Cas9质粒不同比例混合后电转细胞基因编辑效率，来确定最佳比例。
具体步骤如下：
(1)猪原代成纤维细胞制备
1)分别取刚出生丛江香猪雌性4只(1 2 3 4)，雄性4只(A B C D)的猪耳组织0.5g，去除外部组织，75﹪酒精浸泡30-40s。
2)用含5％P/S(Gibco Penicillin-Streptomycin)的PBS洗涤5次，不含P/S的PBS洗一次：
其中5％P/S的PBS配方为：5％P/S(Gibco Penicillin-Streptomycin)+95％PBS，5％、95％为体积百分比。
3)用剪刀将组织剪碎，加入5mL1％的胶原酶(Sigma)溶液，37℃摇床消化1h。
4)500g离心5min，去上清，将沉淀用1mL完全培养基重悬，铺入含10mL完全培养基并已用0.2％明胶(VWR)封盘的9cm细胞培养皿中。
其中，细胞完全培养基的配方为：15％胎牛血清(Gibco)+83％DMEM培养基(Gibco)+1％P/S(Gibco Penicillin-Streptomycin)+1％HEPES(Solarbio)，15％、83％、1％、1％为体积百分比。
5)置于37℃，5％CO 2(体积百分比)、5％O 2(体积百分比)的恒温培养箱中进行培养。
6)将细胞培养至长满皿底60％左右时使用0.25％(Gibco)的胰蛋白酶将细胞消化下来，然后加入完全培养基终止消化，将细胞悬液转入15mL离心管中，400g离心4min，弃去上清，使用细胞冻存液(90％完全培养基+10％DMSO，体积比)将细胞冻存备用。
(2)gRNA和Cas9表达载体不同摩尔比混合电转猪原代细胞
分别将Cas9表达载体pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO与上述构建的gRNA表达载体pKG-U6gRNA(MSTN-1)和pKG-U6gRNA(MSTN-2)共转染猪原代成纤维细胞。
使用哺乳动物核转染试剂盒(Neon kit,Thermofisher)与Neon TM transfection system电转仪进行电转实验。
分组如下：
组B(对照)：pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO，质粒实际用量2ug
组1：pKG-U6gRNA(MSTN-1)+pKG-U6gRNA(MSTN-2)+pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO，摩尔比为0.5：0.5：1，质粒实际用量为0.22ug+0.22ug+1.56ug，总量2ug
组2：pKG-U6gRNA(MSTN-1)+pKG-U6gRNA(MSTN-2)+pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO，摩尔比为1：1：1，质粒实际用量为0.36ug+0.36ug+1.27ug，总量2ug
组3：pKG-U6gRNA(MSTN-1)+pKG-U6gRNA(MSTN-2)+pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO，摩尔比为1.5：1.5：1，质粒实际用量为0.46ug+0.46ug+1.08ug，总量2ug
组4：pKG-U6gRNA(MSTN-1)+pKG-U6gRNA(MSTN-2)+pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO，摩尔比为2：2：1，质粒实际用量为0.53ug+0.53ug+0.93ug，总量2ug
配制电转反应液，体系如下：
Figure PCTCN2019114157-appb-000005
混匀过程中注意切勿产生气泡；
猪原代细胞电转及细胞收集：
1)将细胞用胰酶消化，得到的细胞悬液用PBS磷酸缓冲液(Solarbio)洗一遍，600g离心6min，弃去上清，使用7uL电转基本溶液R重悬细胞，重悬过程中要避免气泡的产生；
2)吸取7uL细胞悬液，加入步骤1)中的电转反应液中混匀，混匀过程中注意切勿产生气泡；
3)将试剂盒带有的电转杯放置于Neon TM transfection system电转仪杯槽内，加入3mL E Buffer；
4)用电转枪吸取10uL步骤3)得到的混合液，插入点击杯内，选择电转程序(1450V 10ms3pulse)，电击转染后立即在超净台内将电转枪中混合液转入到6孔板中，每孔含2mL15％胎牛血清(Gibco)+83％DMEM培养基(Gibco)+1％P/S(Gibco Penicillin-Streptomycin)+1％HEPES(Solarbio)的完全培养液；
5)混匀后放置于37℃，5％CO 2、5％O 2的恒温培养箱中进行培养；
6)电转6-12h换液，电转48h使用胰蛋白酶消化并收集细胞到1.5ml EP管中，后期进行突变效率PCR检测。
(3)PCR检测MSTN基因缺失突变效率
1)向上步收集在1.5mL离心管中的细胞中(根据细胞量，细胞过多需要适当稀释后取部分裂解)加入10uL配制的裂解液(KapaBiosystems:Kapa hotstart mouse genotyping kit,货号KK7352)裂解细胞，粗提细胞基因组DNA。
KAPA2G裂解液配制体系如下：
10X extract Buffer          1uL
Kapa Express extract enzyme 0.2uL
ddH 2O                       8.8uL
裂解过程：75℃15min—95℃5min—4℃，反应结束后基因组DNA于-20℃保存；
2)MSTN组使用MSTN-F896/MSTN-R1351引物进行PCR检测，PCR反应体系如下：
Figure PCTCN2019114157-appb-000006
反应条件如下
Figure PCTCN2019114157-appb-000007
Figure PCTCN2019114157-appb-000008
电泳分析结果如图16所示：gRNA1:gRNA2:cas9的摩尔比为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:无gRNA对照。从结果判断456bp条带为野生型条带(WT)，329bp(456bp-理论缺失127bp)左右条带为缺失突变条带(MT)。
根据公式：基因缺失突变效率＝100*(MT灰度/MT条带bp数)/(WT灰度/WT条带bp数+MT灰度/MT条带bp数)％，分别计算得到MSTN组1基因缺失突变效率为28.6％，MSTN组2基因缺失突变效率为77.8％，MSTN组3基因缺失突变效率为86.8％，MSTN组4基因缺失突变效率为81.5％，其中组3基因编辑效率最高，确定两个gRNA质粒与Cas9质粒最适用量为摩尔比1.5：1.5：1，质粒实际用量为0.46ug+0.46ug+1.08ug。
1.4 Cas9表达载体基因编辑效果测试
为了检测改造后的pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO载体相对于改造前的pX330-U6-Chimeric_BB-CBh-hSpCas9载体基因编辑效率。本发明利用构建的猪MSTN基因和FNDC5基因各两个gRNA靶点载体及改造的cas9载体，并通过电转猪原代成纤维细胞，通过PCR检测各基因发生的缺失突变效率来确定载体的基因编辑效率。
具体步骤：
(1)电转猪原代细胞
分别将Cas9表达载体pX330或改造的pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO与gRNA表达载体pKG-U6gRNA(MSTN-1)和pKG-U6gRNA(MSTN-2)或pKG-U6gRNA(FNDC5-1)和pKG-U6gRNA(FNDC5-2)共转染猪原代成纤维细胞。
使用哺乳动物核转染试剂盒(Neon)与Neon TM transfection system电转仪进行电转实验。
MSTN组B：pKG-U6gRNA(MSTN-1)和pKG-U6gRNA(MSTN-2)
MSTN组330：pX330+pKG-U6gRNA(MSTN-1)和pKG-U6gRNA(MSTN-2)
MSTN组KG：pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO+pKG-U6gRNA(MSTN-1)和pKG-U6gRNA(MSTN-2)
FNDC5组B：pKG-U6gRNA(FNDC5-1)和pKG-U6gRNA(FNDC5-2)
FNDC5组330：pX330+pKG-U6gRNA(FNDC5-1)和pKG-U6gRNA(FNDC5-2)
FNDC5组KG：pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO+pKG-U6gRNA(FNDC5-1)和pKG-U6gRNA(FNDC5-2)
配制电转反应液，体系如下：
Figure PCTCN2019114157-appb-000009
混匀过程中注意切勿产生气泡；
猪原代细胞电转及细胞收集方法请参看以上(1.3gRNA载体及Cas9载体最佳摩尔比筛选)部分。
(2)PCR检测MSTN和FNDC5基因突变效率
按以上(1.3gRNA载体及Cas9载体最佳摩尔比筛选)所描述的步骤，进行细胞裂解，PCR检测及电泳。
取PCR产物3ul，进行琼脂糖凝胶电泳分析，分析结果如图17和图18所示。图17为 MSTN基因编辑效率的对比，KG组比330组突变条带/野生型条带占比更多，说明KG基因编辑效率比330更高。图18为FNDC5基因编辑效率的对比，KG组比330组突变条带/野生型条带占比更多，说明KG基因编辑效率比330更高。
根据公式：基因缺失突变效率＝100*(MT灰度/MT条带bp数)/(WT灰度/WT条带bp数+MT灰度/MT条带bp数)％，分别计算得到MSTN-330组基因缺失突变效率为27.6％，MSTN-KG组基因缺失突变效率为86.5％。FNDC5-330组基因缺失突变效率为18.6％，FNDC5-KG组基因缺失突变效率为81.7％，改造后的载体pU6gRNA eEF1a-mNLS-hSpCas9-EGFP-PURO基因编辑效率提高明显(约3～4倍)。
2.F8基因的敲除
2.1 F8基因敲除靶点gRNA设计及构建
(1)猪耳组织基因组DNA的提取
使用Vazyme公司FastPure Cell/Tissue DNA Isolation Mini Kit(Vazyme Cat.DC102-01)分别进行8只小猪(雄性A B C D雌性1 2 3 4)猪耳组织的基因组DNA的柱式提取，最终基因组DNA溶解于灭菌的去离子水中，使用NanoDrop进行定量，-20℃保存备用。
(2)F8基因敲除预设靶点外显子及邻近基因组序列保守性分析
1)查找猪F8基因信息，如下
F8 coagulation factor VIII[Sus scrofa(pig)]Gene ID:397339
Location:chromosome:X Exon count:26
F8基因转录形式如图19(深色宽线条为编码外显子，浅色宽线条为非编码外显子)。包括26个外显子，其中第15外显子最大，为2454bp，(血友病人F8基因发生突变也经常发生在最大外显子上)，因此本发明计划将靶点设计在F8基因第15外显子内靠前位置。
2)F8基因敲除预设靶点外显子及邻近基因组序列PCR扩增引物设计
根据查到的猪F8基因组序列
( https://www.ncbi.nlm.nih.gov/nuccore/NC_010461.5？report＝genbank&from＝125265330&t o＝125350853&strand＝true)，设计引物扩增8只猪基因组样品F8基因外显子15靠前位点。
使用Primer3进行引物设计，设计结果如下：
Figure PCTCN2019114157-appb-000010
3)F8基因组PCR扩增引物筛选
使用猪(雌2)耳朵组织提取的基因组为模板，使用设计的两条上游和两条下游组合，Max酶(Vazyme公司货号：P505)进行PCR，并电泳，筛选好的扩增引物，结果如图20，图中47为F8-GT-F491/F8-GT-R1463引物扩增条带；48为F8-GT-F491/F8-GT-R1490引物扩增条带；49为F8-GT-F572/F8-GT-R1463引物扩增条带；50为F8-GT-F572/F8-GT-R1490引物扩增条带，确定采用48即F8-GT-F491/F8-GT-R1490为引物。
4)8只猪F8基因片段PCR扩增
分别以8个基因组模板(雄A B C D雌1 2 3 4)，引物F8-GT-F491/F8-GT-R1490，Max酶进行F8基因组片段的扩增，结果如图21。
5)F8基因序列保守性分析
以上PCR扩增产物，使用扩增引物进行测序(通用生物公司测序)。测序结果使用Snapgene与网上发表的F8基因序列进行比对分析，结果如图22，白色竖线代表突变或缺失突变，黑色竖线代表插入突变，设计靶点及检测引物需避免设计在突变序列上。
(3)在预设靶点外显子上保守区设计双gRNA靶点，可造成编码外显子发生移码突变
(1)使用synthego进行靶点gRNA设计
设计靶点需避开可能的突变位点，使用synthego进行靶点gRNA设计：
https://www.synthego.com/products/bioinformatics/crispr-design-tool
F8基因敲除靶点设计如下：
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)
各靶点组合及造成的理论缺失见表10。
表10靶点组合及造成的理论缺失
Figure PCTCN2019114157-appb-000011
根据比对结果，避开可能的突变位点，设计后期用于突变检测的引物如表11：
表11 F8检测引物
B1-F843 ctgggctgtgagtaaccagagtt(SEQ ID NO：80)
B1-R1258 GGCTTCTTGAAGATCAGATGAGG(SEQ ID NO：81)
合成的F8共4个靶点的插入序列互补DNA oligo如表12：
表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)
每个靶点合成2对互补的DNA oligo，通过退火可以形成与pKG-U6gRNA载体BbsI酶切后粘性末端互补的DNA双链(如图15)。
合成的F8基因靶点的插入序列互补DNA oligo退火后形成的双链插入序列如图23～图26所示：
B1-gRNA1插入序列如图23；B1-gRNA2插入序列如图24；B1-gRNA3插入序列如图25；B1-gRNA4插入序列如图26。
参看上述gRNA表达载体构建过程(1.2构建MSTN和FNDC5基因gRNA靶点载体来检测所改造的cas9载体的效率)进行F8 gRNA表达载体构建。
正确克隆分别命名为pKG-U6gRNA(B1-1)，pKG-U6gRNA(B1-2)，pKG-U6gRNA(B1-3)，pKG-U6gRNA(B1-4)，质粒小抽，-20℃保存备用。
2.2不同靶点gRNA载体组合与Cas9载体电转猪耳原代成纤维细胞
使用构建好的4个gRNA表达载体pKG-U6gRNA(B1)与Cas9表达载体pKG-GE3共转染猪原代成纤维细胞。使用哺乳动物成纤维细胞核转染试剂盒(Neon)与Neon TM transfection system电转仪进行电转实验。
组合1：pKG-U6gRNA(B1-1)+pKG-U6gRNA(B1-3)+pKG-GE3理论缺失136bp
组合2：pKG-U6gRNA(B1-1)+pKG-U6gRNA(B1-4)+pKG-GE3理论缺失151bp
组合3：pKG-U6gRNA(B1-2)+pKG-U6gRNA(B1-3)+pKG-GE3理论缺失86bp
组合4：pKG-U6gRNA(B1-2)+pKG-U6gRNA(B1-4)+pKG-GE3理论缺失101bp
组5：未电转细胞
按gRNA1:gRNA2:cas9＝1.5:1.5:1的摩尔比配制电转反应液，体系如下：
Figure PCTCN2019114157-appb-000012
Figure PCTCN2019114157-appb-000013
混匀过程中注意切勿产生气泡；
按照以上(1.3gRNA载体及Cas9载体最佳摩尔比筛选)中提到的电转方法进行猪原代细胞电转及细胞收集。
2.3对电转后细胞进行PCR检测，确定高效编辑靶点组合
按以上(1.3gRNA载体及Cas9载体最佳摩尔比筛选)所描述的步骤，进行细胞裂解，PCR检测(检测引物为：B1-F843/B1-R1258)及电泳分析，结果如下。
F8 gRNA组合突变效率PCR检测结果如图27，B1-51为组合1(gRNA 1,3)；B1-52为组合2(gRNA 1,4)；B1-53为组合3(gRNA 2,3)；B1-54为组合4(gRNA 2,4)。
较大条带为野生型条带，较小条带为缺失突变后条带，较小条带(突变条带)相对较大条带(野生型条带)越亮，则突变效率越高。实验结果显示组合2(pKG-U6gRNA(B1-1)+pKG-U6gRNA(B1-4)+pKG-GE3)理论缺失151bp，突变效率最高。
2.4制备猪耳原代成纤维细胞F8基因编辑单克隆细胞
(1)使用高效靶点gRNA组合与Cas9载体电转猪耳原代成纤维细胞
按照以上靶点gRNA载体组合与Cas9载体电转猪耳原代成纤维细胞的方法(见1.3gRNA载体及Cas9载体最佳摩尔比筛选)进行细胞电转，使用2号细胞(雌性，血型AO)，分成三组进行电转：
F8突变：pKG-U6gRNA(B1-1)(0.46ug)+pKG-U6gRNA(B1-4)(0.46ug)+pKG-GE3(1.08ug)。各成分摩尔比为：pKG-U6gRNA(B1-1):pKG-U6gRNA(B1-4):pKG-GE3＝1.5：1.5：1。
(2)分离单克隆，放大培养
1)将电转后细胞培养36-48小时后，进行单克隆分选，使用胰蛋白酶进行消化，完全培养基中和，500g离心5min，去上清，将沉淀用1mL完全培养基重悬，并适当稀释，用口吸管挑取单克隆转移到含200ul完全培养基的96孔板中，每组细胞挑取一板96个单克隆，放置于37℃，5％CO 2、5％O 2的恒温培养箱中进行培养，每2～3天换一次细胞培养基，期间用显微镜观察每孔细胞生长情况，排除无细胞及非单克隆细胞的孔；
2)待96孔板的孔中细胞长满孔底(大约2周左右)，使用胰蛋白酶消化并收集细胞，其中2/3细胞接种到含有完全培养基的6孔板中，剩余的1/3的细胞收集在1.5mL离心管中；
3)待6孔板细胞长至50％丰满度时使用0.25％(Gibco)的胰蛋白酶消化并收集细胞，使用细胞冻存液(90％完全培养基+10％DMSO，体积比)将细胞冻存。
(3)单克隆细胞培养后进行PCR检测，测序确定突变类型
1)PCR检测
收集在1.5mL离心管中的细胞，按照以上(四高效靶点gRNA组合筛选中2对电转后细胞进行PCR检测，确定高效编辑靶点组合)方法进行单克隆PCR检测。检测结果如图28。图28是组-1 F8突变单克隆的F8 PCR(引物B1-F843/B1-R1258)检测结果，从结果判断3,4,10,11,12,14,15为纯合缺失突变单克隆，5,6,7,16,19,22为杂合缺失突变单克隆，1,2,8,9,13,17,18,20,21未发生大片段缺失。
2)测序进一步确定各单克隆突变类型
以上PCR产物，通过胶回收(多带需都回收)用PCR引物进行测序(通用生物公司)，对测序结果进行分析，进一步确定各单克隆F8基因突变情况。
将测序结果分成两类，无套峰和有套峰，无套峰可通过测序结果直接与F8对应基因组序列进行比对，前面无套峰，后面出现2个套峰，需根据野生型序列或理论缺失后序列进行确定套峰中含有的两种序列，如果前面无套峰，后面套峰较乱，可以判断该克隆为非单克隆细胞。图29为无套峰的单克隆F8正反测序结果(1,2,3,10,12,13,14,15,18,20,21,WT)与 F8野生型序列比对结果，白色空格代表缺失突变，插入线条代表插入突变，从结果可以看出3,10,12,14,15,18发生了大片段缺失，1,20,21发生了两靶点处的小片段的插入或缺失突变，2,13为野生型。
含套峰测序结果需结合胶图与野生型序列及理论缺失序列等进行序列推测，也可通过克隆载体，克隆大量测序来确定基因型。以4号单克隆为例，4号克隆B1-F843测序结果与WT的B1-F843测序结果对比图如图30所示，从结果看出，4号克隆双峰中没有野生型序列存在(与胶图中4号发生纯合缺失突变判断一致)，结合图31和图32，F8-gRNA1和F8-gRNA4理论造成的缺失，发生理论缺失后的序列(空格处为缺失点)：
TTGTGACAGGGA ACGTGGAGCTTGACCCGCAGTCTGGAGAGA(SEQ ID NO：90)
其中一条序列为：TTGTGACAGGGANNACGTGGAGCTTGACCC(SEQ ID NO：91)
推测另一条序列为：TTGTGACAGGGANNTTGACCCGCAGTCT(SEQ ID NO：92)根据序列推测NN为GC；
这两条序列分别为：TTGTGACAGGGATGACGTGGAGCTTGACCC(SEQ ID NO：93)发生149bp片段缺失和TTGTGACAGGGAGCTTGACCCGCAGTCT(SEQ ID NO：94)，发生158bp片段缺失。
则4号克隆B1基因编码序列发生-149bp和-158bp突变。
按照此类方法对所有套峰克隆进行序列分析。
(4)Miseq测序进一步确定单克隆细胞突变类型
1)高通量测序文库构建及测序
通过高通量测序(Miseq测序)进一步准确确定每个单克隆的基因型。单克隆Miseq测序文库的构建、文库质检及上机测序均委托第三方测序服务公司(金唯智公司)完成。
2)高通量测序结果及分析
图33是F8单克隆1-23的Miseq测序结果与野生型序列比对结果图(每个克隆有1～4种序列)，从结果可以判断大部分克隆发生了大片段缺失，有的序列分别在两个靶点中的一个发生了缺失或插入突变，有的序列为野生型序列。
通过具体序列的分析，各克隆基因型如表13：
表13
Figure PCTCN2019114157-appb-000014
Figure PCTCN2019114157-appb-000015
Miseq分析结果与PCR产物测序分析结果一致。F8基因编辑细胞单克隆1-23中，纯合突变细胞克隆有：1,3,4,10,11,12,14,15,16,18,19,20,21,22；野生型细胞克隆有：2,13；杂合突变克隆有：5,6,7；非单克隆有：8,9,17。
3.F9基因的敲除
3.1 F9基因敲除靶点gRNA设计及构建
(1)猪耳组织基因组DNA的提取
方法见2.1 F8基因敲除靶点gRNA设计及构建。
(2)F9基因敲除预设靶点外显子及邻近基因组序列保守性分析
1)查找猪F9基因信息，如下：
F9 coagulation factor IX[Sus scrofa(pig)]Gene ID:397518
Location:X Exon count:8
F9基因转录图谱如图34所示(图中深色宽线条为编码外显子，浅色宽线条为非编码外显子)，包括8个外显子，第2外显子164bp，本发明计划将靶点设计在F9基因第2外显子内。
2)F9基因敲除预设靶点外显子及邻近基因组序列PCR扩增引物设计
根据查到的猪F9基因组序列
( https://www.ncbi.nlm.nih.gov/nuccore/NC_010461.5？report＝genbank&from＝114218704& to＝114250436)，设计引物扩增8只猪基因组样品F9基因外显子2及两侧序列。
使用Primer3进行引物设计，设计结果如下：
Figure PCTCN2019114157-appb-000016
Figure PCTCN2019114157-appb-000017
3)F9基因组PCR扩增引物筛选
使用猪(雌2)耳朵组织提取的基因组为模板，使用设计的两条上游和两条下游组合，Max酶(Vazyme公司货号：P505)进行PCR，并电泳，筛选好的扩增引物。电泳结果如图35所示，与中51为F9-GT-F333/F9-GT-R1074引物扩增条带；52为F9-GT-F333/F9-GT-R1211引物扩增条带；53为F9-GT-F421/F9-GT-R1074引物扩增条带；54为F9-GT-F421/F9-GT-R1211引物扩增条带，筛选52即F9-GT-F333/F9-GT-R1211作为扩增引物。
4)8只猪基因组模板F9基因组片段PCR扩增
分别以8个基因组模板(雄A B C D雌1 2 3 4)，引物F9-GT-F333/F9-GT-R1211，Max酶进行F9基因组片段的扩增，结果如图36。
5)F9基因序列保守性分析
以上PCR扩增产物，使用扩增引物进行测序(通用生物公司测序)。测序结果使用Snapgene与网上发表的F9基因序列进行比对分析，结果如图37，白色竖线代表突变或缺失突变，黑色竖线代表插入突变，设计靶点及检测引物需避免设计在突变序列上。
(3)在预设靶点外显子上保守区设计双gRNA靶点，可造成编码外显子发生移码突变(1)使用synthego进行靶点gRNA设计
设计靶点需避开可能的突变位点，使用synthego进行靶点gRNA设计
https://www.synthego.com/products/bioinformatics/crispr-design-tool
F9基因敲除靶点设计如下：
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)
靶点组合及造成的理论缺失见表14。
表14靶点组合及造成的理论缺失
5端靶点序列 方向 编号 3端靶点序列 方向 编号 缺失长度bp
TGGTGGCATTTTCACGATCA - 1 AACTGGAAGAGTTTGTTCGA + 4 70
ATGCCACCAAAATTCTGCAT + 2 AAACTGGAAGAGTTTGTTCG + 3 44
根据比对结果，避开可能的突变位点，设计后期用于突变检测的引物，见表15。
表15 F9检测引物
B2-F539 gggattaccgggattaaaaacaa(SEQ ID NO：103)
B2-R853 tttttattctttgctccgcatgt(SEQ ID NO：104)
合成的F9(命名为B2)共4个靶点的插入序列互补DNA oligo如表16：
表16 F9的4个靶点的插入序列互补DNA oligo
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)
每个靶点合成2对互补的DNA oligo，通过退火可以形成与pKG-U6gRNA载体BbsI酶切后粘性末端互补的DNA双链(如图18)。
合成的F9基因靶点的插入序列互补DNA oligo通过退火得到的双链插入序列如图38～图 41所示：
B2-gRNA1插入序列如图38；B2-gRNA2插入序列如图39；B2-gRNA3插入序列如图40；B2-gRNA4插入序列如图41。
参看上述gRNA表达载体构建过程(1.2构建MSTN和FNDC5基因gRNA靶点载体来检测所改造的cas9载体的效率)进行F9gRNA表达载体构建。
正确克隆分别命名为pKG-U6gRNA(B2-1)，pKG-U6gRNA(B2-2)，pKG-U6gRNA(B2-3)，pKG-U6gRNA(B2-4)，质粒小抽，-20℃保存备用。
3.2不同靶点gRNA载体组合与Cas9载体电转猪耳原代成纤维细胞
使用构建好的4个gRNA表达载体pKG-U6gRNA(B2)与Cas9表达载体pKG-GE3共转染猪原代成纤维细胞。使用哺乳动物成纤维细胞核转染试剂盒(Neon kit,Thermofisher)与Neon TM transfection system电转仪进行电转实验。
组合1：pKG-U6gRNA(B2-1)+pKG-U6gRNA(B2-4)+pKG-GE3理论B2缺失70bp
组合2：pKG-U6gRNA(B2-2)+pKG-U6gRNA(B2-3)+pKG-GE3理论B2缺失44bp
组3：未电转细胞
按gRNA1:gRNA2:cas9＝1.5:1.5:1的摩尔比配制电转反应液，体系如下：
Figure PCTCN2019114157-appb-000018
混匀过程中注意切勿产生气泡；
按照以上(1.3gRNA载体及Cas9载体最佳摩尔比筛选)中提到的电转方法进行猪原代细胞电转及细胞收集。
3.3对电转后细胞进行PCR检测，确定高效编辑靶点组合
按以上(1.3gRNA载体及Cas9载体最佳摩尔比筛选)所描述的步骤，进行细胞裂解，PCR检测(检测引物为：B2-F539/B2-R853)及电泳分析，结果如下。
F9组合gRNA突变效率PCR检测结果如图42；B2 1,4为组合1；B2 2,3为组合2；B2 WT为组3。
较大条带为野生型条带，较小条带为缺失突变后条带，较小条带(突变条带)相对较大条带(野生型条带)越亮，则突变效率越高。根据实验结果判断B2(即F9)中，组合2pKG-U6gRNA(B2-2)+pKG-U6gRNA(B2-3)+pKG-GE3，理论B2缺失44bp突变效率最高。
3.4制备猪耳原代成纤维细胞F9基因编辑单克隆细胞
(1)使用高效靶点gRNA组合与Cas9载体电转猪耳原代成纤维细胞
按照以上靶点gRNA载体组合与Cas9载体电转猪耳原代成纤维细胞的方法(见1.3gRNA载体及Cas9载体最佳摩尔比筛选)进行细胞电转，使用2号细胞(雌性，血型AO)，分成三组进行电转：
组2 F9突变：pKG-U6gRNA(B2-2)(0.46ug)+pKG-U6gRNA(B2-3)(0.46ug)+pKG-GE3(1.08ug)。各成分摩尔比为：pKG-U6gRNA(B2-2)：pKG-U6gRNA(B2-3)：pKG-GE3＝1.5：1.5：1。
(2)分离单克隆，放大培养
方法步骤参见上述2.4制备猪耳原代成纤维细胞F8基因编辑单克隆细胞。
(3)单克隆细胞培养后进行PCR检测，测序确定突变类型
1)PCR检测
收集在1.5mL离心管中的细胞，按照以上(1.3gRNA载体及Cas9载体最佳摩尔比筛选) 方法进行单克隆细胞裂解，PCR检测及电泳分析。组2 F9突变单克隆的F9 PCR(引物B2-F539/B2-R853)检测结果如图43，从结果判断24,26,29,31,37,38,40,49,50为纯合缺失突变单克隆，25,28,39,45,46,48为杂合缺失突变单克隆，27,30,32,33,34,35,36,41,42,43,44,47未发生大片的缺失。
2)测序进一步确定各单克隆突变类型
以上PCR产物，通过胶回收(多带需都回收)用PCR引物进行测序(通用生物公司)，对测序结果进行分析，进一步确定各单克隆F9基因突变情况。
将测序结果分成两类，无套峰和有套峰，无套峰可通过测序结果直接与F9对应基因组序列进行比对，前面无套峰，后面出现2个套峰，需根据野生型序列或理论缺失后序列进行确定套峰中含有的两种序列，如果前面无套峰，后面套峰较乱，可以判断该克隆为非单克隆细胞。
图44为无套峰的单克隆F9测序结果(24,26,27,28,29,30 31,33,34,35,36,38,40,41,42,43,44,47,WT)与F9野生型序列比对结果图，从比对结果判断24,26,28,29,31,38,40发生了纯合缺失突变，剩余单克隆27,30,33,34,35,36,41,42,43,44,47为野生型，没有出现在两靶点之一处发生小片段插入或缺失突变的克隆。
含套峰测序结果需结合胶图与野生型序列及理论缺失序列等进行序列推测，也可通过PCR产物克隆测序来确定基因型。
(4)Miseq测序进一步确定单克隆细胞突变类型
1)高通量测序文库构建及测序
通过高通量测序(Miseq测序)进一步准确确定每个单克隆的基因型。单克隆Miseq测序文库的构建、文库质检及上机测序均委托第三方测序服务公司(金唯智公司)完成。
2)高通量测序结果及分析
图45是F9基因编辑单克隆24-50Miseq测序结果与野生型序列比对结果(每个克隆有1-2种序列)，从结果可以判断大部分克隆发生了大片段缺失，有的序列分别在两个靶点中的一个发生了缺失或插入突变，有的序列为野生型序列。
通过具体序列的分析，各克隆基因型如表17：
表17
Figure PCTCN2019114157-appb-000019
Figure PCTCN2019114157-appb-000020
从以上分析结果看出，成功构建得到了F9基因突变细胞株，其中F9纯合突变细胞克隆有：24,25,26,28,29,31,37,38,40,48,49,50；杂合突变克隆有：32,39,45,46。
4.制备F8和F9基因联合敲除的单克隆细胞
(1)使用B1、B2高效靶点gRNA组合与Cas9载体电转猪耳原代成纤维细胞
使用上述实验筛选出的最佳B1、B2靶点gRNA组合，按上述电转猪耳原代成纤维细胞的方法(见1.3gRNA载体及Cas9载体最佳摩尔比筛选)将gRNA组合与Cas9载体进行细胞电转，使用2号细胞(雌性，血型AO)，分成三组进行电转：
F8突变：pKG-U6gRNA(B1-1)(0.23ug)+pKG-U6gRNA(B1-4)(0.23ug)+pKG-GE3(0.54ug)；
F9突变：pKG-U6gRNA(B2-2)(0.23ug)+pKG-U6gRNA(B2-3)(0.23ug)+pKG-GE3(0.54ug)。
上述F8突变和F9突变各基因编辑成分混合在一起，电转猪原代成纤维细胞。
上述各成分摩尔比为：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)分离单克隆，放大培养
方法步骤参见上述2.4制备猪耳原代成纤维细胞F8基因编辑单克隆细胞。
(3)单克隆细胞培养后进行PCR检测，测序确定突变类型
1)PCR检测
收集在1.5mL离心管中的细胞，按照以上(1.3gRNA载体及Cas9载体最佳摩尔比筛选)方法进行单克隆细胞裂解，PCR检测及电泳分析。检测结果如图46和图47。
从组3 F8+F9突变单克隆的F8(引物B1-F843/B1-R1258)和F9(引物B2-F539/B2-R853)PCR检测结果判断51,53,57,70为F8 F9双基因缺失突变单克隆，52,55,59,67,75为F8、F9均未发生缺失突变单克隆，54,56,61,66,69,73为F8纯合缺失突变F9未发生缺失突变单克隆，62,63,64,68,74为F8杂合缺失突变F9未发生缺失突变单克隆，65为F8发生杂合缺失突变F9发生纯合缺失突变单克隆，71,72为F8纯合缺失突变F9杂合缺失突变单克隆。
2)测序进一步确定各单克隆突变类型
以上PCR产物，通过胶回收(多带需都回收)用PCR引物进行测序(通用生物公司)，对测序结果进行分析，进一步确定各单克隆F8及F9基因突变情况。
F8+F9单克隆的F8基因PCR产物测序结果分析图如图48，将测序结果分成两类，无套峰和有套峰，无套峰可通过测序结果直接与F8和F9对应基因组序列进行比对，前面无套峰，后面出现2个套峰，需根据野生型序列或理论缺失后序列进行确定套峰中含有的两种序列，如果前面无套峰，后面套峰较乱，可以判断该克隆为非单克隆细胞。
图48为F8测序结果无套峰单克隆正反向测序结果(51,52,53,56,57,58,66,67,69,70,71,72,75,WT)与F8野生型序列比对结果，白色空格代表缺失突变，插入线条代表插入突变，从结果可以看克隆51,53,56,57,58,66,69,70,71,72发生F8基因大片段缺失纯合突变，克隆52,67发生F8基因靶点1或靶点4处纯合小片段缺失，75为F8基因野生型。
F8+F9单克隆的F9基因产物测序结果分析如图49。图49是F9无套峰单克隆正反测序结果(52,53,54,55,56,59,62,64,65,66,68,69,75,WT)与F9野生型序列比对结果，白色空格代表缺失突变，插入线条代表插入突变，从结果可以看克隆53,65发生F9基因大片段缺失纯合突变，克隆52,54,55,56,59,62,64,66,68,69,75为F9基因野生型。含套峰测序结果需结合胶图与野生型序列及理论缺失序列等进行序列推测，也可通过PCR产物克隆测序来确定基因型。
(4)Miseq测序进一步确定单克隆细胞突变类型
1)高通量测序文库构建及测序
通过高通量测序(Miseq测序)进一步准确确定每个单克隆的基因型。单克隆Miseq测序文库的构建、文库质检及上机测序均委托第三方测序服务公司(金唯智公司)完成。
2)高通量测序结果及分析
不同单克隆(51-75)的F8 Miseq测序结果分析图谱见图50，除60(未得到文库产物)外F8的Miseq测序结果与F8野生型序列比对结果，从结果可以判断大部分克隆发生F8基因组片段缺失，有部分序列分别在两个靶点之一存在插入或缺失突变，有部分克隆含有野生型序列。
不同单克隆(51,53,57,60,61,63,65,67,70,71,72,73,74)的F9 Miseq测序结果与F9野生型序列比对结果见图51。从结果可以判断大部分克隆发生F9基因组片段缺失，有部分序列分别在2个靶点中的一个发生了插入或缺失突变，有部分克隆含有野生型序列。
通过具体序列的分析，各克隆基因型如表18。
表18 F8+F9单克隆miseq测序结果分析
Figure PCTCN2019114157-appb-000021
Figure PCTCN2019114157-appb-000022
在F8和F9基因同时编辑的单克隆细胞(51-75)中，成功构建F8和F9同时纯合突变的细胞克隆有：51,53,57,61,65,70,73。

Claims (13)

  1. gRNA靶点组合在构建血友病模型猪细胞系中的应用，所述gRNA靶点组合由第一gRNA靶点和第二gRNA靶点，和/或，第三gRNA靶点和第四gRNA靶点组成，其特征在于，其中，所述第一gRNA靶点的碱基序列为TATAGTTGTGACAGGGACAT，第二gRNA靶点的碱基序列为CACAAGTCCAGAAGATGACG；
    第三gRNA靶点的碱基序列为ATGCCACCAAAATTCTGCAT；第四gRNA靶点的碱基序列为AAACTGGAAGAGTTTGTTCG。
  2. 双链DNA分子，包括粘性末端和靶点片段，其特征在于，所述靶点片段的碱基序列分别为：
    TATAGTTGTGACAGGGACAT；
    或CACAAGTCCAGAAGATGACG；
    或ATGCCACCAAAATTCTGCAT；
    或AAACTGGAAGAGTTTGTTCG。
  3. 包含如权利要求2所述双链DNA分子的表达盒或gRNA表达载体。
  4. CRISPR/Cas9系统，包括第一gRNA表达载体、第二gRNA表达载体和Cas9表达载体，其特征在于，
    所述第一gRNA表达载体的靶点序列为TATAGTTGTGACAGGGACAT，所述第二gRNA表达载体的靶点序列为CACAAGTCCAGAAGATGACG。
  5. 根据权利要求4所述的CRISPR/Cas9系统，其特征在于，所述第一gRNA表达载体、第二gRNA表达载体和Cas9表达载体的摩尔比为1.5～2:1.5～2:1。
  6. CRISPR/Cas9系统，包括第三gRNA表达载体、第四gRNA表达载体和Cas9表达载体，其特征在于，
    所述第三gRNA表达载体的靶点序列为ATGCCACCAAAATTCTGCAT，所述第四gRNA表达载体的靶点序列为AAACTGGAAGAGTTTGTTCG。
  7. 根据权利要求6所述的CRISPR/Cas9系统，其特征在于，所述第三gRNA表达载体、第四gRNA表达载体和Cas9表达载体的摩尔比为1.5～2:1.5～2:1。
  8. CRISPR/Cas9系统，包括第一gRNA表达载体、第二gRNA表达载体、第三gRNA表达载体、第四gRNA表达载体和Cas9表达载体，其特征在于，
    所述第一gRNA表达载体的靶点序列为TATAGTTGTGACAGGGACAT，所述第二gRNA表达载体的靶点序列为CACAAGTCCAGAAGATGACG；
    所述第三gRNA表达载体的靶点序列为ATGCCACCAAAATTCTGCAT，所述第四gRNA表达载体的靶点序列为AAACTGGAAGAGTTTGTTCG。
  9. 根据权利要求8所述的CRISPR/Cas9系统，其特征在于，所述第一gRNA表达载体、第二gRNA表达载体、第三gRNA表达载体、第四gRNA表达载体和Cas9表达载体的摩尔比为0.75～1:0.75～1:0.75～1:0.75～1:1。
  10. 根据权利要求4-9任一所述的CRISPR/Cas9系统，其特征在于，所述Cas9表达载体的碱基序列如SEQ ID NO:70所示。
  11. 根据权利要求4-9任一所述的CRISPR/Cas9系统，其特征在于，gRNA表达载体的原始载体为pKG-U6gRNA。
  12. 构建血友病模型猪细胞系的方法，其特征在于，包括以下步骤：将权利要求4-9任一所述的CRISPR/Cas9系统转入猪耳原代成纤维细胞中，筛选基因突变单克隆细胞。
  13. 根据权利要求12所述的方法构建的A、B和A&B型血友病模型猪细胞系。
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