| CRISPR/Cas9 technology is a new transgenic technology.With the research on the structure and working principle of CRISPR/Cas9 in recent years,CRISPR/Cas9 has been successfully applied in animal husbandry production,disease treatment,organ transplantation,environmental protection,drug research and development,gene research and other fields.Parotid Secretory Protein(PSP)gene is located in the 17 th chromosome of the pig,can express a lot in the parotid gland.As an exocrine organ,the secreted protein of the parotid gland has the advantages of strong tissue specificity,few expressed protein species,large secretion,no influence on gender and developmental period,easy collection and purification,etc.,which makes the parotid gland as a bioreactor has great research potential.Although there are studies on transgenic pigs that use the parotid gland to express foreign genes,most of them use traditional transgenic technology,and there are problems such as random integration of foreign genes,low integration efficiency,poor genetic stability,poor expression stability,and the expression level of exogenous genes in different transgenic lines varied greatly due to the differences in the number of integrative copies,integration sites and integration modes.Through gene targeting,exogenous genes are connected with endogenous genes through 2A sequence or IREs sequence,so that exogenous genes are expressed in single copy form under the regulation of complete endogenous gene regulation elements,which not only ensures the normal expression of endogenous genes,but also solves the above problems of traditional transgenic technology.Based on the PSP gene model of pigs,this study aims to establish a method system for site-directed integration of porcine PSP gene of pigs by using CRISPR/Cas9 system,screen out sgRNA with high knock-in activity of targeted PSP gene,and analyze the off-target effect of CRISPR/Cas9 on PSP gene integration,laying a foundation for the preparation of transgenic pigs of the above types.Firstly,the cDNA sequence of PSP gene was obtained by searching the NCBI gene library.Zhang Lab online sgRNA design website was used to screen out 5 pairs of sgRNA sequences targeting PSP gene of pigs,and the corresponding sgRNA expression plasmid vector was constructed.Vector plasmids were used as the template for PCR amplification and purification of sgRNA in vitro transcription template,and then sgRNA in vitro transcription kit was used to transcribe the corresponding sgNRA.Pig genomic DNA was used as template to PCR amplify in vitro cleavage substrate and purify.The Cas9 protein from Cas9 in vitro enzyme cutting kit,in vitro cutting substrate and sgRNA were mixed and incubated according to the instructions of Cas9 in vitro enzyme cutting kit.The cutting efficiency was observed and detected by 1% agarose gel electrophoresis.The results of in vitro digestion showed that sgRNA1,sgRNA3,sgRNA4 and sgRNA5 could guide Cas9 protein to cleave the in vitro cleavage substrate into two segments,indicating that sgRNA1,sgRNA3,sgRNA4 and sgRNA5 had higher activity to guide Cas9 protein in vitro enzymatic digestion,which could be used in target experiments.The sgRNA2 failed to cut the substrate in vitro,indicating that sgRNA2 did not have activity to guide Cas9 protein in vitro enzymatic digestion.The sgRNA targeting PSP gene with high in vitro digestion activity targeting PSP gene was screened to construct the sgRNA and Cas9 co-expression vector pCas9-sgRNA.The upstream and downstream homology arms of the targeting vector were amplified by PCR using pig genomic DNA as a template,and the pCMV-NeoR-bGH plasmid was cleaved by restriction endonucleases Mlu? and Xho? to obtain the expression structure of neomycin resistance gene(NeoR).The expression structure of NeoR was ligated with the upstream and downstream homology arms to construct the targeting vector pMD19-5'arm-NeoR-3'arm.The sgRNA and Cas9 co-expression vectors and targeting vectors were co-transfected into porcine kidney cells(PK-15 cells),and after screening with G418,single cell clones were isolated by infinite dilution.The positive monoclonal cells were identified by PCR amplification and sequencing,which were site-directed integration by NeoR.The results of PCR amplification identification and sequencing of the selected monoclonal cells showed that 5 of the 22 cell clones screened with sgRNA1 as the guidance sgRNA were positive monoclonal cells,with the target efficiency of 22.7%.Twelve of the 24 cell clones screened by sgRNA3 as the guidance sgRNA were positive monoclonal cells,and the target efficiency was 50.0%.eight of the 19 cell clones screened by sgRNA4 as the guidance sgRNA were positive monoclonal cells,and the target efficiency was 42.1%.Six of the 23 cell clones screened by sgRNA5 as the guidance sgRNA were positive monoclonal cells,with a target efficiency of 26.1%.In order to further analyze the potential off-target sites of the screened positive monoclonal cells,the online sgRNA design software CCTop was used for online analysis of the screened sgRNA,and 5 potential off-target sites with high possibility of miss were selected for each.The designed primers used the positive monoclonal cells as the template for PCR amplification and sequencing.The results showed that the first potential off-target site sgRNA1-1 occurred off-target effect,which was one of the five potential off-target sites guided by sgRNA1.No off-target effects occurred in the five potential off-target sites guided by sgRNA3.No off-target effects occurred in the five potential off-target sites guided by sgRNA4.the fifth potential off-target site sgRNA5-5 occurred off-target effect,which was one of the five potential off-target sites guided by sgRNA5.In summary,through in vitro screening,target experiment and off-target analysis,two pairs of sgRNA(sgRNA3 and sgRNA4)that effectively guide exogenous genes to site-specific integrate into pig PSP gene were finally screened out,and a method for gene editing of pig PSP gene was successfully established,providing basic data for in-depth study of transgenic pigs. |
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