Construction Of TLR3,TLR4 Gene Knockout Cell Line And Chicken Model By CRISPR/Cas9

CRISPR/Cas9 technology is a new generation of gene editing technology developed in recent years,composed of Cas9 nuclease and target-specific sequence of sgRNA,widely used in animal and plant gene editing research,has been successful in cynomolgus monkey,Rat,Mice,rice and Arabidopsis thaliana and other species to achieve accurate gene modification.Innate immunity is the first line of defense against the pathogenic microbial infection,and can quickly start to invade the pathogenic microorganisms.TLR3 and TLR4 as pattern recognition receptors(PRRs),recognize specific pathogen-associated molecular patterns(PAMPs),and activate natural immune responses.In this study,the CRISPR/Cas9 system was used to edit the TLR3 and TLR4 genes in poultry DF1 cell line and chicken,which was of great significance to the study of the innate immune response and the pathogenic mechanism of infectious diseases.In this study,the recombinant plasmids p UC19-TLR3-sgRNA1,2,pUC19-TLR4-sgRNA1,2 were constructed by targeting the TLR3 and TLR4 in the first exon using the CRISPR/Cas9 double plasmid system according to the sgRNA design principle,which was with pCAG-Cas9-EGFP co-transfected into DF-1 cells.Cells of expression GFP were sorted by flow cytometry.The genomic,PCR,cloning and sequencing were used to estimate the activity of sgRNA.And the single cell clones were cultured.The TLR3 and TLR4 knockout DF1 cell lines were screened by genotype and sequencing.Each one of the DF1 cell lines with TLR3 and TLR4 knockout was obtained,the results showed that CRISPR/Cas9 double plasmid system could be used to edit the DF1 cell line.Lv-EF1a-Cas9-U6-sgRNA,which was started with EF1 a as the promoter,was used to construct.The recombinant promoter CAG of CMV and avian ?-actin was used as promoter to start the lentivirus expression vector Lv-CAG-Cas9-U6-sgRNA.Each one of the DF1 cell lines with TLR3 and TLR4 knockout was obtained,by transfection of DF1 cell line,flow sorting,culture single cell cloning,genotype and sequencing analysis.The results showed that the strategy of co-expression of Cas9 and sgRNA in lentiviral vector could be used for gene editing in avian DF1 cell lines.The lentiviral expression vector was packaged into lentivirus,and the lentivirus was injected into the hypocotyls of the chicken embryo through the equatorial window method.346 fertilized eggs targeting the TLR3 gene were injected and 40 chicken were obtained,TLR4 gene fertilized eggs 307,32 chicken were obtained;hatching rates were 11.6% and 10.4%.348 development end of chicken embryo were detected by PCR,polyacrylamide gel electrophoresis and sequencing.Two of the chickens' embryo were found to have gene deletion.A total of 10 chickens exist in the presence of Cas9 and sgRNA integration,but did not find the gene deletion chickens.This study demonstrated that the CR9PR/Cas9 double plasmid system,co-expressing the Cas9-sgRNA lentiviral vector startegy can be in the avian DF-1 cell lines to achieve gene editing;exploring CRISPR/Cas9 lentivirus system for gene editing of poultry,which laid the foundation for the study of gene knockout Chicken model.