Construction And Verification Of A Novel CRISPR/SpCas9-hRed? Gene Editing System

CRISPR gene editing technology is widely favored because of its simple design,high activity,and can meet the editing needs of most gene regions.Animal gene editing and breeding based on CRISPR technology has become a research hotspot in recent years.The CRISPR/Cas9 system is the most widely used in scientific research due to its earliest development,high activity and wide application range.At present,researchers have developed a variety of improved CRISPR/Cas9 systems,which have greatly enriched the CRISPR gene editing technology system.Among them,fusion expression of functional protein and Cas9 protein to strengthen or expand its function is a universal and effective improvement strategy.In this study,the codon humanized optimized phage single-stranded annealing protein gene h Red? was introduced into the CRISPR/Sp Cas9 system,and a new CRISPR/Sp Cas9-h Red? gene editing system was constructed,and the systemic gene editing function verification was carried out.The main research contents are as follows:(1)First synthesize the codon-humanized optimized h Red? gene and construct the corresponding expression vector;combine the h Red? and CRISPR/Sp Cas9 expression vector with the reporter vector based on NHEJ or SSA repair(NHEJ-PG/SSA-PG)Transfect HEK293 T cells to detect the effect of co-expression of h Red? and Sp Cas9 on the effect of gene editing.The results show that co-expression of h Red? and Sp Cas9 can effectively increase the repair efficiency of NHEJ by 20% and the repair efficiency of SSA by 1.4 times.(2)To further target the genomic EMX1 locus,co-transfect HEK293 T cells with h Red?and CRISPR/Sp Cas9 expression vectors and the donor DNA template to detect and analyze the NHEJ and HDR editing efficiency of the genomic EMX1 target.The results were similar to the level of the reporter vector.The editing efficiency of NHEJ in the co-expression group of h Red? and Sp Cas9 was increased by about 1.7 times,while the editing efficiency of HDR was reduced by 29%.(3)Using three different fusion expression strategies,h Red?-MHGGS-Sp Cas9,h Red?-XTEN-Sp Cas9 and Sp Cas9-h Red?,h Red? and Sp Cas9 were fused and expressed to establish a CRISPR/Sp Cas9-h Red? gene editing system.The validation results of gene editing experiments show that the three fusion expression strategies can improve the editing efficiency of NHEJ mediated by the CRISPR/Sp Cas9 system to varying degrees,and the editing efficiency of NHEJ mediated by Sp Cas9-h Red? is 3.6 times that of Sp Cas9.In summary,this study has established a CRISPR/Sp Cas9-h Red? gene editing system that can improve the efficiency of NHEJ editing,providing new tools for editing low-efficiency sites in gene editing research,and further enriching the CRISPR gene editing technology.