Effect Of Co-expression And Fusion Expression Of YRad52,Ad4 E1B55K,Ad4 E4orf6 And Cas9 On HDR Efficiency

Since the advent of the CRISPR/Cas9 system as a new generation of genome editing tools,it has caused a frenzy in the field of life sciences,especially in mammalian cells.Mammalian cells repair double strand breaks(DSBs)through a variety of pathways,including non-homologous end joining(NHEJ)and homology-directed repair(HDR)approach.After the DSB is generated by CRISPR/Cas,it is repaired mainly through the NHEJ pathway,which is easy to introduce random insertions or deletions and is an inexact repair approach.In the case of providing a exogenous DNA template with homology to the target locus,the cells repair DSB through the precise HDR pathway.However,the efficiency of HDR in DSB repair is much lower than NHEJ.The inefficiency of HDR is still the bottleneck of percise genome editing.How to improve the efficiency of HDR has become a hot field for scientists.In recent years,researchers have developed a variety of strategies to improve HDR efficiency,such as inhibiting the NHEJ pathway,enhancing the HDR pathway and controlling the cell cycle,all of which can improve HDR efficiency to varying degrees.Based on related research,this study successfully integrated the yeast Rad52(yRad52),adenovirus 4(Ad4)E4orf6,and Ad4 E1B55 K proteins into the CRISPR/Cas9 system,and compared the effects of the Cas9 protein with the recombinant proteins yRad52?Ad4 E4orf6 or Ad4 E1B55 K protein co-expression strategies and fusion expression strategies on HDR efficiency are expected to provide new technical means for improving the efficiency of precise genome editing.The main results of this study are as follows:1.In order to verify our hypothesis in a reporter system,a HDR efficiency detection fluorescence report system was designed and constructed.Cas9 with yRad52 protein,Ad4 E1B55 K,Ad4 E4orf6 protein,fusion expression vector,reporter vector and donor vector were co-transfected into HEK293 T cells,and the fluorescence of the cells were analyzed by flow cytometric analysis.Statistical analysis of FACS data showed that the fusion expression Cas9-Rad52 could significantly improve the HDR efficiency by 1.5 times,and the co-expression Cas9-T2A-E4 increased HDR efficiency by 1.5 times at the reporter system.2.To further verify the conclusions at the genome level,Cas9 and yRad52,Ad4 E1B55 K,Ad4 E4orf6 co-expression vector and fusion expression vector were co-transfected into HEK293 T cells with HDR-USR1 reporter vector and donor vector,respectively,HDR-USR1 is a new universal screening report carrier developed by the laboratory,which can screen and enrich HDR-positive cells.The results of the enzyme digestion products showed that the HDR efficiency of Cas9-Rad52 fusion expression was 17.1±0.5% at the EMX1 locus in HEK293 T cells,which was 2 times of that in the control group.The HDR efficiency of the co-expression strategy Cas9-T2A-Rad52/Cas9-T2A-E1 B was 12.3±0.7% and 12.1±0.7%,which is 1.4 times of that in the control group.3.The Cas9-Rad52 fusion expression vector was used in conjunction with the Ad 4 E1B55 K protein.Gray-scale analysis showed that its HDR efficiency was 16.3%,which was a 3.9-fold improvement than the control group,indicating that the yRad52 protein and Ad4 E1B55 K protein have a synergistic effect at the EMX1 locus of HEK293 T cells.This shows that the combination of Cas9-Rad52 and Ad4 E1B55 K can further improve HDR efficiency.In conclusion,the co-expression and fusion expression of Cas9 with yRad52 and the Cas9-T2A-E1 B co-expression strategy can enhance the HDR efficiency in the mammalian cell.Among them,the fusion expression Cas9-Rad52 greatly improved HDR efficiency.And the combination of yRad52 and Ad4 E1B55 K protein has a synergistic effect on promoting HDR efficiency.This research provides an effective strategy for improving the efficiency of HDR precision genome editing,and helps to promote the application of gene editing in basic functional research,precision medicine,and animal genetic and breeding.