The Co-transfection Of CRISPR Plasmid And Homologous Recombination Template Reduces The Gene Editing Efficiency Of The CRISPR/Cas9 System

CRISPR/Cas9 system can recognize and cut sequence-specific DNA.It is a widely used gene editing tool.Although CRISPR/Cas9 system can generate gene knockout efficiently,the efficiency of accurate gene editing is still low.It is mainly due to the low efficiency of homology-directed repair(HDR).Many methods have improved the efficiency of CRISPR/Cas9-mediated homologous recombination to some extent,such as suppressing non-homologous end-joining(NHEJ)repair pathway,optimizing the design of homologous templates.A recent study has reported that the sequential delivery of Cas9 RNP and homologous templates by electroporation increased HDR efficiency.But the mechanism behind it is unknown.Therefore,this study explored the effect of simultaneous transfection with CRISPR plasmid and HDR donors on the gene editing efficiency of CRISPR/Cas9 system and a feasible solution to reduce the influence.T7E1 assay and RT-qPCR were used to compare the cleavage efficiency of Cas9 protein,Cas9 RNA and DNA level between cells transfected with CRSIPR plasmid only and cells co-transfected with CRISPR plasmid and HDR templates.The changes of intracellular Cas9 DNA and RNA level were detected in the condition of reducing the quantity of co-transfected template DNA.Imaging the green fluorescence intensity in cells transfected with GFP plasmid and single-stranded DNA(ssDNA)or double-stranded DNA(dsDNA).Finally,to identify whether sequential delivery of CRISPR plasmid and HDR donors can reduce the above influence,we delivered CRISPR plasmid first and then HDR donors,and measured the changes in DNA level,RNA level and cleavage activity of Cas9.Compared with CRISPR plasmid-only transfected group,co-transfection of CRISPR plasmid and HDR donors significantly reduced the transfection efficiency of CRISPR plasmid and the cleavage activity of Cas9.The complementary fragments of double-stranded DNAs also reduced the transfection efficiency of co-transfected GFP plasmid.But groups sequentially transfected with CRISPR plasmid and HDR donors had no significant difference from CRISPR plasmid-only transfected group in transfection efficiency of CRISPR plasmid and cutting efficiency of Cas9 protein.To conclude,co-transfection of CRISPR plasmid and HDR donors reduced the gene editing efficiency of CRISPR/Cas9 system and this problem can be solved by delivering CRISPR plasmid followed by HDR templates.These results help to optimize transfection methods of CRISPR/Cas9 system and further improve the efficiency of CRISPR/Cas9 system-mediated homologous recombination.