Abstract: Objective To explore the effect of co-transfection of CRISPR plasmid and HDR donors on the gene editing efficiency of CRISPR/Cas9 system and a feasible solution to the problem. Methods 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; RT-qPCR were used to detect the changes of intracellular Cas9 DNA and RNA level when reducing the quantity of co-transfected template DNA; RT-qPCR and T7E1 assay were used to measure the DNA level, RNA level and cleavage activity of Cas9 when delivering CRISPR plasmid followed by HDR donors. Results Compared with the group only CRISPR plasmid transfected, co-transfection of CRISPR plasmid and HDR donors significantly reduced the transfection efficiency of CRISPR plasmid (P<0.001) and the cleavage activity of Cas9 (P<0.001). But there was no significant difference in transfection efficiency of CRISPR plasmid and cutting efficiency of CRISPR/Cas9 system between only CRISPR plasmid transfected group and groups sequentially transfected with CRISPR plasmid and HDR donors. Conclusions Co-transfection of CRISPR plasmid and HDR donors can reduce the gene editing efficiency of CRISPR/Cas9 system and this problem can be solved by delivering CRISPR plasmid first and then HDR templates.

Key words: CRISPR/Cas9 system, co-transfection, sequential transfection, homology-directed repair template