CRISPR质粒和同源重组模板的共转染可降低CRISPR/Cas9系统的基因编辑效率

Co-delivery of CRISPR plasmid and HDR donor can reduce the gene editing efficiency of CRISPR/Cas9 system

目的探讨CRISPR质粒和同源重组模板的共转染对CRISPR/Cas9系统的基因编辑效率的影响以及可行的解决办法。方法用T7E1内切酶实验和RT-qPCR检测只转染CRISPR质粒组和质粒和模板共转染组细胞内Cas9的切割效率,Cas9 RNA和DNA水平的差异;RT-qPCR检测降低共转染的模板DNA的数量时,细胞内Cas9 DNA和RNA水平的变化;RT-qPCR和T7E1内切酶实验检测先转染CRISPR质粒后转染同源重组模板时,细胞内Cas9 DNA和RNA水平以及切割效率的变化。结果与只转染CRISPR质粒组相比,CRISPR质粒和同源重组模板的共转染显著降低了CRISPR质粒的转染效率(P<0.001)和Cas9的切割效率(P<0.001)。而先转染质粒后转染同源重组模板组和只转染质粒组在CRISPR质粒的转染效率和Cas9的切割效率差别无统计学意义。结论CRISPR质粒和同源重组模板的共转染降低了CRISPR/Cas9系统的基因编辑效率,而先转染质粒后转染同源重组模板可以解决这一问题。

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 was 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 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 among 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.