细胞周期因子Geminin对CRISPR/Cas9编辑效率的影响

Effect of cell cycle regulator Geminin on the genome editing efficiency of CRISPR/Cas9

[目的]旨在探究细胞周期因子Geminin对Cas9在细胞基因组中的编辑效率是否有影响。[方法]将Gemi-nin的前40个氨基酸序列对应的cDNA序列克隆到含靶向COSMC基因的sgRNA的pX459质粒pX459-COSMC-Cas9(wtCas9).上得到能在Cas9蛋白N端融合表达Geminin片段的pX459-COSMC-Geminin-Cas9质粒(Geminin-Cas9)。在人胚胎肾细胞HEK293T或人结肠癌细胞HT29细胞中同时表达wtCas9和Geminin-Cas9蛋白质,检测并比较细胞中Cas9的表达水平和Cas9编辑COSMC基因的效率。[结果]Westerm Blotting检测发现相比wtCas9质粒转染Geminin-Cas9质粒的细胞中Cas9表达水平更低,Geminin-Cas9载体对COSMC基因的编辑效率在编辑前期也相对较低,但随着编辑时间的延长,两者的编辑效率趋于一致。[结论]细胞周期因子Geminin的存在能降低Cas9在细胞中的总体表达水平,在编辑前期会在一定程度上降低Cas9的编辑效率,而延长编辑时间又能消除这一影响。

[Objective]The aim of this study was to explore whether the cell cycle regulator Geminin has an effect on the ge-nome editing efficiency of Cas9 in cells.[Method]The cDNA sequences corresponding to the first 40 amino acids of Geminin were cloned into the pX459 plasmid,which contains a COSMC targeting sgRNA(wtCas9),to obtain the Geminin-Cas9 plas-mid.The Geminin-Cas9 could express the Cas9 fused with Geminin at the N-terminus.The wtCas9 and Geminin-Cas9 plas-mids were expressed in HEK293T and HT29 cells,and the expression level of Cas9 in cells and the editing eficiency of Cas9 on COSMC gene were detected and compared.[Result]W estern Blotting analysis showed that the expression level of Cas9 in the cells transfected with Geminin-Cas9 was lower than the cells transfected with wtCas9,and the editing efficiency of Geminin-Cas9 on COSMC gene was also relatively lower in the early stage of genome editing.However,the editing fficiency of wtCas9 and Geminin-Cas9 tended to be the same as the editing time goes on.[Conclusion]The existence of cell cycle regulator Gemi-nin can reduce the overall expression level of Cas9 in cells,and the editing efficiency of Geminin-Cas9 is found to reduce to some extent in the early stage of editing.However,the fficiency of Geminin-Cas9 is restored to the level of wtCas9 in the ex-tension of editing time.