应用Surrogate报告载体提高CRISPR/Cas9对TMEM215基因的打靶效率

Improving Gene Targeting Efficiency on TMEM215 Mediated by CRISPR/Cas9 by Using Surrogate Reporter

目的:构建Surrogate报告载体,并利用Surrogate报告载体提高CRISPR/Cas9对HEK293T细胞TMEM215基因打靶效率。方法:构建针对人TMEM215的CRISPR/Cas9表达载体及相应Surrogate报告载体,两者共转HEK293T细胞,通过流式分析、T7EI检测、TA克隆测序等明确Surrogate报告载体对不同sgRNA打靶效率的检测及对基因修饰细胞的筛选富集作用。结果:流式分析结果表明,Surrogate报告载体成功检测出不同sgRNA的打靶效率,并筛选出高效率sgRNA;T7EI检测及TA克隆测序显示,外加嘌呤霉素抗性筛选时,Surrogate报告载体可有效富集基因修饰细胞。结论:成功构建Surrogate报告载体,并利用Surrogate报告载体提高CRISPR/Cas9对HEK293T细胞TMEM215基因的打靶效率。

Objective： This work aims to construct the surrogate reporter and use it to improve gene targeting efficiency on human TMEM215 in HEK293T ceils. Methods： The CRISPR/Cas9 expressing vectors targeting human TMEM215 and the related surrogate reporter vectors were constructed and co-transfected into HEK293T cell line. The gene targeting efficiency was determined by Fluorescence-activated cell sorting （FACS）, T7EI assay and sequencing analysis. Results： FACS results showed that the surrogate reporters can be used as an indicator of the gene targeting efficiency of each sgRNA. T7EI assay and sequencing analysis further confirmed the results and showed that puromycin selected cell populations are highly enriched with cells containing mutations induced by Cas9/sgRNA complex. Conclusions： Surrogate reporter can improve gene targeting efficiency mediated by CRISPR/Cas9 on TMEM215 in HEK293T cell line.