利用CRISPR/Cas9n系统敲除人源SNF5基因

Knock-out of Human SNF5 Gene by CRISPR/Cas9n System

目的:建立敲除人源基因组中SNF5基因的CRISPR/Cas9n系统。方法:设计一对靶向人源SNF5基因第1个外显子的sg RNA,分别克隆至p X461、p X462表达载体后,转入人胚肾293T细胞,通过Western印迹检测细胞株中SNF5基因的敲除效果。结果:测序证明构建的靶向SNF5基因CRISPR/Cas9n重组质粒与设计吻合。Western印迹结果显示,重组质粒p X461-h SNF5sg RNA转染293T细胞后24 h,细胞内SNF5表达水平明显降低;重组质粒p X462-h SNF5sg RNA转染293T细胞后48 h,细胞内SNF5表达水平显著降低。结论:通过CRISPR/Cas9n系统获得了靶向SNF5基因的重组质粒,构建的重组质粒能有效敲除SNF5基因的表达。

Objective：To knock out human SNF5 gene by CRISPR/Cas9 n system.Methods：A pair of sgRNAs targeting exon 1 of human SNF5 gene were designed and subcloned into the pX461 and pX462 vectors,and then transfected into human embryo kidney 293 T cells.The effect of recombination plasmids on the expression of SNF5 gene was identified by western blotting.Results：CRISPR/Cas9 n plasmids targeting SNF5 gene were confirmed by DNA sequencing.Western blotting showed that SNF5 protein decreased obviously when 293 T cells were transfected with recombinant plasmid pX461-hSNF5 sgRNAs for 24 h,and decreased significantly when 293 T cells were transfected with recombinant plasmid pX462-hSNF5 sgRNAs for 48 h.Conclusion：CRISPR/Cas9 n palsmids targeting human SNF5 gene were successfully constructed and could knock out the expression of SNF5 gene effectively.