运用CRISPR/Cas9技术敲除人源黏着斑蛋白基因

摘要

Objective:To knock out human vinculin(VCL) gene by CRISPR/Cas9 system.Methods:A singleguide RNA(sgRNA) targeting the exon 3 of human VCL gene was designed and cloned into vector,then by lentivirus it was transfected into human MDA-MB-231 cells.The effect of recombination plasmid on the expression of VCL gene was identified by Western blot and PCR assays.Results:The PCR assay and DNA sequencing indicated that the CRISPR/Cas9 system could knock down VCL gene in this study.Western blotting showed that vinculin significantly decreased in Cas9-VCL group.Conclusion:The CRISPR/Cas9 palsmid targeting human VCL gene has been constructed and knocked out the expression of VCL gene effectively.%目的:建立CRISPR/Cas9n系统,用于敲除人源黏着斑蛋白(VCL)基因.方法:设计一个靶向人源VCL基因第3个外显子的单向导RNA (sgRNA),分别克隆表达载体后,通过慢病毒转入人MDA-MB-231细胞,通过PCR及Western印迹检测细胞株中VCL基因的敲除效果.结果:测序结果显示靶向VCL基因CRISPR/Cas9重组质粒构建成功;PCR产物测序结果表明本次设计的Cas9/sgRNA能够对VCL基因进行编辑敲除;Western印迹显示Cas9-VCL组的MDA-MB-231细胞内VCL表达水平较对照组显著降低.结论:通过CRISPR/Cas9系统获得了靶向VCL基因的重组质粒,构建的重组质粒能有效敲除VCL.