利用CRISPR/Cas9系统构建稳定敲除CBX2基因的A549细胞系

Construction of CBX2 gene knockout stable A549 cell line with CRISPR/Cas9 system

目的:利用CRISPR/Cas9系统构建稳定敲除CBX2基因的A549细胞系。方法:设计针对敲除CBX2的short guide RNA(sgRNA),并克隆到载体PX459中。将测序正确的重组质粒转染到A549细胞中,利用嘌呤霉素筛选转染阳性细胞并分离得到5个单克隆细胞系,通过Western blot方法检测构建的细胞系中CBX2蛋白表达情况。结果:成功构建了靶向CBX2的CRISPR/Cas9重组质粒,筛选出的5个单克隆细胞系中CBX2蛋白表达水平均显著下降。结论:成功利用CRISPR/Cas9系统构建了稳定敲除CBX2基因的A549细胞系。

Objective： To construct CBX2 gene knockout stable A549 cell line with CRISPR/Cas9 system. Methods：The single guide RNA （sgRNA） targeting CBX2 gene was designed and inserted into the expression vector PX459 using molecular cloning technique. The recombined plasmid was verified by sequencing. The plasmid was transfected into A549 cell line. After puromycine selection, monoclonal cell line was picked for expansion. Finally, the expression of CBX2 protein in A549 CBX2 knockout cell line was detected by Western blot. Results：The CRISPR / Cas9 recombinant plasmid targeting CBX2 was successfully constructed, and the expression of CBX2 protein was significantly decreased. Conclusion：The A549 CBX2 knockout cell line could be successfully constructed by CRISPR/Cas9 system.