基于CRISPR/Cas9技术构建SIRT6基因敲除的A549肺腺癌细胞系

Construction of A549 lung adenocarcinoma cell lines with SIRT6 knockout based on CRISPR/Cas9 technology

目的应用CRISPR/Cas9技术构建SIRT6基因敲除的人肺腺癌A549稳转细胞系。方法根据CRISPR/Cas9技术靶点设计原则,通过生物信息学设计向导RNA(single-guide RNA sgRNA),构建sgRNA-SIRT6质粒并包装成慢病毒感染A549细胞,使用嘌呤霉素筛选SIRT6基因敲除的A549细胞并行基因组测序鉴定,应用Western Blot检测SIRT6基因的敲除情况。结果DNA测序结果显示sgRNA-SIRT6重组质粒构建成功,Western Blot结果显示,转染sgRNA-SIRT6重组质粒的细胞无SIRT6表达。结论通过CRISPR/Cas9技术成功构建了SIRT6基因敲除的A549细胞系,为后续研究SIRT6基因在肺腺癌细胞中的作用机制和功能奠定了基础。

Objective To construct A549 lung adenocarcinoma cell lines with SIRT6 knockout by CRISPR/Cas9 technology.Methods Based on the CRISPR/Cas9 target design principles,the sgRNA-SIRT6 plasmid was constructed by single-guide RNA and turned into lentivirus to infect A549 cells.The A549 cells with SIRT6 gene knockout were screened out by puromycin and identified by genome sequencing,and the SIRT6 gene knockout was detected by Western Blot.Results DNA sequencing results showed that sgRNA-SIRT6 recombinant plasmid was successfully constructed,and Western Blot results showed that SIRT6 was not expressed in cells transfected with sgRNA-SIRT6 recombinant plasmid.Conclusion The A549 cell lines with SIRT6 gene knockout are successfully constructed with CRISPR/Cas9 technology,which lays a foundation for further research on the mechanism and function of SIRT6 gene in lung adenocarcinoma cells.