利用可诱导表达Cas9的HeLa细胞系进行顺铂耐药性基因筛选

Screening of cisplatin resistance genes in HeLa cell line with inducible expression of Cas9

目的:利用PiggyBac转座子系统,构建可诱导表达Cas9的稳定HeLa细胞系,并针对人核受体基因构建sg RNA文库,进行顺铂耐药性基因筛选。方法:构建PB-TRE-Cas9-NLS-IRES-hrGFP-Zeo载体,与PB-CAG-rtTA以及转座酶载体一同电转到HeLa细胞中,通过药筛和挑选单克隆得到稳定细胞系;构建核受体基因sgRNA病毒文库,并感染可诱导表达Cas9的HeLa细胞系,筛选对顺铂具有抵抗性的克隆,并进行目的基因的测序鉴定。结果:挑选出可诱导表达Cas9的8E单克隆细胞株,在5个基因位点上可以进行高效编辑;感染sgRNA文库之后,挑选顺铂耐药的克隆,并鉴定出大部分克隆含有VDR sg RNA,经测序发现这些克隆中的VDR基因发生突变。结论:利用PiggyBac转座子系统,构建了可诱导表达Cas9的稳定HeLa细胞系,利用这个细胞系可以在多位点进行高效基因编辑,并可用于高通量文库筛选顺铂耐药性基因。

Objective:Taking advantage of the PiggyBac transposon system to generate a stable HeLa cell line that can inducibly express Cas9,and to construct a sgRNA library for human nuclear receptor genes to screen for cisplatin resistance genes. Methods:PB-TRE-Cas9-NLS-IRES-hrGFP-Zeo vector was constructed and transferred into HeLa cells together with PB-CAG-rtTA and the transposase vectors. Stable cell lines were obtained by drug screening and picking up single clones. Constructed the sgRNA virus library targeting nuclear receptor genes to infect the stable cell line,screened the clones resistant to cisplatin,and carried out the sequencing and identification of the target genes. Results:Five gene loci were edited efficiently in the stable cell line. Clones resistant to cisplatin were obtained after infection of sgRNA library,and most clones harbored VDR sgRNA and mutant VDR gene. Conclusion:Using PiggyBac transposon system,stable HeLa cell line with inducible expression of Cas9 was successfully constructed. The cell line can be used for efficient gene editing at multiple sites and high-throughput library screening for cisplatin resistance genes.