用于CRISPR文库筛选的N2a-Cas9细胞系的构建

Construction of N2a-Cas9 cell line for genome-wide CRISPR screening

为了进行CRISPR文库筛选,本研究采用慢病毒转导的方法利用N2a细胞构建表达Cas9蛋白的N2a-Cas9单克隆细胞系。实验将LentiCas9-Blast、pSPA X2和pMD 2.G 3种质粒共转染人胚胎肾细胞(HEK293T)获取高滴度的重组慢病毒,收集重组慢病毒并感染N2a细胞,经杀稻瘟菌素(Blasticidin)筛选,通过有限稀释法获得含有Cas9基因的多个单克隆细胞株。Western blot结果显示候选细胞系高水平表达Cas9蛋白;利用慢病毒表达报告基因载体系统检测显示候选细胞系的Cas9蛋白具有很高的切割活力;利用CellTiter-Glo试剂检测结果显示,Cas9基因在候选细胞系内高表达但不影响细胞增殖活性。本研究利用慢病毒转导系统构建了稳定表达Cas9蛋白的N2a-Cas9单克隆细胞系,为基于CRISPR/Cas9全基因组高通量筛选技术探究神经细胞内关键宿主基因调控狂犬病病毒嗜神经性提供研究基础。

In this study,mouse neuroblastoma(N2a)cells were used to construct N2a-Cas9 cell line which stably expresses Cas9 protein by using lentivirus transfection technology.This constrcted cells line laid the foundation for CRISPR library screening.First,three plasmids including LentiCas9-Blast,pSPA X2,and pMD 2.G were co-transfected into human renal epithelial cells(HEK293T)to obtain recombinant lentivirus with high titers.N2a cells were infected with the recombinant lentivirus,and then screened by Blasticidin.Finally,several monoclonal cell lines expressing Cas9 protein were isolated by limiting dilution.The expression of Cas9 expression in N2a-Cas9 cell line was determined by western blot,and the high cleavage activity of Cas9 was also examined by using lentivirus-based reporter vector system.By using CellTiter-Glo®reagent,the cell proliferation activity was well detectable and not decreased in N2a-Cas9 cell lines,where Cas9 gene was expressed in a high level.Therefore,N2a-Cas9 cell line was successfully constructed using a lentiviral transfection system,which can provide a basis for genome-wide CRISPR screening to identify crucial host factors regulating Rabies virus neuro-tropism.