利用CRISPR—Cas9系统鉴定丙型肝炎病毒感染必需的胞内宿主因子

Identification of intracellular host factors essential for hepatitis C virus infection with CRISPR-Cas9 method

目的 鉴定丙型肝炎病毒（HCV）感染过程必需的胞内宿主因子,为发现新的抗病毒靶点提供科学依据.方法 利用涵盖19 050个人类基因的CRISPR-Cas9文库小向导RNA（sgRNA）慢病毒感染人肝癌细胞Huh7.5（共计1×108个细胞）,然后利用杀稻瘟菌素和嘌呤霉素抗性标记筛选出成功敲除基因的细胞克隆群,再将细胞克隆群感染HCVJFH1株（感染复数为0.30）,通过与未敲除的Huh7.5细胞（对照组）感染水平比较,筛选获得较对照组感染水平明显降低的细胞克隆群.最后通过Illumina测序获得该克隆被敲除的基因序列,利用短发夹RNA（shRNA）技术对单个基因分别验证,明确HCV感染必需的胞内宿主因子.结果 利用杀稻瘟菌素和嘌呤霉素抗性标记成功筛选出经CRISPR-Cas9文库敲除基因的细胞克隆,利用免疫荧光检测获得了与对照组相比HCV感染水平明显下降的细胞克隆,将这些克隆进行Illumina测序,结果提示至少存在10个可能与HCV感染水平降低相关的基因.shRNA敲低单个基因验证结果显示,与未敲除的Huh7.5细胞相比,单独敲低MTCP1和RPS14基因的Huh7.5细胞的HCV感染率均降低,差异均具有统计学意义（均P＜0.05）,表明MTCP1和RPS14是HCV感染必需的两个胞内宿主因子.结论 建立了利用CRISPR-Cas9系统鉴定HCV感染必需的胞内宿主因子的新方法,筛选出了MTCP1和RPS14这两个HCV感染必需的胞内宿主因子,为深入研究其作用机制奠定了基础,也为发现新的抗病毒靶点提供了科学依据.

Objective To identify the intracellular host factors necessary for the hepatitis C virus （HCV） infection process so as to provide a scientific basis for the discovery of new antiviral targets.Methods In total 1×108 human liver cancer cells Huh7.5 were infected with a CRISPR Cas9 lentiviral small guide RNA （sgRNA） library which covers 19 050 human genes.Cell clonal populations with genes knockout were screened using blasticidin and puromycin resistance markers,and then were infected with HCV JFH1 strain （multiplicity of infection of 0.30）.By comparing the infection levels with the unknocked Huh7.5 cells （control group）,the cell populations with significantly reduced infection levels were screened and obtained.Finally,the cloned gene sequence of cell clonal populations was obtained by Illumina sequencing,and the single gene was separately verified by short hairpin RNA （shRNA） technology to identify the intracellular host factor necessary for HCV infection.Results The gene knockout cell populations by CRISPR-Cas9 library were successfully screened by blasticidin and puromycin resistance markers.Cell populations with significantly decreased HCV infection levels compared with the control group were obtained by immunofluorescence assay.These populations were sequenced by Illumina and the results suggest that there are at least 10 genes that may be associated with a reduction in HCV infection.The shRNA knockdown single gene verification results showed that compared with the unknocked Huh7.5 cells,the HCV infection rates were decreased in MTCP1 or RPS14 genes knockout Huh7.5 cells,and the differences were statistically significant （all P〈0.05）.That indicates that MTCP1 and RPS14 are two intracellular host factors essential for HCV infection.Conclusion A new method for identifying intracellular host factors essential for HCV infection was established.The two intracellular host factors essential for HCV infection,MTCP1 and RPS14,were screened.This study lays a foundation for further studies on related action mechanism,and also provides a scientific basis for discovering new antiviral targets.