A CRISPR-based screen identifies genes essential for West Nile virus-induced cell death. Homo sapiens

West Nile virus (WNV) causes an acute neurological infection attended by massive neuronal cell death. However, the mechanism(s) behind the virus-induced cell death is poorly understood. Using a library containing 77,406 sgRNAs targeting 20,121 genes, we performed a genome-wide screen using CRISPR/Cas9. HEK 293FT cells were infected with lentivirus expressing sgRNAs and then transfected with a Cas9 expressing construct. WNV infection killed most cells during a 12d selection. Survivor cells were harvested, from which DNA was isolated. The sgRNAs integrated in genome of survivor cells were amplified with PCR. The PCR product was sequenced with Illumina MiSeq to profile the sgRNA population in the survivor cells. Three replicates were conducted. Similarly, a second round of screen was conducted. Among the genes identified, seven genes, EMC2, EMC3, SEL1L, DERL2, UBE2G2, UBE2J2, and HRD1, stood out as having the strongest phenotype, whose knockout conferred strong protection against WNV-induced cell death with two different WNV strains and in three cell lines. Interestingly, knockout of these genes did not block WNV replication. Thus, these appear to be essential genes that link WNV replication to downstream cell death pathway(s). In addition, the fact that all of these genes belong to the endoplasmic reticulum-associated protein degradation (ERAD) pathway suggests that this might be the primary driver of WNV-induced cell death. Overall design: Examination of sgRNA populations in survival 293FT cells

西尼罗河病毒（West Nile virus, WNV）可引发急性神经系统感染，并伴随大量神经元细胞死亡。然而，病毒诱导细胞死亡的具体机制尚未完全阐明。本研究依托靶向20121个基因的77406条单引导RNA（single guide RNA, sgRNA）文库，利用CRISPR/Cas9技术开展全基因组筛选。具体流程为：将表达sgRNA的慢病毒感染HEK 293FT细胞，随后转染表达Cas9的重组构建体。经过12天的筛选培养后，西尼罗河病毒感染可杀死绝大多数细胞，收集存活细胞并提取其基因组DNA。采用PCR技术扩增整合于存活细胞基因组中的sgRNA序列，再通过Illumina MiSeq测序平台对存活细胞中的sgRNA群体进行谱分析。本实验共设置3次生物学重复，并开展了第二轮筛选实验。在筛选得到的基因中，EMC2、EMC3、SEL1L、DERL2、UBE2G2、UBE2J2及HRD1这7个基因表现出最为显著的表型效应；敲除这些基因可使细胞在两种不同西尼罗河病毒毒株感染以及三种细胞系中，均获得针对病毒诱导细胞死亡的强烈保护作用。值得注意的是，敲除上述基因并未阻断西尼罗河病毒的复制，因此这些基因似乎是连接病毒复制与下游细胞死亡通路的关键必需基因。此外，上述所有基因均属于内质网相关蛋白降解（endoplasmic reticulum-associated protein degradation, ERAD）通路，这提示该通路可能是西尼罗河病毒诱导细胞死亡的核心驱动因素。整体实验设计：分析存活293FT细胞中的sgRNA群体特征。