CRISPR-Cas9 Screen for SARS-CoV-2 Host Dependency Factors

该项目于2020年春季启动，现已准备好迎接病毒挑战阶段。我们已经从Juan Bonifacino获得了CRISPR-Cas9文库，该文库可以通过慢病毒载体传递给不同类型的细胞。Scarlett Shih是该实验室的研究员，负责领导这一项目的工作。她已经将携带CRISPR-Cas9文库的慢病毒载体应用于HeLa、Vero和HEK293T-hACE2细胞，并使用嘌呤霉素选择了具有不同基因失活的细胞。这些细胞被捐赠给USAMRIID的Rajini Mudhasani，用于感染野生型SARS-CoV-2。缺少感染所需的关键因子的细胞将在生产性SARS感染的其他细胞病变效应中存活下来，导致随着时间的推移，培养中的抗病毒细胞逐渐丰富。对这些细胞进行批量RNA测序，以确定与细胞生存相关的引导RNA序列，从而确定编码宿主依赖因子的基因。筛查的初步结果之后将进行有针对性的分析，以确认特定基因在SARS-CoV-2感染过程中的功能作用。

This project was initiated in spring of 2020 and is now ready for the virus challenge stage. We have obtained CRISPR-Cas9 libraries from Juan Bonifacino that can be delivered to diverse cell types by lentiviral vectors. Scarlett Shi, a Research Fellow in the lab, is leading the work on this project. She has applied lentiviral vectors carrying CRISPR-Cas9 libraries to HeLa, Vero, and HEK293T-hACE2 cells and has selected cells with diverse gene inactivations using puromycin. These cells were given to Rajini Mudhasani of USAMRIID for infection with wild-type SARS-CoV-2. Cells that are missing a crucial factor needed for infection will survive the otherwise cytopathic effects of productive SARS infection, resulting in the progressive enrichment of virus-resistant cells in culture over time. These cells will be subjected to bulk RNA sequencing in order to identify the guide RNA sequences associated with cell survival, thereby identifying the genes encoding host dependency factors. The initial results of the screen will be followed with targeted assays to confirm the functional role of a given gene during SARS-CoV-2 infection.