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

该项目于2020年春季启动，现已为病毒挑战阶段做好准备。我们从Juan Bonifacino获得了CRISPR-Cas9文库，可以通过慢病毒载体传递到不同的细胞类型。该实验室的研究员斯佳丽·施（Scarlett Shi）领导了这个项目的工作。她将携带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多种基因失活的细胞。这些细胞被给予USAMRIID的Rajini Mudhasani感染野生型SARS-CoV-2。缺少感染所需的关键因子的细胞将在非典型肺炎感染的其他细胞病变效应中存活下来，从而导致培养中的病毒抗性细胞随着时间的推移逐渐富集。这些细胞将进行大量RNA测序，以鉴定与细胞存活相关的引导RNA序列，从而鉴定编码宿主依赖因子的基因。筛选的初步结果之后将进行靶向测定，以确认特定基因在SARS-CoV-2感染期间的功能作用。

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.