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

Division Of Basic Sciences - Nci

Investigators

Abstract

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.