$199,245
DIVISION OF BASIC SCIENCES - NCI
National Cancer Institute (NCI)
This project was initiated in spring of 2020 and is now in the preparatory phase. We have obtained CRISPR-Cas9 libraries from Juan Bonifacino that can be delivered to diverese cell types by lentiviral vectors. We have 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 will be 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. We anticipate that infections of our genetically modified cell lines will begin in fall of 2020.