CRISPR- Cas9 genetic screening in ACE2 293T cells
Lentiviral Brunello CRISPR KO library was purchased from Addgene at a titer of 1.4*10^7 TU/mL (a gift from David Root and John Doench Addgene #73178) (Doench et al., 2016). 40*10^6 ACE2 293T cells were transduced with the Brunello library at an MOI of 0.3 to enable ≥100-fold gRNA representation. 72h after transduction with the viral library the cells were selected with puromycin at 1ug/mL for 5 days and cultured for 5 more days to allow for the depletion of proteins from the cells. After that, cells were plated in 10cm dishes for transduction with the viral particles for screening. 2*10^6 cells in each 10cm dish were plated for 10 dishes for each of the screening condition the day before transduction. The spike pseudovirus was concentrated using the LV concentrator (Catalog #631213, Takara Bio) on the day of transduction. After 48h of transduction with the pseudoviruses the cells were FACS sorted based on mNG or GFP fluorescence into infected and uninfected groups.