Genome-wide CRISPR screening strategy to selectively identify genes promoting and inhibiting envelope mediated entry.
Our strategy for genome-wide loss of function screens to identify genes facilitating and inhibiting viral entry is illustrated in Figure 1A. 293T cells or variants (described below) were mutagenized by transduction with the Brunello pooled sgRNA library containing 76,441 single gRNA targeting 19,114 human genes (Doench et al. 2016). After 72h of infection with the Brunello library, we then selected these cells with puromycin for 5 days and cultured 5 more days to allow for protein depletion. Reporter viruses consisted of lentiviral vectors that deliver mNeonGreen pseudotyped with SARS-CoV-2 spike (Spike-mNG) or VSV-G (VSVG-mNG), with the rationale that comparative analysis of genes associated with entry would be distinct between screens and that genes influencing expression of the reporter, such as viral integration would be common (Figure 1A). Additionally, a Gag-Cre fusion protein, that undergoes cleavage upon viral maturation, thus upon fusion with the target cell membrane, Cre is able to activate a floxed dsRED/eGFP reporter allowing further separation of host genes associated with viral entry from those that may influencing reporter expression (Figure 1B) (Esposito et al., 2016; P. E. Mangeot et al., 2019). After 48h of infection with the virus we collected the fractions of the total transduced population and cells sorted on high and low/no GFP fluorescence by fluorescent activated cell sorting (FACS). We then extracted genomic DNA from the sorted cells and PCR amplified the sgRNA for next generation high throughput sequencing (Figure 1A). Analysis using MAGeCK provided sgRNA frequencies in the high and low fractions for statistical comparison of gene-function associations (Chen et al., 2018; Li et al., 2014). The raw read counts from these screens can be found in NCBI GEO (accession number: GSE206996 ).