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 ).