Validation of candidate hits using one vector system shows the
strength of the CRISPR-Cas9 whole genome screening.
To validate the candidate hits from our screen we generated single gene
knockouts in ACE2-293T and ACE2-293T-Cre cells by introducing a one
vector Cas9 endonuclease and single gRNA (Stringer et al., 2019) Figure
4A). We evaluated this approach by targeting and staining cells for ACE2
expression. Knockout of ACE2 in both ACE2-293T and ACE2-293T-Cre
reporter cells was highly efficient, >95% (Figure 4A and
4B). As expected, knockout of ACE2 from ACE2-293T and ACE2-293T-Cre
abolished the infection by both Spike-mNG and Spike-Cre (Figure 4). ACE2
KO did not affect VSVG-mNG which was anticipated; however, it did lead
to a slight drop in infectivity of VSVG-Cre (Figure 4E and 4F).
We evaluated gene disruptions of SLC35B2, HSP90B1 and EP300, which
reflect three distinct molecular steps in viral infection and reporter
expression, with SLC35B2 affecting broad recruitment of viral particles
to the cell surface and HSP90B1 affecting LDLR expression and hence
VSV-G attachment and EP300 affecting lentiviral integration and reporter
gene expression. As expected, the SLC35B2 KO dropped the infection of
both Spike and VSVG mediated entry (Figure 4), consistent with heparan
sulfate proteoglycan being important for general viral attachment to the
cell surface (Guimond et al., 2022). The ER chaperone Gp96 (HSP90B1) was
identified in both VSVG-mNG (Rank#3) and VSVG-CRE (Rank#2) as top hit
but not in Spike-mNG (Rank#607). As anticipated, HSP90B1 KO reduced
VSVG-mNG and VSVG-Cre mediated entry without effect on Spike-mediated
infection (Figure 4). The acetyltransferase EP300 was identified as a
top hit in both Spike-mNG and VSVG-mNG screens, but not a hit in
VSVG-Cre consistent with its previously identified role in lentiviral
integration (Cereseto et al., 2005). As expected, EP300 KO reduced
VSVG-mNG reporter expression. However, it unexpectedly decreased
VSVG-Cre reporter expression slightly and had no effect on Spike-mNG
reporter expression (Figure 4). We are unclear as to the reason for this
reproducible discrepancy; however, one possibility is that the titers
used in the screen may have been slightly lower than in the validation
experiment, which somehow allowed Spike-mNG to bypass the effect of the
EP300 knockout.