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.