Fig. 5: Characterization of the impact of identified SARS-CoV-2 dependency factors.
Calu-3-Cas9 cells were transduced to express two sgRNAs (g1, g2) per gene or non-targeting, control sgRNAs (CTRL g1, g2). a, Cells were infected with SARS-CoV-2 mNG, and infection efficiency was scored 48 h later by flow cytometry. b, Expression levels of ACE2 were analyzed by immunoblot. Actin served as a loading control. A representative experiment (from two independent experiments) is shown. c, Relative surface ACE2 expression was measured using a Spike-RBD-mFc fusion followed by flow cytometry analysis. d, Cells were incubated with SARS-CoV-2 for 2 h, treated with Subtilisin A followed by RNA extraction and RdRp RT-qPCR analysis. e. Cells were infected with Spike del19 and VSV-G pseudotyped, GFP-expressing VSV, and infection efficiency was analyzed 24 h later by flow cytometry. f, Cells were infected with SARS-CoV-2 and, 24 h later, lysed for RNA extraction and RdRp RT-qPCR analysis. g, Supernatants from panel f were harvested and plaque assays performed. PFU, plaque-forming units. h, Cells were infected with MERS-CoV, and 16 h later, viral production in the supernatant was measured by the 50% tissue culture infectious dose (TCID50). i, Cells were pretreated (or not) with camostat mesylate (cam.) or remdesivir (RDV), incubated with SARS-CoV-2 for 30 min on ice and washed. Spike was then primed with trypsin or not and the media replaced, and 7 h later, cells were lysed for RNA extraction and RdRp RT-qPCR analysis. j, Similar to panel i, with Spike-pseudotyped, Firefly-expressing VSV. Cells were lysed and relative infection measured by monitoring Firefly activity 24 h later. The mean and s.e.m. of five or more (a) or three or more (c–f,h,i; except for EP300 and ATP8B1 KO in panel c and for ATP8B1 and TMPRSS2 KO in panel e, n = 2) or four (g) independent experiments, or the mean of two independent experiments (j), are shown. Statistical significance was analyzed using a two-sided t test with no adjustment for multiple comparisons (a,c,i) or a one-way ANOVA with Dunnett’s test (d–h) (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Exact numbers and P values are indicated in Supplementary Data 17. The red and dark red dashed lines represent 50% and 80% inhibition, respectively (a,c–f).
