Extended Data Fig. 6: Proviral gene candidate validation in other cell lines.
a. Caco-2-Cas9 cells expressing 2 sgRNAs (g1, g2) per indicated gene were infected with SARS-CoV-2 mNG reporter. Infection efficiency scored 48 h later by flow cytometry (left panel). In parallel, supernatants were collected and virus production determined by plaque assays (right panel). Mean and s.e.m. of 3 (left) and mean of 2 (right) independent experiments are shown, respectively. b. Similar to a, with A549-ACE2 cells. Mean and s.e.m. of 5 (left) and mean of 2 (right) independent experiments. c. Similar to a, with Huh7.5.1-ACE2 cells. Mean and s.e.m. of 4 independent experiments (left) or a representative experiment (with technical duplicates) is shown (right). a-c. One-way ANOVA with Dunnett’s test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Of note, ACE2 g1 targets an intron-exon junction on ACE2 endogenous sequence and cannot target ACE2 ectopic CDS in ACE2-transduced cells. d-f. Expression levels of AP1G1, AP1B1 and AAGAB in Caco-2 (d), A549-ACE2 (e) and Huh7.5.1-ACE2 (f) KO cells were analyzed by immunoblot, Actin served as a loading control. A representative immunoblot is shown (from 3 independent experiments). g. RNA samples from 9 (HAE from different donors22), 8 (Calu-3), or 3 (Caco-2, A549-ACE2) independent experiments were analyzed by RT-qPCR. Statistical significance was determined by unadjusted, two-sided Mann-Whitney test (**** p < 0.0001). h. Candidate gene expression levels in cell types from the respiratory epithelium (from Chua et al.33). Expression levels in COVID-19 versus healthy patients are color coded; the percentage of cells expressing the respective gene is size coded, as indicated. i. Cells were pretreated for 1 h with camostat mesylate or not, incubated with SARS-CoV-2 mNG on ice and washed. The media was replaced with serum-free media containing trypsin in order to prime Spike, or no trypsin. Infection efficiency was analyzed by flow cytometry after 24 h (for the + camostat conditions) or after 48 h (for CTRL). Statistical significance was determined by two-sided t-tests with no adjustment for multiple comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). The exact n and p-values (a, b, c, g, i) are indicated in Supplementary Data 17.
