Fig. 8. Impact of the proviral genes identified by CRISPRa on coronaviruses SARS-CoV-2, HCoV-229E, HCoV-NL63 and on orthomyxovirus influenza A.

Calu-3-dCas9-VP64 cells were stably transduced to express 2 different sgRNAs (g1, g2) per indicated gene promoter and selected. a. Cells were non infected (N.I.) or incubated with SARS-CoV-2 bearing NLuc reporter and the infection efficiency was scored 30 h later by monitoring NLuc activity. b. Cells were infected by SARS-CoV-2 at MOI 0.05 and 5 days later stained with crystal violet. Representative images from 2 independent experiments are shown. c. Cells were infected with influenza A virus bearing NLuc reporter and 10h later, relative infection efficiency was measured by monitoring NLuc activity. d. Cells were infected with HCoV-NL63 and 5 days later, infection efficiency was determined using RT-qPCR. e. Cells were infected with HCoV-229E-Renilla and 72h later, relative infection efficiency was measured by monitoring Renilla activity. f. Cells were incubated with SARS-CoV-2 for 2h and then treated with Subtilisin A followed by RNA extraction and RdRp RT-qPCR analysis as a measure of viral internalization. g. Cells were infected with Spike del19 and VSV-G pseudotyped, Firefly-expressing VSV and infection efficiency was analyzed 24h later by monitoring Firefly activity. h. Cells were infected with SARS-CoV-2 and, 24h later, lysed for RNA extraction and RdRp RT-qPCR analysis. i. Aliquots of the supernatants from h were harvested and plaque assays were performed to evaluate the production of infectious viruses in the different conditions. The mean and SEM of ≥ 3 (a, c, e, f), ≥ 4 (d, g, h), or 2 (i) independent experiments are shown. Statistical significance was determined by a two-sided t-test with (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001) (a, c-h). Exact n numbers and p-values are indicated in Supplementary Data 17. The green and dark green dashed lines indicate 150% and 300% increase in infection efficiency, respectively (a, c-e, g-h).