Fig. 1: ISG-focused CRISPR/Cas9 screening approach to identify restriction factors for SARS-CoV-2.

a CRISPR/Cas9 screen outline. A549-ACE2 cells were transduced with lentivectors encoding the ISG CRISPR/Cas9 library and selected by puromycin treatment for 20 days. Library cells were then pre-treated with 200 U/mL of IFNα for 16 h, and infection with SARS-CoV-2 at an MOI of 1. At 24 h p.i., infected cells were fixed with formalin treatment, permeabilized by saponin treatment and stained with a monoclonal anti-spike antibody. After secondary staining, infected cells were sorted and harvested. Non-infected, non-IFNα treated cells were harvested as a control. DNA was extracted from both cellular fractions and sgRNA loci amplification was carried out by PCR. Following NGS, bio-informatic analysis using the MAGeCK package was conducted. This figure was created with BioRender.com. b Screen results. By taking into account the enrichment ratios of each of the 8 different sgRNAs for every gene, the MAGeCK analysis provides a positive score for KO enriched in infected cells (i.e. restriction factor, represented in the top fraction of the graph) and a negative score for KO depleted in infected cells (i.e. proviral factors, represented in the bottom portion of the graph). Genes with an FDR < 0.05 are represented in black. 3 genes with a FDR > 0.05, but with a p value < 0.005 were additionally selected and are represented in red. c Individual sgRNA enrichment. For the indicated genes, the enrichment ratio of the 8 sgRNAs present in the library was calculated as the MAGeCK normalized read counts in infected cells divided by those in the original pool of cells and is represented in log2 fold change. As a control, the enrichment ratio of the 200 non-targeting control (NTCs) is also represented.