Genome-wide CRISPR Screen Identifies Host Dependency Factors of RSV Infection

To create a more comprehensive portrait of RSV infection and its genetic requirements, we performed two whole-genome CRISPR/Cas9 knockout screens. First, A549 cells were transduced with the GeCKOv2 library, and infected with RSV-EGFP for 24 hours. To enrich for host dependency factors, two different iterations of the screen were performed. For the first iteration (the "viability screen"), EGFP negative cells were sorted and cultured for 1 week, thus enriching for cells that were not infected at the time of the sort and remained viable after one week in culture. For the second iteration of the screen (the "early infection screen"), EGFP negative and EGFP-low cells were sorted 24 hours post infection and immediately harvested. For both screens, enriched sgRNAs, that presumably conferred protection from RSV infection, were PCR amplified and sequenced.