Fig. 2

Workflow of CRISPR-Cas9 functional genetic screens. a sgRNA libraries are ligated onto plasmid backbones are then transformed into electrocompetent bacterial cells. The amplified sgRNA library is purified from a bacterial lysate and transfected into virus-producing cells to generate a sgRNA library. b The sgRNA library is transduced into target cells, which are subsequently subjected to phenotype selection. Genomic DNA is then harvested, and embedded sgRNAs are amplified by PCR and identified by NGS. Hits are determined and ranked by their relative enrichment or depletion of the respective sgRNAs in the selected versus non-selected control cells. c The initial validation of screen hits typically relies on: I. small-scale repeat analysis targeting genes of interest with sgRNAs that had been used in the original screen, plus additional sgRNAs directed toward the same gene; II. genomic sequencing-based verification that the targeted gene was indeed sequence-altered; and III. verification that restoring the wild-type gene sequence rescues the selection phenotype