Genome-wide CRISPR KO screen in BMDMs [Bulk CIRSPR Screen]

Macrophages adopt dynamic functional states to maintain tissue homeostasis or respond to environmental challenges. Here we performed a genome-wide CRISPR screen in primary BMDMs identifiying negative regulators of iNOS expression in response to IFNg stimulation. We performed multiple pooled genome-wide CRISPR knockout (KO) screens using an optimized Streptococcus pyogenes Cas9 single guide RNA (sgRNA) library paired with delivery of recombinant Cas9 protein via electroporation. Our library consisted of 8 combined lentivirus sgRNA library pools expressing 152 339 sgRNAs targeting 18776 genes and non-targeting control sgRNAs. BMDMs were sorted and sgRNAs isolated and sequenced from 3 sources: (1) iNOS high cells post-IFNγ stimulation (iNOS_high); (2) cells from the last day of differentiation prior to IFNγ stimulation (reference); and (3) cells collected three days after lentiviral transduction as a library control (input)