Infections were performed to achieve a representation of at least 500 cells per sgRNA per replicate (500X; ~40 x 10^6 cells per replicate). Cells were split upon confluence, re-seeded back in culture and maintained at a density of 500X over six population doublings (PD6). After PD6, ~40 x 10^6 cells per replicate were harvested and processed for genomic DNA extrac-tion, barcode amplification and next-generation sequencing, as described previously21. LNCaP_FGC cells were transduced with low MOI lentivirus (~0.3) of the human Brunello CRISPR KO pooled library (Addgene # 73179) and selected with puromycin for 5 days to enrich for the positive transductants.