library_construction_protocol: Genomic DNA extraction and Illumina sequencing of gRNAs were described previously (Tzelepis et al., 2016). 19bp single-end sequency was performed with the custom sequencing primer 5′-TCTTCCGATCTCTTGTGGAAAGGACGAAACACCG-3′. Enrichment and depletion of guides and genes were analysed using the MAGeCK statistical package63by comparing read counts from each cell line with counts from the plasmid as the initial population. For the screen we used our murine v2 lentiviral guideRNA library containing 90 230 gRNAs against 18 424 genes. 3*107cells were then transduced with a pre-determined volume of the mouse v2 genome-wide gRNA lentiviral supernatant that gave rise to 30% transduction efficiency measured by BFP expression.