The authors describe a pooled, loss-of-function genetic screening approach using the CRISPR/Cas9 system in human cells. They used a genome-scale lentiviral single guide RNA (sgRNA) library to generate knockout cell lines and performed screens in two human cell lines. The screens identified all expected members of the DNA mismatch repair pathway and the DNA topoisomerase II (TOP2A) poison etoposide, as well as cyclin-dependent kinase 6 (CDK6). A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes. The authors also found that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs. These results establish Cas9/sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells.The authors describe a pooled, loss-of-function genetic screening approach using the CRISPR/Cas9 system in human cells. They used a genome-scale lentiviral single guide RNA (sgRNA) library to generate knockout cell lines and performed screens in two human cell lines. The screens identified all expected members of the DNA mismatch repair pathway and the DNA topoisomerase II (TOP2A) poison etoposide, as well as cyclin-dependent kinase 6 (CDK6). A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes. The authors also found that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs. These results establish Cas9/sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells.