The bacterial clustered regularly interspaced short palindromic repeats 
(CRISPR)-Cas9 system for genome editing has greatly expanded the toolbox for 
mammalian genetics, enabling the rapid generation of isogenic cell lines and 
mice with modified alleles. Here, we describe a pooled, loss-of-function genetic 
screening approach suitable for both positive and negative selection that uses a 
genome-scale lentiviral single-guide RNA (sgRNA) library. sgRNA expression 
cassettes were stably integrated into the genome, which enabled a complex mutant 
pool to be tracked by massively parallel sequencing. We used a library 
containing 73,000 sgRNAs to generate knockout collections and performed screens 
in two human cell lines. A screen for resistance to the nucleotide analog 
6-thioguanine identified all expected members of the DNA mismatch repair 
pathway, whereas another for the DNA topoisomerase II (TOP2A) poison etoposide 
identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6. A 
negative selection screen for essential genes identified numerous gene sets 
corresponding to fundamental processes. Last, we show that sgRNA efficiency is 
associated with specific sequence motifs, enabling the prediction of more 
effective sgRNAs. Collectively, these results establish Cas9/sgRNA screens as a 
powerful tool for systematic genetic analysis in mammalian cells.