The CRISPR/Cas9 system has revolutionized mammalian genetics by enabling efficient genome editing. This study describes a pooled, loss-of-function genetic screening approach using a genome-scale lentiviral single guide RNA (sgRNA) library. sgRNA expression cassettes were stably integrated into the genome, allowing for the tracking of a complex mutant pool via massively parallel sequencing. A library containing 73,000 sgRNAs was used to generate knockout collections and perform screens in two human cell lines. A screen for resistance to 6-thioguanine identified all expected members of the DNA mismatch repair pathway, while another for etoposide identified TOP2A and CDK6. A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes. The study also showed 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 study addresses the need for efficient, large-scale loss-of-function screening in mammalian cells. Traditional methods, such as insertional mutagenesis and RNAi, have limitations, including integration biases and off-target effects. The CRISPR/Cas9 system, which uses a single guide RNA (sgRNA) to direct the Cas9 nuclease to cause double-stranded cleavage of target DNA sequences, offers a more efficient and precise method. The study demonstrates the feasibility of using the CRISPR/Cas9 system to perform large-scale, loss-of-function screens in mammalian cells. The approach involves using a pool of sgRNA-expressing lentivirus to generate a library of knockout cells that can be screened under both positive and negative selection. Each sgRNA serves as a distinct DNA barcode that can be used to count the number of cells carrying it using high-throughput sequencing. The study tested the approach in the near-haploid human KBM7 CML cell line, creating a clonal derivative expressing the Cas9 nuclease under a doxycycline-inducible promoter. Transduction of these cells with a lentivirus expressing an sgRNA targeting the endogenous AAVS1 locus revealed substantial cleavage at the AAVS1 locus. The study also analyzed off-target activity of CRISPR/Cas9 and found minimal cleavage at potential off-target sites. The study then tested the ability to simultaneously screen tens of thousands of sgRNAs, designing a library with 73,151 members. The library included 10 sgRNAs for each of 7033 genes and all possible sgRNAs for each of the 84 genes encoding ribosomal proteins. The study performed screens for genes involved in DNA mismatch repair and found that sgRNAs targeting the genes encoding the four components of the MMR pathway were dramatically enriched. The study also addressed the challenge of lossThe CRISPR/Cas9 system has revolutionized mammalian genetics by enabling efficient genome editing. This study describes a pooled, loss-of-function genetic screening approach using a genome-scale lentiviral single guide RNA (sgRNA) library. sgRNA expression cassettes were stably integrated into the genome, allowing for the tracking of a complex mutant pool via massively parallel sequencing. A library containing 73,000 sgRNAs was used to generate knockout collections and perform screens in two human cell lines. A screen for resistance to 6-thioguanine identified all expected members of the DNA mismatch repair pathway, while another for etoposide identified TOP2A and CDK6. A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes. The study also showed 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 study addresses the need for efficient, large-scale loss-of-function screening in mammalian cells. Traditional methods, such as insertional mutagenesis and RNAi, have limitations, including integration biases and off-target effects. The CRISPR/Cas9 system, which uses a single guide RNA (sgRNA) to direct the Cas9 nuclease to cause double-stranded cleavage of target DNA sequences, offers a more efficient and precise method. The study demonstrates the feasibility of using the CRISPR/Cas9 system to perform large-scale, loss-of-function screens in mammalian cells. The approach involves using a pool of sgRNA-expressing lentivirus to generate a library of knockout cells that can be screened under both positive and negative selection. Each sgRNA serves as a distinct DNA barcode that can be used to count the number of cells carrying it using high-throughput sequencing. The study tested the approach in the near-haploid human KBM7 CML cell line, creating a clonal derivative expressing the Cas9 nuclease under a doxycycline-inducible promoter. Transduction of these cells with a lentivirus expressing an sgRNA targeting the endogenous AAVS1 locus revealed substantial cleavage at the AAVS1 locus. The study also analyzed off-target activity of CRISPR/Cas9 and found minimal cleavage at potential off-target sites. The study then tested the ability to simultaneously screen tens of thousands of sgRNAs, designing a library with 73,151 members. The library included 10 sgRNAs for each of 7033 genes and all possible sgRNAs for each of the 84 genes encoding ribosomal proteins. The study performed screens for genes involved in DNA mismatch repair and found that sgRNAs targeting the genes encoding the four components of the MMR pathway were dramatically enriched. The study also addressed the challenge of loss