SUMMARY Most human epithelial tumors harbor numerous alterations, making it difficult to predict which genes are required for tumor survival. To systematically identify cancer dependencies, we analyzed 501 genome-scale loss-of-function screens performed in diverse human cancer cell lines. We developed DEMETER, an analytical framework that segregates on-from off-target effects of RNAi. 769 genes were differentially required in subsets of these cell lines at a threshold of six standard deviations from the mean. We found predictive models for 426 dependencies (55%) by nonlinear regression modeling considering 66,646 molecular features. Many dependencies fall into a limited number of classes, and unexpectedly, in 82% of models, the top biomarkers were expression-based. We demonstrated the basis behind one such predictive model linking hypermethylation of the UBB ubiquitin gene to a dependency on UBC. Together, these observations provide a foundation for a cancer dependency map that facilitates the prioritization of therapeutic targets.

The CRISPR-Cas9 system has revolutionized gene editing both on single genes and in multiplexed loss-of-function screens, enabling precise genome-scale identification of genes essential to proliferation and survival of cancer cells1,2. However, previous studies reported that a gene-independent anti-proliferative effect of Cas9-mediated DNA cleavage confounds such measurement of genetic dependency, leading to false positive results in copy number amplified regions3,4. We developed CERES, a computational method to estimate gene dependency levels from CRISPR-Cas9 essentiality screens while accounting for the copy-number-specific effect. As part of our efforts to define a cancer dependency map, we performed genome-scale CRISPR-Cas9 essentiality screens across 342 cancer cell lines and applied CERES to this dataset. We found that CERES reduced false positive results and estimated sgRNA activity for both this dataset and previously published screens performed with different sgRNA libraries. Here, we demonstrate the utility of this collection of screens, upon CERES correction, in revealing cancer-type-specific vulnerabilities.

Major efforts using loss-of-function genetic screens to systematically identify genes essential to the proliferation and survival of cancer cells have been reported [1][2][3][4][5][6][7][8][9] . Genes identified by these approaches may represent specific genetic vulnerabilities of cancer cells, suggesting treatment strategies and directing the development of novel therapeutics. The CRISPR-Cas9 genome editing system has proven to be a powerful tool to interrogate gene essentiality in cancer cell lines. Its relative ease of application, high rates of target validation, and increased specificity compared to RNA interference technology make it an ideal instrument for use in high-throughput functional genomic screening 10 .However, we and others have recently observed that measurements of genetic dependency in genome-scale CRISPR-Cas9 loss-of-function screens are influenced by the genomic copy number (CN) of the region targeted by the sgRNA-Cas9 complex [1][2][3][4] . Targeting Cas9 to DNA sequences within regions of high CN gain creates multiple DNA double-strand breaks (DSBs), inducing a gene-independent DNA damage response and a G2 cell-cycle arrest phenotype 2 .This systematic, sequence-independent effect due to DNA cleavage (copy-number effect)confounds the measurement of the consequences of gene deletion on cell viability (geneknockout effect) and is detectable even among low-level CN amplifications and deletions. In particular, this phenomenon hinders interpretation of CRISPR-Cas9 experiments in cancer cell

The CRISPR-Cas9 system enables genome editing and somatic cell genetic screens in mammalian cells. We performed genome scale loss-of-function screens in 33 cancer cell lines to identify genes essential for proliferation/survival and found a strong correlation between increased gene copy number and decreased cell viability after genome editing. Within regions of copy number gain, CRISPR-Cas9 targeting of both expressed and unexpressed genes, as well as intergenic loci, led to significantly decreased cell proliferation through induction of a G2 cell cycle arrest. By examining single guide RNAs that map to multiple genomic sites, we found that this cell response to CRISPR-Cas9 editing correlated strongly with the number of target loci. These observations indicate that genome targeting by CRISPR-Cas9 elicits a gene-independent anti-proliferative cell response. This effect has important practical implications for interpretation of CRISPR-Cas9 screening data and confounds the use of this technology for identification of essential genes in amplified regions.

Genome-scale CRISPR-Cas9 viability screens performed in cancer cell lines provide a systematic approach to identify cancer dependencies and new therapeutic targets. As multiple large-scale screens become available, a formal assessment of the reproducibility of these experiments becomes necessary. We analyze data from recently published pan-cancer CRISPR-Cas9 screens performed at the Broad and Sanger Institutes. Despite significant differences in experimental protocols and reagents, we find that the screen results are highly concordant across multiple metrics with both common and specific dependencies jointly identified across the two studies. Furthermore, robust biomarkers of gene dependency found in one data set are recovered in the other. Through further analysis and replication experiments at each institute, we show that batch effects are driven principally by two key experimental parameters: the reagent library and the assay length. These results indicate that the Broad and Sanger CRISPR-Cas9 viability screens yield robust and reproducible findings.