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

Pro-inflammatory cytokines produced in the tumor microenvironment facilitate tumor development and metastatic progression. In particular, TNF-α promotes cancer invasion and angiogenesis associated with epithelial-mesenchyme transition (EMT), however, the mechanisms underlying its induction of EMT in cancer cells remain unclear. Here we show that EMT and cancer stemness properties induced by chronic treatment with TNFα̣ are mediated by the upregulation of the transcriptional repressor Twist1. Exposure to TNF-α rapidly induced Twist1 mRNA and protein expression in normal breast epithelial and breast cancer cells. Both IKK-β and NF-κB p65 were required for TNF-α-induced expression of Twist1, suggesting the involvement of canonical NF-κB signaling. In support of this likelihood, we defined a functional NF-κB binding site in the Twist1 promoter and overexpression of p65 was sufficient to induce transcriptional upregulation of Twist1 along with EMT in mammary epithelial cells. Conversely, suppressing Twist1 expression abrogated p65-induced cell migration, invasion, EMT and stemness properties, establishing that Twist1 is required for NF-κB to induce these aggressive phenotypes in breast cancer cells. Taken together, our results establish a signaling axis through which the tumor microenvironment elicits Twist1 expression to promote cancer metastasis. We suggest that targeting NF-κB-mediated Twist1 upregulation may offer an effective a therapeutic strategy for breast cancer treatment.