Activating mutations of KRAS are among the most common mutations found in human cancers, and cancers that harbor KRAS clearly depend on the activity of this oncogene for tumor maintenance. However, despite considerable effort, direct targeting of KRAS or known KRAS effector pathways has not yet led to effective therapies in cancers that harbor mutant KRAS. An alternative approach to direct targeting of known cancer alleles is to exploit the genetic concept of synthetic lethality, in which gene products are identified that, when suppressed or inhibited, result in cell death only in the presence of anothernon-lethal mutation. Synthetic phenotype screens in model organisms have provided insights into a broad spectrum of biological processes and in principle; this strategy provides a means to target currently "undruggable" proteins while simultaneously reducing the potential for side effects. Over the past several years, we and others have used RNAi-mediated suppression of gene expression to identify genes whose expression is required in cell lines that depend on mutant KRAS for survival. Inhibitors to some of these synthetic lethal candidates are now the subject of clinical trials in KRAS-driven cancers. However, these early studies used different cells and experimental systems and were limited by scale, technological issues or context. In addition, the discovery and development of new gene manipulation technologies such as Cas9-CRISPR, and methods to isolate and propagate human tumors now provide the opportunity to comprehensively identify novel genes and pathways that are required for the survival of KRAS-dependent cancers. In this application, we propose to use new genome scale gene manipulation technologies, potentially more relevant human and murine experimental models and advanced analytical approaches in an integrated approach to systematically identify KRAS synthetic lethal relationships in cell, organoid and animal models. Specifically, we will performedgenome scale CRISPR mediated loss of function experiments to identify KRAS co-dependencies in both in vitro and in vivo model systems and identify genes and pathways that when inhibited synergize with known KRAS effector pathways to induce tumor regression in KRAS driven cancers. These studies will permit us to define the signaling network perturbed by oncogenic KRAS necessary for tumor maintenance and progression. Targets identified by these approaches will form the basis of translational studies to develop novel therapeutic approaches.