A major goal since the completion of the Human Genome Project has been to understand all functional elements in the human genome and the role they play in normal biological processes and disease. To that end, large pooled libraries of RNA interference (RNAi) reagents have been developed for genome-wide loss-of-function screens but have been hindered by 3 problems: 1) the incompleteness of protein depletion inherent in partial knock-down; 2) off-target effects from the seed sequence; and 3) genetic elements that are not transcribed are inaccessible to manipulation. Genome engineering using precisely targeted nucleases has emerged as an innovative technology to modify the genome and causally interrogate the role of different functional elements. Recently, I developed a new technology for functional genomic screening using the RNA- guided CRISPR/Cas9 nuclease (Shalem*, Sanjana*, et al., Science, 2014). Since CRISPR works on the DNA level, it is possible to manipulate non-coding elements that are inaccessible to RNAi. The research goal of this proposal is to develop new biological tools and analysis techniques for functional annotation of non-coding elements using pooled CRISPR screens. Mentored phase: First, I plan to develop and optimize high-throughput CRISPR non-coding mutagenesis libraries targeting introns, UTRs, promoters, non-coding RNAs, and intergenic regions to enable screening at high-resolution with megabase-scale coverage. Next, I will validate functional non-coding elements and use this large dataset to find unifying principles of how non-coding elements regulate gene expression. Independent phase: I plan to develop a novel CRISPR architecture for tiled deletion screens capable of deleting many segments over entire chromosomes or even entire genomes. With this technology and the increased screening throughput it enables, I will be able to develop a long-term independent research program in several possible directions, including further genome biology, personalized functional genomics, and predictive diagnostics for drug-genome interactions. The two primary areas of training needed to help me succeed in my research goals are 1) CRISPR technology development (mentor: Dr. Feng Zhang) and 2) knowledge of human genetics and non-coding variation (mentor: Dr. David Altshuler). Each mentor is an established expert in these fields. My career development plan integrates additional laboratory training, specialized tutorials in human genetics from world experts, local and national presentations of my research, and courses in scientific writing, grantsmanship and job search strategies. To assist with science- and career-related decisions, I have assembled an Advisory Committee with a team of established, senior genomics experts: Drs. Eric Lander, Steven Hyman, and David Root. The Broad Institute is an ideal environment: All Mentors and Advisors are located in one building and there are facilities for high-throughput functional screening in th RNAi Platform (Director: Dr. Root).