PROJECT SUMMARY Gene expression enhancers are critical for establishing transcriptional programs in numerous contexts throughout metazoans. There have been incredible advancements over the last decade in our ability to predict enhancers and recent consortium efforts have identified millions of candidate enhancers across the human genome. Unfortunately, it is difficult to test candidate enhancers at their endogenous locus with current approaches requiring a large amount of effort to assess the activity of a single enhancer. Enhancers often work in combination to regulate a single gene and current methods are not amenable to studying combinatorial enhancer usage on a large-scale. To address the need for high-throughput methods for studying enhancers in their native genomic context, we have adapted the CRISPR/Cas9 system to target enhancers and plan to test the necessity and sufficiency of hundreds of genomic loci in driving transcriptional responses. By targeting multiple distal regulatory regions simultaneously, we also have the ability to assess how enhancers work in concert. During this project we will develop methods for high-throughput testing of enhancers in situ, using estrogen receptor α (ER) and estrogen signaling in endometrial cancer cells as a model system. There are three aims of the proposed project: (1) Determination of combinations of candidate enhancers that are necessary for proper transcription regulation; (2) Identification of combinations of regulatory regions that are sufficient to recapitulate an ER-driven transcriptional response; (3) Comparison of genetic deletion of enhancers to epigenetic engineering. The successful completion of this project will lead to the discovery of regulatory regions that are important for transcription regulation and provide an understanding of how combinations of enhancers work in concert to regulate transcription. This project will also establish methods for high-throughput testing of enhancers in their endogenous locus, which will be useful for studying enhancers in a number of additional biological systems.