Project Summary The human genome encodes over 2 million DNA regulatory elements called enhancers that control gene expression in specific cell types and states. Enhancers harbor tens of thousands of genetic variants that influence risk for common diseases and traits. Each of these enhancer variants could reveal insights into the molecular mechanisms of human diseases. Yet, we have lacked tools to systematically map which enhancers regulate which genes in each of the thousands of cell types in the human body. To address this challenge, we have recently developed CRISPR tools to experimentally test thousands of enhancers in parallel, and discovered a simple computational model that can predict enhancer-gene regulation from chromatin state. These nascent technologies suggest a new strategy to map enhancers across many cell types to connect noncoding variants to target genes. Here we will develop and extend these new technologies to map and predict enhancer-gene connections at single-cell resolution. First, we will characterize how enhancer function changes across developmental trajectories, by combining our CRISPR tool with a new single-cell readout method to survey thousands of enhancer-gene connections in differentiating vascular cells. Second, we will develop a computational model that can predict enhancer-gene regulation from single-cell measurements of chromatin accessibility. Third, we will apply these tools to build maps of enhancer-gene regulation in the adult human heart, and demonstrate the utility of these maps by characterizing genetic variants associated with coronary artery disease. These technologies will enable mapping enhancer-gene regulation in many cell types, building a foundational resource for connecting noncoding genetic variants to their molecular functions. This approach will be broadly applicable to any common, complex disease. This proposal builds on the PI’s experiences in genomics and team science with the ENCODE Consortium and Variant- to-Function Initiative. This R35 Genomic Innovator Award will help the PI launch a career at the interface of human genomics and cardiovascular disease that will include significant contributions to team science efforts. The environment at Stanford University in the Department of Genetics and Children’s Heart Center is ideal for supporting these scientific and leadership roles.