Transcriptional enhancers are the most common class of noncoding regulatory sequence in the human genome, where hundreds of thousands of enhancers far outnumber our ~20,000 protein-coding genes. There is now irrefutable evidence from experimental studies for critical roles of enhancers in human disease, suggesting that a significant proportion of the thousands of non-coding GWAS findings is due to altered enhancer functions. At the dawn of the personalized genome sequencing era, with nearly 100,000 whole genomes sequenced by NHGRI’s Genome Sequencing Program alone, determining causal links between enhancers and human disease has become a Grand Challenge in biology. Over the past three funding cycles of this R01, our group spearheaded the combined use of comparative genomics, chromatin profiling, and high-throughput mouse engineering to create transformative approaches and unique resources for enhancer discovery and characterization. Through our VISTA Enhancer Browser and a large series of associated experimental studies, we have identified ~300,000 putative enhancers from mouse tissues, performed ~3,000 transgenic mouse assays to determine in vivo enhancer activities, deleted >50 enhancers from the mouse genome, assisted hundreds of outside investigators, and provided data access to a global community of >2,000 unique VISTA users/month. Despite this substantial progress, significant remaining questions preclude the interpretation of genetic variants affecting enhancers, impeding efforts to elucidate the pathophysiological consequences of enhancer mutations. Specific barriers include a lack of understanding for how variants alter the activity of any given enhancer, and the lack of cellular resolution and thus cell type specificity information for most in vivo enhancers identified to date. In this competitive renewal, we propose to: (1) Generate a large panel of 4,500 human mutation alleles of validated VISTA enhancers and determine their impact on function in vivo through a novel, high-throughput, site-specific transgene integration system in mice, (2) Determine the expression and phenotypic impact of the most severe mutations identified in Aim 1 through 100 CRISPR knockins of human wild-type and mutant enhancers to the orthologous mouse loci, (3) Perform single-cell RNA sequencing from mouse tissues marked by active enhancers in purpose-engineered reporter mice to determine the in vivo cell type specificities of 500 validated VISTA enhancers, and (4) Provide continued community access to genomic enhancer resources including high-throughput transgenic capabilities to characterize 500 enhancers in vivo. Through these studies, we expect to gain significant insight into the role of enhancers in mammalian biology, revealing the impact of enhancer mutation on gene expression and downstream phenotypes, and the cell type specificity of enhancer activity in animals. With the now realized growth of clinical whole-genome sequencing, this work will be vital ...