Multiscale genome engineering to map cis-regulatory variants in human and mouse

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PROJECT SUMMARY Genome-wide association studies (GWAS) have linked 1000s of genomic loci with human traits and diseases. However, the mechanistic inner workings of these loci are largely unknown, leaving the principal goal of GWAS – illuminating the causal biological etiology of heritable phenotypes – unfulfilled. Most GWAS loci occur within noncoding regions of the genome whose functional impact on gene regulation is difficult to unravel. Here, we propose to develop a high-throughput, integrated genome engineering toolbox to build context-specific maps of enhancers and variants for immune traits and autoimmune disorders. Our multi-PI team consists of experts in complementary fields: molecular genomics and CRISPR screens, large-scale human genetics and functional genomics data analysis, immunology, statistical modeling, and single-cell multiomics. Specifically, we propose to: 1) Identify genes and cis-regulatory elements (CREs) relevant for T cell function. T cells are a central cell type implicated in multiple autoimmune diseases. We will first perform genome-wide loss- and gain-of-function screens for 9 phenotypes reflecting T-cell differentiation and activation using primary human T cells. For top- ranked genes, we will interrogate CREs near each gene and explore their mechanisms via single-cell profiling and saturation mutagenesis. 2) Build a context-specific enhancer map of GWAS loci in T cells. We will test 1,000 candidate CREs that overlap GWAS loci using CRISPRi/a screens in the same primary T-cell system, complemented by single-cell ECCITE-seq to measure effects on the transcriptome and surface proteome. This will produce a comprehensive map of regulatory elements for a large number of loci, and their context-specific impact on transcriptomic and cellular phenotypes. Then, we will construct a context-specific variant map of regulatory elements in T cells by inserting specific alleles via base editing at 100 validated CREs. This will produce a fine-resolution map of regulatory sites within CREs. 3) Test 100 syntenic CREs from in mouse models of gut homeostasis and inflammation in vivo. We will focus on T-cell tissue accumulation (reflecting activation and migration) and alterations in transcriptional and cell surface phenotypes of the migrating cells. By doing so, we will determine if the relevant variants have similar roles in human disease and provide pathways towards targeting the pathogenic functions of those genes. Through these Aims, we will build a highly-generalizable toolkit for multi-scale interrogation of noncoding elements and an accessible, open, and reusable resource of enhancer and variant effects on molecular, cellular, and physiological traits. Altogether, we will analyze the regulatory architecture of the genome, leveraging our diverse perturbations and phenotypic layers, and characterize functional mechanisms of loci associated with autoimmune disorders.