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

项目总结 全基因组关联研究已将数千个基因组位点与人类特征和疾病联系起来。 然而，这些基因座的机制内部工作在很大程度上是未知的，留下了GWAs的主要目标 -阐明可遗传表型的因果生物病因学-未完成。大多数GWA基因座都发生在 基因组的非编码区，其对基因调控的功能影响很难解开。在这里，我们 建议开发一个高通量、集成的基因组工程工具箱，以构建特定于背景的地图 免疫特性和自身免疫性疾病的增强剂和变种。我们的多PI团队由以下专家组成 互补领域：分子基因组学和CRISPR筛查、大规模人类遗传学和功能 基因组数据分析、免疫学、统计建模和单细胞多组学。具体来说，我们建议 目的：1)确定与T细胞功能相关的基因和顺式调节元件(CRE)。T细胞是一个中心细胞 与多种自身免疫性疾病有关。我们将首先在全基因组范围内进行功能丧失和获得 利用原代人类T细胞筛选出反映T细胞分化和激活的9种表型。对于顶端- ，我们将询问每个基因附近的CRE，并通过单细胞图谱来探索它们的机制 饱和诱变。2)构建T细胞中Gwas基因座的特定背景增强子图。我们将测试1,000 在同一初级T细胞系统中使用CRISPRi/a筛选重叠Gwas基因座的候选CRE， 辅以单细胞ECCITE-SEQ，以测量对转录组和表面蛋白质组的影响。这 将为大量的基因座及其特定于环境的调控因素生成一张全面的地图 对转录和细胞表型的影响。然后，我们将构建一个特定于上下文的变体映射 通过碱基编辑在100个有效的Cres插入特定的等位基因，在T细胞中调节元件。这将是 制作一张CRES内监管地点的精细分辨率地图。3)在小鼠模型中测试100个Syntenic Cres 体内肠道动态平衡和炎症的影响。我们将重点关注T细胞组织的聚集(反映激活 和迁移)以及迁移细胞的转录和细胞表面表型的改变。通过这样做， 我们将确定相关的变异是否在人类疾病中具有类似的作用，并提供途径 以这些基因的致病功能为目标。通过这些目标，我们将建立一个高度可概括的 用于多尺度询问非编码元素的工具包以及可访问的、开放的和可重用的资源 增强子和变异对分子、细胞和生理特性的影响。总之，我们将分析 基因组的调控架构，利用我们不同的扰动和表型层，以及 描述与自身免疫性疾病相关的基因座的作用机制。

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.