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

多尺度基因组工程绘制人类和小鼠顺式调控变异图谱

• 批准号: 10737026
• 负责人: Tuuli Lappalainen
• 金额: $ 113.33万
• 依托单位: NEW YORK GENOME CENTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-15 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10737026
• 关键词: Alleles; Architecture; Autoimmune; Autoimmune Diseases; Biological; CRISPR interference; CRISPR screen; Cell Survival; Cell physiology; Cell surface; Cells; Chromatin; Chromosome Mapping; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Complex; Crohn' s disease; Data; Data Analyses; Dimensions; Disease; Elements; Enhancers; Etiology; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Complementation Test; Genetic Transcription; Genetic Variation; Genome; Genome Mappings; Genome engineering; Genomics; Goals; Heritability; Homeostasis; Human; Human Genetics; Immune; Immune response; Immunology; Immunophenotyping; In Vitro; Inflammation; Inflammatory; Infusion procedures; Insulin-Dependent Diabetes Mellitus; Intervention; Large Intestine; Link; Maps; Measurable; Measurement; Measures; Methods; Molecular; Mus; Mutagenesis; Nucleotides; Open Reading Frames; Organ; Pathogenicity; Pathway interactions; Phenotype; Physiological; Proteome; Psoriasis; Regulation; Regulator Genes; Regulatory Element; Regulatory Pathway; Repression; Research; Resolution; Resources; Role; Shapes; Site; Small Intestines; Statistical Models; Surface; System; Systemic Lupus Erythematosus; T cell differentiation; T-Lymphocyte; Technology; Testing; Tissues; Ulcerative Colitis; Umbilical Cord Blood; Untranslated RNA; Variant; base editing; candidate selection; causal variant; cell motility; cell type; chromatin immunoprecipitation; disease phenotype; disorder risk; functional genomics; gain of function; genetic association; genetic variant; genome editing; genome wide association study; genome-wide; genome-wide analysis; genomic data; genomic locus; genomic variation; gut homeostasis; gut inflammation; high throughput analysis; human disease; immune function; in vivo; in vivo Model; insight; knowledgebase; loss of function; migration; molecular phenotype; mouse model; multi-scale atlas; multiple omics; new technology; next generation; novel; peripheral blood; prevent; technological innovation; therapeutic development; trait; transcriptome; transcriptomics

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.

项目摘要 全基因组关联研究（GWAS）已经将1000多个基因组位点与人类性状和疾病联系起来。 然而，这些位点的内部机制在很大程度上是未知的，留下了GWAS的主要目标 - 阐明遗传表型的因果生物病因学-未实现。大多数GWAS位点发生在 基因组的非编码区，其对基因调控的功能影响难以阐明。这里我们 建议开发一个高通量的综合基因组工程工具箱，以建立特定背景的地图， 增强剂和变体用于免疫性状和自身免疫性疾病。我们的多PI团队由以下领域的专家组成： 互补领域：分子基因组学和CRISPR筛选，大规模人类遗传学和功能 基因组学数据分析、免疫学、统计建模和单细胞多组学。具体来说，我们建议 1）鉴定与T细胞功能相关的基因和顺式调节元件（克雷斯）。T细胞是一种中枢细胞 与多种自身免疫性疾病有关。我们将首先进行全基因组功能的丧失和获得 使用原代人T细胞筛选反映T细胞分化和活化的9种表型。对于顶部- 我们将询问每个基因附近的克雷斯，并通过单细胞分析探索其机制 和饱和诱变。2)构建T细胞中GWAS基因座的上下文特异性增强子图谱。我们将测试1000个 在相同的原代T细胞系统中使用CRISPRi/a筛选与GWAS基因座重叠的候选克雷斯， 通过单细胞ECCITE-seq补充，以测量对转录组和表面蛋白质组的影响。这 将产生大量基因座的调控元件的综合图谱，以及它们的环境特异性 对转录组和细胞表型的影响。然后，我们将构建一个上下文特定的变体映射， 通过在100个经验证的克雷斯处经由碱基编辑插入特定等位基因，在T细胞中插入调节元件。这将 产生克雷斯内调控位点的精细分辨率图。3)测试来自小鼠模型的100个同线克雷斯 肠道稳态和炎症的影响。我们将重点关注T细胞组织积累（反映活化 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， 我们将确定相关变异在人类疾病中是否具有相似的作用，并提供 针对这些基因的致病功能。通过这些目标，我们将建立一个高度概括性的 一个工具包，用于非编码元素的多尺度询问，以及一个可访问的，开放的和可重复使用的资源， 增强子和变异体对分子、细胞和生理性状的影响。总而言之，我们将分析 基因组的调控结构，利用我们的多样性扰动和表型层， 表征与自身免疫性疾病相关的基因座的功能机制。