High-Throughput Functional Annotation of Gene Regulatory Elements and Variants Critical to Complex Cellular Phenotypes

对复杂细胞表型至关重要的基因调控元件和变异体的高通量功能注释

• 批准号: 10689190
• 负责人: GREGORY E CRAWFORD
• 金额: $ 191.9万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10689190
• 关键词: 3-Dimensional; Biological; Biological Assay; Biological Process; CRISPR screen; CRISPR/Cas technology; Cataloging; Catalogs; Cell Lineage; Cell Survival; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complex; Coupled; Cultured Cells; Data; Development; Disease; Disease Progression; Disease susceptibility; Elements; Environment; Environmental Risk Factor; Gene Expression; Gene Expression Regulation; Gene Order; Genes; Genetic Polymorphism; Genetic Variation; Genome; Genomics; Genotype-Tissue Expression Project; Goals; Guide RNA; Hepatocyte; Homeostasis; Human; Human Development; Human Genetics; Human Genome; Learning; Libraries; Link; Linkage Disequilibrium; Maintenance; Maps; Methods; Mus; Muscle Cells; Natural regeneration; Neurons; Pharmaceutical Preparations; Phenotype; Play; Positioning Attribute; Protocols documentation; Quantitative Trait Loci; Reagent; Regulator Genes; Regulatory Element; Reporter; Resources; Role; Shapes; Specific qualifier value; Stimulus; Suspensions; Tissues; Transgenic Organisms; Undifferentiated; Untranslated RNA; Variant; Viral; Viral Vector; causal variant; cell fate specification; cell growth; cell type; delivery vehicle; epigenome editing; epigenomics; fitness; gene function; genome annotation; genome-wide; genomic variation; in vitro Model; in vivo; induced pluripotent stem cell; mouse model; novel; pharmacologic; population based; response; single-cell RNA sequencing; tissue regeneration; tool; trait; whole genome

ABSTRACT Large scale genome annotation consortia such as ENCODE, Epigenomics Roadmap, and others have identified millions of putative regulatory elements. We now need to focus efforts on comprehensively characterizing and quantifying the function of those elements, and noncoding variants that map within these regions, on gene expression and cell phenotypes. Our long-term goal is to assign function to every regulatory element and noncoding variant in the human genome, understand how that function changes in different contexts, and use that information to better understand cell fitness, disease mechanisms, cell lineage specification, and tissue homeostasis. To accomplish this goal, we have developed multiple novel high-throughput CRISPR-based technologies for characterizing the function of putative gene regulatory elements by perturbing their activity in their endogenous, native context. We have coupled these methods with single-cell RNA-seq to identify the target gene(s) for each regulatory element. We have also developed dCas9 effector mice to characterize elements in their natural in vivo context. In addition, we have developed population-based high-throughput reporter assays (POP-STARR) to characterize the impact of noncoding genetic variation across the entire genome. The objective of this proposal is to apply and share our compendium of complementary, robust, scaleable, and well-characterized methods by working collaboratively to support the IGVF Consortium goals of understanding how genomes and genomic variation function and orchestrate complex phenotypes. Our track record in developing, applying, and sharing these high-throughput characterization methods, as well as providing access to all data, supports that we will be successful in accomplishing our objective via the following specific aims: Aim 1. Characterize all gene regulatory elements essential for cell survival. Aim 2. Characterize all gene regulatory elements essential to cell lineage specification. Aim 3. Characterize all gene regulatory elements in select eQTL regions. Aim 4. Characterize all non- coding elements essential to tissue homeostasis in a mouse model. We will make all data immediately available, as well as share comprehensive protocols, reagents, and analysis tools to the scientific community. Together, the diverse approaches of this Characterization Center will lead to transformative progress in understanding the role of regulatory elements and noncoding variants across many diverse phenotypes.

摘要 大型基因组注释联合体，如ENCODE、表观基因组学路线图和 其他人已经确定了数百万个假定的监管要素。我们现在需要把精力集中在 全面表征和量化这些元素的功能，并进行非编码 在这些区域内映射的变体，对基因表达和细胞表型有影响。我们的长期合作 目标是为人类中的每个调节元件和非编码变体分配功能 基因组，了解该功能如何在不同的环境中变化，并使用该信息来 更好地了解细胞适合性、疾病机制、细胞谱系规范和组织 动态平衡。为了实现这一目标，我们开发了多个新颖的高通量 基于CRISPR的基因调控功能鉴定技术 通过扰乱元素在其内生的、本地的上下文中的活动。我们把这些连在一起了 方法用单细胞RNAseq方法确定每个调控元件的靶基因(S)。我们 还培育了dCas9效应小鼠来表征它们体内天然的元素 背景。此外，我们还开发了基于人群的高通量记者检测方法 (POP-STAR)来表征非编码遗传变异在整个 基因组。这项建议的目的是应用和分享我们的概要 通过协作实现互补性、健壮性、可伸缩性和良好特性的方法 支持IGVF联合会的目标，了解基因组和基因组变异 功能和编排复杂的表型。我们在开发、应用和 共享这些高通量表征方法，以及提供对所有数据的访问， 支持我们通过以下具体措施成功实现我们的目标 目的：目的1.鉴定细胞生存所必需的所有基因调控元件。目标2. 描述对细胞谱系规定至关重要的所有基因调控元件。目标3. 确定选定eQTL区域中所有基因调控元件的特征。目标4.描述所有非 在小鼠模型中编码组织动态平衡所必需的元件。我们将把所有数据 立即可用，并共享全面的方案、试剂和分析工具 向科学界致敬。总而言之，该特性中心的不同方法将 导致在理解调控元件和非编码的作用方面取得变革性的进展 许多不同表型的变异体。