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

基本信息

批准号：
10475750
负责人：
GREGORY E CRAWFORD
金额：
$ 223.83万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
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/10475750
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 Pharmacology Phenotype Play Positioning Attribute Protocols documentation Quantitative Trait Loci Reagent Regenerative response Regulator Genes Regulatory Element Reporter Role Shapes Stimulus Suspensions Tissues Transgenic Organisms Undifferentiated Untranslated RNA Variant Viral Viral Vector base causal variant cell fate specification cell growth cell type data resource 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 population based response single-cell RNA sequencing tissue regeneration tool trait whole genome

项目摘要

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.

抽象的大型基因组注释联盟，例如编码，表观基因组路线图和其他人已经确定了数百万个推定的监管要素。我们现在需要专注于全面表征和量化这些元素的功能，而非编码这些区域内映射的变体，基因表达和细胞表型。我们的长期目标是将功能分配给人类的每个监管元素和非编码变体基因组，了解该功能在不同上下文中的变化，并将这些信息使用更好地了解细胞健身，疾病机制，细胞谱系规格和组织稳态。为了实现这一目标，我们开发了多个新颖的高通量基于CRISPR的技术来表征推定基因调节的功能通过在内源性的本地环境中扰动其活性来通过元素。我们耦合了这些具有单细胞RNA-Seq的方法可确定每个调节元件的靶基因。我们还开发了DCAS9效应子小鼠以表征其自然体内元素语境。此外，我们开发了基于人群的高通量记者测定法（流行音乐）表征整个不编码遗传变异的影响基因组。该提案的目的是申请并分享我们的汇编通过合作，互补，健壮，可扩展性和良好的方法支持IGVF财团的目标，以了解基因组和基因组变异功能和编排复杂表型。我们在开发，应用和共享这些高通量表征方法，并提供对所有数据的访问支持我们将通过以下特定的特定来成功实现目标目的：目标1。表征所有基因调节元件对于细胞存活所必需的。目标2。表征所有基因调节元件对细胞谱系规范必不可少的。目标3。表征所选eqtl区域中所有基因调节元件。目标4。表征所有非 - 在小鼠模型中对组织稳态必不可少的编码元素。我们将制作所有数据立即可用，以及共享全面的协议，试剂和分析工具向科学界。在一起，这个特征中心的多种方法将导致变革性进步在理解监管要素和非编码的作用方面许多不同表型的变体。