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：
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
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/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

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.

抽象的大规模基因组注释联盟，例如 ENCODE、Epigenomics Roadmap 和其他人已经确定了数百万个假定的监管要素。我们现在需要集中精力全面表征和量化这些元素的功能，以及非编码在这些区域内绘制基因表达和细胞表型的变体。我们的长期目标是为人类的每个调控元件和非编码变体分配功能基因组，了解该功能在不同环境中如何变化，并使用该信息更好地了解细胞适应性、疾病机制、细胞谱系规范和组织体内平衡。为了实现这一目标，我们开发了多种新颖的高通量基于 CRISPR 的技术用于表征假定基因调控的功能通过扰乱元素在内源性、原生环境中的活动来控制元素。我们已经将这些结合起来使用单细胞 RNA-seq 方法来识别每个调控元件的靶基因。我们还开发了 dCas9 效应小鼠来表征其体内自然元素语境。此外，我们还开发了基于人群的高通量报告分析（POP-STARR）来表征非编码遗传变异对整个物种的影响基因组。该提案的目的是应用和分享我们的纲要通过协作，形成互补、稳健、可扩展且特征明确的方法支持 IGVF 联盟了解基因组和基因组变异的目标发挥作用并协调复杂的表型。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，支持我们通过以下具体措施成功实现我们的目标目标：目标 1. 表征细胞生存所必需的所有基因调控元件。目标2。表征细胞谱系规范所必需的所有基因调控元件。目标3。表征选定 eQTL 区域中的所有基因调控元件。目标 4. 表征所有非小鼠模型中组织稳态所必需的编码元件。我们将把所有数据立即可用，并共享全面的实验方案、试剂和分析工具给科学界。该表征中心的多种方法共同将在理解调控元件和非编码的作用方面带来变革性进展许多不同表型的变异。