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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：
10297406
负责人：
GREGORY E CRAWFORD
金额：
$ 95.64万
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/10297406
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 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.

摘要