Massively parallel characterization of variants and elements impacting transcriptional regulation in dynamic cellular systems

影响动态细胞系统转录调控的变异体和元件的大规模并行表征

基本信息

批准号：
10676325
负责人：
Nadav Ahituv
金额：
$ 179.83万
依托单位：
UNIVERSITY OF WASHINGTON
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/10676325
关键词：
Affect Biological Assay Biological Models Calibration Cardiac Cardiac Myocytes Catalogs Cells Characteristics Chromatin Computer software Coupling Data Data Set Development Disease Distal Elements Embryo Enhancers Gene Expression Gene Expression Regulation Genes Genetic Genomics Goals Heritability Human Human Biology Human Development Human Genetics Human Genome Individual Leadership Learning Libraries Link Measurement Mediating Methods Modeling Mus Mutagenesis Mutate Mutation Neurologic Organoids Penetrance Phenotype Population Protocols documentation Rare Diseases Regulatory Element Reporter Rest Risk Role Severities System Technology Teratoma Testing Therapeutic Transcriptional Regulation Untranslated RNA Validation Variant Work cell type combinatorial data sharing directed differentiation disorder risk experience experimental study falls genetic variant genome editing genome-wide genomic variation human disease in vitro Model in vivo indexing nerve stem cell open source predictive modeling prime editing rare variant

项目摘要

SUMMARY / ABSTRACT A major fraction of heritability for common diseases, as well as for the penetrance and expressivity of rare diseases, partitions to distal regulatory elements in the human genome, overwhelmingly cell type-specific enhancers. However, a rate-limiting challenge for the field has been how to identify the specific variants, elements and regulated genes that mediate these effects on disease liability. Towards the overall goals of the Impact of Genomic Variation on Function (IGVF) Consortium, we propose to test over one million human regulatory elements or variants for their functional effects on transcriptional regulation, as well as to query over 100,000 distal regulatory elements for the gene(s) that they regulate. A first theme of our proposal is the diversity of multiplex technologies that we will employ to these ends, including massively parallel reporter assays (MPRAs), crisprQTL, saturation genome editing, multiplex prime editing and single cell combinatorial indexing, many of which we pioneered. A second theme is a focus on dynamic cellular systems that enable a given library of variants and/or elements to be tested across a broad range of cell types and states within a single experiment; these will include ESC-derived neuronal progenitors, cardiomyocytes, embryoid bodies, gastruloids and organoids, and in select cases, mice. A third theme involves leveraging our experience (e.g. CADD, a widely used, genome-wide catalog of variant effect predictions) to support the overarching goals of IGVF. Specifically, we envision using functional measurements generated by us and others to produce well-calibrated predictions of enhancer activity and variant effects that are continuous along the branching trajectories that comprise human development. Our specific aims are as follows: (1) To perform massively parallel validation and functional characterization of candidate human enhancers in a broad range of cell type contexts. (2) To perform massively parallel characterization of human genetic variants with potential roles in human disease. (3) To contribute to a comprehensive variant-element-phenotype catalog while taking a leadership role in synergistic interactions within IGVF, in the dissemination of methods, data and predictions, and in the overarching goals of the consortium.

摘要/摘要常见疾病遗传力的主要部分，以及罕见疾病的外显率和表现力疾病，人类基因组中远端调控元件的分区，绝大多数是细胞类型特异性的增强剂。然而，该领域的一个限制速率的挑战是如何识别特定的变体，介导这些对疾病易感性影响的元素和调节基因。为实现总体目标基因组变异对功能的影响（IGVF）联盟，我们建议测试超过一百万人调控元件或变体对转录调控的功能影响，以及查询它们所调节的基因有 100,000 个远端调节元件。我们提案的第一个主题是多样性我们将为此目的采用的多重技术，包括大规模并行报告基因检测 (MPRA)、crisprQTL、饱和基因组编辑、多重引物编辑和单细胞组合索引，其中许多都是我们开创的。第二个主题是关注支持给定库的动态细胞系统在单个实验中要在广泛的细胞类型和状态中测试的变体和/或元件；这些将包括 ESC 衍生的神经元祖细胞、心肌细胞、胚状体、原肠胚细胞和类器官，在某些情况下，还有小鼠。第三个主题涉及利用我们的经验（例如 CADD，一个广泛使用全基因组变异效应预测目录）来支持 IGVF 的总体目标。具体来说，我们设想使用我们和其他人生成的功能测量来产生经过良好校准的预测增强子活性和变异效应沿着组成人类的分支轨迹连续发展。我们的具体目标如下：（1）执行大规模并行验证和功能在广泛的细胞类型背景下候选人类增强子的表征。（二）大规模演出人类遗传变异在人类疾病中的潜在作用的平行表征。 (3) 为促进全面的变异元素表型目录，同时在协同相互作用中发挥领导作用在 IGVF 内部，在方法、数据和预测的传播中，以及在 IGVF 的总体目标中财团。