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：
10471968
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/10471968
关键词：
Affect Biological Assay Biological Models 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 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 /摘要共同疾病的遗传力的主要部分以及稀有的渗透率和表现力疾病，人类基因组中远端调节元件的分区，绝大部分的细胞类型特异性增强剂。但是，对该领域的限制挑战是如何识别特定变体，介导这些影响疾病责任的元素和调节基因。朝着总体目标基因组变异对功能（IGVF）财团的影响，我们建议测试超过一百万人类其功能对转录调控的功能影响的调节元素或变体，以及查询它们调节的基因的100,000个远端调节元素。我们提案的第一个主题是多样性我们将采用这些目的的多重技术，包括大量平行的记者测定（MPRA），CRISPRQTL，饱和基因组编辑，多重质量编辑和单细胞组合索引，我们开创了许多。第二个主题是专注于启用给定库的动态蜂窝系统在单个实验中，要在各种细胞类型和状态上测试的变体和/或元素；这些将包括ESC衍生的神经元祖细胞，心肌细胞，胚胎体，胃类和器官，在某些情况下，小鼠。第三个主题涉及利用我们的经验（例如CADD，广泛使用的，全基因组的变异效应预测目录）以支持IGVF的总体目标。具体来说，我们设想使用美国和其他人生成的功能测量来产生良好的预测增强剂活性和变体效应，这些效应沿分支轨迹连续，包括人类发展。我们的具体目的如下：（1）执行大量并行验证和功能在广泛的细胞类型环境中的候选人增强子的表征。（2）大量表现人类遗传变异的平行表征在人类疾病中具有潜在作用。（3）为在协同互动中扮演领导角色的同时，全面的变体 - 元素 - 表型目录在IGVF中，在传播方法，数据和预测以及在总体目标中财团。