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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：
10831639
负责人：
Nadav Ahituv
金额：
$ 3.39万
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：
2021-09-09 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10831639
关键词：
ATAC-seq Affect Base Sequence Biological Assay Biological Markers Calibration Cancer cell line Cardiac Myocytes Catalogs Cause of Death Cell Proliferation Cells ChIP-seq Characteristics Clustered Regularly Interspaced Short Palindromic Repeats Code Computer Models DNA Sequence DNase I hypersensitive sites sequencing Data Data Set Development Disease Distal Elements Enhancers Epithelium Gene Expression Gene Expression Regulation Gene Targeting Genes Genetic Genome Goals Grant Heritability Human Human Development Human Genetics Human Genome Individual Interruption Leadership Learning Lentivirus Libraries Malignant Neoplasms Malignant neoplasm of liver Malignant neoplasm of prostate Measurement Mediating Mesenchymal Methods Migration Assay Modeling Mus Mutation Neoplasm Metastasis Oncogenes Organoids Patients Penetrance Phenotype Proliferating Proliferation Marker Prostate Rare Diseases Recurrence Regulator Genes Regulatory Element Reporter Risk Role Severities Somatic Mutation System Technology Testing The Cancer Genome Atlas Therapeutic Transcriptional Regulation Untranslated RNA Validation Variant Work cancer type cell type combinatorial computerized tools disorder risk driver mutation experience experimental study falls functional genomics genetic variant genome editing genome-wide genomic data genomic variation human disease indexing nerve stem cell novel predictive tools prime editing promoter prostate cancer cell line tumor

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.

总结/摘要常见病的遗传率，以及罕见病的遗传率和表现率，疾病，人类基因组中远端调控元件的分区，压倒性的细胞类型特异性增强剂。然而，该领域的一个限速挑战是如何识别特定的变体，元件和调节基因介导这些疾病的易感性。实现联合国千年发展目标的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作用，以及查询 100，000个远端调控元件用于它们所调控的基因。我们提案的第一个主题是多样性我们将采用多重技术来达到这些目的，包括大规模平行报告分析，（MPRAs）、crisprQTL、饱和基因组编辑、多重引物编辑和单细胞组合索引，其中许多是我们开创的。第二个主题是关注动态细胞系统，在单个实验中跨广泛的细胞类型和状态测试的变体和/或元件; 这些细胞将包括ESC衍生的神经元祖细胞、心肌细胞、胚状体、类胃细胞和类器官，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，一个广泛的使用，全基因组范围的变异效应预测目录），以支持IGVF的总体目标。具体地说，我们设想使用我们和其他人产生的功能测量来产生校准良好的预测增强子活性和变体效应的连续沿着分支轨迹，包括人类发展我们的具体目标如下：（1）进行大规模并行验证和功能验证。在广泛的细胞类型背景下，候选人增强子的表征。(2)大规模地表演平行表征在人类疾病中具有潜在作用的人类遗传变异。(3)支持必要的全面的变异-元件-表型目录，同时在协同相互作用中发挥领导作用在IGVF中，在方法、数据和预测的传播中，以及在财团