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

摘要/摘要 常见疾病的遗传力的很大一部分，以及罕见疾病的外显性和表现力 疾病，人类基因组中远端调控元件的分离，绝大多数是细胞类型特有的 增强剂。然而，该领域面临的一个限速挑战是如何识别特定的变种， 调节这些对疾病易感性的影响的元素和调控基因。迈向新世纪的总体目标 基因组变异对功能的影响(IGVF)联盟，我们建议对100多万人进行测试 调控元件或变体对转录调控的功能影响，以及对 100,000个远端调控元件，用于它们调控的基因(S)。我们建议的第一个主题是多样性 我们将为这些目的而采用的多路技术，包括大规模并行的报告分析 (MPRAs)、CRISprQTL、饱和基因组编辑、多重质粒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图书馆的动态蜂窝系统 在一次实验中测试多种细胞类型和状态的变异体和/或元素； 这些细胞包括胚胎干细胞来源的神经前体细胞、心肌细胞、类胚体、原肠和 有机化合物，在某些情况下，还有老鼠。第三个主题涉及利用我们的经验(例如，CADD、广泛的 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， 我们设想使用由我们和其他人生成的功能测量来产生校准良好的预测 沿着构成人类的分支轨迹连续的增强子活性和变化效应 发展。我们的具体目标如下：(1)执行大规模并行验证和函数 在广泛的细胞类型背景下对候选人类增强剂的表征。(二)大手笔演出 人类基因变异与人类疾病潜在作用的平行表征。(3)对 全面的变异元素-表型目录，同时在协同互动中发挥领导作用 在政府间气候变化框架内，在方法、数据和预测的传播方面，以及在全球气候变化框架的总体目标中 财团。

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.