Massively parallel reporter assays and genome editing of ENCODE predicted regulatory elements

ENCODE 预测调控元件的大规模并行报告分析和基因组编辑

• 批准号: 10238522
• 负责人: Nadav Ahituv
• 金额: $ 140.95万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-02-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10238522
• 关键词: 3' Untranslated Regions; ATAC-seq; Address; Adopted; Architecture; Biochemical; Biological Assay; Biology; CRISPR/Cas technology; Catalogs; Cell Line; Cells; ChIP-seq; Chromatin; Communities; Computer Models; DNase I hypersensitive sites sequencing; Data; Development; Disease; Dissection; Elements; Encyclopedia of DNA Elements; Encyclopedias; Enhancers; Experimental Designs; Gene Expression Regulation; Gene Silencing; Genes; Genetic; Genetic Transcription; Genetic Variation; Genome; Genomics; Genotype; Goals; HepG2; Human Genome; Infection; K-562; Lentivirus; Libraries; Link; Linkage Disequilibrium; Maps; Massive Parallel Sequencing; Methods; Modeling; Molecular Profiling; Motivation; Mutation; Pattern; Population; Protocols documentation; Regulatory Element; Reporter; Repression; Research Design; Research Personnel; Resources; Sampling; System; Techniques; Technology; Testing; Tissues; Validation; Variant; Work; base; cell type; design; disorder risk; experimental study; genetic variant; genome editing; genome wide association study; genome-wide; human disease; insight; interest; parent grant; promoter; repaired; transcriptome sequencing

Since its inception in 2003, the Encyclopedia of DNA Elements (ENCODE) Consortium has made remarkable progress towards the identification of all functional elements in the human genome. However, major limitations of the current catalog are that the vast majority of elements have not been functionally characterized, the impact of genetic variation on their function is poorly defined, the precise levels of activation or repression that they confer remain unmeasured, and the specific gene(s) that they regulate are not definitively known. To address these gaps, we will implement ‘in genome’ massively parallel functional assays to characterize over 100,000 ENCODE-based candidate regulatory elements, to confirm and quantify their activities as well as to link many of them to their target genes. In a systematic comparison of episomal vs. genomic massively parallel reporter assays (MPRA), we show that episomal assays fail to accurately capture the full patterns of regulatory activity that are observed in the context of chromatin. We therefore focus exclusively on methods that test candidate regulatory elements in an integrated, ‘in genome’ context. First, using lentivirus-based massively parallel reporter assays, we will characterize at least 100,000 ENCODE-based regulatory elements for their promoter/enhancer activity while integrated into the genome (lentiMPRA; Aim 1a). Importantly, lentiMPRA can be carried out in almost every cell type and leverages ongoing developments in lentivirus technology. Early results will be used to iteratively develop models that make better selections for subsequent rounds of functional characterization. Second, we will use CRISPR/Cas9 and multiplex homology directed repair to integrate a subset of candidate enhancers to the 3’ UTR of transcriptionally inactive genes, allowing us to further validate and characterize their ability to activate transcription in a natural genomic context (‘in genome’ STARR-seq; Aim 1b). Finally, we will implement a new paradigm involving CRISPR/Cas9-based multiplex genome editing followed by RNAseq/ ATAC-seq molecular profiling to characterize a genome-wide subset of candidate regulatory elements in their native genomic context for the functional consequences of mutations on them, while also determining the target gene(s) that they regulate (massively parallel genome editing; Aim 2). Although we will initially focus our efforts on K562 and HepG2 cells, we will also perform work in other cell lines as appropriate for the needs of the ENCODE Consortium, with 25% of our capacity dedicated to a common set of elements. Combined with the efforts of the other functional characterization centers, our work will provide unprecedented ‘in genome’ validation and characterization of ENCODE-defined candidate regulatory elements, while also facilitating insights into our understanding of the basic biology of gene regulation and how regulatory variants contribute to human disease risk.

自 2003 年成立以来，DNA 元素百科全书 (ENCODE) 联盟取得了令人瞩目的成就 人类基因组中所有功能元件的鉴定取得进展。然而，主要的局限性 当前目录中的绝大多数元素尚未进行功能表征，影响 遗传变异对其功能的影响尚不清楚，但它们激活或抑制的精确水平 赋予的作用仍未测量，并且它们调节的特定基因尚不清楚。致地址 这些差距，我们将实施“基因组内”大规模并行功能测定，以表征超过 100,000 基于 ENCODE 的候选监管要素，以确认和量化其活动并将许多 他们到他们的目标基因。游离型与基因组大规模平行报告基因的系统比较 分析（MPRA），我们表明附加型分析无法准确捕获监管活动的完整模式 在染色质背景下观察到的。因此，我们只关注测试候选人的方法 整合的“基因组”环境中的调控元件。首先，使用基于慢病毒的大规模并行报告基因 分析中，我们将表征至少 100,000 个基于 ENCODE 的启动子/增强子调控元件 活性，同时整合到基因组中（lentiMPRA；目标 1a）。重要的是，lentiMPRA 可以在 几乎所有细胞类型，并利用慢病毒技术的持续发展。早期结果将被使用 迭代开发模型，为后续几轮功能表征做出更好的选择。 其次，我们将使用 CRISPR/Cas9 和多重同源定向修复来整合候选的子集 转录失活基因 3' UTR 的增强子，使我们能够进一步验证和表征它们 在自然基因组环境中激活转录的能力（“基因组中”STARR-seq；目标 1b）。最后，我们将 实施一种新范例，涉及基于 CRISPR/Cas9 的多重基因组编辑，然后进行 RNAseq/ ATAC-seq 分子分析，用于表征候选调控元件的全基因组子集 他们的原生基因组背景决定了突变对他们的功能影响，同时也决定了 它们调节的目标基因（大规模并行基因组编辑；目标 2）。尽管我们最初会集中精力 在K562和HepG2细胞的努力中，我们还将根据需要在其他细胞系中进行工作 ENCODE 联盟，将 25% 的产能专门用于一组通用元素。结合 在其他功能表征中心的努力下，我们的工作将提供前所未有的“基因组内”验证 和 ENCODE 定义的候选监管要素的表征，同时还有助于深入了解我们的 了解基因调控的基础生物学以及调控变异如何导致人类疾病 风险。

项目成果

Genetic variation in the SIM1 locus is associated with erectile dysfunction.

• DOI: 10.1073/pnas.1809872115
• 发表时间: 2018-10-23
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Jorgenson E;Matharu N;Palmer MR;Yin J;Shan J;Hoffmann TJ;Thai KK;Zhou X;Hotaling JM;Jarvik GP;Ahituv N;Wessells H;Van Den Eeden SK
• 通讯作者: Van Den Eeden SK

MPRAbase: A Massively Parallel Reporter Assay Database.

MPRAbase：大规模并行报告分析数据库。

• DOI: 10.1101/2023.11.19.567742
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Zhao,Jingjing;Baltoumas,FotisA;Konnaris,MaxwellA;Mouratidis,Ioannis;Liu,Zhe;Sims,Jasmine;Agarwal,Vikram;Pavlopoulos,GeorgiosA;Georgakopoulos-Soares,Ilias;Ahituv,Nadav
• 通讯作者: Ahituv,Nadav

The power of multiplexed functional analysis of genetic variants.

• DOI: 10.1038/nprot.2016.135
• 发表时间: 2016-10
• 期刊: Nature protocols
• 影响因子: 14.8
• 作者: Gasperini M;Starita L;Shendure J
• 通讯作者: Shendure J

Massively parallel jumping assay decodes Alu retrotransposition activity.

大规模并行跳跃测定解码 Alu 逆转录转座活性。

• DOI: 10.1101/2024.04.16.589814
• 发表时间: 2024
• 期刊: bioRxiv : the preprint server for biology
• 作者: Matharu,Navneet;Zhao,Jingjing;Sohota,Ajuni;Deng,Linbei;Hung,Yan;Li,Zizheng;Sims,Jasmine;Rattanasopha,Sawitree;Meyer,Josh;Carbone,Lucia;Kircher,Martin;Ahituv,Nadav
• 通讯作者: Ahituv,Nadav

Genome-wide strand asymmetry in massively parallel reporter activity favors genic strands.

• DOI: 10.1101/gr.270751.120
• 发表时间: 2021-05
• 期刊: Genome research
• 影响因子: 7
• 作者: Roberts BS;Partridge EC;Moyers BA;Agarwal V;Newberry KM;Martin BK;Shendure J;Myers RM;Cooper GM
• 通讯作者: Cooper GM