Multiscale exploration of the functional non-coding genome

功能性非编码基因组的多尺度探索

• 批准号: 10170407
• 负责人: Luca Pinello
• 金额: $ 50.76万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10170407
• 关键词: Affect; BCL11A gene; Cells; Cellular Assay; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complex; Development; Disease; Dissection; Elements; Enhancers; Foundations; Funding; Gene Expression; Genetic; Genetic Transcription; Genetic Variation; Genome; Goals; Hemoglobin; Induced Mutation; Link; National Human Genome Research Institute; Nucleic Acid Regulatory Sequences; Nucleotides; Phenotype; Play; Regulatory Element; Research; Resolution; Role; Severity of illness; Sickle Cell Anemia; Structure; System; Therapeutic; Therapeutic Intervention; Translating; Untranslated RNA; Variant; Vision; Work; beta Thalassemia; cell type; computerized tools; developmental disease; experience; genetic variant; genome wide association study; hemoglobin B; human disease; improved; insight; novel; programs; promoter; therapeutic target; trait; translational impact; user-friendly

PROJECT SUMMARY Most disease- or trait-associated genetic variants lie within non-coding sequences of the genome such as enhancers, promoters and insulators. Those sequences regulate gene expression, play critical roles in determining disease severity and may serve as targets for novel rational therapeutic interventions. However, we still lack insights on the exact mechanisms by which non-coding sequences are translated into function and on the impact of genetic variation within them. CRISPR perturbations of non-coding elements, offer unprecedented opportunities for assessing their function in a myriad of developmental and disease settings. We hypothesize that an improved understanding of non-coding sequences by direct perturbation in their endogenous context, and with a direct readout of the genetic perturbations, will offer new opportunities to therapeutically intervene in human disease. The long-term goal of our work is to overcome the limitations of the current strategies to study the non-coding genome. Our overall vision is to push our understanding of the mechanisms of action of non-coding sequences on gene expression at nucleotide resolution and single-cell level. The main objective of this proposal is a multi-scale discovery and dissection of regulatory elements by combining CRISPR targeted perturbations and single cell assays with two key goals: (1) Uncover functional non-coding elements with unbiased and generalizable approaches for different cell types and elucidate their regulatory grammar and mechanisms of action on gene expression. (2) Study how endogenous or perturbation induced mutations in non-coding sequences are reflected in gene expression programs at a single-cell level. To pilot our conceptual framework, we will study the non- coding regulatory elements within the BCL11A gene, a master regulator of the hemoglobin switch and a therapeutic target for sickle cell disease (SCD) and β-thalassemia. In fact, several clinical trials (e.g. NCT03432364) are underway aiming to disrupt regulatory sequences at BCL11A as a therapy for these β- hemoglobin disorders. This is one of the most well-studied loci identified by GWAS and is a system in which we have extensive experience and expertise. However, our approaches will also be generally applicable to other loci linked to traits or diseases. At the end of this project we will provide a general framework and user-friendly computational tools to study the function and the structure of non-coding regulatory sequences generalizable to different perturbation screens, regulatory regions, cell types and phenotypes. Importantly, all the computational tools developed in this proposal will be shared with NHGRI funded consortia with similar goals such as ENCODE, and with the broader scientific community. We anticipate that the proposed research could have a positive translational impact providing the foundation to develop strategies involving non-coding sequence perturbations with direct therapeutic potential for human disease.

项目摘要 大多数疾病或性状相关的遗传变异位于基因组的非编码序列内，例如 增强剂、促进剂和绝缘剂。这些序列调节基因表达，在人类基因组中发挥关键作用。 确定疾病的严重程度，并可作为新的合理的治疗干预的目标。 然而，我们仍然缺乏对非编码序列翻译的确切机制的见解 以及它们内部遗传变异的影响。CRISPR非编码干扰 要素，提供了前所未有的机会，以评估其在各种发展和 疾病设置。我们假设，通过直接测序来提高对非编码序列的理解， 干扰在其内源性背景下，并与遗传干扰的直接读出，将提供 为人类疾病的治疗提供了新的机会。我们工作的长期目标是 克服了目前研究非编码基因组策略的局限性。我们的总体愿景是 推动我们对非编码序列对基因表达的作用机制的理解 核苷酸分辨率和单细胞水平。该提案的主要目标是多尺度发现， 通过将CRISPR靶向扰动和单细胞测定与 两个关键目标：（1）用无偏和可推广的方法揭示功能性非编码元素， 不同的细胞类型，并阐明其调控语法和基因表达的作用机制。 (2)研究非编码序列中内源性或扰动诱导的突变如何反映在 在单细胞水平上的基因表达程序。为了试行我们的概念框架，我们将研究非 BCL 11 A基因内的编码调节元件、血红蛋白开关的主调节子和 镰状细胞病（SCD）和β-地中海贫血的治疗靶点。事实上，一些临床试验（例如， NCT 03432364）正在进行中，旨在破坏BCL 11 A的调控序列，作为这些β- 血红蛋白紊乱这是由GWAS鉴定的最充分研究的基因座之一，并且是一个系统， 我们拥有丰富的经验和专业知识。然而，我们的方法通常也会 适用于与性状或疾病相关的其他基因座。在这个项目结束时，我们将提供一个一般 框架和用户友好的计算工具，研究非编码的功能和结构 可推广到不同干扰筛选、调节区、细胞类型和 表型重要的是，本提案中开发的所有计算工具将与NHGRI共享 资助了具有类似目标的财团，如ENCODE，以及更广泛的科学界。我们 预计拟议的研究可能会产生积极的转化影响，为 开发涉及对人类具有直接治疗潜力的非编码序列扰动的策略 疾病