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: 10656290
• 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/10656290
• 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; experience; genetic variant; genome wide association study; 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.

项目总结 大多数与疾病或性状相关的遗传变异存在于基因组的非编码序列中，例如 增强剂、促进剂和绝缘体。这些序列调节基因表达，在基因表达中发挥关键作用 确定疾病严重程度，并可作为新的合理治疗干预措施的目标。 然而，我们仍然缺乏对非编码序列翻译的确切机制的深入了解 它们的功能以及它们内部遗传变异的影响。非编码的CRISPR扰动 元素，提供了前所未有的机会来评估它们在无数发育和 疾病设置。我们假设，通过直接对非编码序列的理解的改进 在它们的内生环境中的扰动，以及通过直接读出遗传扰动，将提供 对人类疾病进行治疗干预的新机会。我们工作的长期目标是 克服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。这项提议的主要目标是多尺度的发现和 通过结合CRISPR靶向扰动和单细胞分析来剖析调控元件 两个关键目标：(1)用无偏见和可推广的方法发现功能性非编码元素 不同的细胞类型，阐明它们对基因表达的调控语法和作用机制。 (2)研究内源性或扰动诱导的非编码序列突变如何反映在 单细胞水平的基因表达程序。为了试验我们的概念框架，我们将研究非 编码BCL11A基因内的调节元件，血红蛋白开关的主调节因子和 镰状细胞病和β-地中海贫血的治疗目标。事实上，一些临床试验(例如， NCT03432364)正在进行中，目的是扰乱BCL11A的调控序列，作为治疗这些β- 血红蛋白紊乱。这是GWAS发现的研究最充分的基因座之一，是一个系统 我们拥有丰富的经验和专业知识。然而，我们的方法也将是一般的 适用于其他与性状或疾病连锁的基因座。在这个项目结束时，我们将提供一个一般的 框架和用户友好的计算工具，用于研究非编码的功能和结构 可概括为不同扰动筛选、调控区域、细胞类型和 表型。重要的是，该提案中开发的所有计算工具都将与NHGRI共享 资助具有类似目标的财团，如ENCODE，并与更广泛的科学界合作。我们 预期拟议的研究可能会产生积极的翻译影响，从而为 开发对人类具有直接治疗潜力的非编码序列扰动策略 疾病。

项目成果

Benchmarking computational methods to identify spatially variable genes and peaks.

对计算方法进行基准测试以识别空间可变基因和峰值。

• DOI: 10.1101/2023.12.02.569717
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Li,Zhijian;Patel,ZainM;Song,Dongyuan;Yan,Guanao;Li,JingyiJessica;Pinello,Luca
• 通讯作者: Pinello,Luca

Precise DNA cleavage using CRISPR-SpRYgests.

使用CRISPR-Sprygests精确的DNA裂解。

• DOI: 10.1038/s41587-022-01492-y
• 发表时间: 2023-03
• 期刊: NATURE BIOTECHNOLOGY
• 影响因子: 46.9
• 作者: Christie, Kathleen A.;Guo, Jimmy A.;Silverstein, Rachel A.;Doll, Roman M.;Mabuchi, Megumu;Stutzman, Hannah E.;Lin, Jiecong;Ma, Linyuan;Walton, Russell T.;Pinello, Luca;Robb, G. Brett;Kleinstiver, Benjamin P.
• 通讯作者: Kleinstiver, Benjamin P.