Programmable RNA-targeting CRISPR-Cas tools to study RNA biology

用于研究 RNA 生物学的可编程 RNA 靶向 CRISPR-Cas 工具

• 批准号: 10581919
• 负责人: Mitchell O'Connell
• 金额: $ 14.53万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10581919
• 关键词: Address; Alternative Splicing; BCAR1 gene; Binding Proteins; Biology; Biomedical Research; Cells; Cellular biology; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Defect; Deposition; Development; Disease; Enzymes; Floods; Flow Cytometry; Genetic Transcription; Goals; Guide RNA; Health; Human; Knowledge; Label; Location; Machine Learning; Malignant Neoplasms; Methods; MicroRNAs; Microprocessor; Modeling; Molecular; Molecular Biology; National Institute of General Medical Sciences; Neurodegenerative Disorders; Process; Proteins; RNA; RNA Binding; RNA Processing; RNA Sequences; RNA Splicing; RNA analysis; RNA-Protein Interaction; Research; Strategic Planning; System; Transcript; base; clinical application; design; innovation; knock-down; nervous system disorder; next generation sequencing; programs; single cell analysis; tool; tool development; transcriptome; virtual

Summary Technological advances such as next-generation sequencing and single-cell analysis have opened the flood gates for RNA analysis, revealing that the transcriptome is significantly more complex than previously thought, both with respect to the diversity of RNA transcripts, their temporal expression dynamics, and their cellular location. Moreover, thousands of dysregulated RNAs have been observed in diseases including cancer and neurodegenerative disorders. These observations underscore the dire need for new molecular tools to precisely dissect RNA function in health and disease. The goal of my research program is to harness the unprecedented power of CRISPR-Cas systems to create a versatile range of methods to precisely manipulate virtually any RNA, and to use these tools to explore fundamental knowledge gaps in RNA biology. This proposal specifically focuses on addressing the following knowledge gaps: 1) Despite the technological advances in harnessing RNA-targeting CRISPR-Cas enzymes Cas9 and Cas13 for research and clinical applications, we still do not understand the principles of gRNA selection for efficient and tunable RNA-targeting. This problem precludes the facile development of a number of RNA-targeting applications and underscores the need for a thorough interrogation of gRNA selection. Our goal here is to develop a model to predict highly-active gRNAs for Cas9/Cas13 RNA- targeting in human cells. We will use a combination of high-throughput RNA:protein interactome methods, flow- cytometry based gRNA screens and machine learning to precisely define features of highly active RNA-targeting gRNAs for RNA-binding and knockdown for Cas9 and Cas13. 2) The vast majority of human RNAs are alternatively spliced, and RNA splicing defects are common in cancers and neurological diseases. However, our understanding of the downstream functional effects of splicing in a majority of cases is rudimentary. To address this issue, we will develop a toolbox of robust, multiplexable Cas-based splicing factors to offer an unprecedented opportunity to study the functional consequences of alternative splicing. that can determine in a single step both the identity of proteins bound to a And 3) There is a paucity of methods specific RNA and their location on that RNA. The development of such an approach will enable us to determine the spatial arrangement of proteins on specific RNAs to dissect their dynamic deposition in range of RNA processes such as transcription, 3ʹ-end and micro-RNA processing, and lncRNA function. To address this issue, we propose to develop a genetically encodable Cas-based RNA proximity-labeling strategy to precisely identify proteins that bind in close proximity to a specific RNA sequence. We will then use this approach to identify protein factors involved in regulating microprocessor (Drosha/DGCR8) activity at specific primary micro-RNA loci. These research goals fully align with the NIGMS 5 Year Strategic Plan, through the development of essential research tools to study and RNA function for biomedical research.

总结 下一代测序和单细胞分析等技术进步打开了洪水 RNA分析的门，揭示了转录组比以前认为的要复杂得多， 这两个方面的多样性RNA转录本，其时间表达动力学，和他们的细胞 位置.此外，在包括癌症和癌症在内的疾病中已经观察到数千种失调的RNA。 神经退行性疾病这些观察结果强调了对新分子工具的迫切需要， 剖析RNA在健康和疾病中的功能。我的研究项目的目标是利用前所未有的 CRISPR-Cas系统的力量，创造了一系列多功能的方法来精确操纵几乎任何RNA， 并利用这些工具来探索RNA生物学中的基本知识缺口。该提案特别关注 解决以下知识差距：1）尽管在利用RNA靶向方面取得了技术进步 CRISPR-Cas酶Cas9和Cas 13的研究和临床应用，我们仍然不了解 gRNA选择的原理，用于有效和可调的RNA靶向。这个问题排除了容易的 开发了一些RNA靶向应用程序，并强调了彻底询问的必要性 gRNA选择我们的目标是开发一个模型来预测Cas9/Cas 13 RNA的高活性gRNA。 靶向于人体细胞。我们将使用高通量RNA：蛋白质相互作用组方法，流式细胞术， 基于细胞术的gRNA筛选和机器学习可精确定义高活性RNA靶向的特征 用于RNA结合和Cas9和Cas 13的敲低的gRNA。2)绝大多数人类RNA是 选择性剪接和RNA剪接缺陷在癌症和神经疾病中是常见的。但我们的 在大多数情况下，对剪接的下游功能效应的理解是基本的。解决 在这个问题上，我们将开发一个强大的，可复用的基于Cas的剪接因子的工具箱，以提供前所未有的 有机会研究选择性剪接的功能后果。 它可以在一个步骤中确定与蛋白质结合的蛋白质的身份， 3）缺乏方法 特异性RNA及其在 核糖核酸这种方法的发展将使我们能够确定蛋白质的空间排列， 特异性RNA，以剖析它们在一系列RNA过程中的动态沉积，如转录、3 ′-末端和 micro-RNA加工和lncRNA功能。为了解决这个问题，我们建议开发一种基因 可编码的基于Cas的RNA邻近标记策略，以精确识别紧密结合的蛋白质 一个特定的RNA序列。然后，我们将使用这种方法来确定参与调节的蛋白质因子。 微处理器（Drosha/DGCR 8）在特定的主要micro-RNA基因座上的活性。这些研究目标完全一致 与NIGMS 5年战略计划，通过开发必要的研究工具，研究和RNA 生物医学研究的功能。