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

摘要 下一代测序和单细胞分析等技术进步开启了这股洪流 盖茨的RNA分析，揭示了转录组比之前认为的要复杂得多， 无论是关于RNA转录本的多样性，它们的时间表达动力学，还是它们的细胞 地点。此外，在包括癌症和癌症在内的疾病中观察到了数千种调控失调的RNA。 神经退行性疾病。这些观察结果强调了对新的分子工具的迫切需求，以精确地 剖析RNA在健康和疾病中的作用。我的研究计划的目标是利用前所未有的 CRISPR-CAS系统创造了一系列通用的方法来精确操作几乎任何RNA的能力， 并使用这些工具来探索RNA生物学中的基本知识差距。这项提案特别关注 关于解决以下知识差距：1)尽管在利用RNA靶向方面取得了技术进步 CRISPR-Cas酶Cas9和Cas13用于研究和临床应用，我们仍然不了解 用于有效和可调的RNA靶向的gRNA选择原则。这个问题妨碍了 开发了一些RNA靶向应用程序，并强调需要进行彻底的审问 GRNA选择。我们的目标是开发一个模型来预测Cas9/Cas13 RNA的高活性gRNA- 以人类细胞为靶点。我们将使用高通量RNA：蛋白质相互作用组方法、Flow- 基于细胞学的gRNA筛选和机器学习，以精确定义高活性RNA靶向的特征 Cas9和Cas13的RNA结合和击倒的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系统疾病中很常见。然而，我们的 在大多数情况下，对剪接的下游功能影响的了解是初步的。致信地址 这一期，我们将开发一个强大的、可复用的基于CAS的剪接因子工具箱，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一个 3)方法匮乏 特定RNA及其在其上的位置 核糖核酸。这种方法的发展将使我们能够确定蛋白质在 特定的RNA来剖析它们在一系列RNA过程中的动态沉积，如转录、3ʹ-End和 MicroRNA的加工，以及lncRNA的功能。为了解决这个问题，我们建议开发一种基因 基于可编码CA的RNA邻近标记策略精确识别紧密结合的蛋白质 结合到特定的RNA序列上。然后，我们将使用这种方法来确定参与调控的蛋白质因子 微处理器(DROSHA/Dgcr8)在特定初级微RNA基因座的活性。这些研究目标完全一致 配合NIGMS的5年战略计划，通过开发必要的研究工具来研究和RNA 生物医学研究的功能。

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.