Manipulating and Interrogating Spatial Transcriptomics

操纵和询问空间转录组学

• 批准号: 10702050
• 负责人: Lei Stanley Qi
• 金额: $ 108.08万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10702050
• 关键词: Amyotrophic Lateral Sclerosis; Atlases; Axon; Biology; Cell physiology; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Computer Analysis; Development; Disease; Embryo; Fluorescent in Situ Hybridization; Foundations; Fragile X Syndrome; Goals; Image; In Situ; In Vitro; Mediating; Messenger RNA; Methods; Monitor; Neurons; Pathologic; Pathology; Physiological; Play; Protein Biosynthesis; RNA; RNA-Binding Proteins; Regulation; Resolution; Role; Site; Spinal Muscular Atrophy; Synapses; Technology; Therapeutic; Time; Transcript; Untranslated RNA; axon guidance; cell type; deep learning; in vivo; insight; machine learning algorithm; nervous system disorder; novel strategies; single molecule; spatiotemporal; transcriptomics

ABSTRACT Spatial mRNA organization plays a fundamental role in diverse cellular processes and disease. In large, compartmentalized cells (e.g., neurons and embryos), subcellular mRNA localization offers a core mechanism for the spatiotemporal regulation of protein synthesis. Since the initial discovery of subcellular mRNA distribution in 1983, high-throughput imaging and sequencing methods have revealed that, in many cell types, thousands of RNAs are localized to distinct compartments. For example, many axonal-related mRNAs in neurons will transport to the “site of needed” along the very long (>100μm) axon, which likely play an important role in axon development and local synaptic activities. Furthermore, mounting evidence shows a correlation between aberrant spatial RNA organization and an increasing number of diseases, including amyotrophic lateral sclerosis (ALS), fragile X syndrome (FXS), and spinal muscular atrophy (SMA). However, due to a lack of technologies that allow for the tracking and manipulation of the spatial localization of endogenous mRNAs in primary cells and in vivo, the mechanism and functional relevance of spatial organization has only been explored for a small number of mRNAs. In this proposal, we seek to establish a set of technologies as a new foundation to study spatial RNA biology, by developing an integrated framework that allows for sophisticated computational analysis, real-time RNA tracking, and programmable spatial manipulation of any endogenous mRNA(s) in situ and in vivo, on a high-throughput (>1,000 mRNAs in parallel) scale. To achieve this goal, we will start by building a deep learning framework that can analyze spatially localized RNAs in different cell types and predict their associated regulatory factors (e.g., RNA motifs, RNA binding proteins). This will provide an atlas of spatial RNA organization as well as candidate RNAs for functional studies. Next, we will develop two novel approaches, RNA live-cell fluorescent in situ hybridization (RNA-LiveFISH) for single-molecule, real-time dynamic tracking, and CRISPR- mediated transcript organization (CRISPR-TO) for programmable manipulation of any target mRNA localization. The two approaches form a new framework that enables us to study the regulatory mechanism and functional relevance of subcellular mRNA localization with unprecedented ease and spatiotemporal resolution. Third, we seek to apply this framework to study the function of mRNA localization in primary neurons, via high-throughput manipulation of >1,000 mRNAs to uncover functions for axon guidance, growth cone development, and synaptic activities. Selected functional mRNAs (>100) will be verified in vivo. Finally, we will apply the framework to investigate the pathological mechanisms of aberrant RNA localization underlying the neurological disease spinal muscular atrophy (SMA) in vitro and in vivo. We will not only dissect the relationship between mRNA organization and SMA pathology, but also explore the strategy of modulating RNA localization for potential therapeutics. We envision that the proposal will lead to new groundbreaking insights into the mechanism and functional roles of whole-cell mRNA spatial organization for cellular and physiological functions that has not been revealed before.

抽象的 空间 mRNA 组织在多种细胞过程和疾病中发挥着重要作用。 区室化细胞（例如神经元和胚胎），亚细胞 mRNA 定位提供了核心机制 自从首次发现亚细胞 mRNA 分布以来 1983 年，高通量成像和测序方法表明，在许多细胞类型中，有数千种细胞 RNA 定位于不同的区室，例如，神经元中的许多轴突相关 mRNA 都会进行运输。 沿着很长（>100μm）的轴突到达“需要的部位”，这可能在轴突中发挥重要作用 此外，越来越多的证据表明，发育和局部突触活动之间存在相关性。 异常的空间 RNA 组织和越来越多的疾病，包括肌萎缩侧索硬化症 (ALS)、脆性 X 综合征 (FXS) 和脊髓性肌萎缩症 (SMA) 然而，由于缺乏技术。 允许跟踪和操纵原代细胞中内源 mRNA 的空间定位， 在体内，空间组织的机制和功能相关性仅被探索了一小部分。 在本提案中，我们寻求建立一套技术作为研究的新基础。 空间RNA生物学，通过开发一个允许复杂计算分析的集成框架， 实时 RNA 追踪，以及对原位和体内任何内源 mRNA 进行可编程空间操作， 在高通量（> 1,000 个并行 mRNA）规模上为了实现这一目标，我们将从构建深度模型开始。 可以分析不同细胞类型中空间定位的 RNA 并预测其相关的学习框架 调控因子（例如 RNA 基序、RNA 结合蛋白）这将提供空间 RNA 组织图谱。 以及用于功能研究的候选 RNA，接下来，我们将开发两种新方法：RNA 活细胞。 用于单分子、实时动态跟踪的荧光原位杂交 (RNA-LiveFISH) 和 CRISPR- 介导的转录组织 (CRISPR-TO)，用于对任何目标 mRNA 定位进行可编程操作。 这两种方法形成了一个新的框架，使我们能够研究调控机制和功能 亚细胞 mRNA 定位的相关性具有前所未有的轻松性和时空分辨率。 寻求应用该框架通过高通量研究初级神经元中 mRNA 定位的功能 操纵 > 1,000 个 mRNA 以揭示轴突引导、生长锥发育和突触的功能 选定的功能性 mRNA（>100）将在体内进行验证，最后，我们将应用该框架。 研究神经系统疾病脊髓异常RNA定位的病理机制 我们不仅会剖析 mRNA 组织之间的关系。 和 SMA 病理学，同时还探索调节 RNA 定位的策略以实现潜在的治疗。 预计该提案将带来对机制和功能作用的新的突破性见解 全细胞 mRNA 的细胞和生理功能空间组织，以前从未被揭示过。