RNA-programmable cell type targeting and manipulation across vertebrate nervous systems

跨脊椎动物神经系统的 RNA 可编程细胞类型靶向和操作

基本信息

批准号：
10350096
负责人：
Z JOSH HUANG
金额：
$ 58.63万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-13 至 2024-09-12
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10350096
关键词：
Animals BRAIN initiative Basal Ganglia Base Pairing Behavior Bioinformatics Biomedical Engineering Birds Brain Cells Cerebral cortex Cerebrum Clinical Clustered Regularly Interspaced Short Palindromic Repeats Code Cognition Communities Complex Corpus striatum structure DNA Data Dependovirus Development Engineering Enzymes Event Foundations Functional disorder Genes Genetic Genome Genomics Glutamates Goals Health Human Human Cell Line Interneurons Knowledge Link Macaca Mammals Methods Molecular Biology Molecular Genetics Monitor Monkeys Mus Nervous system structure Neurobiology Neurons Neurosciences Neurosciences Research Neurosurgeon Nucleotides Outcome Perception Physicians Pilot Projects Primates Prosencephalon Psyche structure RNA Reagent Research Research Personnel Resources Rodent Scientist Songbirds System Technology Testing Thalamic structure Tissues Translating Translations Transplantation Validation Vertebrates Viral Viral Vector adenosine deaminase base behavioral study brain cell brain tissue cell type combinatorial data management data sharing design epigenomics experience expression vector flexibility gamma-Aminobutyric Acid genetic approach human disease in vivo interest neural circuit neuropsychiatric disorder new technology novel programs sensor single-cell RNA sequencing tool transcriptomics vector vocal learning zebra finch

项目摘要

Systematic experimental access to diverse neuronal cell types is a prerequisite to deciphering brain circuit organization, function, and dysfunction. Thus fundamental progress in neuroscience urgently needs cell type access technologies that are specific, comprehensive, easy to use, affordable, scalable, and general across animal species. Most if not all current genetic approaches to cell type targeting are based on genome and DNA engineering, which has inherent limitations in achieving the desired tool features. We have developed a paradigm-shifting technology for cell type targeting and manipulation based on RNA engineering. This technology builds upon the universal RNA sensing and editing system within metazoan cells, centered around the enzyme adenosine deaminase acting on RNA (ADAR). We term this method CellREADR: Cell access through RNA sensing by Endogenous ADAR. CellREADR can be deployed as a single RNA molecule that detects specific cellular RNAs through Watson-Crick base pairing and switches on the translation of markers, sensors, and effectors through a single base editing event; these RNA molecules can be delivered to animals via viral expression vectors. As such, CellREADR is highly specific and comprehensive, fast, cheap, easy to use, scalable, and in principle should apply to all animals. Importantly, CellREADR is inherently programmable, with unprecedented versatility for combinatorial and multiplexed targeting and editing of cell types in complex tissues. In this proposal, we will apply CellREADR to target and validate a large set of neuron types of the broadly defined cerebral cortex and basal ganglia in several mammalian and avian species. Our proposal is grounded on the evolutionary conservation as well as divergence of these forebrain cell types, which may underlie conserved and divergent circuit function and behavior across species. We have assembled an interdisciplinary team of investigators with expertise in molecular genetics, systems neuroscience, human and clinical neuroscience, bioengineering, and computation genomics. First, we will further optimize the CellREADR method and develop a comprehensive set of AAV tools for targeting and manipulating all major transcriptomic types of glutamatergic (GLU) and GABAergic neurons of the mouse cerebral cortex. Second, we will extend CellREADR to target and validate a large set of GLU and GABA neuron types in human ex vivo cortical tissues, macaque monkey cerebral cortex, and zebra finch cortex and basal ganglia. Third, we will establish a central CellREADR Portal for computational design of CellREADR reagents across vertebrate species and dissemination of technology and resource throughout the neuroscience community. By using cell-specific RNA profiles as the basis for genetic access and manipulation, CellREADR cell-editing technology is poised to transform the scale and rate of discovery in neuroscience and across biomedical fields. Impacts on the BRAIN Initiative will be immediate and far-reaching by translating the massive progress in transcriptomic cell types to understanding brain circuit function and dysfunction.

对不同神经元细胞类型进行系统实验是破译大脑回路的先决条件组织、功能和功能障碍。因此，神经科学的根本性进展迫切需要细胞类型访问特定的、全面的、易于使用的、负担得起的、可扩展的和通用的技术动物种类。目前大多数（如果不是全部）细胞类型靶向遗传方法都是基于基因组和 DNA 工程在实现所需的工具功能方面具有固有的局限性。我们开发了一个基于 RNA 工程的细胞类型靶向和操作的范式转换技术。这技术建立在后生动物细胞内的通用 RNA 传感和编辑系统的基础上，以作用于 RNA 的腺苷脱氨酶 (ADAR)。我们将此方法称为 CellREADR：单元访问通过内源性 ADAR 的 RNA 传感。 CellREADR 可以作为单个 RNA 分子部署通过 Watson-Crick 碱基配对检测特定的细胞 RNA 并开启标记的翻译，通过单个碱基编辑事件的传感器和效应器；这些RNA分子可以传递给动物通过病毒表达载体。因此，CellREADR 具有高度特异性和全面性、快速、廉价、易于使用使用、可扩展，原则上应适用于所有动物。重要的是，CellREADR 本质上是可编程，具有前所未有的多功能性，可用于细胞的组合和多重靶向和编辑复杂组织中的类型。在本提案中，我们将应用 CellREADR 来定位和验证大量神经元几种哺乳动物和鸟类的广义大脑皮层和基底神经节的类型。我们的该提案基于进化保守性以及这些前脑细胞类型的分歧，这可能是跨物种保守且不同的电路功能和行为的基础。我们有组建了一支跨学科研究团队，具有分子遗传学、系统方面的专业知识神经科学、人类和临床神经科学、生物工程和计算基因组学。首先，我们将进一步优化CellREADR方法并开发一套全面的AAV工具用于靶向和操纵小鼠谷氨酸能 (GLU) 和 GABA 能神经元的所有主要转录组类型大脑皮层。其次，我们将扩展 CellREADR 以定位和验证大量 GLU 和 GABA 人类离体皮质组织、猕猴大脑皮质和斑胸草雀皮质中的神经元类型基底神经节。第三，我们将建立一个中央CellREADR Portal，用于CellREADR的计算设计跨脊椎动物物种的试剂以及在整个世界范围内传播技术和资源神经科学界。通过使用细胞特异性 RNA 谱作为遗传获取和操作的基础， CellREADR 细胞编辑技术有望改变神经科学和医学领域的发现规模和速度横跨生物医学领域。通过翻译 BRAIN 计划，其影响将是立竿见影且深远的。转录组细胞类型在理解脑回路功能和功能障碍方面取得了巨大进展。