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

• 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
• 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

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.

系统的实验访问多种神经元细胞类型是破译脑电路的先决条件 组织，功能和功能障碍。因此，神经科学的基本进展急需细胞类型 特定，全面，易于使用，负担得起，可扩展性和一般性的访问技术 动物物种。大多数当前的细胞类型靶向遗传方法（如果不是全部）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。我们已经开发了 基于RNA工程的细胞类型靶向和操作的范式转移技术。这 技术建立在后生细胞内的通用RNA感应和编辑系统上，以围绕 作用于RNA（ADAR）的腺苷脱氨酶。我们称此方法CellReadr：单元格访问 通过内源性ADAR传感的RNA传感。 CellReadr可以作为单个RNA分子部署 通过Watson-Crick底座配对检测特定的细胞RNA，并在标记的翻译上进行开关， 传感器和通过单个基础编辑事件的效应器；这些RNA分子可以传递到动物 通过病毒表达向量。因此，CellReadr高度具体，全面，快速，便宜，易于 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，用于组合和多重靶向和编辑单元格 在复杂的组织中类型。在此提案中，我们将应用CellReadr对目标并验证大量神经元 几种哺乳动物和禽类中广泛定义的大脑皮层和基底神经节的类型。我们的 提案基于进化保护以及这些前脑细胞类型的差异， 这可能是跨物种的保守和发散的电路功能和行为的基础。我们有 组建了一个研究人员的跨学科团队，具有分子遗传学，系统的专业知识 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，我们会的 进一步优化CellReadr方法，并开发一套全面的AAV工具来定位和 操纵小鼠的所有主要转录组类型的谷氨酸能（GLU）和GABA能神经元 大脑皮层。其次，我们将延长CellReadr以靶向并验证大量的GLU和GABA 人体外皮质组织中的神经元类型，猕猴的脑皮质和斑马芬奇皮质和 基底神经节。第三，我们将建立一个用于CellReadR计算设计的Central CellReadr门户 跨脊椎动物物种的试剂以及整个整个技术和资源的传播 神经科学社区。通过使用细胞特异性的RNA谱作为遗传获取和操纵的基础， CellReadr细胞编辑技术有望改变神经科学和发现的尺度和发现率 跨生物医学领域。对大脑倡议的影响将是直接和深远的，通过翻译 转录组细胞类型的大规模进展，以了解脑电路功能和功能障碍。