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

对不同类型神经元细胞进行系统的实验是破译脑回路的先决条件 组织、功能和功能障碍。因此，神经科学的根本进展迫切需要细胞类型 访问技术，这些技术是特定的、全面的、易于使用的、经济实惠的、可扩展的，并且在整个 动物种类。大多数（如果不是全部的话）当前细胞类型靶向的遗传方法是基于基因组和 DNA工程在实现所需工具特征方面具有固有的局限性。我们已经开发出一种 基于RNA工程的细胞类型靶向和操纵的范式转换技术。这 这项技术建立在后生动物细胞内的通用RNA传感和编辑系统之上， 作用于RNA的腺苷脱氨酶（阿达尔）。我们将这种方法称为CellREADR：Cell access 通过内源性阿达尔的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， 传感器和效应器通过一个单一的碱基编辑事件;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、可扩展，原则上应适用于所有动物。重要的是，CellREADR本质上是 可编程的，具有前所未有的多功能性，用于细胞的组合和多重靶向和编辑 复杂组织中的类型。在这个提议中，我们将应用CellREADR来定位和验证一个大的神经元集合， 在几种哺乳动物和鸟类中广泛定义的大脑皮层和基底神经节的类型。我们 该提议是基于这些前脑细胞类型的进化保守性和分化， 这可能是跨物种的保守和不同的电路功能和行为的基础。我们有 组建了一个跨学科的研究团队，他们在分子遗传学、系统 神经科学、人类和临床神经科学、生物工程和计算基因组学。一是 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，我们将扩展CellREADR，以针对和验证大量的GLU和GABA 人离体皮质组织、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门户，用于CellREADR的计算设计 试剂在脊椎动物物种和传播的技术和资源在整个 神经科学社区通过使用细胞特异性RNA谱作为遗传访问和操作的基础， CellREADR细胞编辑技术有望改变神经科学的发现规模和速度， 在生物医学领域的应用通过翻译《世界知识产权宣言》，将对BRAIN倡议产生直接和深远的影响。 在转录组细胞类型方面取得了巨大进展，以了解脑回路功能和功能障碍。

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.