CRISPR/Cas9 tools for identifying and manipulating diverse neuronal populations

描述（由申请人提供）：大脑是由各种功能不同的神经元组成的，这些神经元以电路的形式连接在一起。对神经元多样性和已定义的神经元亚型在神经回路中的作用的更深入了解，将增强我们对大脑的基本认识，并促进神经和精神健康疾病治疗方面的进展。一个主要的障碍是缺乏表征神经元亚型并在体内操纵它们以评估其功能的工具。神经元的身份是由产生特定神经元特性的基因表达指定的，而这些特性反过来又决定了连接回路内的神经元功能。除了基因表达，神经元蛋白的亚细胞区隔化是功能连接的关键组成部分。因此，确定连接蛋白的精确细胞和亚细胞表达模式，如神经递质受体、离子通道和细胞粘附分子，将大大促进我们对神经回路分子和生化逻辑的理解。在这里，我们提出了优化CRISPR/Cas9介导的果蝇HDR（目标1）的实验，并开发了一个创新的基于CRISPR/Cas9的工具包，同时标记神经元连接的内源性基因（目标2），并获得对表达目标基因的神经元的实验控制（目标3）。

DESCRIPTION (provided by applicant): The brain is composed of a great assortment of functionally diverse neurons connected in circuits. A greater understanding of neuronal diversity and the role of defined neuronal subtypes in neural circuits will enhance our fundamental understanding of the brain and foster progress in the treatment of neurological and mental health disorders. A major obstacle is a lack of tools for characterizing neuronal subtypes and manipulating them in vivo to assess their function Neuronal identity is specified by the expression of genes that give rise to specific neuronal properties, which, in turn, determine neuronal function within connected circuits. Beyond gene expression, the subcellular compartmentalization of neuronal proteins is a critical component of functional connectivity. Thus, determining the precise cellular and subcellular expression patterns of the proteins that underlie connectivity, such as neurotransmitter receptors, ion channels and cell adhesion molecules, will substantially advance our understanding of the molecular and biochemical logic of neural circuits. Here, we propose experiments to optimize CRISPR/Cas9-mediated HDR in Drosophila (Aim 1) and develop an innovative CRISPR/Cas9- based toolkit to simultaneously tag the endogenous genes that underlie neuronal connectivity (Aim 2) and gain experimental control of the neurons that express the targeted gene (Aim 3).