CRISPR/Cas9 tools for identifying and manipulating diverse neuronal populations

描述（由申请人提供）：大脑由各种功能不同的神经元组成，这些神经元连接在电路中。更好地了解神经元的多样性和神经回路中定义的神经元亚型的作用将增强我们对大脑的基本理解，并促进神经和精神健康疾病治疗的进展。一个主要的障碍是缺乏表征神经元亚型和在体内操纵它们以评估其功能的工具。神经元身份由引起特定神经元特性的基因的表达来指定，这反过来又决定了连接电路内的神经元功能。除了基因表达，神经元蛋白质的亚细胞区室化是功能连接的关键组成部分。因此，确定精确的细胞和亚细胞表达模式的蛋白质的连接，如神经递质受体，离子通道和细胞粘附分子，将大大提高我们的神经回路的分子和生化逻辑的理解。在这里，我们提出了在果蝇中优化CRISPR/Cas9介导的HDR的实验（目标1），并开发了一种创新的基于CRISPR/Cas9的工具包，以同时标记神经元连接基础的内源性基因（目标2），并获得表达靶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).