Nicotinic receptor gene editing vectors

项目概要 乙酰胆碱 (ACh) 是一种重要的神经递质，参与注意力、唤醒、视觉处理、运动 控制和动机行为。胆碱能传输在许多毁灭性的人类疾病中受到干扰 紊乱/疾病，包括阿尔茨海默病、帕金森病、重度抑郁症和毒瘾。 毒蕈碱 ACh 受体是 GPCR，而烟碱 ACh 受体（nAChR；本提案的主题） 是半胱氨酸环、配体门控阳离子通道家族。 nAChR 以包含 5 个 a7 的同五聚体形式存在 亚基或杂五聚体，需要 2 个 a 亚基、2 个 b 亚基和第 5 个亚基，可以是 a 或 a b 亚基。旨在探索脊椎动物大脑中 nAChR 功能的研究主要依赖于啮齿类动物 研究中，小鼠遗传技术提供了重要的见解。该领域的一个关键差距是缺乏 nAChR 基因编辑的合适工具。 R21 项目将通过两个独立的科学目标来填补这一空白。在 目标 1，我们将为每个关键的 nAChR 亚基基因创建一个能够催化 CRISPR 介导的单一载体 基因编辑导致功能丧失突变。指导 RNA 种类将在体外进行验证 矢量构建。载体将通过基因表达分析和膜片钳在体内进一步验证 电生理学。这组载体可用于任何脑区域特异性 nAChR 基因编辑。 受体小鼠品系，包括选择性繁殖的品系，其遗传特性不会因杂交而受到干扰 C57BL/6 或其他常见背景。在目标 2 中，我们将创建一系列并行向量以允许细胞 类型特异性 nAChR 基因编辑。来自 Aim 1 的经过审查的 gRNA 将被整合到一组载体中，这些载体将 被引入一组专门的小鼠品系中，这些小鼠品系以 Cre 依赖性方式产生 Cas9 核酸酶。 与目标 1 一样，我们将使用基因表达分析和膜片钳在体内验证该系统 电生理学。最终，这些新载体将极大地扩展我们的分子工具箱，从而实现更广泛的应用。 在各种遗传背景和特定电路中控制 nAChR 基因编辑。

PROJECT SUMMARY Acetylcholine (ACh) is an important neurotransmitter involved in attention, arousal, visual processing, motor control, and motivated behavior. Cholinergic transmission is perturbed in a number of devastating human disorders/diseases, including Alzheimer's disease, Parkinson's disease, major depression, and drug addiction. Muscarinic ACh receptors are GPCRs, whereas nicotinic ACh receptors (nAChRs; the subject of this proposal) are a family of Cys-loop, ligand-gated cation channels. nAChRs exist either as homopentamers containing 5 a7 subunits, or heteropentamers requiring 2 a subunits, 2 b subunits, and a 5th subunit that may be either an a or a b subunit. Studies aimed at probing nAChR function in the vertebrate brain have relied principally on rodent studies, where mouse genetic techniques have permitted key insights. A key gap in the field is the lack of suitable tools for nAChR gene editing. This R21 project will fill that gap via two independent scientific aims. In Aim 1, we will create – for each key nAChR subunit gene – a single vector that will catalyze CRISPR-mediated gene editing that results in a loss-of-function mutation. Guide RNA species will be validated in vitro prior to vector construction. Vectors will be further validated in vivo via gene expression analysis and patch clamp electrophysiology. This set of vectors will be useful for brain-region specific nAChR gene editing in any recipient mouse strain, including selectively-bred strains whose genetics cannot be disturbed by crosses to C57BL/6 or other common backgrounds. In Aim 2, we will create a parallel series of vectors to allow for cell type-specific nAChR gene editing. Vetted gRNAs from Aim 1 will be incorporated into a set of vectors that will be introduced into a specialized set of mouse strains that produce Cas9 nuclease in a Cre-dependent manner. As in Aim 1, we will validate this system in vivo using gene expression analyses and patch clamp electrophysiology. Ultimately, these new vectors will greatly expand our molecular toolbox, allowing for wide control over nAChR gene editing in various genetic backgrounds and in specific circuits.