Nicotinic receptor gene editing vectors

项目总结 乙酰胆碱(ACh)是一种重要的神经递质，参与注意、觉醒、视觉处理、运动 控制力和动机行为。胆碱能传递在一些毁灭性的人类中受到干扰 障碍/疾病，包括阿尔茨海默氏症、帕金森氏症、严重抑郁症和毒瘾。 毒鼠型ACh受体是GPCRs，而烟碱型ACh受体(nAChRs；本提案的主题) 是一类Cys环、配体门控的阳离子通道。NAChRs以含有5个A7的同型五聚体形式存在 亚基，或需要2a亚基、2b亚基和第5亚基的异戊异构体，可以是a或 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动物 研究，在这些研究中，老鼠基因技术允许获得关键的见解。该领域的一个关键差距是缺乏 适用于nAChR基因编辑的工具。这个R21项目将通过两个独立的科学目标来填补这一空白。在……里面 目标1，我们将为每个关键的nAChR亚单位基因创建一个单独的载体，该载体将催化CRISPR介导的 导致功能丧失突变的基因编辑。指南RNA物种将在体外进行验证，然后 向量构造。载体将通过基因表达分析和膜片钳在体内得到进一步验证 电生理学。这组载体将用于脑区域特异的nAChR基因编辑 受体小鼠品系，包括选育的品系，其遗传学不会受到杂交的干扰 C57BL/6或其他常见背景。在目标2中，我们将创建一系列并行的向量以允许细胞 特定类型的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， 被引入一组专门的小鼠品系，以依赖于Cre的方式产生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，允许广泛的 在不同的遗传背景和特定的电路中控制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.