Peng Can, Yan Yijin, Kim Veronica J, Engle Staci E, Berry Jennifer N, McIntosh J Michael, Neve Rachael L, Drenan Ryan M
Department of Pharmacology, Northwestern University Feinberg School of Medicine, 320 East Superior Street, Searle 5-450, Chicago, IL, 60611, USA.
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN, USA.
Eur J Neurosci. 2019 Aug;50(3):2224-2238. doi: 10.1111/ejn.13957. Epub 2018 Jul 25.
Nicotinic acetylcholine receptors (nAChRs), prototype members of the cys-loop ligand-gated ion channel family, are key mediators of cholinergic transmission in the central nervous system. Despite their importance, technical gaps exist in our ability to dissect the function of individual subunits in the brain. To overcome these barriers, we designed CRISPR/Cas9 small guide RNA sequences (sgRNAs) for the production of loss-of-function alleles in mouse nAChR genes. These sgRNAs were validated in vitro via deep sequencing. We subsequently targeted candidate nAChR genes in vivo by creating herpes simplex virus (HSV) vectors delivering sgRNAs and Cas9 expression to mouse brain. The production of loss-of-function insertions or deletions (indels) by these 'all-in-one' HSV vectors was confirmed using brain slice patch clamp electrophysiology coupled with pharmacological analysis. Next, we developed a scheme for cell type-specific gene editing in mouse brain. Knockin mice expressing Cas9 in a Cre-dependent manner were validated using viral microinjections and genetic crosses to common Cre-driver mouse lines. We subsequently confirmed functional Cas9 activity by targeting the ubiquitous neuronal protein, NeuN, using adeno-associated virus (AAV) delivery of sgRNAs. Finally, the mouse β2 nAChR gene was successfully targeted in dopamine transporter (DAT)-positive neurons via CRISPR/Cas9. The sgRNA sequences and viral vectors, including our scheme for Cre-dependent gene editing, should be generally useful to the scientific research community. These tools could lead to new discoveries related to the function of nAChRs in neurotransmission and behavioral processes.
烟碱型乙酰胆碱受体（nAChRs）是半胱氨酸环配体门控离子通道家族的原型成员，是中枢神经系统中胆碱能传递的关键介质。尽管它们很重要，但我们在剖析大脑中单个亚基功能的能力方面仍存在技术差距。为了克服这些障碍，我们设计了CRISPR/Cas9小向导RNA序列（sgRNAs），用于在小鼠nAChR基因中产生功能丧失等位基因。这些sgRNAs通过深度测序在体外得到验证。随后，我们通过创建将sgRNAs和Cas9表达传递到小鼠大脑的单纯疱疹病毒（HSV）载体，在体内靶向候选nAChR基因。使用脑片膜片钳电生理学结合药理学分析，证实了这些“一体化”HSV载体产生的功能丧失插入或缺失（indels）。接下来，我们开发了一种在小鼠大脑中进行细胞类型特异性基因编辑的方案。使用病毒显微注射和与常见Cre驱动小鼠品系的遗传杂交，验证了以Cre依赖方式表达Cas9的敲入小鼠。随后，我们通过使用腺相关病毒（AAV）递送sgRNAs靶向普遍存在的神经元蛋白NeuN，证实了功能性Cas9活性。最后，通过CRISPR/Cas9成功地在多巴胺转运体（DAT）阳性神经元中靶向了小鼠β2 nAChR基因。sgRNA序列和病毒载体，包括我们的Cre依赖基因编辑方案，应该对科学界普遍有用。这些工具可能会带来与nAChRs在神经传递和行为过程中的功能相关的新发现。
