Gene editing vectors for studying nicotinic acetylcholine receptors in cholinergic transmission

• Published in: The European journal of neuroscience Vol. 50; no. 3; pp. 2224 - 2238
• Main Authors: Peng, Can, Yan, Yijin, Kim, Veronica J., Engle, Staci E., Berry, Jennifer N., McIntosh, J. Michael, Neve, Rachael L., Drenan, Ryan M.
• Format: Journal Article
• Language: English
• Published: France Wiley Subscription Services, Inc 01-08-2019
• Subjects: Acetylcholine receptors (nicotinic); Brain slice preparation; Cas9; Central nervous system; Cholinergic transmission; CRISPR; Dopamine transporter; Electrophysiology; Expression vectors; Genes; Genome editing; Herpes simplex; mouse; Neurotransmission; nicotine; Ribonucleic acid; RNA; virus; CRISPR; mouse; nicotine; Cas9; virus
• ISSN: 0953-816X; 1460-9568
• DOI: 10.1111/ejn.13957

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.
Peng et al. introduce a set of viral vectors for nicotinic acetylcholine receptor (nAChR) gene editing. Herpes simplex virus vectors allow all‐in‐one delivery of CRISPR/Cas9 elements for gene editing in any mouse strain, and adeno‐associated virus vectors allow cell type‐specific nAChR gene editing. These vectors will be useful for next‐generation molecular‐level queries into the contribution of nAChR subunits in addiction and psychiatric disorders.