Gene editing vectors for studying nicotinic acetylcholine receptors in cholinergic transmission

Can Peng, Yijin Yan, Veronica J. Kim, Staci E. Engle, Jennifer N. Berry, J. Michael McIntosh, Rachael L. Neve, Ryan M. Drenan*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

7 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)2224-2238
Number of pages15
JournalEuropean Journal of Neuroscience
Issue number3
StatePublished - Aug 1 2019


  • Cas9
  • mouse
  • nicotine
  • virus

ASJC Scopus subject areas

  • General Neuroscience


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