Conditional Single Vector CRISPR/SaCas9 Viruses for Efficient Mutagenesis in the Adult Mouse Nervous System

Avery C. Hunker, Marta E. Soden, Dasha Krayushkina, Gabriel Heymann, Rajeshwar Awatramani, Larry S. Zweifel*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Scopus citations

Abstract

Mice engineered for conditional, cell type-specific gene inactivation have dominated the field of mouse genetics because of the high efficiency of Cre-loxP-mediated recombination. Recent advances in CRISPR/Cas9 technologies have provided alternatives for rapid gene mutagenesis for loss-of-function (LOF) analysis. Whether these strategies can be streamlined for rapid genetic analysis with the efficiencies comparable with those of conventional genetic approaches has yet to be established. We show that a single adeno-associated viral (AAV) vector containing a recombinase-dependent Staphylococcus aureus Cas9 (SaCas9) and a single guide RNA (sgRNA) are as efficient as conventional conditional gene knockout and can be adapted for use in either Cre- or Flp-driver mouse lines. The efficacy of this approach is demonstrated for the analysis of GABAergic, glutamatergic, and monoaminergic neurotransmission. Using this strategy, we reveal insight into the role of GABAergic regulation of midbrain GABA-producing neurons in psychomotor activation.

Original languageEnglish (US)
Pages (from-to)4303-4316.e6
JournalCell reports
Volume30
Issue number12
DOIs
StatePublished - Mar 24 2020

Keywords

  • AAV
  • CRISPR
  • GABA
  • SaCas9
  • VTA
  • dopamine
  • glutamate

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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