Transgene

Recombinase

Phenotype

Receptor

Serotonin

Gene knockin

Cre-Lox recombination

Biology

Guide RNA

Gene

Cre recombinase

Insertional mutagenesis

Recombination

Cas9

Genetically modified mouse

Genome

Subgenomic mRNA

Mutagenesis

Conditional gene knockout

GABAergic

Glutamatergic

Recombinant DNA

Mutation

Computational biology

CRISPR

Viral vector

Monoaminergic

Genetics

Glutamate receptor

Gene knockout

Cell Reports

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.

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