Cell-Specific Single Viral Vector CRISPR/Cas9 Editing and Genetically Encoded Tool Delivery in the Central and...

CRISPR/Cas9 gene editing represents an exciting avenue to study genes of unknown function and can be combined with genetically encoded tools such as fluorescent proteins, channelrhodopsins, DREADDs, and various biosensors to more deeply probe the function of these genes in different cell types. However, current strategies to also manipulate or visualize edited cells are challenging due to the large size of Cas9 proteins and the limited packaging capacity of adeno-associated viruses (AAVs). To overcome these constraints, we developed an alternative gene editing strategy using a single AAV vector and mouse lines that express Cre-dependent Cas9 to achieve efficient cell-type specific editing across the nervous system. Expressing Cre-dependent Cas9 from a genomic locus affords space to package guide RNAs for gene editing together with Cre-dependent, genetically encoded tools to manipulate, map, or monitor neurons using a single virus. We validated this strategy with three common tools in neuroscience: ChRonos, a channelrhodopsin, for studying synaptic transmission using optogenetics, GCaMP8f for recording Ca²⁺ transients using photometry, and mCherry for tracing axonal projections. We tested these tools in multiple brain regions and cell types, including GABAergic neurons in the nucleus accumbens, glutamatergic neurons projecting from the ventral pallidum to the lateral habenula, dopaminergic neurons in the ventral tegmental area, and proprioceptive neurons in the periphery. This flexible approach could help identify and test the function of novel genes affecting synaptic transmission, circuit activity, or morphology with a single viral injection.