A NEW PARKINSON’S DISEASE MODEL USING TARGETED GENETIC MANIPULATION TO ENABLE DOPAMINERGIC NEURON–SPECIFIC IN VIVO CRISPR SCREENING

Parkinson’s disease (PD) features progressive motor and cognitive impairment linked to dopaminergic (DA) neuron loss in the substantia nigra pars compacta (SNpc) and accumulation of phosphorylated α-synuclein. Existing mouse models showed high variability and did not recapitulate disease-relevant molecular pathology, or produced motor phenotypes but lacked robust aging-associated neurodegeneration, and were poorly suited to functional genomics. To address these gaps, we established an in vivo CRISPR screening platform in an optimized murine PD model to identify genes and pathways that drive DA neurodegeneration and represent therapeutic targets. We stereotactically delivered into the SNpc of adult Dat-Cre; LSL-Cas9-GFP mice a novel AAV1/2 vector carrying an sgRNA cassette and a Cre-dependent “inverted” α-synuclein construct (InvαSyn: AAV-U6sgRNA-lox71-SNCA^A53T-HA-P2A-dTom-lox66), enabling selective expression of mutant human α-synuclein (A53T) and dTomato in DA neurons. We profiled pathology across disease stages by harvesting tissue at 4, 8, and 12 weeks post-injection and performed spatial transcriptomics with NanoString Digital Spatial Profiler to define molecular programs in and around the SNpc and nominate candidate genes. We cloned these candidates as an sgRNA library into the InvαSyn backbone, produced AAV, injected cohorts, and harvested tissue at 3–4 months for DeepSeq-based quantification of sgRNA representation. Because Cre recombinase and Cas9 are restricted to dopaminergic neurons in Dat-Cre; LSL-Cas9-GFP mice, both α-synuclein expression and genome editing occurred selectively within DA neurons in the SNpc, ensuring cell-type specificity in the region of interest. This integrated strategy identified promising modifiers of DA neuron vulnerability, providing a functional roadmap toward new PD therapeutic targets.