CRISPR screening by AAV episome-sequencing (CrAAVe-seq) is a highly scalable cell type-specific in vivo screening...

There is a significant need for scalable CRISPR-based genetic screening methods that can be applied directly in mammalian tissues in vivo while enabling cell type-specific analysis. To address this, we developed an adeno-associated virus (AAV)-based CRISPR screening platform, CrAAVe-seq, that incorporates a Cre-sensitive sgRNA construct for pooled screening within targeted cell populations in mouse tissues. We demonstrate the utility of this approach by screening two distinct large sgRNA libraries, together targeting over 5,000 genes, in mouse brains to create a robust profile of neuron-essential genes. We validate two genes as strongly neuron-essential in both primary mouse neurons and in vivo, confirming the predictive power of our platform. By comparing results from individual mice and across different cell populations, we highlight the reproducibility and scalability of the platform and show that it is highly sensitive even for screening smaller neuronal subpopulations. We systematically characterize the impact of sgRNA library size, mouse cohort size, the size of the targeted cell population, viral titer, and multiplicity of infection on screen performance to establish general guidelines for large-scale in vivo screens.