2025/08/22 by Biswarathan Ramani, Indigo V.L. Rose, Noam Teyssier +8 · 1 voice
Biochemistry, Genetics and Molecular Biology · #Advanced biosensing and bioanalysis techniques #CRISPR and Genetic Engineering #RNA and protein synthesis mechanisms
paper · pdf · doi:10.1038/s41593-025-02043-9
openalex publication_date 2025/08/22 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/28
There is a substantial need for scalable CRISPR-based genetic screening methods that can be applied in mammalian tissues in vivo while enabling cell-type-specific analysis. Here 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 used this approach to screen two large sgRNA libraries, which collectively target over 5,000 genes, in mouse brains and uncovered genes essential for neuronal survival, of which we validated Rabggta and Hspa5. We highlight the reproducibility and scalability of the platform and show that it is sufficiently sensitive for screening in a restricted subset of neurons. We systematically characterize the impact of sgRNA library size, mouse cohort size, the size of the targeted cell population, viral titer, and coinfection rate on screen performance to establish general guidelines for large-scale in vivo screens.