Massively parallel in vivo Perturb-seq reveals cell type-specific transcriptional networks in cortical development
Leveraging AAV's versatile tropism and labeling capacity, we expanded the scale of in vivo CRISPR screen with single-cell transcriptomic phenotyping across embryonic to adult brains and peripheral nervous systems. Through extensive tests of 86 AAV serotypes, combined with transposon systems, we substantially amplified labeling and accelerated in vivo gene delivery from weeks to days. Our proof-of-principle in utero screen identified the pleiotropic effects of Foxg1, highlighting its tight regulation of distinct networks essential for cell fate specification of Layer 6 corticothalamic neurons. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% by lentivirus, to achieve analysis of over 30,000 cells in one experiment and enable massively parallel in vivo Perturb-seq. Compatible with various phenotypic measurements (single-cell or spatial multi-omics), it presents a flexible approach to interrogate gene function across cell types in vivo, translating gene variants to their causal function. Overall design: AAV transduced cells were dissociated and Fluorescent-activated sorted (FACS) to analyze the AAV transduction efficiency, gRNA capturing efficiency or perturbation effects with scRNA-seq Please note that the Perturb_* samples are the standard scRNA-seq sample, while the Perturb_*_Foxgdialout samples are enriched library on Foxg1 only with a separate library prep and sequencing.