Massively parallel Perturb-seq reveals cell type-specific transcriptional networks in cortical development.

bioRxiv : the preprint server for biology
Authors
Abstract

Systematic analysis of gene function across diverse cell types is hindered by two challenges: obtaining sufficient cells from live tissues and accurately identifying each cell's perturbation in high-throughput single-cell assays. Leveraging AAV's versatile cell type tropism and high labeling capacity, we expanded the resolution and scale of CRISPR screens: allowing phenotypic analysis at single-cell resolution across a multitude of cell types in the embryonic brain, adult brain, and peripheral nervous system. We undertook extensive tests of 86 AAV serotypes, combined with a transposon system, to substantially amplify labeling and accelerate gene delivery from weeks to days. Using this platform, we performed an genetic screen as proof-of-principle and identified pleiotropic regulatory networks of in cortical development, including Layer 6 corticothalamic neurons where it tightly controls distinct networks essential for cell fate specification. Notably, our platform can label >6% of cerebral cells, surpassing the current state-of-the-art efficacy at <0.1% (mediated by lentivirus), and achieve analysis of over 30,000 cells in one experiment, thus enabling massively parallel Perturb-seq. Compatible with various perturbation techniques (CRISPRa/i) and phenotypic measurements (single-cell or spatial multi-omics), our platform presents a flexible, modular approach to interrogate gene function across diverse cell types , connecting gene variants to their causal functions.

Year of Publication
2023
Journal
bioRxiv : the preprint server for biology
Date Published
09/2023
DOI
10.1101/2023.09.18.558077
PubMed ID
37790302
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