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. We performed a proof-of-principle in utero screen and identified pleiotropic effects of Foxg1, featured by its tight regulation of distinct networks essential for cell fate specification of Layer 6 corticothalamic neurons.
See PerturbSeqAnalysis and the associated documentation for the code used in upstream analysis of the Perturb-seq data (starting with CellRanger output through clustering and cell type identification).
aav_downstream.R: Main driver script that calls each of the below
propeller.R: Method to detect cell type proportion changes
run_sva_edger.R: Identify DEGs using edgeR with 1 surrogate variable from sva
Enrich_FGSEA_new.R: Identify enriched GO terms
mod.hidden.mult.R: Elastic net based method to find the most affected cells for each perturbation
fastq_barcodemapping.py: Count number of barcodes for each AAV variant
deseq2.R: Identify significantly enriched AAV variants
annotations.R: annotate insertion sites
GFPcount.cpproj: Cell profiler script to quantify the GFP intensity in HT22 cells