GSE157977
In vivo Perturb-Seq reveals neuronal and glial abnormalities associated with autism risk genes
We developed and applied a scalable genetic screening approach, in vivo Perturb-Seq, to functionally evaluate 35 autism spectrum disorder/neurodevelopmental delay (ASD/ND) de novo loss-of-function risk genes (see https://www.biorxiv.org/content/10.1101/791525v1 for complete list). Using CRISPR-Cas9, we introduced frameshift mutations in these risk genes in pools, within the developing mouse brain in utero, followed by single-cell RNA sequencing of perturbed cells in the postnatal brain. We identified cell type-specific and evolutionarily conserved gene modules from both neuronal and glial cell classes. Recurrent gene modules and cell types are affected across this cohort of perturbations, representing key cellular effects across sets of ASD/ND risk genes. Overall design: All animal experiments were performed according to protocols approved by the Institutional Animal Care and Use Committees (IACUC) of Harvard University and of the Broad Institute of MIT and Harvard. In utero lentiviral injection into the lateral ventricles was performed at E12.5 in Cas9 transgenic mice (4-6 month old, Jax #026179), and each single-cell library was made by combining the BFP+ cells from 1-3 litters (4-20 animals) of P7 animals harvested on the same day. The FACS-purified cells were sorted into cold Hibernate A/B27 medium and subjected to single-cell RNA sequencing library preparation. Our analysis comprises 18 independent libraries of Perturb-Seq cells (17 after filtering). Single-cell RNA sequencing libraries were created using the Chromium Single Cell 3' Solution v2 kit (10x Genomics) following the manufacturer's protocol. Each library was sequenced with Illumina NextSeq high-output 75-cycle kit with sequencing saturation above 70%. Dial-out PCR was performed to extract the perturbation barcode in each cell.