In vivo Perturb-Seq reveals neuronal and glial abnormalities associated with Autism risk genes

Abstract

The thousands of disease risk genes and loci identified through human genetic studies far outstrip our current capacity to systematically study their functions. New experimental approaches are needed for functional investigations of large panels of genes in a biologically relevant context. Here, we developed a scalable genetic screen approach, in vivo Perturb-Seq, and applied this method to the functional evaluation of 35 autism spectrum disorder (ASD) de novo loss-of-function risk genes. Using CRISPR-Cas9, we introduced frameshift mutations in these risk genes in pools, within the developing brain in utero, and then performed single-cell RNA-Seq in the postnatal brain. We identified cell type-specific gene signatures from both neuronal and glial cell classes that are affected by genetic perturbations, and pointed at elements of both convergent and divergent cellular effects across this cohort of ASD risk genes. In vivo Perturb-Seq pioneers a systems genetics approach to investigate at scale how diverse mutations affect cell types and states in the biologically relevant context of the developing organism.

Experience-independent transformation of single-cell 3D genome structure and transcriptome during postnatal development of the mammalian brain

“…The other 8 were inhibitory: 5 known interneuron types (SST: Sst+; PV: Pvalb+; VIP: Vip+; LAMP5: Lamp5+, named as in (Tasic et al, 2018), some cells of which are called NDNF interneurons by (Tasic et al, 2016) and others; MEIS2: higher expression of Meis2 than in other neurons, named as in (Di Bella et al, 2020;Jin et al, 2019;Tasic et al, 2018)), medium spiny neurons (MSN: Tac1+, also Meis2+ but lower than in MEIS2 interneurons; also seen in (Jin et al, 2019)), and 2 unknown interneuron types ( Figure 1A, Figure 1B, Figure S3).…”

Autism spectrum disorder at the crossroad between genes and environment: contributions, convergences, and interactions in ASD developmental pathophysiology

“…The impact of these experimental strategies on developmental neuroscience will be substantial, with the imminent application of CRISPR multiplexed perturbations on brain organoids shedding light on the precise contribution of multiple genomic regions to cellular phenotypes throughout the different stages of brain development. A first applicative example, although in a murine model, is the study of 35 ASD risk genes [171] in which in utero pooled genome editing of forebrain progenitors was followed by single cell transcriptional analysis at postnatal stages to uncover their effect in specific cell populations.…”

Genetically Engineering the Nervous System with CRISPR-Cas

“…While most screens are performed in vitro or ex vivo, two CRISPR-Cas9 mediated in vivo screens have recently been reported in the brain (Chow et al, 2017;Jin et al, 2019). A recent in vivo screen (Perturb-Seq, first described by Dixit et al, 2016) aimed at systematically uncovering the phenotypes of a large panel of ASD-related genes was performed by coupling cell-type specific transcriptomics and a lentivirus-mediated sgRNA library targeting 35 putative ASD-risk genes.…”