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© 2020 American Association for the Advancement of Science. All rights reserved. The number of disease risk genes and loci identified through human genetic studies far outstrips the capacity to systematically study their functions. We 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. Using CRISPR-Cas9, we introduced frameshift mutations in these risk genes in pools, within the developing mouse brain in utero, followed by single-cell RNAsequencing 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. In vivo Perturb-Seq allows us to investigate how diverse mutations affect cell types and states in the developing organism.

© 2020 American Association for the Advancement of Science. All rights reserved. The number of disease risk genes and loci identified through human genetic studies far outstrips the capacity to systematically study their functions. We 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. Using CRISPR-Cas9, we introduced frameshift mutations in these risk genes in pools, within the developing mouse brain in utero, followed by single-cell RNAsequencing 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. In vivo Perturb-Seq allows us to investigate how diverse mutations affect cell types and states in the developing organism.

The neocortex contains an unparalleled diversity of neuronal subtypes, each defined by distinct traits that are developmentally acquired under the control of subtype-specific and pan-neuronal genes. The regulatory logic that orchestrates the expression of these unique combinations of genes is unknown for any class of cortical neuron. Here, we report that Fezf2 is a selector gene able to regulate the expression of gene sets that collectively define mouse corticospinal motor neurons (CSMN). We find that Fezf2 directly induces the glutamatergic identity of CSMN via activation of Vglut1 (Slc17a7) and inhibits a GABAergic fate by repressing transcription of Gad1. In addition, we identify the axon guidance receptor EphB1 as a target of Fezf2 necessary to execute the ipsilateral extension of the corticospinal tract. Our data indicate that co-regulated expression of neuron subtype–specific and pan-neuronal gene batteries by a single transcription factor is one component of the regulatory logic responsible for the establishment of CSMN identity.

Many studies are uncovering functional roles for long noncoding RNAs (lncRNAs), yet few have been tested for in vivo relevance through genetic ablation in animal models. To investigate the functional relevance of lncRNAs in various physiological conditions, we have developed a collection of 18 lncRNA knockout strains in which the locus is maintained transcriptionally active. Initial characterization revealed peri- and postnatal lethal phenotypes in three mutant strains (Fendrr, Peril, and Mdgt), the latter two exhibiting incomplete penetrance and growth defects in survivors. We also report growth defects for two additional mutant strains (linc–Brn1b and linc–Pint). Further analysis revealed defects in lung, gastrointestinal tract, and heart in Fendrr[superscript −/−] neonates, whereas linc–Brn1b[superscript −/−] mutants displayed distinct abnormalities in the generation of upper layer II–IV neurons in the neocortex. This study demonstrates that lncRNAs play critical roles in vivo and provides a framework and impetus for future larger-scale functional investigation into the roles of lncRNA molecules., National Science Foundation (U.S.) (Postdoctoral Fellowship in Biology)

The ability to direct functional proteins to specific DNA sequences is a long-sought goal in the study and engineering of biological processes. Transcription activator–like effectors (TALEs) from Xanthomonas sp. are site-specific DNA-binding proteins that can be readily designed to target new sequences. Because TALEs contain a large number of repeat domains, it can be difficult to synthesize new variants. Here we describe a method that overcomes this problem. We leverage codon degeneracy and type IIs restriction enzymes to generate orthogonal ligation linkers between individual repeat monomers, thus allowing full-length, customized, repeat domains to be constructed by hierarchical ligation. We synthesized 17 TALEs that are customized to recognize specific DNA-binding sites, and demonstrate that they can specifically modulate transcription of endogenous genes (SOX2 and KLF4) in human cells., Harvard University. Society of Fellows, National Human Genome Research Institute (U.S.) (Center for Excellence in Genomics Science P50 HG003170), United States. Dept. of Energy (Genomes to Life DE-FG02-02ER63445), United States. Defense Advanced Research Projects Agency (W911NF-08-1-0254, G.M.C.), Wyss Institute of Biologically Inspired Engineering, National Institutes of Health (U.S.) (Transformative R01 (R01 NS073124-01)), European School of Molecular Medicine (predoctoral fellowship)

GABAergic interneurons of the cerebral cortex represent one of the most diversified populations of cells of the Central Nervous System. It is well established that different subtypes of interneurons populate and integrate into the cortical layers, where they critically modulate the firing activity of their excitatory projection neuron partners. The developmental origin of interneurons has been extensively studied, and elegant prior work has demonstrated that in rodents, the vast majority of interneurons are born in the ventral telencephalon within the medial and caudal ganglionic eminences (MGE and CGE). Although great progress has been made over the last decade in understanding the molecular diversity and fate-specification of MGE-derived interneuron subtypes, the mechanisms controlling the birth and diversification of CGE-derived interneurons, as well as their functional roles within cortical circuitry, remain poorly understood. Here, I addressed this question directly, by investigating a potential role over CGE-derived interneurons of COUP-TFI, a transcription factor highly expressed in the embryonic CGE. I generated and studied conditional, null-mutant mice where the COUP-TFI gene was selectively deleted from the ventral telencephalon (COUP-TFIfl;fl x Dlx5/6CRE-IRES-GFP). In this mouse model, all GABAergic interneurons that arise from the subventricular zone of the ventral telencephalon lack COUP-TFI, without loss of this gene from neurons within the cerebral cortex. These conditional mutants are viable and survive to adulthood, thus enabling investigation of the birth and differentiation of interneuronal subtypes through postnatal stages of development, when local cortical microcircuitry is built. I found that conditional loss-of-function of COUP-TFI in subventricular precursors and post-mitotic cells of the basal ganglia led to a decrease within the cortex of late-born, CGE-derived, VIP- and CR-expressing bipolar interneurons, which was compensated by a concur