Scalable functional analysis of neuropsychiatric risk genes with spatially integrated in vivo Perturb-seq

Although numerous genes and loci associated with autism spectrum disorder and neurodevelopmental delay (ASD/NDD) have been identified through genome sequencing efforts, the precise mechanisms by which most of these genetic variants lead to the condition remain largely unknown. We propose to combine high-content in vivo

genetic screening with whole brain cytoarchitecture spatial information from neurons with loss-of-function mutations in ASD/NDD risk genes to bridge the gap between genetic insights and mechanistic understanding. This approach, which can be scaled to interrogate large panels of genetic variants in parallel, has the power to

reveal how these diverse gene variants converge to produce ASD/NDD, including identifying the specific brain regions, cell types, neural circuits, developmental time windows, and molecular networks involved in the pathogenesis of these disorders. To achieve this, we propose to use high-resolution and multimodal phenotypic characterizations to

comprehensively map the functions in the neocortex and striatum of a set of 72 high-confidence ASD/NDD risk genes, many of which encode transcriptional regulators. We will adapt in vivo Perturb-seq to allow high efficiency screening across multiple developmental time points with both single-nucleus transcriptome and chromatin

accessibility readouts. These rich datasets will enable us to build gene regulatory networks (GRNs) that will reveal shared and divergent molecular signatures associated with this set of ASD/NDD risk genes. In parallel, we will explore how perturbation of 5 high-confidence ASD/NDD risk genes impacts cellular migration,

morphology, and long-range connectivity. Here, we will use Perturb-CAST (cytoarchitecture see-through) to combine sparse genetic perturbations in vivo with whole mount brain clearing and light-sheet imaging to examine brain-wide changes in cytoarchitecture across developmental time points. This work will expand two major technologies, in vivo Perturb-seq and Perturb-CAST, which will be broadly

impactful tools for understanding the genetic basis of ASD/NDD and other complex brain disorders. By focusing on corticostriatal pathways and integrating spatial information, the proposal seeks to uncover commonalities and shared mechanisms among ASD/NDD risk genes, ultimately contributing to a more comprehensive

understanding of the disorder and potentially guiding future therapeutic approaches.