In vivo Perturb-map: scalable genetic screens with single-cell and spatial resolution in intact tissues

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Human genetic studies have provided lists of genes and loci associated with risk for neuropsychiatric disorders including autism spectrum disorder and neurodevelopmental delay (ASD/ND), but to systematically evaluate their functions and mechanisms remains a dauting task. What brain regions, cell types, and neural circuits are involved in the pathological state? Do different risk genes and genetic variants affect the same sets of brain regions, cell types, neuronal projection networks and circuits? To address these questions, we will develop scalable genomic technologies to allow high-throughput, in vivo genetic screening with single-cell and meso-scale spatial resolution readout from intact tissues. First, we aim establish a method, named Perturb-map, to readout the spatial location, cytoarchitecture, and projectome of each perturbed cell from intact, whole brains without sectioning (Aim 1). We will perform sparse perturbation using the CRISPR-Cas9 system in vivo and harvest the brain with pooled genetic perturbations. Next, we will use hybridization-based detection and whole- brain tissue clearing methods, to allow iterative rounds of light-sheet imaging to deduce gRNA perturbation identities within an intact tissue. We will further incorporate imaging of fluorescent reporters and endogenous markers to ask how the perturbation changes the cell type identity, anatomical distribution, morphology and neuronal projectome from intact tissues. In parallel, this approach will be complemented with an improved in vivo Perturb-seq with unprecedented scalability (Aim 2a-b). We will establish an AAV-based Perturb-seq system to allow high scalability and flexible tissue-specificity. We will then apply it to study a panel of ASD/ND risk genes across two brain regions. Pooled, sparse perturbation will be introduced during embryogenesis, and single-cell multi-omic analysis and in situ Perturb-map analysis will reveal how each perturbation affect gene expression, cellular migration, cytoarchitecture, and neuronal projectome for each specific cell type across brain regions including neocortex and striatum, which are implicated in the disorders. This will allow the first in vivo screen with 3-dimensional, meso-scale optical readout with high spatial resolution. The PI has extensive experience and has pioneered the in vivo Perturb-seq technology, and she has brought together a diverse team and expertise with long-standing relationships and collaborations across the fields of tissue clearing and high-throughput imaging, computational genomics, and human genetics (including non-Caucasian populations) for equitable biomedicine (co-I Dr. Zhuhao Wu, LoS from Drs. Pejman Mohammadi, Joshua Levin, and Mireille Kamariza). Altogether, we will establish Perturb-Map as a scalable and generalizable approach to study gene function in complex tissues. We expect to identify the cell types, molecular networks, and projectomes affected by diverse ASD/ND risk gene variants with unprecedented scale and resolution. Perturb-Seq and Perturb-Map will be modular and broadly applicable genomic tools contributing to not only neuroscience but also many other tissue types and fields as we move from gene variant to their mechanisms and functions.