Abstract:

The thousands of disease risk genes and loci identified through human genetic studies far outstrip our current capacity to systematically assign them with function. To address the need to achieve biological understanding of large numbers of gene variants, we developed Perturb-Seq for in vivo scalable genetic screens, and demonstrated it for 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, followed by single-cell RNA-Seq in the postnatal brain as the phenotypic read-out. We derived cell type-specific gene programs affected by these genetic perturbations and showed that both neuronal and glial cell classes are affected by loss of function mutation of selected ASD risk genes; the phenotype in developmental maturation of oligodendrocytes is validated by a germline mouse model. In vivo Perturb-Seq pioneers a systems genetic approach to investigate how diverse mutations affect cell types and states associated with neurodevelopmental and other pathologies.