Jackson, C. R.; Borsos, M.; Appling, N.; Jackson, C. R.; Coughlin, G. M.; Gradinaru, V.

Congenital disorders of cortical development arise from genetic lesions that disrupt neurogenesis and neuronal migration. Unfortunately, tools to model or correct these defects before birth are limited. Here we establish a platform for systemic in utero gene delivery and genome editing in the mouse cortex at midgestation. By microdissecting a uterine window over the vitelline vein at embryonic day 12.5 (E12.5), we achieve fetal circulation access, enabling robust AAV-mediated transduction of the central nervous system (CNS) while reducing off-target expression in peripheral organs. Barcoded capsid screens reveal that AAV9 exhibits developmental stage-dependent tropism, with higher CNS penetrance and lower liver transduction at E12.5 than at E15.5. Leveraging this window, we provide a proof-of-concept of efficient cortical editing, using Cre-lox and CRISPR/Cas9 strategies to recapitulate prenatal reeler-like cortical misordering phenotypes following Reln knockout. We further use homology-directed repair to demonstrate precise genome modification, epitope-tagging the endogenous Reln and Actb loci, and installing a human-derived pathogenic allele of PDHA1. Importantly, we show that edited cells span neural progenitors and differentiated neurons across the cortex and hippocampus. These results define a permissive midgestational window for prenatal genome editing, providing a platform for functional modeling of congenital CNS disorders and exploration of early therapeutic interventions with minimized peripheral exposure.