Defining a Midgestational Window for In Utero Genome Editing of the Fetal Murine Cortex

Abstract

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.