SYSTEMATIC IN UTERO CRISPR SCREENING IDENTIFIES TRANSCRIPTIONAL CONTROL OF CLASS-SPECIFIC NEURONAL DIFFERENTIATION IN THE NEOCORTEX

The mammalian neocortex contains a diverse array of neuronal subtypes that underlie higher-order sensory, motor, and cognitive functions. This diversity is generated during embryonic development through tightly regulated gene expression programs, yet how large numbers of regulatory genes collectively coordinate neuronal subtype specification remains poorly understood. A major barrier has been the difficulty of performing large-scale functional screens directly in the developing brain in utero. Here, we developed and applied a massively high-throughput in utero CRISPR/Cas9 loss of function screening platform to systematically interrogate transcriptional regulators of neocortical development. We focused on Callosal Projection Neurons (CPNs), a major class of excitatory cortical neurons that has expanded and diversified during mammalian evolution. Guide RNAs targeting 2,881 mouse transcription factors were delivered to neocortical progenitors across hundreds of embryos in utero. Targeted neurons were subsequently recovered postnatally and subjected to molecular profiling to assess effects on CPN development. This unbiased screen identified multiple previously uncharacterized transcription factors, including several Zinc Finger family members, as modulators of CPN differentiation. Targeted perturbation of these factors in vivo revealed altered molecular signatures consistent with disrupted CPN identity. Together, this work establishes a scalable in utero functional genomics framework that enables systematic dissection of gene regulatory programs in the developing brain. By coupling large-scale CRISPR screening with molecular phenotyping, this approach provides a powerful strategy to uncover the genetic logic underlying neuronal diversity in the mammalian neocortex.