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Summary: Genetic loss and gain of function in mice have typically been studied by using knockout or knockin mice that take months to years to generate. To address this problem for the nervous system, we developed NEPTUNE (NEural Plate Targeting by in Utero NanoinjEction) to rapidly and flexibly transduce the neural plate with virus prior to neurulation, and thus manipulate the future nervous system. Stable integration in >95% of cells in the brain enabled long-term overexpression, and conditional expression was achieved by using cell-type-specific MiniPromoters. Knockdown of Olig2 by using NEPTUNE recapitulated the phenotype of Olig2−/− embryos. We used NEPTUNE to investigate Sptbn2, mutations in which cause spinocerebellar ataxia type 5. Sptbn2 knockdown induced dose-dependent defects in the neural tube, embryonic turning, and abdominal wall closure, previously unreported functions for Sptbn2. NEPTUNE thus offers a rapid and cost-effective technique to test gene function in the nervous system and can reveal phenotypes incompatible with life. Motivation: Current methods used to alter gene expression in mouse embryos to study brain development lack efficacy and are time consuming. NEPTUNE provides an addition to the molecular toolbox, allowing rapid and widespread manipulation of gene expression before neurulation in vivo. This technique can be adapted to specifically target central nervous system (CNS) cell subtypes such as neurons or glia. Functional analysis can be performed within days after injection. NEPTUNE can thus reduce the financial and ethical costs of animal research, while accelerating the process from hypothesis to results in vivo.