Neural plate targeting by in utero nanoinjection (NEPTUNE) reveals a role forSptbn2in neurulation and abdominal wall closure

Gene variants associated with disease are efficiently identified with whole genome sequencing or GWAS, but validationin vivolags behind. We developed NEPTUNE (<underline>ne</underline>ural<underline>p</underline>late targeting by in<underline>u</underline>tero<underline>n</underline>anoinj<underline>e</underline>ction), to rapidly and flexibly introduce gene expression-modifying viruses to the embryonic murine neural plate prior to neurulation, to target the future adult nervous system. Stable integration in >95% of cells in the brain enabled long-term gain- or loss-of-function, and conditional expression was achieved using mini-promotors for cell types of interest. Using NEPTUNE, we silencedSptbn2, a gene associated with Spinocerebellar ataxia type 5 (SCA5) in humans. Silencing ofSptbn2induced severe neural tube defects and embryo resorption, suggesting thatSPTBN2in-frame and missense deletions in SCA5 reflect hypomorphic or neomorphic functions, not loss of function. In conclusion, NEPTUNE offers a novel, rapid and cost-effective technique to test gene function in brain development, and can reveal loss of function phenotypes incompatible with life.