Neurodevelopmental disability can be caused by pathogenic variants in any one of numerous genes, many of which are still being identified. The identification of a variant of uncertain significance (VUS) in an NDD gene in a proband leads to families, clinicians, and scientists wondering if the VUS is causal or a “red herring.” Ideally, reliable, cell-based, protein-based, or biochemical assays would test functional consequences of such variants, helping add experimental evidence for variant reclassification under ACMG criteria. However, for many NDD genes, there are no in vitro or ex vivo assays, and the effect of a VUS must be tested in vivo. This exploratory grant proposes an efficient and innovative strategy to evaluate VUSs in genes that cause NDD when haploinsufficient in humans, that cause no phenotype or a subtle phenotype when haploinsufficient in mice, and that cause a clear fetal phenotype when knocked out (KO) in mice. This is the situation for many human disease genes, including the NDD gene ANKRD17. De novo heterozygous nonsense, frameshift, and canonical splice site variants in ANKRD17 found in multiple probands with NDD indicate the mechanism of mutational effect is haploinsufficiency. However, Ankrd17 haploinsufficient mice are viable and fertile, without a reported neurodevelopmental phenotype. In contrast, Ankrd17 KO mice exhibit a lethal cardiovascular phenotype at E10.5. ANKRD17 and Ankrd17 are 96% identical and 98% similar at the amino acid level. The advantage of our strategy is that is simpler to determine if a hemizygous or homozygous variant is a loss-of- function because it causes a fetal KO phenotype than it is to determine if that variant causes a subtle heterozygous phenotype postnatally. The former only requires efficient zygotic editing, and fetal phenotyping and genotyping. The latter requires newly generated mouse strains and postnatal neurobehavioral testing. As proof of concept, we will use i-GONAD to introduce Ankrd17 pathogenic and benign variants in E0.7 zygotes. i-GONAD is an efficient in vivo zygote gene-editing method for introducing hemizygous, heterozygous, homozygous, and compound heterozygous variants. We will phenotype fetuses at E10.5 and correlate phenotypes with genotypes. We expect fetuses that are hemizygous and homozygous for pathogenic variants to exhibit the same phenotype as KO fetuses, while fetuses hemizygous or homozygous for benign variants will appear normal. We will then test VUSs in ANKRD17 that were identified in patients with NDD and make the results of these experiments accessible via the NIH-supported ClinVar portal. Successful completion of these experiments will establish a new paradigm for testing VUSs in genes that cause highly penetrant haploinsufficiency phenotypes in humans, subtle or no haploinsufficiency phenotype in mice, and highly penetrant fetal phenotypes in knockout mice.