The Zebrafish Resource Core (Zf-Core) will be an integral component of the Washington University in St. Louis School of Medicine Model Organism Screening Center (wuMOSC), a multi-model organism platform for functional analysis of Undiagnosed Diseases Network (UDN) variants. It will serve as a resource for gene variant modeling, particularly in cases where analysis is not feasible in invertebrate model organisms, such as D. melanogaster and/or C. elegans. We anticipate that our bioinformatic analyses will designate ~65 variants per year to be analyzed in the zebrafish model. We will leverage the relatively short generation time, high fecundity, and external development of transparent zebrafish embryos to develop and implement a rapid, efficient, and disease variant-tailored pipeline for evaluating the variant pathogenicity using CRISPR/Cas9 approaches and/or RNA overexpression. The Zf-Core has improved on previous methods of fish husbandry by employing large-scale robotic feeding to allow for rapid growth of animals. This effectively halves the typical zebrafish generation time and thus will accelerate screening UDN variants proposed here. The PI laboratory also recently improved homologous recombination-based genome editing methods in zebrafish using TALEN or CRISPR/Cas9 strategies. For UDN gene-variants where the null allele manifests a phenotype prior to day 3 post fertilization, we will assess the degree to which the mutant phenotype can be rescued by injection into one-celled zygotes of synthetic RNA encoding the human protein variant, or zebrafish protein with this variant introduced. These experiments provide a rapid assessment as to whether the variant has normal, reduced, elevated or otherwise abnormal activity. If lines harboring nonsense or other deleterious mutations in the disease gene of interest already exist they will be imported to our Zf-Core. Alternatively, we will generate small insertions/deletions (indel) mutations using CRISPR/Cas9 strategy by designing a guide RNA that would also allow us to introduce the disease variant into the endogenous locus. In a second strategy, for genes with phenotypes detectable after day 3 post fertilization, the variant will be knocked-into the zebrafish ortholog by CRISPR/Cas9 editing. The phenotype of the resulting loss of function and knock-in mutants will be analyzed to verify any reported defects or to uncover them. Transparent embryos and larvae will be evaluated at the level of overall morphology, formation of specific organs and tissues using in vivo microscopy, and adults by microCT imaging to assess skeleton and soft tissues. Observation of a phenotype with a variant knock-in suggests that the variant is deleterious to the model organism and provid...