Generate Zebrafish Conditional Knockout Model for Ciliopathy Research

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Abstract Cilia are evolutionarily conserved microtubule-based organelles that project from the surface of the cells and plays an important role in protein trafficking, signaling cascade regulation, and mechanical movement of fluids. Multiple signaling pathways have been described to be regulated through the cilia, including Hedgehog, Wnt, Notched, and PDGF. Mutations that affect ciliary components are associated with a multitude of human diseases, together called ciliopathies. The medical conditions that result from ciliary dysfunction include, but are not limited to, retinal degeneration, anosmia, neural tube closure, polydactyly, cardiac malformations, obesity, polycystic kidney disease and cancer. Ciliopathies include rare human disorders such as, but not limited to, Meckel Syndrome (MKS), Joubert Syndrome (JBTS), Nephronophthisis (NPHP), and Bardet-Biedl Syndrome (BBS). With hopes of treating these diseases and disorders it is important understand how ciliary dysfunction contributes to the pathology of disease. While many of the structural aspects of cilia can be studies in cell culture, to understand the pathology animal models are required. Zebrafish is a powerful model for studying human disease and ciliopathies; however, they have been historically limited to morpholino knockdown. We have recently generated a number zygotic knockout in cilia associated genes, and discovered a number of disease phenotypes. However many of these knockouts are embryonic lethal preventing analyses of adult disease phenotypes. For this type of analysis, there is a need for zebrafish conditional knockouts. Further, a conditional knockout would also provide a tool for embryonic analysis as far as cell of origin of a phenotypes or isolation of one phenotype outside the context of multiple phenotypes. However, the generation of zebrafish conditional alleles are technically challenging and cumbersome to make. We hypothesis that using CRISPR/Cas9 mediated knock-in of an invertible FlEx gene disruption cassette we will generate ift88, mks5, and bbs5 conditional allele. We will validate that in the permissive orientation they act like a wild type allele, and in the non-permissive orientation, they act like a null allele. Further, we will validate the ability to induce temporal and spatial Cre induced recombination with these alleles. Successful completion of this proposal will provide a highly useful animal model to the research community.