Zinc finger nuclease induced knockout and knock-in models in zebrafish

DESCRIPTION (provided by applicant): Zebrafish are an important animal model system for both basic science research and preclinical disease modeling. Complementary to mammalian models, the zebrafish system facilitates methods that are not practical (e.g.,large-scale forward genetic screens), not possible (in vivo imaging of embryonic development), or not cost-effective (high-throughput chemical screens for drug discovery) in mice or rats. Until recently, however, methods for targeted genetic manipulations(e.g., knockout) have eluded the zebrafish field. Targeted genetic modifications stand as the single most desired methodology of the rapidly growing zebrafish market. The advent of zinc finger nuclease (ZFN)-based genome modification has brought gene knockout, and potentially knock-in, strategies to zebrafish researchers. Yet, the process of identifying appropriate ZFN pairs for a given gene target is not trivial, resulting in only a small number of labs that have successfully applied this approach, to date. Here we propose to test whether customized ZFN pairs from Sigma's Advanced Genetic Engineering (SAGE) group improve the efficiency of creating targeted genetic modifications in zebrafish. If proven effective, we will partner with SAGE - which holds anexclusive license from Sangamo Biosciences for creating ZFN-based animal models - to create a catalog of knockout and knock-in zebrafish. Proprietary techniques that SAGE has applied to other species (e.g. rodents) will be employed both in terms of identifying optimal ZFN targets/pairs and in terms of facilitating ZFN-based modifications. Aim 1: Create three knockout disease models in zebrafish using customized ZFN pairs from SAGE targeting 1three genetic loci: 1) sapje, 2) pink1, 3) kif1b, corresponding to knockout models for Muscular Dystrophy, Parkinson's Disease, and Multiple Sclerosis, respectively. Aim 2: Test knock-in efficiency using customized ZFN pairs from SAGE. Although ZFN-based knockout methods have been validated in zebrafish, ZFN-basedknock-in success has not been demonstrated. For this pilot study, proprietary information from SAGE, our own insights regarding transgenesis, and data from groups that have created ZFN-based knock-ins in other systems, we be employed to introduce a fluorescent reporter into the krox20 locus. Successful demonstration of ZFN-induced knock-in would pave the way for the creation of a catalog highly versatile research models. Success of this Phase I proposal will be followed by scale-up initiatives in Phase II and partnering with SAGE in Phase III (see letter of support) to bring 2 both Knockout zebrafish (KOZTM) and Knock-in zebrafish (KIZTM) models to market. We anticipate that development of a strong disease model catalog, coupled with an extremely strongIP position, will promote lucrative relationships with pharmaceutical partners in our efforts to bring insights afforded by the zebrafish system to bear on human disease. PUBLIC HEALTH RELEVANCE: The ability to manipulate genes in a targeted manner revolutionized the fields of molecular genetics and disease modeling but, until recently, was only applicable in the mouse (e.g., gene knockouts). The advent of zinc finger nuclease (ZFN) technology facilitates targeted gene manipulation in any species in which the genome has been sequenced. Accordingly, Luminomics proposes to create ZFN-induced gene knockout and knock-in models in zebrafish, the fastest growing vertebrate model species, as an off-the-shelf product line of broad appeal to both academic and commercial sectors of the zebrafish research community.