The zebrafish has characteristics that make it an ideal model organism for studying genetic determinants that participate in development and disease. The advent of zinc finger nucleases, and more recently TAL-effector nucleases, has provided an accessible methodology for the targeted disruption of practically any gene within the zebrafish genome. However limitations still remain to the application of these site-specific nucleases (SSNs) in zebrafish, in particular for the generation of large deletions or tailor-made modifications to large coding (or non-coding) regions of the genome. Realizing this goal is critical to exploiting the full potential of the zebrafish as a developmental and disease model. In this grant application, we will rely on our established expertise in the field to optimize and exted the use of SSNs in zebrafish. To provide a foundation for our genome editing efforts, initial studies in Aim 1 will focus on increasing the efficiency of double-strand break formation by SSNs through improvements in the nuclease architecture. These improvements will be coupled to new methods to optimize the ratio of germline to somatic lesion frequency. In parallel, we will test the application of improved SSNs to rapidly interrogate gene function through efficient targeted biallelic somatic cell knockout. In particular, we will focus on development of approaches to allow biallelic gene knockout in a restricted somatic cell-type to facilitate analysi of cell autonomy.
In Aim 2, we will apply SSNs to expand the repertoire of desired lesions that can be introduced at targeted sites in the zebrafish genome. This will include the application of SSNs to introduce large deletions and inversions through the use of multiple SSN pairs, as well as incorporation of domains that facilitate chromatin looping to generate efficient deletion of intervening genomic segments. We will also apply a similar approach to replace deleted regions with an exogenously supplied donor DNA to allow tailor-made alteration of the zebrafish genome.
In Aim 3, we will apply improved SSNs to determine the function of non-coding sequences in the zebrafish genome during hematopoiesis and vascular development. In particular, we will introduce targeted deletions in the locus control (LCR) region of the major globin locus to determine its importance for globin switching during embryonic development. In parallel, we will apply SSNs to generate targeted deletions in miR-126a and b to determine the distinct roles of these microRNAs during flow- dependent and -independent vascular morphogenesis. The advances made in the context of the studies proposed in this application will enable the zebrafish community to create a variety of tailored genomic manipulations to facilitate detailed investigation of gene function. As we have in the past, we will continue to share all protocols and reagents that are developed in the course of these studies to facilitate their application within the community.
We are developing technology to allow the genome of the zebrafish to be engineered. This technology will allow researchers to construct models of human disease in this animal to develop new therapies. We will apply this new technology to better understand the underlying biology of blood cell and blood vessel formation.
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