Development of a zebrafish model for inherited cardiac electrical disorders

Long-QT Syndrome (LQTS) is a cardiac electrical disorder distinguished by irregular heart rates and sudden cardiac death. LQTS Type II (LQTS2), which accounts for approximately 40% of cases, is caused by loss-of-function mutations in the Kv11.1 (hERG) channel. LQTS-associated hERG channel variants are typically studied in heterologous expression systems, or induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs); however these single cells/tissue layers do not recapitulate the complexities of a whole organ or organism. Zebrafish have a similar cardiac electrophysiological profile to humans and a well-defined genome, and have thus emerged as a powerful tool to study LQTS-associated variants in an organismal context. In this thesis I explain why zebrafish are such a powerful model for studying both inherited and acquired LQTS. I focus on inherited LQTS2, and the genetic approaches to model this in zebrafish. I describe the development of a CRISPR system that can be used to efficiently edit and detect clinically relevant LQTS2 point mutations, using a novel exon replacement strategy with fluorescent reporter gene incorporation. This approach is paired with an early phenotyping pipeline for efficient characterization of the impact of LQTS variants on cardiac structure and electrical activity. Since some variants may produce embryonic lethal phenotypes, and because compensatory mechanisms may confound measurement of the effect of variants introduced in zebrafish embryos, I then developed an inducible CRISPR system that could be triggered in adult fish. To achieve this, I designed and constructed a photoinducible CRISPR system that could be used to introduce the Cas9 gene into the zebrafish genome so that editing can be triggered in adult stages with endogenously produced inducible Cas nucleases. The final part of my thesis includes a pedagogical research study on teaching the same genetic engineering tools that I use in my thesis, with the goal of facilitating undergraduate student involvement in research. Overall, my thesis work extends the utility of zebrafish as a disease modeling platform, providing knowledge consolidation, novel gene-editing approaches that can account for developmental compensatory influences and can be applied to multiple genes of interest, and opportunities for enhanced undergraduate education in the fundamentals of CRISPR technologies.