Creating a process that would allow for high-throughput and fast correlation of phenotypes to genotypes in a vertebrate model system is a valuable tool for gene discovery and functional confirmation. Zebrafish has many advantages as a model for neurodegenerative disorders and genetic screens, such as easy and fast generation of embryos, ease of direct injection, availability of transgenic lines, as well as measurable locomotive behaviors. Genetic screens in CRISPR/Cas9 induced F0 generations are time and cost-effective, but producing a reliable phenotype has been a challenge.
The inherited movement disorders such as cerebellar ataxia have not been clearly established in a zebrafish model, despite over 50 known ataxia disease genes in humans. It also has not been assessed whether locomotive phenotypes can be measurably distinguished between different movement disorders.
In this study we produce mosaic genotypes in F0 generation in zebrafish using CRISPR/ Cas9 targeting a known ataxia gene snx14, a gene for Parkinsonism disease (pink1), and pla2g6, a gene involved in a complex phenotype with parkinsonism, ataxia and neuroaxonal dystrophy. We perform high-throughput movement analysis using a video tracking system Danio Vision (Noldus Ethovision software). We estimate the differences in locomotive manifestations between Parkinsonism and ataxia. We then evaluate the cerebellar morphology of these mutants in a transgenic line with a Purkinje cell fluorescent marker (Aldoca: GAP-Venus). We then perform next generation deep sequencing to measure the Cas9 cutting efficiency at >10,000 read depth in these mosaic animals. Finally, genotypes are correlated with movement as well as cerebellar morphology. This allows conclusions regarding the necessary levels of genome editing to produce observable and measurable phenotypes, also addressing the question whether genetic mutations linked to movement disorders with distinct clinical symptoms in people produce distinct motor deficits in zebrafish models.