Meeting Abstract

P3-18   -   Using CRISPR-Cas9 to Create Xenopus Models of Neurodegenerative Diseases Davis, MR*; Coppenrath, KE; Horb, ME; University of Alabama at Birmingham, Birmingham, AL; Marine Biological Laboratory, Woods Hole, MA; Marine Biological Laboratory, Woods Hole, MA mdavis08@uab.edu

Animal models of human disease are important research tools used to study disease diagnosis, development, and treatment. Xenopus tropicalis is a popular genetic model and superior in comparison to mice in regard to their large brood size and also compared to zebra fish which, despite having large broods, have less synteny compared to mammals. Provided these reasons, we chose to utilize Xenopus tropicalis to genetically modify two genes involved in neural development. Low expression of either of these genes, ntf3 or nrg1, is associated with neurodevelopmental disorders such as schizophrenia, while mutations of ntf3 are associated with neurodegenerative disorders (NDDs) including Alzheimer’s. Similarly, loss-of-function mutations of nrg1 have been linked to another NDD, ALS. To create animal models with which researchers may use to study NDDs, we used CRISPR-Cas9 gene-editing technology to knock-out these two genes by creating indels in their protein-coding regions. The knockout of these genes involved designing and synthesizing single guide RNA (sgRNA) that Cas9 could use to cut the gene at the start of its protein domain, thereby rendering the translated protein functionless. After injecting both sgRNA and Cas9 into Xenopus tropicalis embryos, surviving tadpoles were screened for guide efficiency and observed to have phenotypic differences in comparison to embryos injected with a control mixture of water and dye. Tadpoles in which ntf3 was edited had different swimming behaviors. Meanwhile, tadpoles in which nrg1 was mutated displayed exogastrulation, a process in which nervous system development is halted due to the endoderm shifting from residing within the embryo to outside the embryo. The prevalence of these phenotypes suggest mutations in these genes affects nervous system development in Xenopus tropicalis and have the potential to be exploited to create model organisms for NDDs.