Investigating the GeneWeld Technique as a Method to Create Humanized TP53 Zebrafish Models of Li-Fraumeni Syndrome

Abstract

Li-Fraumeni syndrome (LFS) is a highly penetrant cancer predisposition disorder caused by germline mutations in the tumour suppressor gene, TP53. Currently, there are no approved cancer prevention treatments for LFS. To address this unmet need, I set out to investigate compounds that restore wildtype p53 functionality using zebrafish as a model organism. I utilized the GeneWeld technique, a CRISPR/Cas9 microhomology mediated transgenesis method, to generate "humanized" TP53 zebrafish models which harbour the human TP53 coding sequence in a tp53⁻ᐟ⁻ background. The generated lines (harbouring TP53ᵂᵀ or TP53ᴿ¹⁷⁵ᴴ) were found to express the transgene mRNA transcript, however, the human p53 protein was not detectable. I theorize this is due to low transcript levels, and future work will be focused on optimizing expression of the transgene. Once finalized, these models will be used to discover new cancer preventative compounds that can be used to improve the lifetime outlook for individuals with LFS.