Alexander Riddell, “Jun Gene Knockout in Danio rerio using CRISPR/ Cas9 Gene Editing”
Mentor: Ava Udvadia, Biological Sciences
Poster #110

For this semester, research will be focused on using CRISPR/Cas9 gene editing to create a knockout of the Jun gene or disrupt its putative regeneration-specific enhancers elements. A recently published protocol demonstrated how high efficiency CRISPR/Cas9-mediated gene targeting can be used in an acute phenotypic screen to identify genes involved in axon regeneration after spinal cord injury (Keatinge et al.,2021). Following this protocol, I will design several guide RNAs to direct targeted deletions in the jun gene or its regulatory elements when injected with the Cas9 nuclease into single-cell stage zebrafish embryos. The goal for this semester is to design and test the efficacy of the Jun guide RNAs and to determine the efficiency of bi-allelic knockout in injected animals. Since we are ultimately interested in investigating the role of Jun in regeneration, it may be necessary to supplement the developing embryos with Jun RNA to enable the embryos to undergo normal development of the optic nerve. This would enable subsequent analysis of Jun knockout after optic nerve injury in larval animals. This semester’s research will help to continue to develop our understanding of Jun’s specific role in axon regeneration and provide pathways for further research on regulation of gene expression involved with regeneration after optic nerve injury.