938A Poster - 15. Models of human disease
Thursday April 07, 2:00 PM - 4:00 PM

A CRISPR-Cas9 Mediated Knockout of RNaseZ in Drosophila Neurons


Authors:
Max Luf; Ekaterina Migunova; Jake Nelson; Edward Dubrovsky

Affiliation: Department of Biological Sciences, Fordham University

Keywords:
a. neural degeneration; b. neural disorder

The RNaseZ gene is a vital and highly conserved gene with homologs in all eukaryotes. It plays an essential role in the maturation of tRNA, being the only enzyme to process the 3’-ends of pre-tRNA in both the nucleus and mitochondria. Point mutations in the human homolog, ELAC2, have been linked to a heterogeneous group of neurological conditions, such as microcephaly and untreatable epilepsy. Because these conditions result in such a poor quality of life for patients, we seek to understand what unique role RNaseZ may play in the nervous system.
Here we report the importance of RNaseZ to the Drosophila nervous system and demonstrate its role using neuron-specific knockouts of RNaseZ. To determine if point mutations of RNaseZ homologous to those in humans could elicit neurological dysfunction in Drosophila we first generated two transgenic lines (gZF154L and gZT520I) carrying mutant alleles of RNaseZ under its natural promoter. When the endogenous RNaseZ is removed these transgenes led to increased neurodegeneration in adult flies, decreased motor ability, and shortened life span. To then determine if this process was cell-autonomous, we generated a Cas9-mediated pan-neuronal knockout (KO) of RNaseZ in all Drosophila neurons. This resulted in developmental delays, debilitating motor deficits, and death shortly after eclosion. Because of the dual role of RNaseZ in the mitochondria and the nucleus, we created a Cas9-resistant rescue construct and used it to restore wt RNaseZ exclusively to the nucleus, creating a mitochondrial KO of RNaseZ in Drosophila neurons. The mitochondrial KO of RNaseZ produced a phenotype in longevity and locomotive ability almost identical to that of flies with the pan-neuronal KO. Overall, our findings improve our understanding of the unique role that RNaseZ plays in the nervous system and demonstrate how tissue-specific Cas9 engineering and Cas9-resistant rescue constructs can be applied to the modeling of neural degeneration in Drosophila.