Conditional Animal Models of mtDNA Disease

Conditional Animal Models of mtDNA Disease: Abstract Mitochondria are critical for metabolism, organ homeostasis, apoptosis, and aging. This wide range of impact is manifested by the enormous biological variation and diverse disorders in patients with mitochondrial disease affecting organs such as the eye, ear, brain, muscle, and kidney. Understanding how mitochondria function in

normal biology - and how human mitochondrial DNA (mtDNA) variations contribute to health and disease - has been hampered by a lack of animal models due to limited approaches to manipulate this powerhouse of the cell. This project aims to resolve this major technical bottleneck in the field to enable us to deploy a full complement

of gene editors to introduce precise edits in the mitochondrial genome in a manner capable of temporal and spatial control and to use these to make the first conditional animal model of mitochondrial disease using zebrafish. CRISPR gene editing tools have enabled the rapid manipulation of the nuclear genome. CRISPR

systems are dual molecular, with Cas protein and gRNA components. They are not currently operational in the mitochondrial compartment however because of a lack of effective methods to deliver exogenous guide RNAs. We have identified an endogenously trafficked RNA that can carry targeted nucleotide changes from the nucleus

into mitochondria, representing a potential novel method for the introduction of programmed RNAs suitable for use as guide molecules for CRISPR Cas proteins in mitochondria. When combined with a Cre-regulated mitochondrial Cas12a base editor, we will generate a conditional zebrafish disease model for multi-systemic

mtDNA-encoded mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). The establishment of this toolkit will not only shed mechanistic insights but will also facilitate the genomic nosology of a large class of mitochondrial disorders. If successful, the versatile mtRNA chimeric

gRNA delivery system will enable us to model different mitochondrial pathogenic variations. This application harnesses the unique environment of mitochondria to generate a new toolbox to expand the repertoire of tools to edit the mitochondrial genome.