A Primer Genetic Toolkit for Exploring Mitochondrial Biology and Disease Using Zebrafish

Abstract

Mitochondria are a dynamic eukaryotic innovation that play diverse roles in biology and disease. The mitochondrial genome is remarkably conserved in all vertebrates, encoding the same 37-gene set and overall genomic structure, ranging from 16,596 base pairs (bp) in the teleost zebrafish (Danio rerio) to 16,569 bp in humans. Mitochondrial disorders are amongst the most prevalent inherited diseases, affecting roughly 1 in every 5000 individuals. Currently, few effective treatments exist for those with mitochondrial ailments, representing a major unmet patient need. Mitochondrial dysfunction is also a common component of a wide variety of other human illnesses, ranging from neurodegenerative disorders such as Huntington’s disease and Parkinson’s disease to autoimmune illnesses such as multiple sclerosis and rheumatoid arthritis. The electron transport chain (ETC) component of mitochondria is critical for mitochondrial biology and defects can lead to many mitochondrial disease symptoms. Here, we present a publicly available collection of genetic mutants created in highly conserved, nuclear-encoded mitochondrial genes in Danio rerio. The zebrafish system represents a potentially powerful new opportunity for the study of mitochondrial biology and disease due to the large number of orthologous genes shared with humans and the many advanced features of this model system, from genetics to imaging. This collection includes 15 mutant lines in 13 different genes created through locus-specific gene editing to induce frameshift or splice acceptor mutations, leading to predicted protein truncation during translation. Additionally, included are 11 lines created by the random insertion of the gene-breaking transposon (GBT) protein trap cassette. All these targeted mutant alleles truncate conserved domains of genes critical to the proper function of the ETC or genes that have been implicated in human mitochondrial disease. This collection is designed to accelerate the use of zebrafish to study many different aspects of mitochondrial function to widen our understanding of their role in biology and human disease.

Application of Zebrafish in Mitochondrial Dysfunction

“…We can say that mitotoxicity is a new term, used in scientific articles since 2011, when we searched scientific platforms such as PubMed, with 147 scientific publications in total and only 21 review articles. Parallels between human and zebrafish mitochondria extend to their composition and genetic organization, encompassing genes for rRNAs, tRNAs, and proteins integral to oxidative phosphorylation [12,13,41,42]. Consequently, zebrafish serve as an apt model for scrutinizing mitochondrial function, enriching the arena of comparative genomics and providing insights into genetic pathways and key molecular processes among vertebrates [43,44].…”

Engineering TALE-linked deaminases to facilitate precision adenine base editing in mitochondrial DNA

“…Targeted base editing of mammalian mitochondrial DNA (mtDNA) is a powerful and versatile technology 1–7 , which can be used to model mitochondrial genetic diseases in cell lines and animals 8–15 and to develop novel therapeutic modalities that could be used to correct pathogenic mutations in the future 16,17 . Programmable deaminases, which are composed of a custom DNA-binding protein and a nucleobase deaminase, enable mitochondrial DNA editing in a targeted manner 13, 18–25 .…”

“…, which can be used to model mitochondrial genetic diseases in cell lines and animals [8][9][10][11][12][13][14][15] and to develop novel therapeutic modalities that could be used to correct pathogenic mutations in the future 16,…”