Mapping and editing animal mitochondrial genomes: can we overcome the challenges?

Abstract

The animal mitochondrial genome, although small, can have a big impact on health and disease. Non-pathogenic sequence variation among mitochondrial DNA (mtDNA) haplotypes influences traits including fertility, healthspan and lifespan, whereas pathogenic mutations are linked to incurable mitochondrial diseases and other complex conditions like ageing, diabetes, cancer and neurodegeneration. However, we know very little about how mtDNA genetic variation contributes to phenotypic differences. Infrequent recombination, the multicopy nature and nucleic acid-impenetrable membranes present significant challenges that hamper our ability to precisely map mtDNA variants responsible for traits, and to genetically modify mtDNA so that we can isolate specific mutants and characterize their biochemical and physiological consequences. Here, we summarize the past struggles and efforts in developing systems to map and edit mtDNA. We also assess the future of performing forward and reverse genetic studies on animal mitochondrial genomes. This article is part of the theme issue 'Linking the mitochondrial genotype to phenotype: a complex endeavour'.

Keywords: genetic engineering; genotype to phenotype; linkage mapping; mitochondrial DNA.

Figure 1.

Motivation, challenges and opportunities for mapping and editing animal mtDNA. (a) Animal mtDNA can impact health and disease. Sequence variation in mtDNA haplotypes is linked to phenotypic variation. The level of pathogenic mtDNA mutations determines the pathogenicity and severity of symptoms. (b) Mapping of animal mtDNA can be achieved by mixing two mtDNA genotypes with defined phenotypic differences to generate heteroplasmic individuals. Homoplasmic recombinant genomes are then isolated based on spontaneous or induced recombination. Subsequently, individuals carrying different recombinant genomes are assayed for a given phenotype to define trait-associated SNP(s). Current challenges holding back our mapping capacity include the low rate of recombination in animal mitochondria and lack of a system to isolate and select individuals that are homoplasmic for only one type of recombinant genome. (c) Expression of mito-nucleases in heteroplasmic lines can be used to induce and isolate organisms that are homoplasmic for a certain recombinant mtDNA. Expression of chosen mito-nucleases (e.g. mitoRE, mitoTALEN and mitoZFN) introduces double-strand break(s) at different positions of the two parental genomes. The break in each genome will be repaired based on the homologous sequence in the other genome, resulting in the generation of recombinant genomes lacking recognition sites of the targeted nucleases. The mito-nucleases also select against the parental genomes to allow the recombinant mtDNA to take over. The black stop symbol indicates the lack of a recognition site for the expressed mito-nucleases. (d) Multiple challenges remain in order to transform animal mtDNA, including the delivery of external DNA into mitochondria, the low frequency of recombination and the inability to select for transformed genomes. The latter two challenges may be overcome by expression of mito-nucleases, which induces recombination to promote the incorporation of the desired modification(s) presented on the donor template, and selects against the parental genomes to allow the takeover by the transformant.