Rxivist logo

Engineering targeted deletions in the mitochondrial genome

By Jarryd M Campbell, Ester Perales-Clemente, Hirotaka Ata, Noemi Vidal-Folch, Weibin Liu, Karl Clark, Xiaolei Xu, Devin Oglesbee, Timothy J. Nelson, Stephen C. Ekker

Posted 22 Mar 2018
bioRxiv DOI: 10.1101/287342

Mitochondria are a network of critical intracellular organelles with diverse functions ranging from energy production to cell signaling. The mitochondrial genome (mtDNA) consists of 37 genes that support oxidative phosphorylation and are prone to dysfunction that can lead to currently untreatable diseases. Further characterization of mtDNA gene function and creation of more accurate models of human disease will require the ability to engineer precise genomic sequence modifications. To date, mtDNA has been inaccessible to direct modification using traditional genome engineering tools due to unique DNA repair contexts in mitochondria. Here, we report a new DNA modification process using sequence-specific transcription activator-like effector (TALE) proteins to manipulate mtDNA in vivo and in vitro for reverse genetics applications. First, we show mtDNA deletions can be induced in Danio rerio (zebrafish) using site-directed mitoTALE-nickases (mito-nickases). Using this approach, the protein-encoding mtDNA gene nd4 was deleted in injected zebrafish embryos. Furthermore, this DNA engineering system recreated a large deletion spanning from nd5 to atp8, which is commonly found in human diseases like Kearns-Sayre syndrome (KSS) and Pearson syndrome. Enrichment of mtDNA-deleted genomes was achieved using targeted mitoTALE-nucleases (mitoTALENs) by co-delivering both mito-nickases and mitoTALENs into zebrafish embryos. This combined approach yielded deletions in over 90% of injected animals, which were maintained through adulthood in various tissues. Subsequently, we confirmed that large, targeted deletions could be induced with this approach in human cells. In addition, we show that, when provided with a single nick on the mtDNA light strand, the binding of a terminal TALE protein alone at the intended recombination site is sufficient for deletion induction. This 'block and nick' approach yielded engineered mitochondrial molecules with single nucleotide precision using two different targeted deletion sites. This precise seeding method to engineer mtDNA variants is a critical step for the exploration of mtDNA function and for creating new cellular and animal models of mitochondrial disease.

Download data

  • Downloaded 1,752 times
  • Download rankings, all-time:
    • Site-wide: 12,121
    • In molecular biology: 321
  • Year to date:
    • Site-wide: 47,991
  • Since beginning of last month:
    • Site-wide: 74,355

Altmetric data

Downloads over time

Distribution of downloads per paper, site-wide