Engineering targeted deletions in the mitochondrial genome
| Autor: | Jarryd M. Campbell, Weibin Liu, Noemi Vidal-Folch, Karl J. Clark, Xiaolei Xu, Stephen C. Ekker, Timothy J. Nelson, Devin Oglesbee, Hirotaka Ata, Ester Perales-Clemente |
|---|---|
| Rok vydání: | 2018 |
| Předmět: |
0303 health sciences
Mitochondrial DNA biology DNA repair Mitochondrial disease Computational biology biology.organism_classification medicine.disease Genome Genome engineering 03 medical and health sciences 0302 clinical medicine Heavy strand 030220 oncology & carcinogenesis medicine Gene Zebrafish 030304 developmental biology |
| Popis: | Summary ParagraphMitochondria 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 mitochondria1. 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|