Engineered mtZFNs for Manipulation of Human Mitochondrial DNA Heteroplasmy

  • Payam A. GammageEmail author
  • Lindsey Van Haute
  • Michal MinczukEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1351)


Enrichment of desired mitochondrial DNA (mtDNA) haplotypes, in both experimental systems and the clinic, is an end sought by many. Through use of a designer nuclease platform optimized for delivery to mitochondria—the mitochondrially targeted zinc finger-nuclease (mtZFN)—it is possible to discriminate between mtDNA haplotypes with specificity to the order of a single nucleotide substitution. Site-specific cleavage of DNA produces a shift in the heteroplasmic ratio in favor of the untargeted haplotype. Here, we describe protocols for assembly of paired, conventional tail–tail mtZFN constructs and experimental approaches to assess mtZFN activity in mammalian cell cultures.

Key words

Genetic engineering Zinc finger Zinc finger nuclease Mitochondrial disease Heteroplasmy Gene therapy 


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.MRC Mitochondrial Biology UnitCambridgeUK

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