Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Genome editing in mitochondria corrects a pathogenic mtDNA mutation in vivo


Mutations of the mitochondrial genome (mtDNA) underlie a substantial portion of mitochondrial disease burden. These disorders are currently incurable and effectively untreatable, with heterogeneous penetrance, presentation and prognosis. To address the lack of effective treatment for these disorders, we exploited a recently developed mouse model that recapitulates common molecular features of heteroplasmic mtDNA disease in cardiac tissue: the m.5024C>T tRNAAla mouse. Through application of a programmable nuclease therapy approach, using systemically administered, mitochondrially targeted zinc-finger nucleases (mtZFN) delivered by adeno-associated virus, we induced specific elimination of mutant mtDNA across the heart, coupled to a reversion of molecular and biochemical phenotypes. These findings constitute proof of principle that mtDNA heteroplasmy correction using programmable nucleases could provide a therapeutic route for heteroplasmic mitochondrial diseases of diverse genetic origin.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Strategy to eliminate m.5024C>T and in vivo mtDNA heteroplasmy modification.
Fig. 2: Reduction of m.5024C>T mtDNA heteroplasmy results in phenotype rescue.

Data availability

All next-generation sequencing data generated in the present study are available from the BioProject database using accession PRJNA479953. All other datasets and materials are available from the corresponding authors upon reasonable request.


  1. Gorman, G. S. et al. Prevalence of nuclear and mitochondrial DNA mutations related to adult mitochondrial disease. Ann. Neurol. 77, 753–759 (2015).

    Article  CAS  Google Scholar 

  2. Wachsmuth, M., Hubner, A., Li, M., Madea, B. & Stoneking, M. Age-related and heteroplasmy-related variation in human mtDNA copy number. PLoS Genet. 12, e1005939 (2016).

    Article  Google Scholar 

  3. Gorman, G. S. et al. Mitochondrial diseases. Nat. Rev. Dis. Primers 2, 16080 (2016).

    Article  Google Scholar 

  4. Bacman, S. R., Williams, S. L., Pinto, M., Peralta, S. & Moraes, C. T. Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs. Nat. Med. 19, 1111–1113 (2013).

    Article  CAS  Google Scholar 

  5. Gammage, P. A., Rorbach, J., Vincent, A. I., Rebar, E. J. & Minczuk, M. Mitochondrially targeted ZFNs for selective degradation of pathogenic mitochondrial genomes bearing large-scale deletions or point mutations. EMBO Mol. Med. 6, 458–466 (2014).

    Article  CAS  Google Scholar 

  6. Reddy, P. et al. Selective elimination of mitochondrial mutations in the germline by genome editing. Cell 161, 459–469 (2015).

    Article  CAS  Google Scholar 

  7. Gammage, P. A., Moraes, C. T. & Minczuk, M. Mitochondrial genome engineering: the revolution may not be CRISPR-Ized. Trends Genet. 34, 101–110 (2018).

    Article  CAS  Google Scholar 

  8. Alexeyev, M., Shokolenko, I., Wilson, G. & LeDoux, S. The maintenance of mitochondrial DNA integrity—critical analysis and update. Cold Spring Harb. Perspect. Biol. 5, a012641 (2013).

    Article  Google Scholar 

  9. Peeva, V. et al. Linear mitochondrial DNA is rapidly degraded by components of the replication machinery. Nat. Commun. 9, 1727 (2018).

    Article  Google Scholar 

  10. Minczuk, M., Papworth, M. A., Kolasinska, P., Murphy, M. P. & Klug, A. Sequence-specific modification of mitochondrial DNA using a chimeric zinc finger methylase. Proc. Natl Acad. Sci. USA 103, 19689–19694 (2006).

    Article  CAS  Google Scholar 

  11. Minczuk, M., Kolasinska-Zwierz, P., Murphy, M. P. & Papworth, M. A. Construction and testing of engineered zinc-finger proteins for sequence-specific modification of mtDNA. Nat. Protoc. 5, 342–356 (2010).

    Article  CAS  Google Scholar 

  12. Minczuk, M., Papworth, M. A., Miller, J. C., Murphy, M. P. & Klug, A. Development of a single-chain, quasi-dimeric zinc-finger nuclease for the selective degradation of mutated human mitochondrial DNA. Nucleic Acids Res. 36, 3926–3938 (2008).

    Article  CAS  Google Scholar 

  13. Gammage, P. A. et al. Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs. Nucleic Acids Res. 44, 7804–7816 (2016).

    Article  CAS  Google Scholar 

  14. Gaude, E. et al. NADH shuttling couples cytosolic reductive carboxylation of glutamine with glycolysis in cells with mitochondrial dysfunction. Mol. Cell 69, 581–593.e7 (2018).

    Article  CAS  Google Scholar 

  15. Kauppila, J. H. et al. A phenotype-driven approach to generate mouse models with pathogenic mtDNA mutations causing mitochondrial disease. Cell Rep. 16, 2980–2990 (2016).

    Article  CAS  Google Scholar 

  16. Gammage, P. A., Van Haute, L. & Minczuk, M. Engineered mtZFNs for manipulation of human mitochondrial DNA heteroplasmy. Methods Mol. Biol. 1351, 145–162 (2016).

    Article  CAS  Google Scholar 

  17. Pulicherla, N. et al. Engineering liver-detargeted AAV9 vectors for cardiac and musculoskeletal gene transfer. Mol. Ther. 19, 1070–1078 (2011).

    Article  CAS  Google Scholar 

  18. Yarham, J. W., Elson, J. L., Blakely, E. L., McFarland, R. & Taylor, R. W. Mitochondrial tRNA mutations and disease. Wiley Interdiscip. Rev. RNA 1, 304–324 (2010).

    Article  CAS  Google Scholar 

  19. Jazayeri, M. et al. Inducible expression of a dominant negative DNA polymerase-γ depletes mitochondrial DNA and produces a rho0 phenotype. J. Biol. Chem. 278, 9823–9830 (2003).

    Article  CAS  Google Scholar 

  20. Birsoy, K. et al. An essential role of the mitochondrial electron transport chain in cell proliferation is to enable aspartate synthesis. Cell 162, 540–551 (2015).

    Article  CAS  Google Scholar 

  21. Holt, I. J., Harding, A. E. & Morgan-Hughes, J. A. Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature 331, 717–719 (1988).

    Article  CAS  Google Scholar 

  22. Wallace, D. C. et al. Mitochondrial DNA mutation associated with Leber’s hereditary optic neuropathy. Science 242, 1427–1430 (1988).

    Article  CAS  Google Scholar 

  23. Craven, L. et al. Pronuclear transfer in human embryos to prevent transmission of mitochondrial DNA disease. Nature 465, 82–85 (2010).

    Article  CAS  Google Scholar 

  24. Tachibana, M. et al. Towards germline gene therapy of inherited mitochondrial diseases. Nature 493, 627–631 (2013).

    Article  CAS  Google Scholar 

  25. Floros, V. I. et al. Segregation of mitochondrial DNA heteroplasmy through a developmental genetic bottleneck in human embryos. Nat. Cell Biol. 20, 144–151 (2018).

    Article  CAS  Google Scholar 

  26. Yamada, M. et al. Genetic drift can compromise mitochondrial replacement by nuclear transfer in human oocytes. Cell Stem Cell 18, 749–754 (2016).

    Article  CAS  Google Scholar 

  27. Vafai, S. B. & Mootha, V. K. Mitochondrial disorders as windows into an ancient organelle. Nature 491, 374–383 (2012).

    Article  CAS  Google Scholar 

  28. Viscomi, C., Bottani, E. & Zeviani, M. Emerging concepts in the therapy of mitochondrial disease. Biochim. Biophys. Acta 1847, 544–557 (2015).

    Article  CAS  Google Scholar 

  29. Pfeffer, G. et al. New treatments for mitochondrial disease-no time to drop our standards. Nat. Rev. Neurol. 9, 474–481 (2013).

    Article  CAS  Google Scholar 

  30. Bacman, S. R. et al. MitoTALEN reduces mutant mtDNA load and restores tRNAAla levels in a mouse model of heteroplasmic mtDNA mutation. Nat. Med. (2018).

    Article  CAS  Google Scholar 

  31. Beilstein, K., Wittmann, A., Grez, M. & Suess, B. Conditional control of mammalian gene expression by tetracycline-dependent hammerhead ribozymes. ACS Synth. Biol. 4, 526–534 (2015).

    Article  CAS  Google Scholar 

  32. Pearce, S. F. et al. Maturation of selected human mitochondrial tRNAs requires deadenylation. eLife 6, e27596 (2017).

  33. Mackay, G. M., Zheng, L., van den Broek, N. J. & Gottlieb, E. Analysis of cell metabolism using LC–MS and isotope tracers. Methods Enzymol. 561, 171–196 (2015).

    Article  CAS  Google Scholar 

Download references


This work was supported by the Medical Research Council (MC_U105697135 and MC_UU_00015/4 to M.M., MC_UU_12022/6 to C.F. and MC_UU_00015/5 to M.Z.), ERC Advanced Grant (FP7-322424 to M.Z.), NRJ-Institut de France (to M.Z.) and the Max Planck Society (to J.B.S.). P.R.-G. was supported by ‘Fundação para a Ciência e a Tecnologia’ (PD/BD/105750/2014). We acknowledge the important contribution to model development made by N.-G. Larsson, which was essential to this work. We are grateful to the personnel at Phenomics Animal Care Facility, Cambridge, UK, for their technical support in managing our mouse colonies. We are grateful to M. Rice, Phenomics Animal Care Facility, for technical assistance with viral administration. We thank R. Dirksen (MPI, Cologne, Germany) for isolation and immortalization of the MEFs. All FACS experiments were performed at the NIHR BRC Cell Phenotyping Hub, Cambridge, UK, by C. Bowman, E. Perez, J. Markovic Djuric and A. Petrunkina-Harrison.

Author information

Authors and Affiliations



P.A.G. designed the research, performed biochemical, in vitro and in vivo experiments, analyzed the data and wrote the paper. C.V. performed the in vivo experiments. M.-L.S. contributed to model characterization. A.S.H.C. and E.G. performed the mass spectrometry-based metabolomic experiments and analyzed the data. C.A.P. and L.V.H. performed biochemical experiments and analyzed the data. B.J.M. performed biochemical and immunofluorescence experiments. P.R.-G. and R.C. performed the histological experiments. L.Z. designed and assembled the ZFP library. E.J.R. oversaw the ZFP library preparation. M.Z. oversaw the in vivo experiments. C.F. oversaw the mass spectrometry-based metabolomic experiments. J.B.S. provided cell and mouse models and contributed to model characterization. M.M. oversaw the project and co-wrote the paper, with all authors’ involvement.

Corresponding authors

Correspondence to Payam A. Gammage or Michal Minczuk.

Ethics declarations

Competing interests

E.J.R. and L.Z. are full-time employees of Sangamo Therapeutics.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–9 and Supplementary Tables 1 and 2

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gammage, P.A., Viscomi, C., Simard, ML. et al. Genome editing in mitochondria corrects a pathogenic mtDNA mutation in vivo. Nat Med 24, 1691–1695 (2018).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research