Skip to main content

Thank you for visiting nature.com. 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.

  • Review Article
  • Published:

The potential of mitochondrial genome engineering

Nature Reviews Genetics volume 23, pages 199–214 (2022)Cite this article

Subjects

Abstract

Mitochondria are subject to unique genetic control by both nuclear DNA and their own genome, mitochondrial DNA (mtDNA), of which each mitochondrion contains multiple copies. In humans, mutations in mtDNA can lead to devastating, heritable, multi-system diseases that display different tissue-specific presentation at any stage of life. Despite rapid advances in nuclear genome engineering, for years, mammalian mtDNA has remained resistant to genetic manipulation, hampering our ability to understand the mechanisms that underpin mitochondrial disease. Recent developments in the genetic modification of mammalian mtDNA raise the possibility of using genome editing technologies, such as programmable nucleases and base editors, for the treatment of hereditary mitochondrial disease.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Fig. 1: The mammalian mitochondrial genome.
Fig. 2: Milestone timeline in genetic engineering of the human mitochondrial genome.
Fig. 3: Therapeutic strategies for mitochondrial disease.
Fig. 4: Overview of mitochondrial genome-engineering tools.
Fig. 5: Current applications of mtDNA engineering technologies.
Fig. 6: mtDNA mutations and models successfully corrected with mitochondrial nuclease technologies.

Similar content being viewed by others

References

  1. Anderson, S. et al. Sequence and organization of the human mitochondrial genome. Nature 290, 457–465 (1981).

    CAS  PubMed  Google Scholar 

  2. Pagliarini, D. J. et al. A mitochondrial protein compendium elucidates complex I disease biology. Cell 134, 112–123 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Rath, S. et al. MitoCarta3.0: an updated mitochondrial proteome now with sub-organelle localization and pathway annotations. Nucleic Acids Res. 49, D1541–D1547 (2021).

    CAS  PubMed  Google Scholar 

  4. Pfanner, N., Warscheid, B. & Wiedemann, N. Mitochondrial proteins: from biogenesis to functional networks. Nat. Rev. Mol. Cell Biol. 20, 267–284 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Lake, N. J., Compton, A. G., Rahman, S. & Thorburn, D. R. Leigh syndrome: one disorder, more than 75 monogenic causes. Ann. Neurol. 79, 190–203 (2016).

    PubMed  Google Scholar 

  8. McFarland, R., Taylor, R. W. & Turnbull, D. M. A neurological perspective on mitochondrial disease. Lancet Neurol. 9, 829–840 (2010).

    CAS  PubMed  Google Scholar 

  9. Yonova-Doing, E. et al. An atlas of mitochondrial DNA genotype-phenotype associations in the UK Biobank. Nat. Genet. 53, 982–993 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Gorelick, A. N. et al. Respiratory complex and tissue lineage drive recurrent mutations in tumour mtDNA. Nat. Metab. 3, 558–570 (2021).

    CAS  PubMed  Google Scholar 

  11. Kauppila, T. E. S., Kauppila, J. H. K. & Larsson, N. G. Mammalian mitochondria and aging: an update. Cell Metab. 25, 57–71 (2017).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Stewart, J. B. Current progress with mammalian models of mitochondrial DNA disease. J. Inherit. Metab. Dis. 44, 325–342 (2021).

    CAS  PubMed  Google Scholar 

  14. Herbert, M. & Turnbull, D. Progress in mitochondrial replacement therapies. Nat. Rev. Mol. Cell Biol. 19, 71–72 (2018).

    CAS  PubMed  Google Scholar 

  15. Jackson, C. B., Turnbull, D. M., Minczuk, M. & Gammage, P. A. Therapeutic manipulation of mtDNA heteroplasmy: a shifting perspective. Trends Mol. Med. 26, 698–709 (2020).

    CAS  PubMed  Google Scholar 

  16. Magnusson, J., Orth, M., Lestienne, P. & Taanman, J. W. Replication of mitochondrial DNA occurs throughout the mitochondria of cultured human cells. Exp. Cell Res. 289, 133–142 (2003).

    CAS  PubMed  Google Scholar 

  17. Gustafsson, C. M., Falkenberg, M. & Larsson, N. G. Maintenance and expression of mammalian mitochondrial DNA. Annu. Rev. Biochem. 85, 133–160 (2016).

    CAS  PubMed  Google Scholar 

  18. Filograna, R., Mennuni, M., Alsina, D. & Larsson, N. G. Mitochondrial DNA copy number in human disease: the more the better? FEBS Lett. 595, 976–1002 (2021).

    CAS  PubMed  Google Scholar 

  19. Brown, T. A. & Clayton, D. A. Release of replication termination controls mitochondrial DNA copy number after depletion with 2’,3’-dideoxycytidine. Nucleic Acids Res. 30, 2004–2010 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Kazak, L., Reyes, A. & Holt, I. J. Minimizing the damage: repair pathways keep mitochondrial DNA intact. Nat. Rev. Mol. Cell Biol. 13, 659–671 (2012).

    CAS  PubMed  Google Scholar 

  21. Clayton, D. A., Doda, J. N. & Friedberg, E. C. The absence of a pyrimidine dimer repair mechanism in mammalian mitochondria. Proc. Natl Acad. Sci. USA 71, 2777–2781 (1974).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Prakash, A. & Doublie, S. Base excision repair in the mitochondria. J. Cell Biochem. 116, 1490–1499 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

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

    PubMed  PubMed Central  Google Scholar 

  24. Nissanka, N., Bacman, S. R., Plastini, M. J. & Moraes, C. T. The mitochondrial DNA polymerase gamma degrades linear DNA fragments precluding the formation of deletions. Nat. Commun. 9, 2491 (2018).

    PubMed  PubMed Central  Google Scholar 

  25. Hagstrom, E., Freyer, C., Battersby, B. J., Stewart, J. B. & Larsson, N. G. No recombination of mtDNA after heteroplasmy for 50 generations in the mouse maternal germline. Nucleic Acids Res. 42, 1111–1116 (2014).

    PubMed  Google Scholar 

  26. Bacman, S. R., Williams, S. L. & Moraes, C. T. Intra- and inter-molecular recombination of mitochondrial DNA after in vivo induction of multiple double-strand breaks. Nucleic Acids Res. 37, 4218–4226 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Kennedy, S. R., Salk, J. J., Schmitt, M. W. & Loeb, L. A. Ultra-sensitive sequencing reveals an age-related increase in somatic mitochondrial mutations that are inconsistent with oxidative damage. PLoS Genet. 9, e1003794 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Kauppila, J. H. K. et al. Base-excision repair deficiency alone or combined with increased oxidative stress does not increase mtDNA point mutations in mice. Nucleic Acids Res. 46, 6642–6669 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Lynch, M. The lower bound to the evolution of mutation rates. Genome Biol. Evol. 3, 1107–1118 (2011).

    PubMed  PubMed Central  Google Scholar 

  30. Stewart, J. B. & Chinnery, P. F. Extreme heterogeneity of human mitochondrial DNA from organelles to populations. Nat. Rev. Genet. 22, 106–118 (2021).

    CAS  PubMed  Google Scholar 

  31. Thorburn, D. R. Mitochondrial disorders: prevalence, myths and advances. J. Inherit. Metab. Dis. 27, 349–362 (2004).

    CAS  PubMed  Google Scholar 

  32. Lebon, S. et al. Recurrent de novo mitochondrial DNA mutations in respiratory chain deficiency. J. Med. Genet. 40, 896–899 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Zhang, H., Burr, S. P. & Chinnery, P. F. The mitochondrial DNA genetic bottleneck: inheritance and beyond. Essays Biochem. 62, 225–234 (2018).

    PubMed  Google Scholar 

  34. Tatuch, Y. et al. Heteroplasmic mtDNA mutation (T–G) at 8993 can cause Leigh disease when the percentage of abnormal mtDNA is high. Am. J. Hum. Genet. 50, 852–858 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Pickett, S. J. et al. Phenotypic heterogeneity in m.3243A>G mitochondrial disease: the role of nuclear factors. Ann. Clin. Transl. Neurol. 5, 333–345 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Bianco, A. et al. Leber’s hereditary optic neuropathy (LHON) in an Apulian cohort of subjects. Acta Myol. 36, 163–177 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Kirkman, M. A. et al. Gene-environment interactions in Leber hereditary optic neuropathy. Brain 132, 2317–2326 (2009).

    PubMed  PubMed Central  Google Scholar 

  38. Matthews, L. et al. MRI in Leber’s hereditary optic neuropathy: the relationship to multiple sclerosis. J. Neurol. Neurosurg. Psychiatry 86, 537–542 (2015).

    PubMed  Google Scholar 

  39. Giordano, C. et al. Efficient mitochondrial biogenesis drives incomplete penetrance in Leber’s hereditary optic neuropathy. Brain 137, 335–353 (2014).

    PubMed  Google Scholar 

  40. Bianco, A. et al. Mitochondrial DNA copy number differentiates the Leber’s hereditary optic neuropathy affected individuals from the unaffected mutation carriers. Brain 139, e1 (2016).

    PubMed  Google Scholar 

  41. Wei, W. et al. Germline selection shapes human mitochondrial DNA diversity. Science 364, eaau6520 (2019).

    CAS  PubMed  Google Scholar 

  42. Johnston, S. A., Anziano, P. Q., Shark, K., Sanford, J. C. & Butow, R. A. Mitochondrial transformation in yeast by bombardment with microprojectiles. Science 240, 1538–1541 (1988).

    CAS  PubMed  Google Scholar 

  43. Remacle, C., Cardol, P., Coosemans, N., Gaisne, M. & Bonnefoy, N. High-efficiency biolistic transformation of Chlamydomonas mitochondria can be used to insert mutations in complex I genes. Proc. Natl Acad. Sci. USA 103, 4771–4776 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Bonnefoy, N. & Fox, T. D. Directed alteration of Saccharomyces cerevisiae mitochondrial DNA by biolistic transformation and homologous recombination. Methods Mol. Biol. 372, 153–166 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. D’Souza, G. G., Rammohan, R., Cheng, S. M., Torchilin, V. P. & Weissig, V. DQAsome-mediated delivery of plasmid DNA toward mitochondria in living cells. J. Control. Rel. 92, 189–197 (2003).

    Google Scholar 

  46. Yamada, Y. & Harashima, H. Delivery of bioactive molecules to the mitochondrial genome using a membrane-fusing, liposome-based carrier, DF-MITO-Porter. Biomaterials 33, 1589–1595 (2012).

    CAS  PubMed  Google Scholar 

  47. Yu, H. et al. Gene delivery to mitochondria by targeting modified adenoassociated virus suppresses Leber’s hereditary optic neuropathy in a mouse model. Proc. Natl Acad. Sci. USA 109, E1238–E1247 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Bock, F. J. & Tait, S. W. G. Mitochondria as multifaceted regulators of cell death. Nat. Rev. Mol. Cell Biol. 21, 85–100 (2020).

    CAS  PubMed  Google Scholar 

  49. King, M. P. & Attardi, G. Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. Science 246, 500–503 (1989).

    CAS  PubMed  Google Scholar 

  50. Jenuth, J. P., Peterson, A. C., Fu, K. & Shoubridge, E. A. Random genetic drift in the female germline explains the rapid segregation of mammalian mitochondrial DNA. Nat. Genet. 14, 146–151 (1996).

    CAS  PubMed  Google Scholar 

  51. Inoue, K. et al. Generation of mice with mitochondrial dysfunction by introducing mouse mtDNA carrying a deletion into zygotes. Nat. Genet. 26, 176–181 (2000).

    CAS  PubMed  Google Scholar 

  52. Marchington, D. R., Barlow, D. & Poulton, J. Transmitochondrial mice carrying resistance to chloramphenicol on mitochondrial DNA: developing the first mouse model of mitochondrial DNA disease. Nat. Med. 5, 957–960 (1999).

    CAS  PubMed  Google Scholar 

  53. Kasahara, A. et al. Generation of trans-mitochondrial mice carrying homoplasmic mtDNAs with a missense mutation in a structural gene using ES cells. Hum. Mol. Genet. 15, 871–881 (2006).

    CAS  PubMed  Google Scholar 

  54. Fan, W. et al. A mouse model of mitochondrial disease reveals germline selection against severe mtDNA mutations. Science 319, 958–962 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Hyslop, L. A. et al. Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease. Nature 534, 383–386 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Kang, E. et al. Mitochondrial replacement in human oocytes carrying pathogenic mitochondrial DNA mutations. Nature 540, 270–275 (2016).

    CAS  PubMed  Google Scholar 

  57. King, M. P. & Attardi, G. Injection of mitochondria into human cells leads to a rapid replacement of the endogenous mitochondrial DNA. Cell 52, 811–819 (1988).

    CAS  PubMed  Google Scholar 

  58. Shitara, H. et al. Selective and continuous elimination of mitochondria microinjected into mouse eggs from spermatids, but not from liver cells, occurs throughout embryogenesis. Genetics 156, 1277–1284 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Patel, D. et al. Macropinocytic entry of isolated mitochondria in epidermal growth factor-activated human osteosarcoma cells. Sci. Rep. 7, 12886 (2017).

    PubMed  PubMed Central  Google Scholar 

  60. Lightowlers, R. N., Chrzanowska-Lightowlers, Z. M. & Russell, O. M. Mitochondrial transplantation — a possible therapeutic for mitochondrial dysfunction? EMBO Rep. 21, e50964 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT03384420 (2021).

  62. Wu, T. H. et al. Mitochondrial transfer by photothermal nanoblade restores metabolite profile in mammalian cells. Cell. Metab. 23, 921–929 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Patananan, A. N. et al. Pressure-driven mitochondrial transfer pipeline generates mammalian cells of desired genetic combinations and fates. Cell Rep. 33, 108562 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. Patananan, A. N., Wu, T. H., Chiou, P. Y. & Teitell, M. A. Modifying the mitochondrial genome. Cell Metab. 23, 785–796 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Acin-Perez, R. et al. Respiratory complex III is required to maintain complex I in mammalian mitochondria. Mol. Cell 13, 805–815 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Lin, C. S. et al. Mouse mtDNA mutant model of Leber hereditary optic neuropathy. Proc. Natl Acad. Sci. USA 109, 20065–20070 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  67. Valente, W. J. et al. Mitochondrial DNA exhibits resistance to induced point and deletion mutations. Nucleic Acids Res. 44, 8513–8524 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  68. Trifunovic, A. et al. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature 429, 417–423 (2004).

    CAS  PubMed  Google Scholar 

  69. Kujoth, G. C. et al. Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging. Science 309, 481–484 (2005).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  71. McFarland, R. et al. The m.5650G>A mitochondrial tRNAAla mutation is pathogenic and causes a phenotype of pure myopathy. Neuromuscul. Disord. 18, 63–67 (2008).

    PubMed  Google Scholar 

  72. Horvath, R. et al. A tRNA(Ala) mutation causing mitochondrial myopathy clinically resembling myotonic dystrophy. J. Med. Genet. 40, 752–757 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Fayzulin, R. Z. et al. A method for mutagenesis of mouse mtDNA and a resource of mouse mtDNA mutations for modeling human pathological conditions. Nucleic Acids Res. 43, e62 (2015).

    PubMed  PubMed Central  Google Scholar 

  74. Bratic, A. et al. Complementation between polymerase- and exonuclease-deficient mitochondrial DNA polymerase mutants in genomically engineered flies. Nat. Commun. 6, 8808 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  75. Samstag, C. L. et al. Deleterious mitochondrial DNA point mutations are overrepresented in Drosophila expressing a proofreading-defective DNA polymerase gamma. PLoS Genet. 14, e1007805 (2018).

    PubMed  PubMed Central  Google Scholar 

  76. Andreazza, S. et al. Mitochondrially-targeted APOBEC1 is a potent mtDNA mutator affecting mitochondrial function and organismal fitness in Drosophila. Nat. Commun. 10, 3280 (2019).

    PubMed  PubMed Central  Google Scholar 

  77. Srivastava, S. & Moraes, C. T. Manipulating mitochondrial DNA heteroplasmy by a mitochondrially targeted restriction endonuclease. Hum. Mol. Genet. 10, 3093–3099 (2001).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Bayona-Bafaluy, M. P., Blits, B., Battersby, B. J., Shoubridge, E. A. & Moraes, C. T. Rapid directional shift of mitochondrial DNA heteroplasmy in animal tissues by a mitochondrially targeted restriction endonuclease. Proc. Natl Acad. Sci. USA 102, 14392–14397 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Bacman, S. R., Williams, S. L., Duan, D. & Moraes, C. T. Manipulation of mtDNA heteroplasmy in all striated muscles of newborn mice by AAV9-mediated delivery of a mitochondria-targeted restriction endonuclease. Gene Ther. 19, 1101–1106 (2012).

    CAS  PubMed  Google Scholar 

  81. Tanaka, M. et al. Gene therapy for mitochondrial disease by delivering restriction endonuclease SmaI into mitochondria. J. Biomed. Sci. 9, 534–541 (2002).

    CAS  PubMed  Google Scholar 

  82. Alexeyev, M. F. et al. Selective elimination of mutant mitochondrial genomes as therapeutic strategy for the treatment of NARP and MILS syndromes. Gene Ther. 15, 516–523 (2008).

    CAS  PubMed  Google Scholar 

  83. Xu, H., DeLuca, S. Z. & O’Farrell, P. H. Manipulating the metazoan mitochondrial genome with targeted restriction enzymes. Science 321, 575–577 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. Kandul, N. P., Zhang, T., Hay, B. A. & Guo, M. Selective removal of deletion-bearing mitochondrial DNA in heteroplasmic Drosophila. Nat. Commun. 7, 13100 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  85. Kim, Y. G., Cha, J. & Chandrasegaran, S. Hybrid restriction enzymes: zinc finger fusions to FokI cleavage domain. Proc. Natl Acad. Sci. USA 93, 1156–1160 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Urnov, F. D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646–651 (2005).

    CAS  PubMed  Google Scholar 

  87. Christian, M. et al. Targeting DNA double-strand breaks with TAL effector nucleases. Genetics 186, 757–761 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  88. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  89. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  90. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  91. Gaude, E. et al. NADH shuttling couples cytosolic reductive carboxylation of glutamine with glycolysis in cells with mitochondrial dysfunction. Mol. Cell 69, 581–593 e587 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  92. Hashimoto, M. et al. MitoTALEN: a general approach to reduce mutant mtDNA loads and restore oxidative phosphorylation function in mitochondrial diseases. Mol. Ther. 23, 1592–1599 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  93. Yang, Y. et al. Targeted elimination of mutant mitochondrial DNA in MELAS-iPSCs by mitoTALENs. Protein Cell 9, 283–297 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  94. Yahata, N., Boda, H. & Hata, R. Elimination of mutant mtDNA by an optimized mpTALEN restores differentiation capacities of heteroplasmic MELAS-iPSCs. Mol. Ther. Methods Clin. Dev. 20, 54–68 (2021).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  96. Bacman, S. R. et al. MitoTALEN reduces mutant mtDNA load and restores tRNA(Ala) levels in a mouse model of heteroplasmic mtDNA mutation. Nat. Med. 24, 1696–1700 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  97. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  98. Pereira, C. V. et al. mitoTev-TALE: a monomeric DNA editing enzyme to reduce mutant mitochondrial DNA levels. EMBO Mol. Med. 10, e8084 (2018).

    PubMed  PubMed Central  Google Scholar 

  99. Zekonyte, U. et al. Mitochondrial targeted meganuclease as a platform to eliminate mutant mtDNA in vivo. Nat. Commun. 12, 3210 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  100. Phillips, A. F. et al. Single-molecule analysis of mtDNA replication uncovers the basis of the common deletion. Mol. Cell 65, 527–538 e526 (2017).

    CAS  PubMed  Google Scholar 

  101. Persson, O. et al. Copy-choice recombination during mitochondrial L-strand synthesis causes DNA deletions. Nat. Commun. 10, 759 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  102. Mok, B. Y. et al. A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing. Nature 583, 631–637 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  103. Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  104. Nilsen, H. et al. Nuclear and mitochondrial uracil-DNA glycosylases are generated by alternative splicing and transcription from different positions in the UNG gene. Nucleic Acids Res. 25, 750–755 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  105. Lee, H. et al. Mitochondrial DNA editing in mice with DddA-TALE fusion deaminases. Nat. Commun. 12, 1190 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  106. Guo, J. et al. Precision modeling of mitochondrial diseases in zebrafish via DdCBE-mediated mtDNA base editing. Cell Discov. 7, 78 (2021).

    PubMed  PubMed Central  Google Scholar 

  107. Sabharwal, A. et al. The FusX TALE Base Editor (FusXTBE) for rapid mitochondrial DNA programming of human cells in vitro and zebrafish disease models in vivo. Preprint at bioRxiv https://doi.org/10.1101/2021.05.18.444740 (2021).

    Article  Google Scholar 

  108. Russell, O. M., Gorman, G. S., Lightowlers, R. N. & Turnbull, D. M. Mitochondrial diseases: hope for the future. Cell 181, 168–188 (2020).

    CAS  PubMed  Google Scholar 

  109. Yu-Wai-Man, P. et al. Bilateral visual improvement with unilateral gene therapy injection for Leber hereditary optic neuropathy. Sci. Transl Med. 12, eaaz7423 (2020).

    CAS  PubMed  Google Scholar 

  110. Antunes, M. S., Smith, J. J., Jantz, D. & Medford, J. I. Targeted DNA excision in Arabidopsis by a re-engineered homing endonuclease. BMC Biotechnol. 12, 86 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  111. Ratnaike, T. E. et al. MitoPhen database: a human phenotype ontology-based approach to identify mitochondrial DNA diseases. Nucleic Acids Res. 49, 9686–9695 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  112. Gaudelli, N. M. et al. Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  113. US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT03872479 (2021).

  114. Ng, Y. S. et al. Mitochondrial disease in adults: recent advances and future promise. Lancet Neurol. 20, 573–584 (2021).

    CAS  PubMed  Google Scholar 

  115. Wang, D., Tai, P. W. L. & Gao, G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat. Rev. Drug Discov. 18, 358–378 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  116. Minczuk, M. & Gammage, P. A. Methods of optimising expression and delivery of mitochondrial proteins. Patent PCT/GB2019/05080 (2020).

  117. Electronic Medicines Compendium. Zolgensma 2 x 10Exp13 vector genomes/mL solution for infusion. emc https://www.medicines.org.uk/emc/product/11572 (2021).

  118. US National Library of Medicine. ClinicalTrials.gov https://clinicaltrials.gov/ct2/show/NCT02702115 (2021).

  119. Kang, B. C. et al. Chloroplast and mitochondrial DNA editing in plants. Nat. Plants 7, 899–905 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  120. Nakazato, I. et al. Targeted base editing in the plastid genome of Arabidopsis thaliana. Nat. Plants 7, 906–913 (2021).

    CAS  PubMed  PubMed Central  Google Scholar 

  121. 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).

    CAS  PubMed  Google Scholar 

  122. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  123. Nicholls, T. J. & Minczuk, M. In D-loop: 40 years of mitochondrial 7S DNA. Exp. Gerontol. 56, 175–181 (2014).

    CAS  PubMed  Google Scholar 

  124. Wei, W. et al. Nuclear-mitochondrial DNA segments resemble paternally inherited mitochondrial DNA in humans. Nat. Commun. 11, 1740 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  125. Luo, S. et al. Biparental inheritance of mitochondrial DNA in humans. Proc. Natl Acad. Sci. USA 115, 13039–13044 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  126. Pagnamenta, A. T., Wei, W., Rahman, S. & Chinnery, P. F. Biparental inheritance of mitochondrial DNA revisited. Nat. Rev. Genet. 22, 477–478 (2021).

    CAS  PubMed  Google Scholar 

  127. Arnould, S. et al. The I-CreI meganuclease and its engineered derivatives: applications from cell modification to gene therapy. Protein Eng. Des. Sel. 24, 27–31 (2011).

    CAS  PubMed  Google Scholar 

  128. Jantz, D. & Smith, J. J. Recognition sequences for I-CreI-derived meganucleases and uses thereof. US Patent US-9683257-B2 (2017).

  129. Smith, J. J. & Jantz, D. Rationally-designed meganucleases with altered sequence specificity and DNA-binding affinity. US Patent US-8021867-B2 (2013).

  130. Doyon, Y. et al. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures. Nat. Methods 8, 74–79 (2011).

    CAS  PubMed  Google Scholar 

  131. Gillum, A. M. & Clayton, D. A. Mechanism of mitochondrial DNA replication in mouse L-cells: RNA priming during the initiation of heavy-strand synthesis. J. Mol. Biol. 135, 353–368 (1979).

    CAS  PubMed  Google Scholar 

  132. Robberson, D. L. & Clayton, D. A. Replication of mitochondrial DNA in mouse L cells and their thymidine kinase - derivatives: displacement replication on a covalently-closed circular template. Proc. Natl Acad. Sci. USA 69, 3810–3814 (1972).

    CAS  PubMed  PubMed Central  Google Scholar 

  133. Korhonen, J. A., Gaspari, M. & Falkenberg, M. TWINKLE has 5’ -> 3’ DNA helicase activity and is specifically stimulated by mitochondrial single-stranded DNA-binding protein. J. Biol. Chem. 278, 48627–48632 (2003).

    CAS  PubMed  Google Scholar 

  134. Miralles Fuste, J. et al. In vivo occupancy of mitochondrial single-stranded DNA binding protein supports the strand displacement mode of DNA replication. PLoS Genet. 10, e1004832 (2014).

    PubMed  PubMed Central  Google Scholar 

  135. Fuste, J. M. et al. Mitochondrial RNA polymerase is needed for activation of the origin of light-strand DNA replication. Mol. Cell 37, 67–78 (2010).

    CAS  PubMed  Google Scholar 

  136. Yasukawa, T. et al. Replication of vertebrate mitochondrial DNA entails transient ribonucleotide incorporation throughout the lagging strand. EMBO J. 25, 5358–5371 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  137. Holt, I. J., Lorimer, H. E. & Jacobs, H. T. Coupled leading- and lagging-strand synthesis of mammalian mitochondrial DNA. Cell 100, 515–524 (2000).

    CAS  PubMed  Google Scholar 

  138. Falkenberg, M. & Gustafsson, C. M. Mammalian mitochondrial DNA replication and mechanisms of deletion formation. Crit. Rev. Biochem. Mol. Biol. 55, 509–524 (2020).

    CAS  PubMed  Google Scholar 

  139. Taylor, R. W., Chinnery, P. F., Turnbull, D. M. & Lightowlers, R. N. Selective inhibition of mutant human mitochondrial DNA replication in vitro by peptide nucleic acids. Nat. Genet. 15, 212–215 (1997).

    CAS  PubMed  Google Scholar 

  140. Loutre, R., Heckel, A. M., Jeandard, D., Tarassov, I. & Entelis, N. Anti-replicative recombinant 5S rRNA molecules can modulate the mtDNA heteroplasmy in a glucose-dependent manner. PLoS ONE 13, e0199258 (2018).

    PubMed  PubMed Central  Google Scholar 

  141. Naeem, M. M. et al. G-quadruplex-mediated reduction of a pathogenic mitochondrial heteroplasmy. Hum. Mol. Genet. 28, 3163–3174 (2019).

    CAS  PubMed  Google Scholar 

  142. Manfredi, G. et al. Oligomycin induces a decrease in the cellular content of a pathogenic mutation in the human mitochondrial ATPase 6 gene. J. Biol. Chem. 274, 9386–9391 (1999).

    CAS  PubMed  Google Scholar 

  143. Sembongi, H., Di Re, M., Bokori-Brown, M. & Holt, I. J. The yeast Holliday junction resolvase, CCE1, can restore wild-type mitochondrial DNA to human cells carrying rearranged mitochondrial DNA. Hum. Mol. Genet. 16, 2306–2314 (2007).

    CAS  PubMed  Google Scholar 

  144. Suen, D. F., Narendra, D. P., Tanaka, A., Manfredi, G. & Youle, R. J. Parkin overexpression selects against a deleterious mtDNA mutation in heteroplasmic cybrid cells. Proc. Natl Acad. Sci. USA 107, 11835–11840 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  145. Clark, K. M. et al. Reversal of a mitochondrial DNA defect in human skeletal muscle. Nat. Genet. 16, 222–224 (1997).

    CAS  PubMed  Google Scholar 

  146. Filograna, R. et al. Modulation of mtDNA copy number ameliorates the pathological consequences of a heteroplasmic mtDNA mutation in the mouse. Sci. Adv. 5, eaav9824 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  147. Sacconi, S. et al. A functionally dominant mitochondrial DNA mutation. Hum. Mol. Genet. 17, 1814–1820 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank the past and present members of the Mitochondrial Genetics Group at the MRC-MBU, University of Cambridge, for stimulating discussions during the course of the work on the development of the mtZFN technology. They are grateful to P. Nash and C. Mutti for their help with the manuscript. The Medical Research Council UK (MC_UU_00015/4) and The Champ Foundation are acknowledged for their support of the authors’ work.

Author information

Authors and Affiliations

  1. MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK

    Pedro Silva-Pinheiro & Michal Minczuk

Authors
  1. Pedro Silva-Pinheiro
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michal Minczuk
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

The authors contributed equally to all aspects of the article.

Corresponding author

Correspondence to Michal Minczuk.

Ethics declarations

Competing interests

P.S.-P. is a consultant for Pretzel Therapeutics. M.M. is a co-founder, Scientific Advisory Board member and shareholder of Pretzel Therapeutics. M.M. is a co-inventor on the following mtZFN-related patents or patent applications: PCT/GB2006/004755, PCT/US2019/023359, PCT/GB2019/050808.

Additional information

Peer review information

Nature Reviews Genetics thanks Stephen C. Ekker, Jin-Soo Kim, Robert Lightowlers, David R. Thorburn for their contribution to the peer review of this work.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Glossary

Mitochondrial replacement therapy

A form of reproductive in vitro fertilization technique, based upon replacing a mother’s mitochondria that harbour pathogenic mutant mitochondrial DNA with the donor’s healthy mitochondria.

Germline bottleneck

The lowering of mitochondrial DNA (mtDNA) copy number in primordial germ cells followed by rapid replication of this mtDNA population during oocyte maturation, an event that can lead to a random shift of mtDNA mutation load between generations and be responsible for the variable levels of mutant mtDNA in affected offspring from mothers carrying pathogenic mtDNA mutations.

Mutation load

The total genetic burden in a population, for example, within all mitochondrial DNA molecules in the cell, resulting from accumulated deleterious mutations.

Episome

A genetic element inside a cell, for example, vector DNA, that persists independently of the host DNA.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Silva-Pinheiro, P., Minczuk, M. The potential of mitochondrial genome engineering. Nat Rev Genet 23, 199–214 (2022). https://doi.org/10.1038/s41576-021-00432-x

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41576-021-00432-x

This article is cited by

Search

Advanced search

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