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.

  • Primer
  • Published:

Mitochondrial gene editing

Abstract

Mutations in mitochondrial DNA (mtDNA) are responsible for several severe diseases that have no available cures. The multicopy nature of the mitochondrial genome means that mutations often exist in a state known as heteroplasmy, where both mutant and wild-type mtDNA are present in the same cell. The wild-type mtDNA can functionally compensate for the mutant mtDNA until a mutation threshold is reached, beyond which disease symptoms begin to manifest. Despite the interest mitochondrial genetics has generated, the double mitochondrial membrane proved to be a formidable barrier to genetic manipulation. However, in the past two decades, scientists have discovered that mtDNA could be modified by importing gene editing proteins to target specific DNA sequences. Mitochondria-targeted nucleases specifically cleave and eliminate mutant mtDNA in heteroplasmic cells and in animal models. More recently, base editors have been adapted to modify mtDNA via precise C>T or A>G transitions. Therefore, tools to modify mtDNA are, finally, a reality with the promise to revolutionize the mitochondrial genetics field. This Primer delves into mitochondrial gene editing, providing details on the selection of mitochondrial gene editing tools, best practices for designing experiments, relevant types of analyses and specific applications and limitations pertaining to the different technologies and the field.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Fig. 1: Key applications for mitochondrial gene editing.
Fig. 2: Various mitochondrial gene editing technologies.
Fig. 3: Design of mitochondrial gene editing experiments and subsequent experimental analyses.
Fig. 4: Applications of the different mitochondrial gene editing technologies.
Fig. 5: Off-target editing as a result of mitochondrial gene editing.

Similar content being viewed by others

References

  1. Miller, F. J., Rosenfeldt, F. L., Zhang, C., Linnane, A. W. & Nagley, P. Precise determination of mitochondrial DNA copy number in human skeletal and cardiac muscle by a PCR-based assay: lack of change of copy number with age. Nucleic Acids Res. 31, e61 (2003).

    Article  Google Scholar 

  2. Chinnery, P. F. & Turnbull, D. M. Mitochondrial DNA and disease. Lancet 354, 17–21 (1999).

    Article  Google Scholar 

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

    Article  ADS  Google Scholar 

  4. Andrews, R. M. et al. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat. Genet. 23, 147 (1999).

    Article  Google Scholar 

  5. Pfanner, N., Warscheid, B. & Wiedemann, N. Mitochondrial protein organization: from biogenesis to networks and function. Nat. Rev. Mol. Cell Biol. 20, 267–284 (2019).

    Article  Google Scholar 

  6. Chinnery, P. F. & Hudson, G. Mitochondrial genetics. Br. Med. Bull. 106, 135–159 (2013).

    Article  Google Scholar 

  7. Chinnery, P. F., Howell, N., Andrews, R. M. & Turnbull, D. M. Clinical mitochondrial genetics. J. Med. Genet. 36, 425–436 (1999).

    Google Scholar 

  8. Schon, E. A., DiMauro, S. & Hirano, M. Human mitochondrial DNA: roles of inherited and somatic mutations. Nat. Rev. Genet. 13, 878–890 (2012).

    Article  Google Scholar 

  9. Mattman, A. et al. Mitochondrial disease clinical manifestations: an overview. BC Med. J. 53, 183–187 (2011).

    Google Scholar 

  10. Taylor, R. W. & Turnbull, D. M. Mitochondrial DNA mutations in human disease. Nat. Rev. Genet. 6, 389–402 (2007).

    Article  Google Scholar 

  11. Sciacco, M., Bonilla, E., Schon, E. A., Dimauro, S. & Moraes, T. Distribution of wild-type and common deletion forms of mtDNA in normal and respiration-deficient muscle fibers from patients with mitochondrial myopathy. Hum. Mol. Genet. 3, 13–19 (1994).

    Article  Google Scholar 

  12. Russell, O. & Turnbull, D. Mitochondrial DNA disease — molecular insights and potential routes to a cure. Exp. Cell Res. 325, 38–43 (2014).

    Article  Google Scholar 

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

    Article  Google Scholar 

  14. 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  Google Scholar 

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

    Article  ADS  Google Scholar 

  16. 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).

    Article  Google Scholar 

  17. 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).

    Article  Google Scholar 

  18. Pereira, C. V. et al. mitoTev‐TALE: a monomeric DNA editing enzyme to reduce mutant mitochondrial DNA levels. EMBO Mol. Med. 10, 1–11 (2018).

    Article  Google Scholar 

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

    Article  Google Scholar 

  20. Yahata, N., Matsumoto, Y., Omi, M., Yamamoto, N. & Hata, R. TALEN-mediated shift of mitochondrial DNA heteroplasmy in MELAS-iPSCs with m.13513 G>A mutation. Sci. Rep. 7, 15557 (2017).

    Article  ADS  Google Scholar 

  21. Alexeyev, M., Shokolenko, I., Wilson, G. & Ledoux, S. The maintenance of mitochondrial DNA integrity — critical analysis and update. Cold Spring Harb. Perspect. Biol. 5, 1–17 (2013).

    Article  Google Scholar 

  22. Peeva, V. et al. Linear mitochondrial DNA is rapidly degraded by components of the replication machinery. Nat. Commun. https://doi.org/10.1038/s41467-018-04131-w (2018).

    Article  Google Scholar 

  23. Moretton, A. et al. Selective mitochondrial DNA degradation following double-strand breaks. PLoS ONE 12, 1–17 (2017).

    Article  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).

    Article  ADS  Google Scholar 

  25. Carling, P. J., Cree, L. M. & Chinnery, P. F. The implications of mitochondrial DNA copy number regulation during embryogenesis. Mitochondrion 11, 686–692 (2011).

    Article  Google Scholar 

  26. Moraes, C. T. What regulates mitochondrial DNA copy number in animal cells? Trends Genet. 17, 199–205 (2001).

    Article  Google Scholar 

  27. 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  Google Scholar 

  28. Yang, X., Jiang, J., Li, Z., Liang, J. & Xiang, Y. Strategies for mitochondrial gene editing. Comput. Struct. Biotechnol. J. 19, 3319–3329 (2021).

    Article  Google Scholar 

  29. Yin, T., Luo, J., Huang, D. & Li, H. Current progress of mitochondrial genome editing by CRISPR. Front. Physiol. 13, 883459 (2022).

    Article  Google Scholar 

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

    Article  Google Scholar 

  31. Mok, Y. G. et al. Base editing in human cells with monomeric DddA-TALE fusion deaminases. Nat. Commun. 13, 4038 (2022).

    Article  ADS  Google Scholar 

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

    Article  ADS  Google Scholar 

  33. Cho, S. I. et al. Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases. Cell 185, 1764–1776.e12 (2022).

    Article  Google Scholar 

  34. Wiedemann, N. & Pfanner, N. Mitochondrial machineries for protein import and assembly. Annu. Rev. Biochem. 86, 685–714 (2017).

    Article  Google Scholar 

  35. Craven, L., Alston, C. L., Taylor, R. W. & Turnbull, D. M. Recent advances in mitochondrial disease. Annu. Rev. Genomics Hum. Genet. 18, 257–275 (2017).

    Article  Google Scholar 

  36. Rossignol, R. et al. Mitochondrial threshold effects. Biochem. J. 370, 751–762 (2003).

    Article  Google Scholar 

  37. Mikhailov, N. & Hämäläinen, R. H. Modulating mitochondrial DNA heteroplasmy with mitochondrially targeted endonucleases. Ann. Biomed. Eng. https://doi.org/10.1007/s10439-022-03051-7 (2022).

    Article  Google Scholar 

  38. Zekonyte, U., Bacman, S. R. & Moraes, C. T. DNA-editing enzymes as potential treatments for heteroplasmic mtDNA diseases. J. Intern. Med. 287, 685–697 (2020).

    Article  Google Scholar 

  39. Barrera-Paez, J. D. & Moraes, C. T. Mitochondrial genome engineering coming-of-age. Trends Genet. https://doi.org/10.1016/j.tig.2022.04.011 (2022).

    Article  Google Scholar 

  40. Bolender, N., Sickmann, A., Wagner, R., Meisinger, C. & Pfanner, N. Multiple pathways for sorting mitochondrial precursor proteins. EMBO Rep. 9, 42–49 (2008).

    Article  Google Scholar 

  41. Galanis, M., Devenish, R. J. & Nagley, P. Duplication of leader sequence for protein targeting to mitochondria leads to increased import efficiency. FEBS Lett. 282, 425–430 (1991).

    Article  Google Scholar 

  42. Chin, R. M., Panavas, T., Brown, J. M. & Johnson, K. K. Optimized mitochondrial targeting of proteins encoded by modified mRNAs rescues cells harboring mutations in mtATP6. Cell Rep. 22, 2818–2826 (2018).

    Article  Google Scholar 

  43. 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  ADS  Google Scholar 

  44. 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  Google Scholar 

  45. Lei, Z. et al. Mitochondrial base editor induces substantial nuclear off-target mutations. Nature 606, 804–811 (2022).

    Article  ADS  Google Scholar 

  46. Jo, A. et al. Efficient mitochondrial genome editing by CRISPR/Cas9. Biomed. Res. Int. https://doi.org/10.1155/2015/305716 (2015).

    Article  Google Scholar 

  47. Hussain, S. R. A., Yalvac, M. E., Khoo, B., Eckardt, S. & McLaughlin, K. J. Adapting CRISPR/Cas9 system for targeting mitochondrial genome. Front. Genet. 12, 627050 (2021).

    Article  Google Scholar 

  48. Loutre, R., Heckel, A. M., Smirnova, A., Entelis, N. & Tarassov, I. Can mitochondrial DNA be CRISPRized: pro and contra. IUBMB Life 70, 1233–1239 (2018).

    Article  Google Scholar 

  49. Wang, B. et al. CRISPR/Cas9-mediated mutagenesis at microhomologous regions of human mitochondrial genome. Sci. China Life Sci. 64, 1463–1472 (2021).

    Article  ADS  Google Scholar 

  50. Jeong, Y. K., Song, B. & Bae, S. Current status and challenges of DNA base editing tools. Mol. Ther. 28, 1938–1952 (2020).

    Article  Google Scholar 

  51. McDougall, W. M., Okany, C. & Smith, H. C. Deaminase activity on single-stranded DNA (ssDNA) occurs in vitro when APOBEC3G cytidine deaminase forms homotetramers and higher-order complexes. J. Biol. Chem. 286, 30655–30661 (2011).

    Article  Google Scholar 

  52. Mok, B. Y. et al. CRISPR-free base editors with enhanced activity and expanded targeting scope in mitochondrial and nuclear DNA. Nat Biotechnol 40, 1378–1387 (2022).

    Article  Google Scholar 

  53. Lee, S. et al. Enhanced mitochondrial DNA editing in mice using nuclear-exported TALE-linked deaminases and nucleases. Genome Biol. 23, 211 (2022).

    Article  Google Scholar 

  54. Lim, K., Cho, S. I. & Kim, J. S. Nuclear and mitochondrial DNA editing in human cells with zinc finger deaminases. Nat. Commun. 13, 366 (2022).

    Article  ADS  Google Scholar 

  55. Willis, J. C. W., Silva-Pinheiro, P., Widdup, L., Minczuk, M. & Liu, D. R. Compact zinc finger base editors that edit mitochondrial or nuclear DNA in vitro and in vivo. Nat. Commun. 13, 1–16 (2022).

    Article  Google Scholar 

  56. Sabharwal et al. The FusX TALE base editor (FusXTBE) for rapid mitochondrial DNA programming of human cells in vitro and zebrafish disease models in vivo. CRISPR J. 4, 799–821 (2021).

    Google Scholar 

  57. Chen, X. et al. DdCBE-mediated mitochondrial base editing in human 3PN embryos. Cell Discov. 8, 8 (2022).

    Article  Google Scholar 

  58. Wei, Y. et al. Human cleaving embryos enable efficient mitochondrial base-editing with DdCBE. Cell Discov. 8, 7 (2022).

    Article  Google Scholar 

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

    Article  ADS  Google Scholar 

  60. Silva-Pinheiro, P. et al. In vivo mitochondrial base editing via adeno-associated viral delivery to mouse post-mitotic tissue. Nat. Commun. 13, 750 (2022).

    Article  ADS  Google Scholar 

  61. Guo, J. et al. DdCBE mediates efficient and inheritable modifications in mouse mitochondrial genome. Mol. Ther. Nucleic Acids 27, 73–80 (2022).

    Article  Google Scholar 

  62. Qi, X. et al. Precision modeling of mitochondrial disease in rats via DdCBE-mediated mtDNA editing. Cell Discov. 7, 95 (2021).

    Article  Google Scholar 

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

    Article  Google Scholar 

  64. Moraes, C. T., Schon, E. A., DiMauro, S. & Miranda, A. F. Heteroplasmy of mitochondrial genomes in clonal cultures from patients with Kearns–Sayre syndrome. Biochem. Biophys. Res. Commun. 160, 765–771 (1989).

    Article  Google Scholar 

  65. Bacman, S. R., Nissanka, N. & Moraes, C. T. Cybrid Technology. Methods in Cell Biology Vol. 155 (Elsevier, 2020).

  66. Wilkins, H. M., Carl, S. M. & Swerdlow, R. H. Cytoplasmic hybrid (cybrid) cell lines as a practical model for mitochondriopathies. Redox Biol. 2, 619–631 (2014).

    Article  Google Scholar 

  67. Ryytty, S. et al. Varied responses to a high m.3243 A > G mutation load and respiratory chain dysfunction in patient-derived cardiomyocytes. Cells 11, 2593 (2022).

    Article  Google Scholar 

  68. Pavez-Giani, M. G. & Cyganek, L. Recent advances in modeling mitochondrial cardiomyopathy using human induced pluripotent stem cells. Front. Cell Dev. Biol. 9, 1–17 (2022).

    Article  Google Scholar 

  69. 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  Google Scholar 

  70. 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).

    Article  ADS  Google Scholar 

  71. Stadelmann, C. et al. mRNA-mediated delivery of gene editing tools to human primary muscle stem cells. Mol. Ther. Nucleic Acids 28, 47–57 (2022).

    Article  Google Scholar 

  72. Schott, J. W., Morgan, M., Galla, M. & Schambach, A. Viral and synthetic RNA vector technologies and applications. Mol. Ther. 24, 1513–1527 (2016).

    Article  Google Scholar 

  73. Kwon, H. et al. Emergence of synthetic mRNA: in vitro synthesis of mRNA and its applications in regenerative medicine. Biomaterials 156, 172–193 (2018).

    Article  Google Scholar 

  74. Karikó, K., Buckstein, M., Ni, H. & Weissman, D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity 23, 165–175 (2005).

    Article  Google Scholar 

  75. Gallie, D. R. The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency. Genes Dev. 5, 2108–2116 (1991).

    Article  Google Scholar 

  76. Pardi, N., Hogan, M. J., Porter, F. W. & Weissman, D. mRNA vaccines — a new era in vaccinology. Nat. Rev. Drug Discov. 17, 261–279 (2018).

    Article  Google Scholar 

  77. Sork, H. et al. Lipid-based transfection reagents exhibit cryo-induced increase in transfection efficiency. Mol. Ther. Nucleic Acids 5, e290 (2016).

    Article  Google Scholar 

  78. Liu, Y. et al. Factors influencing the efficiency of cationic liposome-mediated intravenous gene delivery. Nat. Biotechnol. 15, 167–173 (1996).

    Article  Google Scholar 

  79. Iversen, N., Birkenes, B., Torsdalen, K. & Djurovic, S. Electroporation by nucleofactor is the best nonviral transfection technique in human endothelial and smooth muscle cells. Genet. Vaccines Ther. 3, 2 (2005).

    Article  Google Scholar 

  80. Distler, J. H. W. et al. Nucleofection: a new, highly efficient transfection method for primary human keratinocytes. Exp. Dermatol. 14, 315–320 (2005).

    Article  Google Scholar 

  81. di Donfrancesco, A. et al. Gene therapy for mitochondrial diseases: current status and future perspective. Pharmaceutics 14, 1287 (2022).

    Article  Google Scholar 

  82. Bulcha, J. T., Wang, Y., Ma, H., Tai, P. W. L. & Gao, G. Viral vector platforms within the gene therapy landscape. Signal Transduct. Target. Ther. 6, 53 (2021).

    Article  Google Scholar 

  83. Nayerossadat, N., Ali, P. & Maedeh, T. Viral and nonviral delivery systems for gene delivery. Adv. Biomed. Res. 1, 27 (2012).

    Article  Google Scholar 

  84. Lundstrom, K. Viral vectors in gene therapy. Diseases 6, 42 (2018).

    Article  Google Scholar 

  85. Xu, C. L., Ruan, M. Z. C., Mahajan, V. B. & Tsang, S. H. Viral delivery systems for CRISPR. Viruses 11, 1–12 (2019).

    Article  Google Scholar 

  86. Hou, X., Zaks, T., Langer, R. & Dong, Y. Lipid nanoparticles for mRNA delivery. Nat. Rev. Mater. 6, 1078–1094 (2021).

    Article  ADS  Google Scholar 

  87. Semple, S. C., Leone, R., Barbosa, C. J., Tam, Y. K. & Lin, P. J. C. Lipid nanoparticle delivery systems to enable mRNA-based therapeutics. Pharmaceutics 14, 398 (2022).

    Article  Google Scholar 

  88. Gutkin, A., Rosenblum, D. & Peer, D. RNA delivery with a human virus-like particle. Nat. Biotechnol. 39, 1514–1515 (2021).

    Article  Google Scholar 

  89. Chau, C., Actis, P. & Hewitt, E. Methods for protein delivery into cells: from current approaches to future perspectives. Biochem. Soc. Trans. 48, 357–365 (2020).

    Article  Google Scholar 

  90. Banskota, S. et al. Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins. Cell 185, 250–265.e16 (2022).

    Article  Google Scholar 

  91. Guo, W., Jiang, L., Bhasin, S., Khan, S. M. & Swerdlow, R. H. DNA extraction procedures meaningfully influence qPCR-based mtDNA copy number determination. Mitochondrion 9, 261–265 (2009).

    Article  Google Scholar 

  92. Tani, H. et al. Aberrant RNA processing contributes to the pathogenesis of mitochondrial diseases in trans-mitochondrial mouse model carrying mitochondrial tRNA Leu (UUR) with a pathogenic A2748G mutation. Nucleic Acids Res. 50, 9382–9396 (2022).

    Article  Google Scholar 

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

    Article  ADS  Google Scholar 

  94. Shimizu, A. et al. Transmitochondrial mice as models for primary prevention of diseases caused by mutation in the tRNALys gene. Proc. Natl Acad. Sci. USA 111, 3104–3109 (2014).

    Article  ADS  Google Scholar 

  95. 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).

    Article  Google Scholar 

  96. 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).

    Article  Google Scholar 

  97. 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).

    Article  Google Scholar 

  98. 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).

    Article  Google Scholar 

  99. Jenuth, J. P., Peterson, A. C. & Shoubridge, E. A. Tissue-specific selection for different mtDNA genotypes in heteroplasmic mice. Nat. Genet. 16, 93–95 (1997).

    Article  Google Scholar 

  100. Naso, M. F., Tomkowicz, B., Perry, W. L. & Strohl, W. R. Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs 31, 317–334 (2017).

    Article  Google Scholar 

  101. 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).

    Article  Google Scholar 

  102. Srivastava, A. In vivo tissue-tropism of adeno-associated viral vectors. Curr. Opin. Virol. 21, 75–80 (2016).

    Article  Google Scholar 

  103. Domenger, C. & Grimm, D. Next-generation AAV vectors — do not judge a virus (only) by its cover. Hum. Mol. Genet. 28, R3–R14 (2019).

    Article  Google Scholar 

  104. Kuzmin, D. A. et al. The clinical landscape for AAV gene therapies. Nat. Rev. Drug Discov. 20, 173–174 (2021).

    Article  Google Scholar 

  105. Deyle, D. R. & Russell, D. W. Adeno-associated virus vector integration. Curr. Opin. Mol. Ther. 11, 442–447 (2009).

    Google Scholar 

  106. Valdmanis, P. N., Lisowski, L. & Kay, M. A. RAAV-mediated tumorigenesis: still unresolved after an AAV assault. Mol. Ther. 20, 2014–2017 (2012).

    Article  Google Scholar 

  107. Manini, A., Abati, E., Nuredini, A., Corti, S. & Comi, G. P. Adeno-associated virus (AAV)-mediated gene therapy for duchenne muscular dystrophy: the issue of transgene persistence. Front. Neurol 12, 814174 (2022).

    Article  Google Scholar 

  108. Kishimoto, T. K. & Samulski, R. J. Addressing high dose AAV toxicity — ‘one and done’ or ‘slower and lower’? Expert. Opin. Biol. Ther. 22, 1067–1072 (2022).

    Article  Google Scholar 

  109. Gray, S. J. & Gray, C. S. J. Timing of gene therapy interventions: the earlier, the better. Mol. Ther. 24, 1017–1018 (2016).

    Article  Google Scholar 

  110. Rapti, K. et al. Neutralizing antibodies against AAV serotypes 1, 2, 6, and 9 in sera of commonly used animal models. Mol. Ther. 20, 73–83 (2012).

    Article  Google Scholar 

  111. Kenjo, E. et al. Low immunogenicity of LNP allows repeated administrations of CRISPR–Cas9 mRNA into skeletal muscle in mice. Nat. Commun. 12, 1–13 (2021).

    Article  Google Scholar 

  112. Kularatne, R. N., Crist, R. M. & Stern, S. T. The future of tissue-targeted lipid nanoparticle-mediated nucleic acid delivery. Pharmaceuticals 15, 897 (2022).

    Article  Google Scholar 

  113. Cheng, Q. et al. Selective organ targeting (SORT) nanoparticles for tissue specific mRNA delivery and CRISPR/Cas gene editing. Nat. Nanotechnol. 15, 313–320 (2020).

    Article  ADS  Google Scholar 

  114. Dilliard, S. A., Cheng, Q. & Siegwart, D. J. On the mechanism of tissue-specific mRNA delivery by selective organ targeting nanoparticles. Proc. Natl Acad. Sci. USA 118, e2109256118 (2021).

    Article  Google Scholar 

  115. Qiu, M. et al. Lung-selective mRNA delivery of synthetic lipid nanoparticles for the treatment of pulmonary lymphangioleiomyomatosis. Proc. Natl Acad. Sci. USA 119, 1–10 (2022).

    Article  Google Scholar 

  116. Kazemian, P. et al. Lipid-nanoparticle-based delivery of CRISPR/Cas9 genome-editing components. Mol. Pharm. 19, 1669–1686 (2022).

    Article  Google Scholar 

  117. Mukai, H., Ogawa, K., Kato, N. & Kawakami, S. Recent advances in lipid nanoparticles for delivery of nucleic acid, mRNA, and gene editing-based therapeutics. Drug Metab. Pharmacokinet. 44, 100450 (2022).

    Article  Google Scholar 

  118. Gillmore, J. D. et al. CRISPR–Cas9 in vivo gene editing for transthyretin amyloidosis. N. Engl. J. Med. 385, 493–502 (2021).

    Article  Google Scholar 

  119. Nooraei, S. et al. Virus-like particles: preparation, immunogenicity and their roles as nanovaccines and drug nanocarriers. J. Nanobiotechnol. 19, 1–27 (2021).

    Article  Google Scholar 

  120. Duan, M., Tu, J. & Lu, Z. Recent advances in detecting mitochondrial DNA heteroplasmic variations. Molecules 23, 323 (2018).

    Article  Google Scholar 

  121. Seroussi, E. Estimating copy-number proportions: the comeback of Sanger sequencing. Genes 12, 1–9 (2021).

    Article  Google Scholar 

  122. Carr, I. M. et al. Inferring relative proportions of DNA variants from sequencing electropherograms. Bioinformatics 25, 3244–3250 (2009).

    Article  Google Scholar 

  123. Huang, T. Next generation sequencing to characterize mitochondrial genomic DNA heteroplasmy. Curr. Protoc. Hum. Genet. https://doi.org/10.1002/0471142905.hg1908s71 (2011).

    Article  Google Scholar 

  124. Legati, A. et al. Current and new next-generation sequencing approaches to study mitochondrial DNA. J. Mol. Diagn. 23, 732–741 (2021).

    Article  Google Scholar 

  125. Santibanez-Koref, M. et al. Assessing mitochondrial heteroplasmy using next generation sequencing: a note of caution. Mitochondrion 46, 302–306 (2019).

    Article  Google Scholar 

  126. Moraes, C. T., Atencio, D. P., Oca-Cossio, J. & Diaz, F. Techniques and pitfalls in the detection of pathogenic mitochondrial DNA mutations. J. Mol. Diagn. 5, 197–208 (2003).

    Article  Google Scholar 

  127. Shoffner, J. M. et al. Myoclonic epilepsy and ragged-red fiber disease (MERRF) is associated with a mitochondrial DNA tRNALys mutation. Cell 61, 931–937 (1990).

    Article  Google Scholar 

  128. Moraes, C. T., Ricci, E., Bonilla, E., DiMauro, S. & Schon, E. A. The mitochondrial tRNALeu(UUR) mutation in mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS): genetic, biochemical, and morphological correlations in skeletal muscle. Am. J. Hum. Genet. 50, 934–949 (1992).

    Google Scholar 

  129. Rong, E. et al. Heteroplasmy detection of mitochondrial DNA A3243G mutation using quantitative real-time PCR assay based on TaqMan-MGB probes. Biomed. Res. Int. 2018, 1286480 (2018).

    Article  Google Scholar 

  130. Bubner, B., Gase, K. & Baldwin, I. T. Two-fold differences are the detection limit for determining transgene copy numbers in plants by real-time PCR. BMC Biotechnol. 4, 14 (2004).

    Article  Google Scholar 

  131. BioRad. Droplet DigitalTM PCR applications guide 145. BioRad http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf (2018).

  132. BioRad. Rare mutation detection best practices guidelines. BioRad https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6628.pdf (2015).

  133. Tytgat, O. et al. Digital polymerase chain reaction for assessment of mutant mitochondrial carry-over after nuclear transfer for in vitro fertilization. Clin. Chem. 67, 968–976 (2021).

    Article  Google Scholar 

  134. Sofronova, J. K. et al. Detection of mutations in mitochondrial DNA by droplet digital PCR. Biochemistry 81, 1031–1037 (2016).

    Google Scholar 

  135. Shoop, W. K., Gorsuch, C. L., Bacman, S. R. & Moraes, C. T. Precise and simultaneous quantification of mitochondrial DNA heteroplasmy and copy number by digital PCR. J. Biological. Chem. 298, 102574 (2022).

    Article  Google Scholar 

  136. Ma, J., Li, N., Guarnera, M. & Jiang, F. Quantification of plasma miRNAs by digital PCR for cancer diagnosis. Biomark Insights 8, 127–136 (2013).

    Article  Google Scholar 

  137. Bacman, S. R., Williams, S. L., Pinto, M. & Moraes, C. T. The use of mitochondria-targeted endonucleases to manipulate mtDNA. Methods Enzymol. 547, 373–397 (2014).

    Article  Google Scholar 

  138. O’Hara, R. et al. Quantitative mitochondrial DNA copy number determination using droplet digital PCR with single-cell resolution. Genome Res. 29, 1878–1888 (2019).

    Article  Google Scholar 

  139. Li, B. et al. Droplet digital PCR shows the D-loop to be an error prone locus for mitochondrial DNA copy number determination. Sci. Rep. 8, 11392 (2018).

    Article  ADS  Google Scholar 

  140. Vercellino, I. & Sazanov, L. A. The assembly, regulation and function of the mitochondrial respiratory chain. Nat. Rev. Mol. Cell Biol. 23, 141–161 (2022).

    Article  Google Scholar 

  141. Agilent. Agilent Seahorse XF Cell Mito Stress Test Kit User Guide. Agilent https://www.agilent.com/cs/library/usermanuals/public/XF_Cell_Mito_Stress_Test_Kit_User_Guide.pdf (2019).

  142. Sasarman, F. & Shoubridge, E. A. Radioactive labeling of mitochondrial translation products in cultured cells. Methods Mol. Biol. 837, 207–217 (2012).

    Article  Google Scholar 

  143. Sasarman, F., Antonicka, H. & Shoubridge, E. A. The A3243G tRNALeu(UUR) MELAS mutation causes amino acid misincorporation and a combined respiratory chain assembly defect partially suppressed by overexpression of EFTu and EFG2. Hum. Mol. Genet. 17, 3697–3707 (2008).

    Article  Google Scholar 

  144. Yousefi, R. et al. Monitoring mitochondrial translation in living cells. EMBO Rep. 22, e51635 (2021).

    Article  Google Scholar 

  145. van Coster, R. et al. Blue native polyacrylamide gel electrophoresis: a powerful tool in diagnosis of oxidative phosphorylation defects. Pediatr. Res. 50, 658–665 (2001).

    Article  Google Scholar 

  146. Fernandez-Vizarra, E. & Zeviani, M. Blue-native electrophoresis to study the OXPHOS complexes. Methods Mol. Biol. 2192, 287–311 (2021).

    Article  Google Scholar 

  147. Held, J. P. & Patel, M. R. Functional conservation of mitochondrial RNA levels despite divergent mtDNA organization. BMC Res. Notes 13, 1–4 (2020).

    Article  Google Scholar 

  148. He, S. L. & Green, R. Northern blotting. Methods Enzymol. 530, 75–87 (2013).

    Article  Google Scholar 

  149. Park, H., Davidson, E. & King, M. P. The pathogenic A3243G mutation in human mitochondrial tRNALeu(UUR) decreases the efficiency of aminoacylation. Biochemistry 42, 958–964 (2003).

    Article  Google Scholar 

  150. McClain, W. H., Jou, Y.-Y., Bhattacharya, S., Gabriel, K. & Schneider, J. The reliability of in vivo structure–function analysis of tRNA aminoacylation. J. Mol. Biol. 290, 391–409 (1999).

    Article  Google Scholar 

  151. Sørensen, M. A. Charging levels of four tRNA species in Escherichia coli Rel+ and Rel strains during amino acid starvation: a simple model for the effect of ppGpp on translational accuracy. J. Mol. Biol. 307, 785–798 (2001).

    Article  Google Scholar 

  152. Varshney, U., Lee, C.-P. & RajBhandary, U. L. Direct analysis of aminoacylation levels of tRNAs in vivo. Application to studying recognition of Escherichia coli initiator tRNA mutants by glutaminyl-tRNA synthetase. J. Biol. Chem. 266, 24712–24718 (1991).

    Article  Google Scholar 

  153. Dittmar, K. A., Sørensen, M. A., Elf, J., Ehrenberg, M. & Pan, T. Selective charging of tRNA isoacceptors induced by amino-acid starvation. EMBO Rep. 6, 151–157 (2005).

    Article  Google Scholar 

  154. Zaborske, J. M. et al. Genome-wide analysis of tRNA charging and activation of the eIF2 kinase Gcn2p. J. Biol. Chem. 284, 25254–25267 (2009).

    Article  Google Scholar 

  155. Zhou, Y., Goodenbour, J. M., Godley, L. A., Wickrema, A. & Pan, T. High levels of tRNA abundance and alteration of tRNA charging by bortezomib in multiple myeloma. Biochem. Biophys. Res. Commun. 385, 160–164 (2009).

    Article  Google Scholar 

  156. Behrens, A., Rodschinka, G. & Nedialkova, D. D. High-resolution quantitative profiling of tRNA abundance and modification status in eukaryotes by mim-tRNAseq. Mol. Cell 81, 1802–1815.e7 (2021).

    Article  Google Scholar 

  157. Evans, M. E., Clark, W. C., Zheng, G. & Pan, T. Determination of tRNA aminoacylation levels by high-throughput sequencing. Nucleic Acids Res. 45, e133 (2017).

    Article  Google Scholar 

  158. Yasukawa, T. et al. Modification defect at anticodon wobble nucleotide of mitochondrial tRNAs(Leu)(UUR) with pathogenic mutations of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes. J. Biol. Chem. 275, 4251–4257 (2000).

    Article  Google Scholar 

  159. Su, D. et al. Quantitative analysis of tRNA modifications by HPLC-coupled mass spectrometry. Nat. Protoc. 9, 828–841 (2014).

    Article  Google Scholar 

  160. Yan, T.-M. et al. Full-range profiling of tRNA modifications using LC–MS/MS at single-base resolution through a site-specific cleavage strategy. Anal. Chem. 93, 1423–1432 (2021).

    Article  Google Scholar 

  161. Saporta, M. A. et al. Axonal Charcot–Marie–Tooth disease patient-derived motor neurons demonstrate disease-specific phenotypes including abnormal electrophysiological properties. Exp. Neurol. 263, 190–199 (2015).

    Article  Google Scholar 

  162. Nunes, G. B. L., Costa, L. M., Gutierrez, S. J. C., Satyal, P. & de Freitas, R. M. Behavioral tests and oxidative stress evaluation in mitochondria isolated from the brain and liver of mice treated with riparin A. Life Sci. 121, 57–64 (2015).

    Article  Google Scholar 

  163. 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  Google Scholar 

  164. Kang, H. Sample size determination and power analysis using the G*Power software. J. Educ. Eval. Health Prof. 18, 1–12 (2021).

    Article  Google Scholar 

  165. Mishra, P., Pandey, C., Singh, U., Keshri, A. & Sabaretnam, M. Selection of appropriate statistical methods for data analysis. Ann. Card. Anaesth. 22, 297–301 (2019).

    Article  Google Scholar 

  166. Battersby, B. J. & Shoubridge, E. A. Selection of a mtDNA sequence variant in hepatocytes of heteroplasmic mice is not due to differences in respiratory chain function or efficiency of replication. Hum. Mol. Genet. 10, 2469–2479 (2001).

    Article  Google Scholar 

  167. Lechuga-Vieco, A. V. et al. Heteroplasmy of wild-type mitochondrial DNA variants in mice causes metabolic heart disease with pulmonary hypertension and frailty. Circulation 145, 1084–1101 (2022).

    Article  Google Scholar 

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

    Article  Google Scholar 

  169. Wang, S., Yi, F. & Qu, J. Eliminate mitochondrial diseases by gene editing in germ-line cells and embryos. Protein Cell 6, 472–475 (2015).

    Article  Google Scholar 

  170. Fu, L. et al. Potential of mitochondrial genome editing for human fertility health. Front. Genet. 12, 673951 (2021).

    Article  Google Scholar 

  171. Pickrell, A. M., Pinto, M., Hida, A. & Moraes, C. T. Striatal dysfunctions associated with mitochondrial DNA damage in dopaminergic neurons in a mouse model of Parkinson’s disease. J. Neurosci. 31, 17649–17658 (2011).

    Article  Google Scholar 

  172. Wang, X. et al. Transient systemic mtDNA damage leads to muscle wasting by reducing the satellite cell pool. Hum. Mol. Genet. 22, 3976–3986 (2013).

    Article  Google Scholar 

  173. Madsen, P. M. et al. Mitochondrial DNA double-strand breaks in oligodendrocytes cause demyelination, axonal injury, and CNS inflammation. J. Neurosci. 37, 10185–10199 (2017).

    Article  Google Scholar 

  174. Fukui, H. & Moraes, C. T. Mechanisms of formation and accumulation of mitochondrial DNA deletions in aging neurons. Hum. Mol. Genet. 18, 1028–1036 (2009).

    Article  Google Scholar 

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

    Article  Google Scholar 

  176. Tigano, M., Vargas, D. C., Tremblay-Belzile, S., Fu, Y. & Sfeir, A. Nuclear sensing of breaks in mitochondrial DNA enhances immune surveillance. Nature 591, 477–481 (2021).

    Article  ADS  Google Scholar 

  177. Castro, R. J. Mitochondrial replacement therapy: the UK and US regulatory landscapes. J. Law Biosci. 3, 726–735 (2016).

    Article  Google Scholar 

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

    Article  ADS  Google Scholar 

  179. Yamada, M. et al. Mitochondrial replacement by genome transfer in human oocytes: efficacy, concerns, and legality. Reprod. Med. Biol. 20, 53–61 (2021).

    Article  Google Scholar 

  180. Reznik, E. et al. Mitochondrial DNA copy number variation across human cancers. eLife 5, e10769 (2016).

    Article  Google Scholar 

  181. Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82 (2011).

    Article  Google Scholar 

  182. Zhang, S., Wang, J. & Wang, J. One-day TALEN assembly protocol and a dual-tagging system for genome editing. ACS Omega 5, 19702–19714 (2020).

    Article  Google Scholar 

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

    Google Scholar 

  184. MacLeod, D. T. et al. Integration of a CD19 CAR into the TCR α chain locus streamlines production of allogeneic gene-edited CAR T cells. Mol. Ther. 25, 949–961 (2017).

    Article  Google Scholar 

  185. Wang, L. et al. Meganuclease targeting of PCSK9 in macaque liver leads to stable reduction in serum cholesterol. Nat. Biotechnol. 36, 717–725 (2018).

    Article  Google Scholar 

  186. Wang, L. et al. Long-term stable reduction of low-density lipoprotein in nonhuman primates following in vivo genome editing of PCSK9. Mol. Ther. 29, 2019–2029 (2021).

    Article  Google Scholar 

  187. Gorsuch, C. L. et al. Targeting the hepatitis B cccDNA with a sequence-specific ARCUS nuclease to eliminate hepatitis B virus in vivo. Mol. Ther. 30, 2909–2922 (2022).

    Article  Google Scholar 

  188. Wei, Y. et al. Mitochondrial base editor DdCBE causes substantial DNA off-target editing in nuclear genome of embryos. Cell Discov. 8, 391–394 (2022).

    Article  Google Scholar 

  189. Lee, S., Lee, H., Baek, G. & Kim, J.-S. Precision mitochondrial DNA editing with high-fidelity DddA-derived base editors. Nat. Biotechnol. https://doi.org/10.1038/s41587-022-01486-w (2022).

    Article  Google Scholar 

  190. Grieger, J. C. & Samulski, R. J. Packaging capacity of adeno-associated virus serotypes: impact of larger genomes on infectivity and postentry steps. J. Virol. 79, 9933–9944 (2005).

    Article  Google Scholar 

  191. Colella, P., Ronzitti, G. & Mingozzi, F. Emerging issues in AAV-mediated in vivo gene therapy. Mol. Ther. Methods Clin. Dev. 8, 87–104 (2018).

    Article  Google Scholar 

  192. Smith, R. H. Adeno-associated virus integration: virus versus vector. Gene Ther. 15, 817–822 (2008).

    Article  Google Scholar 

  193. Hanlon, K. S. et al. High levels of AAV vector integration into CRISPR-induced DNA breaks. Nat. Commun. 10, 4439 (2019).

    Article  ADS  Google Scholar 

  194. Weber, T. Anti-AAV antibodies in AAV gene therapy: current challenges and possible solutions. Front. Immunol. 12, 658399 (2021).

    Article  Google Scholar 

  195. 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).

    Article  Google Scholar 

  196. Abu-Amero, K. K. Lebers hereditary optic neuropathy: the mitochondrial connection revisited. Middle East. Afr. J. Ophthalmol. 18, 17–23 (2011).

    Article  Google Scholar 

Download references

Acknowledgements

The authors thank C. L. Gorsuch for her suggestions related to this work. The work in the Moraes laboratory was funded by National Institute of Health (NIH) Grants 5R01EY010804 and 1R01NS079965, the Florida Biomedical Foundation (21K05), the Muscular Dystrophy Association (MDA 964119) and the Army Research Office (W911NF-21-1-0248).

Author information

Authors and Affiliations

Authors

Contributions

Introduction (W.K.S. and C.T.M.); Experimentation (W.K.S., S.R.B., J.D.B.-P. and C.T.M.); Results (W.K.S., S.R.B., J.D.B.-P. and C.T.M.); Applications (W.K.S., S.R.B., J.D.B.-P. and C.T.M.); Reproducibility and data deposition (C.T.M.); Limitations and optimizations (W.K.S., S.R.B., J.D.B.-P. and C.T.M.); Outlook (W.K.S. and C.T.M.); Overview of the Primer (W.K.S., S.R.B., J.D.B.-P. and C.T.M.).

Corresponding author

Correspondence to Carlos T. Moraes.

Ethics declarations

Competing interests

W.K.S. is employed by Precision BioSciences. All other authors declare no competing interests.

Peer review

Peer review information

Nature Reviews Methods Primers thanks Gino Cortopassi, Giovanni Manfredi, Chengqi Yi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

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

Supplementary information

Glossary

Cytoplasmic hybrid

A cell derived from the fusion of an enucleated patient cell with a cell lacking mitochondrial DNA.

Heteroduplex

A double-stranded DNA sequence that contains a mismatch.

Heteroplasmy

A state in which a cell contains more than one mitochondrial DNA haplotype.

Homoplasmic

Describes a state in which all of the mitochondrial DNA molecules are identical.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Shoop, W.K., Bacman, S.R., Barrera-Paez, J.D. et al. Mitochondrial gene editing. Nat Rev Methods Primers 3, 19 (2023). https://doi.org/10.1038/s43586-023-00200-7

Download citation

  • Published:

  • DOI: https://doi.org/10.1038/s43586-023-00200-7

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