Selective mitochondrial DNA degradation following double-strand breaks

Moretton, Amandine; Morel, Frédéric; Macao, Bertil; Lachaume, Philippe; Ishak, Layal; Lefebvre, Mathilde; Garreau-Balandier, Isabelle; Vernet, Patrick; Falkenberg, Maria; Farge, Géraldine

doi:10.1371/journal.pone.0176795

Cited by 121 publications

(127 citation statements)

References 55 publications

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“…Southern blotting followed by probe hybridization has been a steadfast tool to study restriction fragment length polymorphisms and detect rearrangements in immunoglobulin and T cell receptor genes (Current Protocols article: Brown, ). Southern blotting is one of the best‐described methods for analyzing human mtDNA maintenance (Berglund et al., ; Chen & Cheng, ; Hayashi et al., ; Holt et al., ; Kaukonen et al., ; Kornblum et al., ; Lamantea et al., ; Lehtinen et al., ; Luoma et al., ; Moraes et al., , ; Moretton et al., ; Peeva et al., ; Rocher et al., ; Ronchi et al., ; Schon et al., ; Shokolenko et al., ; Song et al., ; Tengan & Moraes, ; Wallace et al., ). Skeletal muscle mtDNA deletions are associated with the mitochondrial disease progressive external ophthalmoplegia (PEO) and traditionally have been detected via Southern blot analyses (Kaukonen et al., ; Lamantea et al., ; Moraes et al., ; Moslemi, Melberg, Holme, & Oldfors, ).…”

Section: Commentarymentioning

confidence: 99%

“…Studies utilizing Southern blotting have proven to be powerful tools to assess mtDNA maintenance in human cell culture and patient samples (Berglund et al., ; Chen & Cheng, ; Hayashi, Takemitsu, Goto, & Nonaka, ; Holt, Dunbar, & Jacobs, ; Kaukonen et al., ; Kornblum et al., ; Lamantea et al., ; Lehtinen et al., ; Luoma et al., ; Moraes et al., ; Moraes, Atencio, Oca‐Cossio, & Diaz, ; Moretton et al., ; Peeva et al., ; Rocher et al., ; Ronchi et al., ; Schon, Naini, & Shanske, ; Shokolenko et al., ; Song, Wheeler, & Mathews, ; Tengan & Moraes, ; Wallace et al., ) as well as in model organisms such as mice and yeast (Griffiths, Doudican, Shadel, & Doetsch, ; Hance, Ekstrand, & Trifunovic, ; Milenkovic et al., ; Trifunovic et al., ; Tyynismaa et al., , ; Young, Theriault, Li, & Court, ). Here we describe a straightforward Southern blotting and nonradioactive probe hybridization method to estimate the quantity of mtDNA in human genomic DNA samples.…”

Section: Introductionmentioning

confidence: 99%

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Analysis of Human Mitochondrial DNA Content by Southern Blotting and Nonradioactive Probe Hybridization

Wheeler

Young

2019

CP Toxicology

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A single cell can contain several thousand copies of the mitochondrial DNA genome or mtDNA. Tools for assessing mtDNA content are necessary for clinical and toxicological research, as mtDNA depletion is linked to genetic disease and drug toxicity. For instance, mtDNA depletion syndromes are typically fatal childhood disorders that are characterized by severe declines in mtDNA content in affected tissues. Mitochondrial toxicity and mtDNA depletion have also been reported in human immunodeficiency virus–infected patients treated with certain nucleoside reverse transcriptase inhibitors. Further, cell culture studies have demonstrated that exposure to oxidative stress stimulates mtDNA degradation. Here we outline a Southern blot and nonradioactive digoxigenin‐labeled probe hybridization method to estimate mtDNA content in human genomic DNA samples. © 2019 by John Wiley & Sons, Inc.

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Section: Commentarymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Analysis of Human Mitochondrial DNA Content by Southern Blotting and Nonradioactive Probe Hybridization

Wheeler

Young

2019

CP Toxicology

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“…Other mitochondrial genes such as ND1 and Cox3 also had lower transcripts levels but were not significant when compared to the sgRNA11204G treatment group, this lower level may reflect the lower levels of mtDNA overall due to CRISPR/Cas9 mediated cleavage of mitochondrial genome (Fig. 3C) that has been shown to rapidly degrade in vivo (37,38).…”

Section: Inclusion Of the Stem Loop Facilitates Crispr-cas9 Mediatedmentioning

confidence: 94%

Adapting CRISPR/Cas9 System for Targeting Mitochondrial Genome

Hussain

Yalvaç

Khoo

et al. 2020

Preprint

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Gene editing of the mitochondrial genome using CRISPR-Cas9 system is highly challenging mainly due to sub-efficient delivery of guide RNA and Cas9 enzyme complexes into mitochondria. In this study, we were able to perform gene editing in the mitochondrial DNA by appending NADH-ubiquinone oxidoreductase chain 4 (ND4) targeting guide RNA to a RNA transport derived stem loop element (RP-loop) and expressing the Cas9 enzyme with preceding mitochondrial localization sequence. Our results showed mitochondrial colocalization of RP-loop gRNA and a marked reduction of ND4 expression in the cells carrying a A11204G variant in their ND4 sequence coincidently decreasing the mtDNA levels. This proofof-concept study suggests that stem loop element added sgRNA can be transported to the mitochondria and functionally interact with Cas9 to mediate sequence specific mtDNA cleavage. Using this novel approach to target the mtDNA, our results provide further evidence that CRISPR-Cas9 mediated gene editing might potentially be used to treat mtDNA related diseases.

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“…MELAS is one of the most common mitochondrial disorders. It is associated with neurological symptoms and other secondary manifestations such as depression, cardiomyopathy and diabetes mellitus [5][6] [7]. Although this syndrome can be caused by different mutations, 80% of patients harbour a transition of adenine to guanine at the 3243 position in the MT-TL1 gene (tRNALeu (UUR)) of mtDNA.…”

Section: Assessment Of Lbcas12a-based Heteroplasmy Purification In Mementioning

confidence: 99%

Variability in mitochondrial import, mitochondrial health and mtDNA copy number using Type II and Type V CRISPR effectors

Antón

Mullally

Ford

et al. 2020

Preprint

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Current methodologies for targeting the mitochondrial genome for basic research and/or therapeutic strategy development in mitochondrial diseases are restricted by practical limitations and technical inflexibility. The development of a functional molecular toolbox for CRISPR-mediated mitochondrial genome editing is therefore desirable, as this could enable precise targeting of mtDNA haplotypes using the precision and tuneability of CRISPR enzymes; however, published reports of "MitoCRISPR" systems have, to date, lacked reproducibility and independent corroboration. Here, we have explored the requirements for a functional MitoCRISPR system in human cells by engineering several versions of CRISPR nucleases, including the use of alternative mitochondrial protein targeting sequences and smaller paralogues, and the application of gRNA modifications that reportedly induce mitochondrial import. We demonstrate varied mitochondrial targeting efficiencies and influences on mitochondrial dynamics/function of different CRISPR nucleases, with Lachnospiraceae bacterium ND2006 (Lb) Cas12a being better targeted and tolerated than Cas9 variants. We also provide evidence of Cas9 gRNA association with mitochondria in HeLa cells and isolated yeast mitochondria, even in the absence of a targeting RNA aptamer. Finally, we present evidence linking mitochondrial-targeted LbCas12a/crRNA with increased mtDNA copy number dependent upon DNA binding and cleavage activity. We discuss reproducibility issues and the future steps necessary if MitoCRISPR is to be realised.100% of mtDNA copies carry the mutation, or heteroplasmic, where the mutation is carried by a subset of the total mtDNA. In general, mutation load above ~70% is required to present a severe phenotype, although this threshold is disease specific. As a treatment to selectively degrade disease-causing mtDNA haplotypes, the field has developed targeted endonucleases which produce dsDNA breaks in the mutated mtDNA copies, which are then degraded by the mitochondria rather than being repaired [5,6]. The remaining wild type mtDNA then replicates to re-establish mtDNA copy number. This "heteroplasmy purification" has been successfully demonstrated in cell culture and in mouse models using restriction endonucleases (MitoREs), zinc finger nucleases (MitoZFNs), TALENs (MitoTALENs), and homing endonucleases [7,8]. However, although independently validated [9], these tools are impracticable and not widely adopted, because: (i) REs match few clinical mutations and cannot be readily re-engineered [10], and they additionally have high "off-target" cleavage rates [11,12]; (ii) ZFNs require rounds of protein engineering/refinement; (iii) assembly of TALE parts is hindered by problematic cloning of DNA repeats;(iv) TALENs and ZFNs can have low import efficiency and can be mis-trafficked (e.g. ZFs have internal nuclear targeting sequences) [13][14][15]. To overcome some of these difficulties, we have investigated the use of a re-engineered flexible version of the CRISPR method for mitochondrial gen...

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Selective mitochondrial DNA degradation following double-strand breaks

Cited by 121 publications

References 55 publications

Analysis of Human Mitochondrial DNA Content by Southern Blotting and Nonradioactive Probe Hybridization

Analysis of Human Mitochondrial DNA Content by Southern Blotting and Nonradioactive Probe Hybridization

Adapting CRISPR/Cas9 System for Targeting Mitochondrial Genome

Variability in mitochondrial import, mitochondrial health and mtDNA copy number using Type II and Type V CRISPR effectors

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