ArticleScientific reports2022
Site-specific CRISPR-based mitochondrial DNA manipulation is limited by gRNA import.
Article in Scientific reports, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 26 papers.
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Who cites it
26 citing papers in PubMed.
- Current state of the art in the transmission of mitochondrial DNA pathogenic variants: reviewing preventive-therapeutic alternatives.Journal of assisted reproduction and genetics · 2026Review
- Article
- Gene Therapy Tools for Diseases Caused by Mutations of the Mitochondrial Genome.International journal of molecular sciences · 2026Review
- Specific elimination of m.8993T>G mitochondrial haplotype in NARP cybrid cells by CRISPR-Cas9 system.Scientific reports · 2026Article
- Targeting organelle function in T cells for cancer immunotherapy.Nature reviews. Immunology · 2026Review
- Review
- Article
- Advances in gene therapy for mitochondrial genetic disorders: current status and clinical implementation challenges.Journal of translational medicine · 2025Review
- Polycomb Repressive Complex 1 and USP16 localize to the mitochondrion and influence its function.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
- Targeting and tracking mRNA lipid nanoparticles at the particle, transcript and protein level.Nature biomedical engineering · 2025Review
- Rewriting nuclear epigenetic scripts in mitochondrial diseases as a strategy for heteroplasmy control.EMBO molecular medicine · 2025Article
- Lipid nanoparticle delivery of the CRISPR/Cas9 system directly into the mitochondria of cells carrying m.7778G>T mutation in MtDNA (mt-Atp8).Scientific reports · 2025Article
- Genome modification in plant mitochondria.Plant physiology · 2025Review
- Preclinical models of mitochondrial dysfunction: mtDNA and nuclear-encoded regulators in diverse pathologies.Frontiers in aging · 2025Review
- Unconstrained Precision Mitochondrial Genome Editing with αDdCBEs.Human gene therapy · 2024Article
- Guidelines for mitochondrial RNA analysis.Molecular therapy. Nucleic acids · 2024Review
- Tools for editing the mammalian mitochondrial genome.Human molecular genetics · 2024Review
- CRISPR/Cas9-mediated base editors and their prospects for mitochondrial genome engineering.Gene therapy · 2024Review
- Targeted A-to-G base editing in the organellar genomes of Arabidopsis with monomeric programmable deaminases.Plant physiology · 2024Article
- Base editing of organellar DNA with programmable deaminases.Nature reviews. Molecular cell biology · 2024Review
Corrections and comments
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Authors and funding
5 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Achieving CRISPR Cas9-based manipulation of mitochondrial DNA (mtDNA) has been a long-standing goal and would be of great relevance for disease modeling and for clinical applications. In this project, we aimed to deliver Cas9 into the mitochondria of human cells and analyzed Cas9-induced mtDNA cleavage and measured the resulting mtDNA depletion with multiplexed qPCR. In initial experiments, we found that measuring subtle effects on mtDNA copy numbers is challenging because of high biological variability, and detected no significant Cas9-caused mtDNA degradation. To overcome the challenge of being able to detect Cas9 activity on mtDNA, we delivered cytosine base editor Cas9-BE3 to mitochondria and measured its effect (C → T mutations) on mtDNA. Unlike regular Cas9-cutting, this leaves a permanent mark on mtDNA that can be detected with amplicon sequencing, even if the efficiency is low. We detected low levels of C → T mutations in cells that were exposed to mitochondrially targeted Cas9-BE3, but, surprisingly, these occurred regardless of whether a guide RNA (gRNA) specific to the targeted site, or non-targeting gRNA was used. This unspecific off-target activity shows that Cas9-BE3 can technically edit mtDNA, but also strongly indicates that gRNA import to mitochondria was not successful. Going forward mitochondria-targeted Cas9 base editors will be a useful tool for validating successful gRNA delivery to mitochondria without the ambiguity of approaches that rely on quantifying mtDNA copy numbers.
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