Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases

Cho, Sung-Ik; Lee, Seonghyun; Mok, Young Geun; Lim, Kayeong; Lee, Jaesuk; Lee, Ji Min; Chung, Eugene; Kim, Jin-Soo

doi:10.1016/j.cell.2022.03.039

Cited by 122 publications

(137 citation statements)

References 36 publications

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“…Fortunately, we were able to reduce or eliminate mitochondrial genome-wide off-target effects by using mDdCBE-encoding mRNAs rather than plasmid DNA. We believe that, together with dimeric DdCBEs and ZFDs for C-to-T editing and transcriptional activator-like effector nucleases (TALEDs) for A-to-G editing 19 , mDdCBEs can broaden the scope of organellar genome editing.…”

Section: Discussionmentioning

confidence: 99%

Base editing in human cells with monomeric DddA-TALE fusion deaminases

et al. 2022

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Inter-bacterial toxin DddA-derived cytosine base editors (DdCBEs) enable targeted C-to-T conversions in nuclear and organellar DNA. DddAtox, the deaminase catalytic domain derived from Burkholderia cenocepacia, is split into two inactive halves to avoid its cytotoxicity in eukaryotic cells, when fused to transcription activator-like effector (TALE) DNA-binding proteins to make DdCBEs. As a result, DdCBEs function as pairs, which hampers gene delivery via viral vectors with a small cargo size. Here, we present non-toxic, full-length DddAtox variants to make monomeric DdCBEs (mDdCBEs), enabling mitochondrial DNA editing with high efficiencies of up to 50%, when transiently expressed in human cells. We demonstrate that mDdCBEs expressed via AAV in cultured human cells can achieve nearly homoplasmic C-to-T editing in mitochondrial DNA. Interestingly, mDdCBEs often produce mutation patterns different from those obtained with conventional dimeric DdCBEs. Furthermore, mDdCBEs allow base editing at sites for which only one TALE protein can be designed. We also show that transfection of mDdCBE-encoding mRNA, rather than plasmid, can reduce off-target editing in human mitochondrial DNA.

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Section: Discussionmentioning

confidence: 99%

Base editing in human cells with monomeric DddA-TALE fusion deaminases

et al. 2022

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“…Cho and colleagues [164] recently presented TALE-linked deaminases (TALEDs), which are made up of custom-designed TALE DNA-binding arrays, a catalytically impaired full-length DddA variant or split DddA from Burkholderia cenocepacia, and an engineered deoxyadenosine deaminase derived from the E. Coli TadA protein, all of which induce targeted A-to-G editing in human cells. The custom-designed TALEDs catalyzed A-to-G conversions at a total of 17 target sites in diverse mitochondrial genes, with editing frequencies as high as 49%.…”

Section: Mtdna Editingmentioning

confidence: 99%

Gene Therapy for Mitochondrial Diseases: Current Status and Future Perspective

et al. 2022

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Mitochondrial diseases (MDs) are a group of severe genetic disorders caused by mutations in the nuclear or mitochondrial genome encoding proteins involved in the oxidative phosphorylation (OXPHOS) system. MDs have a wide range of symptoms, ranging from organ-specific to multisystemic dysfunctions, with different clinical outcomes. The lack of natural history information, the limits of currently available preclinical models, and the wide range of phenotypic presentations seen in MD patients have all hampered the development of effective therapies. The growing number of pre-clinical and clinical trials over the last decade has shown that gene therapy is a viable precision medicine option for treating MD. However, several obstacles must be overcome, including vector design, targeted tissue tropism and efficient delivery, transgene expression, and immunotoxicity. This manuscript offers a comprehensive overview of the state of the art of gene therapy in MD, addressing the main challenges, the most feasible solutions, and the future perspectives of the field.

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“…Recently, mtDNA editing has witnessed a fresh impetus with the arrival of programmable mitochondrial cytidine and deoxyadenosine deaminases, which have enabled the editing of mtDNA in cellular and model systems, though with sequence constraints for the pathogenic nucleotide residues to be targeted [ 22 , 23 , 24 , 25 , 26 , 27 ]. Unlike the nuclear genome, where most cells have only two copies, each cell can harbor thousands of copies of mtDNA, depending on environmental needs, making mitochondrial genome engineering a population genetics challenge.…”

Section: Introductionmentioning

confidence: 99%

A Primer Genetic Toolkit for Exploring Mitochondrial Biology and Disease Using Zebrafish

Sabharwal

Campbell

Schwab

et al. 2022

Genes

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Mitochondria are a dynamic eukaryotic innovation that play diverse roles in biology and disease. The mitochondrial genome is remarkably conserved in all vertebrates, encoding the same 37-gene set and overall genomic structure, ranging from 16,596 base pairs (bp) in the teleost zebrafish (Danio rerio) to 16,569 bp in humans. Mitochondrial disorders are amongst the most prevalent inherited diseases, affecting roughly 1 in every 5000 individuals. Currently, few effective treatments exist for those with mitochondrial ailments, representing a major unmet patient need. Mitochondrial dysfunction is also a common component of a wide variety of other human illnesses, ranging from neurodegenerative disorders such as Huntington’s disease and Parkinson’s disease to autoimmune illnesses such as multiple sclerosis and rheumatoid arthritis. The electron transport chain (ETC) component of mitochondria is critical for mitochondrial biology and defects can lead to many mitochondrial disease symptoms. Here, we present a publicly available collection of genetic mutants created in highly conserved, nuclear-encoded mitochondrial genes in Danio rerio. The zebrafish system represents a potentially powerful new opportunity for the study of mitochondrial biology and disease due to the large number of orthologous genes shared with humans and the many advanced features of this model system, from genetics to imaging. This collection includes 15 mutant lines in 13 different genes created through locus-specific gene editing to induce frameshift or splice acceptor mutations, leading to predicted protein truncation during translation. Additionally, included are 11 lines created by the random insertion of the gene-breaking transposon (GBT) protein trap cassette. All these targeted mutant alleles truncate conserved domains of genes critical to the proper function of the ETC or genes that have been implicated in human mitochondrial disease. This collection is designed to accelerate the use of zebrafish to study many different aspects of mitochondrial function to widen our understanding of their role in biology and human disease.

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Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases

Cited by 122 publications

References 36 publications

Base editing in human cells with monomeric DddA-TALE fusion deaminases

Base editing in human cells with monomeric DddA-TALE fusion deaminases

Gene Therapy for Mitochondrial Diseases: Current Status and Future Perspective

A Primer Genetic Toolkit for Exploring Mitochondrial Biology and Disease Using Zebrafish

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