A DddA ortholog-based and transactivator-assisted nuclear and mitochondrial cytosine base editors with expanded target compatibility

Guo, Junfan; Yu, Wenxia; Li, Min; Chen, Hongyu; Liu, Jie; Xue, Xiaowen; Lin, Jianxiang; Huang, Shisheng; Shu, Wenjie; Huang, Xingxu; Liu, Zhen; Wang, Shengqi; Qiao, Yong

doi:10.1016/j.molcel.2023.04.012

Cited by 19 publications

(12 citation statements)

References 55 publications

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“…Recently, two studies successfully developed DddA orthology-based cytosine base editors that can efficiently deaminate cytosine in GC context in mtDNA. [13,14] To solve the problem of inaccessible sequence compatibility, there are two commonly used methods in the field, one is to engineer the original deaminase, and the other is to find the new deaminase orthologs. Unlike strategies used in these two studies, our work mainly addressed this challenge by fusing the single-strand activity of cytosine deaminase with DddA11.…”

Section: Introductionmentioning

confidence: 99%

Enhanced C‐To‐T and A‐To‐G Base Editing in Mitochondrial DNA with Engineered DdCBE and TALED

Wei,

Jin,

Huang

et al. 2023

Advanced Science

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Mitochondrial base editing with DddA‐derived cytosine base editor (DdCBE) is limited in the accessible target sequences and modest activity. Here, the optimized DdCBE tools is presented with improved editing activity and expanded C‐to‐T targeting scope by fusing DddA11 variant with different cytosine deaminases with single‐strand DNA activity. Compared to previous DdCBE based on DddA11 variant alone, fusion of the activation‐induced cytidine deaminase (AID) from Xenopus laevis not only permits cytosine editing of 5′‐GC‐3′ sequence, but also elevates editing efficiency at 5′‐TC‐3′, 5′‐CC‐3′, and 5′‐GC‐3′ targets by up to 25‐, 10‐, and 6‐fold, respectively. Furthermore, the A‐to‐G editing efficiency is significantly improved by fusing the evolved DddA6 variant with TALE‐linked deoxyadenosine deaminase (TALED). Notably, the authors introduce the reported high‐fidelity mutations in DddA and add nuclear export signal (NES) sequences in DdCBE and TALED to reduce off‐target editing in the nuclear and mitochondrial genome while improving on‐target editing efficiency in mitochondrial DNA (mtDNA). Finally, these engineered mitochondrial base editors are shown to be efficient in installing mtDNA mutations in human cells or mouse embryos for disease modeling. Collectively, the study shows broad implications for the basic study and therapeutic applications of optimized DdCBE and TALED.

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Section: Introductionmentioning

confidence: 99%

Enhanced C‐To‐T and A‐To‐G Base Editing in Mitochondrial DNA with Engineered DdCBE and TALED

Wei,

Jin,

Huang

et al. 2023

Advanced Science

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“…This approach involves replacing Burkholderia cenocepacia DddAtox (Ddd_ Bc ) with homologous and orthologous proteins identified based on the amino acid sequences of DddA ( Fig. 2G ) ( 43 - 46 ). Specifically, DdCBE_Ss (=FZY2−DdCBE), Q2L7−DdCBE, and Rs DdCBE use a DddA homolog from Simiaoa sunii (Ddd_ Ss = FZY2) ( 43 , 44 ), Streptomyces sp -BK438 (Q2L7) ( 44 ), and Ruminococcus sp .…”

Section: Challenges and Improvements In Mtdna Base Editingmentioning

confidence: 99%

“…Specifically, DdCBE_Ss (=FZY2−DdCBE), Q2L7−DdCBE, and Rs DdCBE use a DddA homolog from Simiaoa sunii (Ddd_ Ss = FZY2) ( 43 , 44 ), Streptomyces sp -BK438 (Q2L7) ( 44 ), and Ruminococcus sp . ( Rs DddA) ( 45 ), respectively; while mitoCBE2.0 uses a DddA ortholog from Roseburia intestinalis (Ddd_ Ri ) ( 46 ). These base editors can even target GC sequences that were previously inaccessible.…”

Section: Challenges and Improvements In Mtdna Base Editingmentioning

confidence: 99%

Mitochondrial genome editing: strategies, challenges, and applications

Lim

2024

BMB Rep

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Mitochondrial DNA (mtDNA), a multicopy genome found in mitochondria, is crucial for oxidative phosphorylation. Mutations in mtDNA can lead to severe mitochondrial dysfunction in tissues and organs with high energy demand. MtDNA muta-tions are closely associated with mitochondrial and age-related disease. To better understand the functional role of mtDNA and work toward developing therapeutics, it is essential to advance technology that is capable of manipulating the mitochondrial genome. This review discusses ongoing efforts in mitochondrial genome editing with mtDNA nucleases and base editors, including the tools, delivery strategies, and applications. Future advances in mitochondrial genome editing to address challenges regarding their efficiency and specificity can achieve the promise of therapeutic genome editing.

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“…2021 ), modified versions of DdCBE, which have an in vitro–evolved DddA or a paralog of DddA isolated from another bacterium, can use ac , GC and CC as substrates in the mammalian mitochondrial genome ( Mok et al. 2022a , Guo et al. 2023 , Mi et al.…”

Section: Crispr-free Protein-based Systems For Organellar Genome Editingmentioning

confidence: 99%

Genome Editing of Plant Mitochondrial and Chloroplast Genomes

Arimura,

Nakazato

2023

Plant and Cell Physiology

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Plastids (including chloroplasts) and mitochondria are remnants of endosymbiotic bacteria yet maintain their own genomes, which encode vital components for photosynthesis and respiration, respectively. Organellar genomes have distinctive features, such as being present as multicopies, being mostly inherited maternally, having characteristic genomic structures, and undergoing frequent homologous recombination. To date, it has proven challenging to modify these genomes. For example, while CRISPR/Cas9 is a widely used system for editing nuclear genes, it has not yet been successfully applied to organellar genomes. Recently, however, precise gene editing technologies have been successfully applied to organellar genomes. Protein-based enzymes, especially TALENs and artificial enzymes utilizing TALEs, have been successfully used to modify these genomes by harnessing organellar targeting signals. This short review introduces and discusses the use of targeted nucleases and base editors on organellar genomes, their effects, and their potential applications in plant science and breeding.

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A DddA ortholog-based and transactivator-assisted nuclear and mitochondrial cytosine base editors with expanded target compatibility

Cited by 19 publications

References 55 publications

Enhanced C‐To‐T and A‐To‐G Base Editing in Mitochondrial DNA with Engineered DdCBE and TALED

Enhanced C‐To‐T and A‐To‐G Base Editing in Mitochondrial DNA with Engineered DdCBE and TALED

Mitochondrial genome editing: strategies, challenges, and applications

Genome Editing of Plant Mitochondrial and Chloroplast Genomes

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