DddA homolog search and engineering expand sequence compatibility of mitochondrial base editing

Li, Mi; Shi, Ming; Li, Yuxuan; Xie, Gang; Rao, Xichen; Wu, Damu; Ai-min, Cheng; Niu, Mengxiao; Xu, Fengli; Yu, Ying; Gao, Ning; Wei, Wensheng; Wang, Xianhua; Wang, Yangming

doi:10.1038/s41467-023-36600-2

Cited by 35 publications

(17 citation statements)

References 25 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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“…The mitochondrial genome can be manipulated using biolistic bombardment and homologous recombination (46). Less precise approaches to mito-genome engineering use base editors, like deaminases, fused to mitochondrial targeting signals (47). A second disadvantage to the mito-TS is the propensity for S. cerevisiae become petite, i.e., to lose mito- genomes when cells lack specific proteins associated with mitochondrial transcription and translation (e.g.…”

Section: Discussionmentioning

confidence: 99%

Translation in a Box: Orthogonal Evolution in theSaccharomyces cerevisiaeMitochondrion

Rothschild-Mancinelli

Álvarez-Carreño

Guo

et al. 2023

Preprint

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The synthesis capabilities of the translation system (TS) provide engineering and directed evolution potential as well as a system for generating novel polymers. Orthogonal translation systems offer a parallel platform for translation engineering, enabling primary and secondary translation systems to operate independently without interfering with each other. However, current translation platforms for engineering lack complete orthogonality between the primary and secondary systems. Using a combination of indirect modifications through antibiotics, truncations of mito-rProteins mL50 and uL23, and replacement of mito-rProtein uL2, we develop and test the mitochondrial translation system in Saccharomyces cerevisiae as a fully orthogonal platform for in vivo directed evolution of translation. Our results show that continuous, comprehensive, creative, directed-evolution of a fully orthogonal TS in vivo appears to be a useful approach for technical control of the TS. This study emphasizes the power of system-wide evolutionary approaches over rational design. Evolution is creative and non-linear and can find unexpected solutions to problems.

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“…DdCBEs with evolved DddA6 show improved C•G-to-T•A editing at T C motifs, while DdCBEs with evolved DddA11 offer significant C•G-to-T•A editing at C C and A C along with T C motifs. Additionally, the recently-established DdCBE_Ss platform is highly effective at catalyzing C•G-to-T•A editing at G C motifs [ 14 ]. Moreover, the TALED platform enables A•T-to-G•C mtDNA editing in a context-independent manner, potentially increasing bystander editing at target loci.…”

Section: Figurementioning

confidence: 99%

Mitochondrial Base Editing: Recent Advances towards Therapeutic Opportunities

Kar

Restrepo-Castillo

Sabharwal

et al. 2023

IJMS

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Mitochondria are critical organelles that form networks within our cells, generate energy dynamically, contribute to diverse cell and organ function, and produce a variety of critical signaling molecules, such as cortisol. This intracellular microbiome can differ between cells, tissues, and organs. Mitochondria can change with disease, age, and in response to the environment. Single nucleotide variants in the circular genomes of human mitochondrial DNA are associated with many different life-threatening diseases. Mitochondrial DNA base editing tools have established novel disease models and represent a new possibility toward personalized gene therapies for the treatment of mtDNA-based disorders.

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DddA homolog search and engineering expand sequence compatibility of mitochondrial base editing

Cited by 35 publications

References 25 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

Translation in a Box: Orthogonal Evolution in theSaccharomyces cerevisiaeMitochondrion

Mitochondrial Base Editing: Recent Advances towards Therapeutic Opportunities

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