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<p>Bystander editing remains a major limitation of current base editors, hindering their precision and therapeutic potential. Here, we present a de novo protein design strategy that creates a structurally rigid interface between a DNA-binding TALE domain and a cytosine deaminase, forming a unified editing module termed TALE-oriented deaminase (TOD). Cryo-EM analysis of TOD–DNA complexes confirms that this precise spatial architecture tightly restricts the deaminase activity window, thereby minimizing unwanted deamination. To further enhance editing specificity, we develop a split version, termed DdCBE–TOD, which virtually eliminates off-target editing. As a proof of concept, we apply DdCBE–TOD to generate a mitochondrial disease mouse model and to correct a pathogenic mutation associated with MERRF syndrome in patient-derived cells, achieving single-nucleotide precision. This work introduces a generalizable and computationally guided approach for ultra-precise base editing, offering a promising platform for both mechanistic studies and therapeutic correction of single-nucleotide mutations.</p>

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Computational design of a high-precision mitochondrial DNA cytosine base editor

  • 期刊论文

  • 发表日期：

    2025年11月17日

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  • Li Mi,
  • Yu-Xuan Li,
  • Xinchen Lv,
  • Zi-Li Wan,
  • Xu Liu,
  • Kairan Zhang,
  • Huican Li,
  • Yue Yao,
  • Leping Zhang,
  • Zhe Xu,
  • Xingyu Zhuang,
  • Kunqian Ji,
  • Min Jiang,
  • Yangming Wang,
  • Peilong Lu

摘要

Bystander editing remains a major limitation of current base editors, hindering their precision and therapeutic potential. Here, we present a de novo protein design strategy that creates a structurally rigid interface between a DNA-binding TALE domain and a cytosine deaminase, forming a unified editing module termed TALE-oriented deaminase (TOD). Cryo-EM analysis of TOD–DNA complexes confirms that this precise spatial architecture tightly restricts the deaminase activity window, thereby minimizing unwanted deamination. To further enhance editing specificity, we develop a split version, termed DdCBE–TOD, which virtually eliminates off-target editing. As a proof of concept, we apply DdCBE–TOD to generate a mitochondrial disease mouse model and to correct a pathogenic mutation associated with MERRF syndrome in patient-derived cells, achieving single-nucleotide precision. This work introduces a generalizable and computationally guided approach for ultra-precise base editing, offering a promising platform for both mechanistic studies and therapeutic correction of single-nucleotide mutations.