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<p>Existing A-to-G base editors for mitochondrial DNA (mtDNA) are limited by low efficiency. We used directed evolution to discover variants of the TadA-8e base editors that have substantially increased activity and expanded targeting compatibility for both nuclear and mitochondrial adenine base editing, especially in previously unfavored sequence contexts. The engineered mtDNA editors (eTd-mtABEs) showed up to 87% editing efficiency in human cells, with greatly reduced DNA and RNA off-target effects. Strand-selective A-to-G editing was enhanced by an average of 3.2-fold with substitution of DddA to DNA nickases in eTd-mtABE backbones compared to mitochondrial ABEs. In rat cells, editing efficiencies of eTd-mtABEs were up to 145-fold higher compared to split DddA transcription activator-like effector-linked deaminase. We also generated rats with sensorineural hearing loss by installing targeted mutations with frequencies of up to 44% through embryonic injection. The developed eTd-mtABEs are efficient and precise mtDNA-engineering tools for basic research and translational studies.</p>

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Efficient mitochondrial A-to-G base editors for the generation of mitochondrial disease models

  • 期刊论文

  • 发表日期：

    2025年6月3日

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  • Liang Chen,
  • Mengjia Hong,
  • Changming Luan,
  • Meng Yuan,
  • Yiming Wang,
  • Xinyuan Guo,
  • Yue Fang,
  • Hao Huang,
  • Xiaohua Dong,
  • Hongyi Gao,
  • Dan Zhang,
  • Xi Chen,
  • Dihao Meng,
  • Molin Huang,
  • Zongyi Yi,
  • Mingyao Liu,
  • Wensheng Wei,
  • Liangcai Gao,
  • Gaojie Song,
  • Xiaoming Zhou,
  • Dali Li

摘要

Existing A-to-G base editors for mitochondrial DNA (mtDNA) are limited by low efficiency. We used directed evolution to discover variants of the TadA-8e base editors that have substantially increased activity and expanded targeting compatibility for both nuclear and mitochondrial adenine base editing, especially in previously unfavored sequence contexts. The engineered mtDNA editors (eTd-mtABEs) showed up to 87% editing efficiency in human cells, with greatly reduced DNA and RNA off-target effects. Strand-selective A-to-G editing was enhanced by an average of 3.2-fold with substitution of DddA to DNA nickases in eTd-mtABE backbones compared to mitochondrial ABEs. In rat cells, editing efficiencies of eTd-mtABEs were up to 145-fold higher compared to split DddA transcription activator-like effector-linked deaminase. We also generated rats with sensorineural hearing loss by installing targeted mutations with frequencies of up to 44% through embryonic injection. The developed eTd-mtABEs are efficient and precise mtDNA-engineering tools for basic research and translational studies.