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.

现有的线粒体 DNA （mtDNA） 的 A-to-G 碱基编辑器受到效率低的限制。我们使用定向进化来发现 TadA-8e 碱基编辑器的变体，这些变体显着增加了核和线粒体腺嘌呤碱基编辑的活性并扩大了靶向兼容性，尤其是在以前不受欢迎的序列环境中。工程化的 mtDNA 编辑器 （eTd-mtABEs） 在人类细胞中的编辑效率高达 87%，DNA 和 RNA 脱靶效应大大降低。与线粒体 ABE 相比，在 eTd-mtABE 骨架中用 DddA 取代 DNA 切口酶，链选择性 A-to-G 编辑平均增强了 3.2 倍。在大鼠细胞中，与分裂的 DddA 转录激活因子样效应子连接的脱氨酶相比，eTd-mtABE 的编辑效率高出 145 倍。我们还通过胚胎注射安装频率高达 44% 的靶向突变，从而产生了患有感音神经性听力损失的大鼠。开发的 eTd-mtABE 是用于基础研究和转化研究的高效、精确的 mtDNA 工程工具。