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

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.