Targeted sequencing data for developing efficient adenine base editor in mitochondrial
Transcription activator-like effector (TALE)-linked deaminases (TALEDs) use their single-stranded DNA (ssDNA)-specific adenosine deaminase TadA8e to mediate A-to-G editing in mitochondrial DNA (mtDNA), but the working mechanism remains elusive, which hinders the development of more effective TALEDs. Here, we revealed that TALED-mediated A-to-G editing relies on the formation of an ssDNA region through base excision repair (BER), which is triggered by double-stranded DNA-specific cytidine deaminase (DddA)-induced C-to-U deamination. On the basis of this mechanism, we developed a series of enhanced TALEDs (eTALED6s) that have significantly increased editing efficiency by replacing DddA with the high-activity variant DddA6 and fusing human uracil DNA glycosylase to TadA8e. By further engineering TadA8e, the resulting eTALED6Rs induced efficient on-target editing with greatly reduced bystander editing and off-target editing at the DNA and RNA levels. Then, we used eTALED6 and eTALED6R to install a pathogenic mutation in mtDNA. In summary, we revealed the mechanism of TALED-mediated A-to-G editing and demonstrated that enhancing BER increased its efficiency, highlighting a new path to improve this editing tool.