Leveraging base excision repair for efficient adenine base editing of mitochondrial DNA

Transcription activator-like effector-linked deaminases (TALEDs) use their single-stranded DNA (ssDNA)-specific adenosine deaminase TadA8e to mediate A-to-G editing in mitochondrial DNA (mtDNA). The working mechanism of this process is unknown, hindering the development of more effective TALEDs. Here we reveal 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. We develop a series of enhanced TALEDs (eTALED6s) with 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 induces efficient on-target editing with reduced bystander editing and off-target editing at the DNA and RNA levels. Lastly, we use eTALED6 and eTALED6R to install a pathogenic mutation in mtDNA. Revealing the mechanism of TALED-mediated A-to-G editing demonstrates that enhancing BER increases editing efficiency.