An engineered mitoCBE facilitates efficient mitochondrial DNA editing and modified mitochondrial transfer.

Double-stranded DNA cytosine deaminase DddA orthologs from multiple types of bacteria have been fused with the transcription-activator-like effector system for mitochondrial DNA (mtDNA) base editing, while the efficiencies remain limited and its nuclear off-targeting activity cannot be ignored yet. Here we identified a DddA ortholog from Burkholderia gladioli (BgDddA) and generated nuclear or mtDNA cytosine base editors (mitoCBEs), exhibiting higher C⋅G-to-T⋅A editing frequencies compared with canonical DdCBE, and fusion with transactivator Rta remarkably improved editing efficiencies by up to 6.4-fold at non-TC targets. Referring to DddA11, we further introduced six substitutions into BgDddA and generated mitoCBE3.2, which efficiently induced disease-associated mtDNA mutations in mouse and human cell lines at both TC and non-TC targets with efficiency reaching up to 99.2%. Using mitoCBE3.2, single clones containing homoplasmic mtDNA mutations or premature stop codons associated with human diseases were generated, and the functions of these mutations have been evaluated upon the treatment of reactive oxygen species inducers. Importantly, mitochondria harboring these homoplasmic mutations were transplanted into wild-type cells, enabling precise base conversions, without the risk of nuclear gene off-targets. Thus, we have engineered an efficient mitoCBE using BgDddA, facilitating mitochondrial disease modeling and potential mutation correction with the aid of mitochondrial transplantation.