Engineering RsDddA as mitochondria base editor with wide target compatibility and enhanced activity

Double-stranded DNA-specific cytidine deaminase (DddA) based editors hold great promise for applications in bio-medical research, medicine, and biotechnology. Strict sequence preference on spacing region presents a challenge for DddA editors to reach their full potential. To overcome this sequence-context constraint, we analyzed metagenomic datasets and identified DddAtox from Ruminococcus sp (RsDddA). We engineered RsDddA for mitochondria base editing experiments in mammalian cell lines and demonstrated RsDddA derived cytosine base editors (RsDdCBE) offered an NC sequence compatibility and exhibited robust editing efficiency. Moreover, our results suggested average frequencies of mtDNA-wide off-target editing arising from RsDdCBE were comparable to canonical DdCBE and its variants. We believe by the suitable application of RsDdCBE, it is a powerful tool for base editing and provides a DddAtox for mutations to further bio-medicine research.