Developing mitochondrial base editors with diverse context compatibility and high fidelity via saturated spacer library.

DddA-derived cytosine base editors (DdCBEs) greatly facilitated the basic and therapeutic research of mitochondrial DNA mutation diseases. Here we devise a saturated spacer library and successfully identify seven DddA homologs by performing high-throughput sequencing based screen. DddAs of Streptomyces sp. BK438 and Lachnospiraceae bacterium sunii NSJ-8 display high deaminase activity with a strong GC context preference, and DddA of Ruminococcus sp. AF17-6 is highly compatible to AC context. We also find that different split sites result in wide divergence on off-target activity and context preference of DdCBEs derived from these DddA homologs. Additionally, we demonstrate the orthogonality between DddA and DddIA, and successfully minimize the nuclear off-target editing by co-expressing corresponding nuclear-localized DddIA. The current study presents a comprehensive and unbiased strategy for screening and characterizing dsDNA cytidine deaminases, and expands the toolbox for mtDNA editing, providing additional insights for optimizing dsDNA base editors.

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

Double-stranded DNA-specific cytidine deaminase (DddA) base 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 a protein dataset and identified a novel DddAtox homolog from Ruminococcus sp. AF17-6 (RsDddA). We engineered RsDddA for mitochondrial base editing in a mammalian cell line and demonstrated RsDddA-derived cytosine base editors (RsDdCBE) offered a broadened NC sequence compatibility and exhibited robust editing efficiency. Moreover, our results suggest the average frequencies of mitochondrial genome-wide off-target editing arising from RsDdCBE are comparable to canonical DdCBE and its variants.