Structural basis for double-stranded DNA cytosine deamination by BaDTF3 and its application in mitochondrial genome editing

Bacterial deaminase toxin family (BaDTF) proteins are weapons used in bacterial warfare, and they are useful tools in base editing, epigenetics analyses, and genomic footprinting applications. Our previous studies revealed the mechanisms of 5'-TC-specific cytosine deamination in double-stranded (ds)DNA by DddA from BaDTF1 and sequence context-independent single-stranded (ss)DNA cytosine deamination by SsdA from BaDTF2. Here, we show that a representative member of BaDTF3, DddB, deaminates cytosines specifically in dsDNA, but with a broad sequence context preference. Our crystal structure of DddB bound to dsDNA reveals a distinct mechanism of substrate engagement, in which a helix-hairpin-helix motif inserted into the minor groove of dsDNA promotes flipping of the target cytosine into the enzyme active site. Based on the structural information, we generate both monomeric and split DddB-derived cytosine base editors (BdCBE) and demonstrate that they can perform CRISPR-free mitochondrial base editing in human cells, with an expanded targeting scope compared to the DddA-derived DdCBEs. Our studies highlight the mechanistic diversity among BaDTF proteins and expand the repertoire of dsDNA deaminase enzymes for genome editing and other applications.