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

Bacterial DNA deaminase toxins are weapons used in bacterial warfare, and they are useful tools in base editing, epigenetics analyses, and genomic footprinting applications. Our previous studies revealed structural bases for 5'-TC-specific cytosine deamination in double-stranded (ds)DNA by DddA, which represents the bacterial deaminase toxin family (BaDTF) 1, and sequence context-independent single-stranded (ss)DNA cytosine deamination by SsdA, which represents BaDTF2. Here, we show that a representative member of BaDTF3 from Taylorella equigenitalis, DddB, deaminates cytosines specifically in dsDNA, but with limited sequence context dependence. Our crystal structure of DddB bound to dsDNA reveals a unique mechanism of substrate engagement, in which a helix-hairpin-helix motif inserted into the minor groove of an underwound dsDNA promotes flipping of the target cytosine into the enzyme active site. DddB also makes key contacts in the DNA major groove, including hydrogen bonds with a base at +2 position 3' of the target cytosine. We further show that DddB-derived cytosine base editor (BdCBE) 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.