Description: Bacterial toxins represent a vast reservoir of biochemical diversity that can be repurposed for biomedical applications. Many toxins function in interbacterial antagonism, yet their modes of action remain largely unknown. Here, we report the discovery, structure, biochemical characterization, and application of DddA, an interbacterial toxin that catalyzes the unprecedented deamination of cytidines within double-stranded DNA. All previously described cytidine deaminases, including those used in base editing, operate on single-stranded DNA and thus when used for genome editing require unwinding of double-stranded DNA by macromolecules such as CRISPR-Cas9 complexed with a guide RNA. The difficulty of delivering guide RNAs into the mitochondria has thus far precluded base editing in mitochondrial DNA (mtDNA). The ability of DddA to deaminate double-stranded DNA raises the possibility of RNA-free precision base editing, rather than simple elimination of targeted mtDNA copies following double-strand DNA breaks. We engineered split-DddA halves that are non-toxic and inactive until brought together to close proximity by adjacent DNA-binding proteins. Fusions of the split-DddA halves, TALE array proteins, and uracil glycosylase inhibitor resulted in RNA-free DddA-derived cytosine base editors (DdCBEs) that efficiently and with high DNA sequence specificity catalyze CG-to-TA conversions at programmable sites within mtDNA or nuclear DNA in human cells. We used DddA-mediated base editing to model disease-associated mtDNA mutations in human cell lines, resulting in changes in rates of respiration and oxidative phosphorylation. CRISPR-free, DddA-mediated base editing enables precision editing of mtDNA, with important basic science and biomedical implications.

Data type: raw sequence reads

Sample scope: Multispecies

Last updated: 2020-01-24