4.8 Article

Precision mitochondrial DNA editing with high-fidelity DddA-derived base editors

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NATURE BIOTECHNOLOGY
卷 41, 期 3, 页码 378-+

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NATURE PORTFOLIO
DOI: 10.1038/s41587-022-01486-w

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Protein engineering has minimized the off-target activity of DddA-derived base editors (DdCBEs), allowing efficient and precise editing of mitochondrial DNA in human cells. This technology may provide a new approach for therapeutic correction of gene mutation diseases.
Bacterial toxin DddA-derived cytosine base editors (DdCBEs)-composed of split DddA(tox) (a cytosine deaminase specific to double-stranded DNA), custom-designed TALE (transcription activator-like effector) DNA-binding proteins, and a uracil glycosylase inhibitor-enable mitochondrial DNA (mtDNA) editing in human cells, which may pave the way for therapeutic correction of pathogenic mtDNA mutations in patients. The utility of DdCBEs has been limited by off-target activity, which is probably caused by spontaneous assembly of the split DddA(tox) deaminase enzyme, independent of DNA-binding interactions. We engineered high-fidelity DddA-derived cytosine base editors (HiFi-DdCBEs) with minimal off-target activity by substituting alanine for amino acid residues at the interface between the split DddA(tox) halves. The resulting domains cannot form a functional deaminase without binding of their linked TALE proteins at adjacent sites on DNA. Whole mitochondrial genome sequencing shows that, unlike conventional DdCBEs, which induce hundreds of unwanted off-target C-to-T conversions in human mtDNA, HiFi-DdCBEs are highly efficient and precise, avoiding collateral off-target mutations, and as such, they will probably be desirable for therapeutic applications. Off-target activity of DddA-derived base editors is minimized through protein engineering.

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