Genetic enhancement

Genome

DNA glycosylase

Cytosine deaminase

Molecular biology

Biochemistry

Mitochondrial DNA

DNA damage

Human mitochondrial genetics

Biology

Gene

Genome editing

Cytosine

Uracil-DNA glycosylase

Computational biology

DNA ligase

Genetics

Nature Biotechnology

Bacterial toxin DddA-derived cytosine base editors (DdCBEs)-composed of split DddAtox (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 DddAtox 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 DddAtox 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.

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