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

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Bacterial toxin DddA-derived cytosine base editors (DdCBEs) enable mitochondrial DNA (mtDNA) editing.
• Off-target activity in DdCBEs limits their therapeutic utility.
• Spontaneous assembly of split DddAtox deaminase is a likely cause of off-target effects.

Purpose of the Study:

• To engineer high-fidelity DdCBEs (HiFi-DdCBEs) with reduced off-target activity.
• To improve the safety and precision of base editing in human mtDNA.
• To enable therapeutic correction of pathogenic mtDNA mutations.

Main Methods:

• Engineered DdCBEs by substituting alanine residues at the interface of split DddAtox halves.
• Designed TALE DNA-binding proteins to recruit the modified deaminase.
• Performed whole mitochondrial genome sequencing to assess editing efficiency and off-target mutations.

Main Results:

• HiFi-DdCBEs exhibit minimal off-target activity compared to conventional DdCBEs.
• The engineered editors avoid unwanted C-to-T conversions in human mtDNA.
• HiFi-DdCBEs demonstrate high efficiency and precision in mtDNA editing.

Conclusions:

• HiFi-DdCBEs offer a precise and safe method for mtDNA editing.
• The engineered base editors hold promise for therapeutic applications.
• This advancement could lead to the correction of genetic mitochondrial diseases.