An engineered mitoCBE facilitates efficient mitochondrial DNA editing and modified mitochondrial transfer.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Bacterial cytosine deaminases fused to transcription-activator-like effectors (TALE) have been explored for mitochondrial DNA (mtDNA) base editing.
• Existing systems show limited efficiency and potential for nuclear off-targeting.

Purpose of the Study:

• To engineer a novel and efficient cytosine base editor for mitochondrial DNA (mtDNA).
• To develop a tool for precise induction of disease-associated mtDNA mutations and explore therapeutic strategies.

Main Methods:

• Identified and engineered a DddA ortholog from Burkholderia gladioli (BgDddA) into a mitochondrial cytosine base editor (mitoCBE).
• Optimized the editor (mitoCBE3.2) through specific substitutions for enhanced activity.
• Utilized the editor to induce homoplasmic mtDNA mutations in cell lines and evaluated their functional impact.
• Employed mitochondrial transplantation to demonstrate targeted base conversion without nuclear off-target effects.

Main Results:

• The engineered BgDddA-based mitoCBEs exhibited higher C•G-to-T•A editing frequencies compared to canonical DdCBEs.
• Fusion with transactivator Rta improved editing efficiency up to 6.4-fold at non-TC targets.
• mitoCBE3.2 achieved up to 99.2% editing efficiency for disease-associated mtDNA mutations in human and mouse cell lines.
• Successfully generated disease-specific homoplasmic mtDNA mutations and demonstrated their functional consequences.
• Mitochondrial transplantation enabled precise base conversions without nuclear off-target concerns.

Conclusions:

• Engineered an efficient BgDddA-based mitoCBE for precise mtDNA base editing.
• The developed system facilitates accurate mitochondrial disease modeling.
• Mitochondrial transplantation offers a potential strategy for mutation correction with high specificity.