Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Mitochondrial DNA (mtDNA) editing is crucial for understanding and treating mitochondrial genetic disorders.
• Current methods, like DddA-derived cytosine base editors (DdCBEs), are limited to specific C-to-T conversions.
• A broader range of mutations is needed for comprehensive disease modeling and therapeutic development.

Purpose of the Study:

• To develop a novel mtDNA editing system capable of inducing targeted A-to-G base conversions in human mitochondria.
• To engineer transcription-activator-like effector (TALE)-linked deaminases (TALEDs) for precise mtDNA modification.

Main Methods:

• Designed custom TALE DNA-binding arrays linked to DddA variants (full-length or split) and an engineered E. coli TadA deaminase.
• Introduced TALEDs into human cells to assess their efficiency and specificity for mtDNA editing.
• Analyzed editing frequencies at various target sites within mitochondrial genes.

Main Results:

• TALEDs successfully induced targeted A-to-G base conversions in human mtDNA.
• High editing efficiencies, up to 49%, were achieved at 17 distinct target sites across multiple mitochondrial genes.
• The system demonstrated effectiveness in human cells, expanding the scope of possible mtDNA mutations.

Conclusions:

• TALEDs represent a significant advancement in mtDNA editing technology, enabling A-to-G conversions.
• This new capability enhances the potential for accurate disease modeling and future gene therapies for mitochondrial disorders.
• The development broadens the mutational landscape accessible for mitochondrial research and therapeutic strategies.