Engineering TALE-linked deaminases to facilitate precision adenine base editing in mitochondrial DNA

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Mitochondrial DNA (mtDNA) editing holds promise for genetic disorders.
• Current base editors like DddA-derived cytosine base editors (DdCBEs) and transcription activator-like effector (TALE)-linked deaminases (TALEDs) enable targeted mtDNA modifications.
• TALEDs, specifically A-to-G editors, have shown significant off-target RNA edits in human cells.

Purpose of the Study:

• To engineer TALED variants with reduced off-target RNA edits.
• To improve the safety and efficacy of base editing for mitochondrial applications.
• To develop a more precise tool for modeling mitochondrial diseases and exploring therapeutic strategies.

Main Methods:

• Engineering the substrate-binding site of TadA8e, the deaminase component of TALEDs.
• Developing TALED variants with fine-tuned deaminase activity.
• Assessing transcriptome-wide off-target edits, mtDNA mutations, and bystander edits.
• Evaluating cytotoxicity and developmental effects in mouse embryos.
• Generating mouse models with pathogenic mtDNA mutations.

Main Results:

• Engineered TALED variants reduced transcriptome-wide off-target edits by over 99%.
• Off-target mtDNA mutations and bystander edits at the target site were minimized.
• TALED variants exhibited reduced cytotoxicity and did not cause developmental arrest in mouse embryos.
• Mice with pathogenic mtDNA mutations (Leigh syndrome) were successfully generated and displayed reduced heart rates.

Conclusions:

• Engineered TALED variants offer a safer and more precise approach to mitochondrial base editing.
• These improved tools are suitable for modeling mitochondrial genetic disorders and developing therapeutic interventions.
• The developed TALED variants overcome limitations of previous base editors, enabling in vivo applications with reduced risks.