Structural insights into DdCBE in action enable high-precision mitochondrial DNA editing

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• DddA-derived cytosine base editors (DdCBE) target mitochondrial DNA (mtDNA) using TALE arrays and DddA deaminase.
• Lack of structural data for DdCBE hinders optimization for precise therapeutic applications.

Purpose of the Study:

• To determine the structures of DdCBE targeting mtDNA loci.
• To develop a predictive model for DdCBE editing outcomes.
• To engineer a highly accurate DdCBE (aDdCBE) for precise mtDNA editing.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine DdCBE structures.
• Systematic design of spacers and analysis of editing data.
• Structure-guided engineering of the DddA deaminase.

Main Results:

• Determined cryo-EM structures of DdCBE at native mitochondrial gene loci.
• Developed WinPred, a model predicting DdCBE editing outcomes and guiding design.
• Engineered aDdCBE with a narrowed editing window (2-3 nt) and minimal off-target edits.
• Successfully introduced a Leber hereditary optic neuropathy (LHON) mutation into mtDNA using aDdCBE.

Conclusions:

• Mechanistic understanding of DdCBE function was achieved.
• WinPred and aDdCBE are valuable tools for modeling and correcting mtDNA mutations.
• This work advances the potential of base editing for mitochondrial diseases.