Unconstrained Precision Mitochondrial Genome Editing with αDdCBEs.

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• DddA-derived cytosine base editors (DdCBEs) are crucial for C•G-to-T•A conversions in mitochondrial DNA (mtDNA).
• A 5'-T constraint limits DdCBE accessibility to over 150 human mtDNA loci.
• Previous methods to bypass this constraint yielded suboptimal specificity.

Purpose of the Study:

• To challenge the 5'-T constraint in DdCBE-mediated mtDNA editing.
• To expand the range of editable motifs in mitochondrial DNA.
• To develop more efficient and specific base editing technologies for mtDNA.

Main Methods:

• Engineered DdCBEs (αDdCBEs) with TALE proteins recognizing all 5' bases were generated.
• The activity and specificity of αDdCBEs were evaluated at various mtDNA loci.
• Compatibility with DddA_tox variants (DddA6, DddA11) and TALE shifting was assessed.

Main Results:

• αDdCBEs demonstrated efficient and specific mtDNA editing across diverse loci, irrespective of the 5' base.
• αDdCBEs outperformed canonical DdCBEs in activity and specificity.
• αDdCBEs are compatible with enhanced DddA_tox variants and TALE shifting for optimized editing.

Conclusions:

• αDdCBEs enable unconstrained, efficient, and specific mitochondrial base editing.
• This technology broadens the scope of potential therapeutic targets for mtDNA disorders.
• αDdCBEs represent a significant advancement in mitochondrial genome engineering.