CRISPR-free base editors with enhanced activity and expanded targeting scope in mitochondrial and nuclear DNA

The all-protein cytosine base editor DdCBE uses TALE proteins and a double-stranded DNA-specific cytidine deaminase (DddA) to mediate targeted C center dot G-to-T center dot A editing. To improve editing efficiency and overcome the strict T (C) under bar sequence-context constraint of DddA, we used phage-assisted non-continuous and continuous evolution to evolve DddA variants with improved activity and expanded targeting scope. Compared to canonical DdCBEs, base editors with evolved DddA6 improved mitochondrial DNA (mtDNA) editing efficiencies at T (C) under bar by 3.3-fold on average. DdCBEs containing evolved DddA11 offered a broadened H (C) under bar (H = A, C or T) sequence compatibility for both mitochondrial and nuclear base editing, increasing average editing efficiencies at A (C) under bar and C (C) under bar targets from less than 10% for canonical DdCBE to 15-30% and up to 50% in cell populations sorted to express both halves of DdCBE. We used these evolved DdCBEs to efficiently install disease-associated mtDNA mutations in human cells at non-T (C) under bar target sites. DddA6 and DddA11 substantially increase the effectiveness and applicability of all-protein base editing.

Automated summary

Evolved DddA variants enhance the editing efficiency and expand the target compatibility of the all-protein base editor DdCBE, allowing efficient editing at non-T(C) target sites and broadening the sequence compatibility for mitochondrial and nuclear base editing.