Coiled-coil heterodimer-mediated split base editing systems enable flexible and robust nucleotide substitutions

Base editors (BEs) enable precise base substitutions, but their size exceeds the packaging capacity of adeno-associated virus (AAV), impeding in vivo applications. Here we design a split BE system that recruits deaminases to Cas9 nickase via coiled-coil heterodimers, resulting in various coiled-coil heterodimers-mediated base editors (CC-BEs), including cytidine base editor (CC-CBE), adenine base editor (CC-ABE), and their derivatives. We reveal that CC-BEs maintain and even improve the editing efficiency of the original unsplit BEs across various cell types and editing scopes, achieving maximum enhancements of 9.6-fold in human immortalized cells and 12.4-fold in primary somatic cells for CC-CBE. Using CC-ABE, we validate in vivo editing efficiency and successfully achieve A-to-G conversion in the Pcsk9 and Dmd genes via dual-AAV vectors in mice. Altogether, we develop a simple and universal strategy to address the challenges posed by the large size of BEs without compromising editing efficiency for base substitutions in vivo. Base editors (BEs) enable precise base substitutions but are limited by their large size. Here, the authors engineer a split BE system utilizing coiled-coil heterodimers (CC-BE) and demonstrate that CC-BEs maintain or even enhance efficiency, enabling gene therapy applications via dual AAV.