Phage-assisted evolution of highly active cytosine base editors with enhanced selectivity and minimal sequence context preference

• DOI: 10.1038/s41467-024-45969-7
• Publication Date: 2024-02-24T11:02:20Z
• Authors: Emily Zhang; Monica E. Neugebauer; Nicholas A. Krasnow; David R. Liu
• Keywords: Cytosine; Stop codon

Abstract TadA-derived cytosine base editors (TadCBEs) enable programmable C•G-to-T•A editing while retaining the small size, high on-target activity, and low off-target activity of TadA deaminases. Existing TadCBEs, however, exhibit residual A•T-to-G•C at certain positions lower efficiencies some sequence contexts with non-SpCas9 targeting domains. To address these limitations, we use phage-assisted evolution to evolve CBE6s from a TadA-mediated dual adenine editor, discovering mutations N46 Y73 in that prevent improve expanded sequence-context compatibility, respectively. In E. coli , CBE6 variants offer no detected any context. human cells, broad Cas domain compatibility retain despite exceeding BE4max previous TadCBEs efficiency. Finally, show selectivity is well-suited for therapeutically relevant stop codon installation without creating unwanted missense editing.