Engineering of Peptide‐Inserted Base Editors with Enhanced Accuracy and Security

Abstract Base editors are effective tools for introducing base conversions without double‐strand breaks, showing broad applications in biotechnological and clinical areas. However, their non‐negligible bystander mutations and off‐target effects have raised extensive safety concerns. To address these issues, a novel method is developed by inserting specific peptide fragments into the substrate binding pocket of deaminases in base editors to modify these outcomes. It is validated that the composition and position of the inserted peptide can significantly impact the performance of A3A‐based cytosine base editor and TadA‐8e‐based adenine base editor, leading to improved editing activity and precision in human HEK293T cells. Importantly, the TadA‐8e variant with DPLVLRRRQ peptide inserted behind S116 residue showed a strong motif preference of Y₄A₅N₆, which can accurately edit the A₅ base in targeted protospacer with minimized bystander and off‐target effects in DNA and RNA‐level. By summarizing the regularity during engineering, a set of systematic procedures is established, which can potentially be used to modify other types of base editors and make them more accurate and secure. In addition, the peptide insertion strategy is also proven to be compatible with traditional amino acid changes which have been reported, exhibiting excellent compatibility.