AbstractBase 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 HEK293... More
AbstractBase 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 Y 4 A 5 N 6 , which can accurately edit the A 5 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.This work developed a novel peptide insertion strategy to enhance the performance of base editors. Through changing the composition and position of the inserted peptide, peptide inserted base editors are engineered to exhibit robust on target editing activity and improved precision. Meanwhile, the harmful bystander editing and off target effects are significantly reduced, thereby enhancing the security and reliability of the application.
