Cytosine base editors (CBEs) use the rat apolipoprotein-B-editing enzyme, catalytic polypeptide-1 APOBEC1 (rAPOBEC1), a cytidine deaminase binding to singlestranded DNA (ssDNA) and is fused to nCas9, to convert cytosine to thymine. CBEs ofer a powerful tool for correcting point mutations, yet their DNA and RNA of-target activities have caused concerns in biomedical applications. We described screens of 23 rationally engineered CBE variants, which revealed that mutation residues in the predicted DNA-binding site can dramatically decrease the Cas9-independent of-target efects. Furthermore, we obtained a CBE variant, YE1-BE3-FNLS, which retains high on-target editing efciency while causing extremely low of-target edits and bystander edits. Our work illustrates examples of how the of-target efects of base editors can be minimized via biologicalinsight-driven engineering to extend the utility of these powerful gene-editing tools for both research and therapeutic applications.

The on-target C-to-T editing efciency of engineered CBEs at 21 target sites