A rationally engineered cytosine base editor retains high on-target activity while reducing both DNA and RNA off-target effects

Cytosine base editors (CBEs) offer a powerful tool for correcting point mutations, yet their DNA and RNA off-target activities have caused concerns in biomedical applications. We describe screens of 23 rationally engineered CBE variants, which reveal mutation residues in the predicted DNA-binding site can dramatically decrease the Cas9-independent off-target effects. Furthermore, we obtained a CBE variant—YE1-BE3-FNLS—that retains high on-target editing efficiency while causing extremely low off-target edits and bystander edits. Structural and biochemical insights help engineer a cytosine base editor variant that possesses improved on-target activity with minimal DNA and RNA off-target editing.