CRISPR DNA base editors with reduced RNA off-target and self-editing activities

Cytosine or adenine base editors (CBEs or ABEs) can introduce specific DNA C-to-T or A-to-G alterations(1–4). However, we recently demonstrated that they can also induce widespread guide RNA-independent RNA base edits(5) and created SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that have reduced unwanted RNA editing activity(5). Here, we describe structure-guided engineering of SECURE-ABE variants with reduced off-target RNA editing and comparable on-target DNA activities that are also among the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. We also tested CBEs with cytidine deaminases other than APOBEC1 and found that the human APOBEC3A (hA3A)-based CBE induces substantial RNA base edits, whereas an enhanced A3A (eA3A)-CBE(6), human activation-induced cytidine deaminase (hAID)-CBE(7), and the petromyzon marinus cytidine deaminase (pmCDA1)-based CBE Target-AID(4) induce reduced RNA edits. Finally, we found that CBEs and ABEs that exhibit RNA off-target editing activity can also self-edit their own transcripts, thereby leading to heterogeneity in base editor coding sequences.