Homo sapiens Raw sequence reads

Cytosine or adenine base editors (CBEs or ABEs) can introduce specific DNA C-to-T or A-to-G alterations. However, we recently demonstrated that they can also induce widespread RNA-independent RNA base edits and created SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that have reduced unwanted RNA editing activity5. 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 the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. We also tested CBEs with cytidine deaminases other than APOBEC1 and found that human APOBEC3A (hA3A) cytidine deaminase CBE induces substantial RNA base edits, whereas our enhanced A3A (eA3A) cytidine deaminase CBE, human activation-induced cytidine deaminase (hAID) CBE induce reduced RNA edits. Finally, we found that CBEs and ABEs that exhibit off-target editing activity can also self-edit their own transcripts leading to a substantial heterogeneity in base editor coding sequences.