Discovered that ABEmax generates low but widespread off-target RNA editing (~15,000 A-to-I edits across transcriptome). Developed V106W mutation in TadA* deaminase that reduces RNA editing 7.2-fold while maintaining DNA editing efficiency. ABEmaxAW (TadA E59A + TadA* V106W) provides 2.7-fold improvement in off-target DNA editing and 3.7-fold reduction in indel formation. Both wild-type TadA and evolved TadA* contribute to RNA editing, requiring mutations in both domains for optimal specificity. V106W acts by sterically blocking accommodation of RNA 2'-hydroxyl groups, decoupling DNA and RNA editing activities.
Analysis and minimization of cellular RNA editing by DNA adenine base editors
Adenine base editors were reported to edit DNA cleanly, but off-target RNA editing was unexamined. Deep sequencing showed ABEmax causes widespread low-level RNA editing, prompting structure-guided TadA mutations. The ABEmaxAW variant cut RNA edits and off-target DNA editing.
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Key findings
Abstract
Adenine base editors (ABEs) enable precise and efficient conversion of target A•T base pairs to G•C base pairs in genomic DNA with a minimum of by-products. While ABEs have been reported to exhibit minimal off-target DNA editing, off-target editing of cellular RNA by ABEs has not been examined in depth. Here, we demonstrate that a current ABE generates low but detectable levels of widespread adenosine-to-inosine editing in cellular RNAs. Using structure-guided principles to design mutations in both deaminase domains, we developed new ABE variants that retain their ability to edit DNA efficiently but show greatly reduced RNA editing activity, as well as lower off-target DNA editing activity and reduced indel by-product formation, in three mammalian cell lines. By decoupling DNA and RNA editing activities, these ABE variants increase the precision of adenine base editing by minimizing both RNA and DNA off-target editing activity.