Precise, minimally evolved Adenine Base Editors through mutation reversion analysis

Initial development of adenine base editors (ABEs), which facilitate A-T to G-C base pair changes in the genome, used directed evolution to install 14 mutations into the wild-type deaminase TadA, producing the first-of-its-kind editor ABE7.10. Here, we study the installed mutation's impact on TadA fitness using comprehensive reversion analysis and apply our results to engineer more efficient, precise editors. By measuring activity in both mammalian tissue culture and E.coli bacterial host systems, we categorize mutations as critical, dispensable, or host-dependent. We show that up to five mutations can be reverted back to wild-type, generating minimally evolved ABEs (ME-ABEs). ME-ABEs demonstrate narrow editing windows (similar to that of ABE7.10), enhanced on-target editing (matching activities of the high activity editor variants ABE8e and ABE8.20 in most sequence contexts), and exhibit low levels of gRNA-dependent and -independent off-target activity. ME-ABEs efficiently target six sites of clinical interest that had previously proved challenging to edit with ABE7.10, ABE8e, or ABE8.20.