Extended Data Fig. 3: ABE7.10 multi-residue reversion variants evaluated in mammalian cells.
From: Precise, minimally evolved adenine base editors generated through mutation reversion analysis
a. Table identifying ABE7.10 multi-residue combination reversions tested. b-c. Activity of combination reversion editors as measured by episomal reporter. Plasmids harboring ABE7.10 or ABE variants listed in a, were transfected into HEK293T cells alongside the EGFP A111V TAC fluorescent turn-on reporter and appropriate gRNA. Cells incubated for 72 h prior to analysis via flow cytometry where BE activity was calculated as described in Fig. 2a. Activity is reported as fold-change in BE activity (b) or fold-change in EGFP MFI, with respect to ABE7.10 (c). Dotted horizontal line shows normalized activity of ABE7.10. Bars and error bars represent the average and associated standard deviation for n = 5 biological replicates for ABE7.10 and C1, n = 3 biological replicates for all others. Data were analyzed using one-way ANOVA with Holm-Šídák multiple comparison test to ABE7.10 with significant p-values reported (p ≥ 0.05 not significant, not indicated) d. Heat map showing activity of a subset of combination reversion editors (those indicated by highlighted cells in a) against three endogenous genomic sites. ABE plasmids were transfected into HEK293T cells alongside one of three gRNA plasmids (protospacers shown in Extended Data Fig. 1a) and allowed to incubate for 72 h. Genomic DNA was then harvested, target amplicons amplified, and BE activity (percent of reads with target A•T converted to G•C) analyzed via NGS. Activity is reported as a fold-change in BE activity for a given editor with respect to ABE7.10 in a given replicate. Heat map colors represent the average fold-change in activity for n = 3 biological replicates, with greyscale indicative of activity less than ABE7.10, white indicative of activity on par with ABE7.10, and cyan indicative of activity greater than ABE7.10.
