Extended Data Fig. 6: Characterization of ABE8 multi-residue reversion variants in mammalian cells.
From: Precise, minimally evolved adenine base editors generated through mutation reversion analysis
a. Naming legend for ABE8-derived reversion editors. b. HEK293T cells were transfected with a single ABE plasmid containing either one of the editors listed in part a, ABE7.10, ABE8e, or ABE8.20, along with one of 27 gRNAs plasmids targeting an endogenous site or a non-targeting (NT) control (See Supplementary Fig. 18). After a 72-h incubation, genomic DNA was harvested, target sites were amplified, and sequences analyzed by NGS. Absolute editing values (percent of reads with a given A•T pair converted to G•C) were normalized to the absolute editing of ABE7.10 at the H2.0 A5 site (the maximally edited A in all experiments) within a given replicate. Editing activity was examined in n = 3 biological replicates for all ABE8 reversion editors, and n = 6 for ABE7.10, ABE8e and ABE8.20. Horizontal dotted line shows the 15% cut-off used to determine the editing window for each editor. c-d. Normalized editing activities of a subset of ABE8-derived editors sorted by 5’ (c) or 3’ (d) motif are shown. Target A’s were limited to ‘editable A’s’ - those A’s in positions where the average normalized editing across all sites for a given editor was greater than 15%. Legend highlights inclusive positions for each editor, resulting in 19 sites interrogated between positions 4–7, 34 sites between positions 3–11, and 26 sites between positions 3–8. The normalized average editing activity for individual sites is shown by points, with each site measured in n = 3 biological replicates for all ABE8-derived reversion editors, and n = 6 for ABE7.10, ABE8e, and ABE8.20. Whiskers show the minimum and maximum average editing for a given editor/motif, boxes show quartiles, and lines show median editing activity. e. Editing activity of a subset of ABE8e-derived combination reversion editors measured by orthogonal R-loop off-target assay, as described in Fig. 4d. x-y plot indicates the average off-target activity for the most edited A position across three independent off-target sites, and average on-target activity at the most edited A position across two independent on-target sites, accounting for n = 3 biological replicates with error bars showing propagated standard deviation.
