Fig. 4: Characterization of ABE7.10 multi-residue reversion variants in mammalian cells.
From: Precise, minimally evolved adenine base editors generated through mutation reversion analysis
a, Renaming legend for a set of ABE7.10 reversion editors. HEK293T cells were transfected with a single ABE plasmid containing either one of the editors listed, ABE7.10, ABE8e or ABE8.20, along with 1 of 27 gRNA plasmids targeting an endogenous site or an NT control (Supplementary Fig. 18). After a 72-h incubation, genomic DNA was collected, target sites were amplified and sequences were analyzed by NGS. Absolute editing values (percentage 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 ABE7.10 reversion editors, and n = 6 for ABE7.10, ABE8e and ABE8.20. b, Normalized editing scores for all 205 A’s were sorted by position and further averaged across all gRNAs to identify editing windows. The number of A’s considered for each position is given below the x-axis. The horizontal dotted line shows the 15% cutoff used to determine editing windows. c, Sequences of all on-target gRNAs and their predicted gRNA-dependent DNA off-target sites. The most editable A base is shown in bold. Mismatches between on-target and off-target site are shown in red. HEK293T cells were transfected with plasmids encoding each of the ABE variants indicated and an on-target gRNA. Both the on-target and off-target loci were amplified and evaluated for A•T to G•C editing with NGS 72 h after transfection. Bars show average editing measured for n = 3 biological replicates, with error bars showing standard deviation. Dashed horizontal lines indicate 0.1% editing activity, demarcating the limit of reliable detection. d, The orthogonal R-loop assay compares ME-ABE gRNA-independent off-target activity with canonical editors. Briefly, this assay uses two orthogonal Cas constructs; Sp-Cas9 ABE and Sp-gRNA edit an on-target site, while Sa-dCas9 and Sa-gRNA expose an off-target ssDNA R-loop for errant deamination (cartoon, above). HEK293T cells were transfected with plasmids encoding an ABE, an on-target Sp-gRNA, Sa-dCas9, and an off-target Sa-gRNA. After 72 h, genomic DNA was collected and on-target and off-target loci were sequenced. The x–y plot indicates the average off-target activity for the most edited A across three independent off-target sites, and average on-target activity at the most edited A across two independent on-target sites, accounting for n = 3 biological replicates with error bars showing propagated standard deviation.
