Fig. 3: ABE7.10 single-residue reversion variants evaluated in bacterial cells.
From: Precise, minimally evolved adenine base editors generated through mutation reversion analysis
a, Base editing in bacteria was measured using an antibiotic survival assay. Sequence encoding a dead chloramphenicol acetyltransferase (CamR) requiring an A•T to G•C correction to restore antibiotic resistance was placed on a carrier plasmid, pCamR. Two versions of this were developed: one with the deactivating mutation H193Y, where the target A had the sequence motif TA6G, and the other a Q122* mutation where the target A had the sequence motif TA6C. S1030 E. coli harboring pCamR were transformed with various ABE7.10 single-residue reversion plasmids optimized for bacterial editing. ABE plasmids also contained appropriate gRNAs for targeting the mutation in CamR to reactivate chloramphenicol resistance, as well as a theophylline-inducible riboswitch to temporally control expression of the base editor. Bacteria were allowed to grow overnight in liquid culture with the base editor expressed, before splitting the culture and dilution plating each sample on +/− chloramphenicol plates (+/− chlor). Colony counts on +/− chlor plates were used to determine the survival rate of a given single-residue reversion editor in bacteria. b,c, Heat maps showing average fold-change values in survival rate for single-residue reversion editors against the H193Y TAG reporter (b) and Q122* TAC reporter (c). d,e, Fold change in survival rates relative to ABE7.10 for bacterial base editing activity assay with the H193Y TAG (d) and Q122* TAC reporters (e). Fold-change survival rate measurements were calculated for n = 9 plating pairs per sample with ROUT 1% outliers removed, and all remaining fold-change values plotted individually. The box plot center illustrates median fold change, hinges show quartiles and whiskers show minimum and maximum fold-change values.
