Fig. 2: Attenuating DarT2 alleviates cytotoxicity while mediating efficient and flexible gene editing in E. coli.
a, Predicted structure of EPEC DarT2. Tested substitutions are in blue. aa, amino acid. b, Impact of tested substitutions on cell viability and kanamycin resistance frequency. The experimental setup can be found in Fig. 1f. Bars and error bars represent the geometric mean ± s.d. of three independent experiments started from separate transformations. Dots represent individual measurements. c, Experimental setup for assessing growth defects caused by editor expression in a ΔrecA strain of E. coli. d, Impact of expressing an append editor with the indicated DarT2 mutant with NT sgRNA in the ΔrecA strain of E. coli. Endpoint OD600 measurements were taken after 12 h of culturing. Growth curves can be found in Supplementary Fig. 2. Bars and error bars represent the mean ± s.d. of three independent experiments started from separate transformations. Dots represent individual measurements. e, Impact of deleting DNA repair genes on cell viability and kanamycin resistance frequency. Bars and error bars represent the geometric mean ± s.d. of three independent experiments starting from separate transformations. Dots represent individual measurements. f, Introducing sequence replacements with ADPr-TA editing. g, Introducing deletions with ADPr-TA editing. h, Introducing insertions with ADPr-TA editing. f–h, Left, size and location of substitutions (orange bar), deletions (dashed box) or insertions (green bar). Numbers (for example, +5/−12) indicate the edited region in relation to the ADP-ribosylated thymine. Right, fraction of screened colonies containing the intended edit. Each bar represents one of two biological replicates starting from separate transformations, screening at least eight colonies per biological replicate. Examples of Sanger sequencing chromatograms indicating edited, mixed and unedited colonies can be found in Extended Data Fig. 4.
