Extended Data Fig. 3: Targeted base editing of the E. coli genome in vitro after DNA payload transformation and antibiotic selection.
From: In situ targeted base editing of bacteria in the mouse gut
a) Adenine base editing (ABE8e, plasmid p2325) of MG1655-mCherry. The experiment was performed in the presence (48 colonies analyzed) or absence (2 colonies analyzed) of a guide RNA targeting the amino acid triad forming the fluorophore of mCherry (tripeptide: M71, Y72, G73). b) Cytosine base editing (CBE = evoAPOBEC1-nCas9-UGI, plasmid p2326) of mCherry on MG1655-mCherry genome. The experiment was performed in presence (48 colonies analyzed) or absence (2 colonies analyzed) of a guide RNA inserting a stop codon at position Q114* into mCherry. Fluorescence of individual colonies was measured by flow cytometry (excitation: 561 nm, emission: 620/15 BP; Attune NxT Thermo Scientific). Dots represent single colonies after overnight incubation on chloramphenicol plates. c) Adenine base editing of β-lactamase (bla) in MG1655-bla in vitro (plasmid p1396). The experiment was performed in presence or absence of a guide RNA targeting the active site of β-lactamase (K71E or K71R). d) Cytosine base editing of β-lactamase in MG1655-bla in vitro (plasmid p2327). The CBE inserts a premature stop codon (Q37*) into the target gene, resulting in the re-sensitization of the bacterial population to β-lactam carbenicillin. The no gRNA controls carried a SapI spacer in the ABE or CBE plasmids. Adenine and cytosine base editing of β-lactamase was analyzed by colony counting after overnight incubation on chloramphenicol (Cm) and Cm/carbenicillin agar plates at 30 °C. The percentage growth was obtained by dividing the number of colonies on Cm/carbenicillin plates by the number on Cm plates. One dot represents one transformation after overnight incubation on plates. Graphs show individual values (dots) and average plus standard deviation. Sequencing data at the target site are shown exemplarily for a single base-edited colony.
