Fig. 2: A non-replicative DNA payload can efficiently edit a target bacterial population.

a, Schematics for the conditional replication of a cosmid. Replication requires both the primase protein and the primase origin of replication (Primase-Ori). b, In vitro plasmid stability time-course assay. Bacteria carrying an inducible primase plasmid were grown either without inducer (blue) or with 100 µM DAPG (yellow). A cosmid harbouring a sfGFP gene (p1324) was delivered at an approximate MOI of 40 (red arrow). Samples from different time points were analysed in a flow cytometer. The graph shows the average and standard deviation of an experiment performed in triplicate. Dashed line indicates background fluorescence of cells before transduction. c, Cells carrying an induced (+) or uninduced (−) primase plasmid were transduced with a payload carrying the conditional origin of replication, incubated for 5 h and plated on either (1) lysogeny broth agar, (2) lysogeny broth agar with chloramphenicol 25 µg ml⁻¹ or (3) lysogeny broth agar with kanamycin 50 µg ml⁻¹, chloramphenicol 25 µg ml⁻¹ and DAPG 100 µM. d, In vitro adenine base editing of bla using a non-replicative payload (p2328). MG1655-bla was transduced in either the presence (blue) or absence (yellow) of primase expression (plasmid p1321). After 2 h, cells transduced at varying MOI were plated on lysogeny broth with carbenicillin. e, Analysis of on- and off-target editing in MG1655-bla by next-generation sequencing. The frequency of mismatches in Illumina sequencing data is shown for all 20 base pair subsequences followed by a NGG PAM in the reference genome with up to seven mismatches to the target sequence, including up to two in the ten PAM-proximal nucleotides. Bars represent the mismatch frequency of the base with the highest frequency in each off-target. The sequence, coordinates and neighbouring genes of each off-target are listed in Supplementary Data 1. Data shown for two biological replicates of samples treated with either ABE or control.

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