Fig. 2

Spacer sequence-mediated lethality of E. coli MC1061::npt cells using npt-targeting cas9 phagemid. (a) Schematic diagrams showing the cas9 genetic construct, with or without npt-targeting crRNA (cas9-npt and cas9-NT, respectively) assembled onto the P1 J72114 phagemid. The presence of npt-targeting crRNA would target the Cas9 endonuclease chromosome of E. coli MC1061::npt cells, causing dsDNA cleavage of the chromosome and cell death. (b) Serial dilutions of transduced E. coli MC1061::npt were plated onto plain LB agar or LB agar supplemented with kanamycin. Data were plotted as change(s) in CFU as compared to input CFU (approximately 10⁷ cells per reaction) used for infection. (c) Quantification of chloramphenicol-resistant CFUs recovered, after treatment with cas9-NT or cas9-npt phagemid lysates. Each data point represents a biological replicate and is the average of four technical repeats. A MOI of 5 P1 transducing units per bacterial cell was used for all infections. Mock infections involved treating E. coli cells with SM buffer. Horizontal bars represent the group mean. The p-values (between non-targeting and targeting phagemid treatments) were determined using a Kruskal–Wallis test, with the significance defined by p < 0.05. p < 0.0005 between targeting phagemid and nontargeting phagemid treatments is shown as ***.