Fig. 4

Cas9-mediated lethality of S. flexneri using chromosomal-targeting J72114 cas9-shiA phagemid. (a) Schematic diagram showing the cas9 genetic construct of P1 J72114 phagemid, with spacer sequence targeting chromosomal gene(s) (i.e., shiA) of S. flexneri (in green). Upon transduction, the presence of crRNA with a spacer sequence complementary to the chromosomal genes of S. flexneri (ct-crRNA) would cause dsDNA cleavage of the chromosome via Cas9 endonuclease, leading to cell death. (b) J72114 cas9 phagemid with spacer sequence(s) targeting the sigA, pic, shiD, and shiA chromosomal genes of S. flexneri 2a 2457O. Lysates were prepared from the wildtype EMG16 cell line. Crude lysates were used for transduction of S. flexneri 2A 2457O, with a MOI of 10 wildtype P1 phages (equivalent to 5 transducing units) to 1 bacterial cell. Data were plotted as change(s) in CFU as compared to input CFU (approximately 1 × 10⁷ cells per reaction) used for infection. (c) Spacer sequence-mediated lethality of S. flexneri 5a MT905 cells with chromosomal-targeting cas9-shiA (in orange) and nontargeting cas9-NT phagemids (in blue) in lysates prepared from the pacA*::npt EMG16 cell line. MOIs of 2.0, 4.0, 6.0, 8.0, and 10.0 were used. Data were plotted as changes in CFU as compared to input CFU (approximately 1 × 10⁷ cells per reaction) used for infection. The number of CFU recovered from the mock infection was shown in black. (d) Cas9 spacer sequence-mediated lethality effect of cas9-shiA phagemid and (e) Nonspacer sequence-mediated lethality effect of cas9 phagemid lysate prepared from wildtype (WT, in black) and pacA*::npt EMG (in orange) cell lines. MOIs of 2, 4, 6, 8, and 10 P1 transducing units to 1 bacterial cell for pacA*::npt EMG lysates were used. For wildtype EMG16 lysates, MOIs of 2.0, 4.0, 6.0 , 8.0, and 10.0 wildtype P1 phages per bacterial cell were used, and the wildtype P1 phage to P1 transducing units is approximately 2 for lysates prepared from the wildtype EMG16 cell line. The cas9-shiA-mediated lethality effect was quantified by measuring the reduction in CFU recovered after treatment with cas9-shiA and cas9-NT phagemids. Mock infections involved treating S. flexneri cells with SM buffer. The reduction in CFU recovered between cas9-NT phagemid treatment and the input cells used for infection (approximately 1 × 10⁷ cells per reaction) would show the nonspacer sequence-mediated lethality effect of P1 phage lysates at all four MOIs tested. Each data point represents a biological repeat and is the average of four technical repeats. Horizontal bars represent the group mean.