Fig. 5

Spacer sequence-mediated lethality of Shigella flexneri 5a M90T in vivo using chromosomal-targeting cas9-shiA phagemid. (a) Enumeration of S. flexneri 5a M90T CFU at 6 h post-infection. n = 9 (targeting); 9 (empty) larvae (cumulated from three independent experiments). (b) Measurement of the survival rate of infected zebrafish larvae at 24, 48, and 72 h post-infection (hpi), treated with cas9-shiA phagemid (orange) or with the nontargeting cas9-NT phagemid (blue). n = 71 (targeting); 74 (empty) larvae (cumulated from three independent experiments). Differences in bacterial load were tested using an unpaired t-test on log 10-transformed data, while differences in survival were tested using a log-rank (Mantel–Cox) test. p < 0.05 is considered statistically significant. p < 0.005 between targeting phagemid and nontargeting phagemid treatments are shown as **. (c) Schematic diagram showing the reduction of S. flexneri infection bacterial load via the spacer sequence-mediated lethality effect of cas9-shiA phagemid. The reduced bacterial load would promote the survival of infected larvae.