Fig. 6: Antimicrobial susceptibility assays accessing wtAcrIIC1 and AcrIIC1-SS activities in vivo.

a Panels 1, 2, assays without wtAcrIIC1 expression: pSG represents negative control carrying no spacer; pSG_KMR represents kmR spacer-introduced plasmid. The gene-editing ability of the two-plasmid system pCas-pSG was confirmed by a significant increase in the size of the inhibition ring relative to the negative control, with a representative diameter of 21 mm vs 17 mm. Panels 3, 4, assays with wtAcrIIC1 induced by IPTG. Co-expression of wtAcrIIC1 restored the inhibition ring because it binds to and deactivates Cas9. Panels 5, 6, assays with wtAcrIIC1 induced and 1 mM H₂O₂ stimulus. wtAcrIIC1 failed to restore the inhibition ring under H₂O₂ stimulus due to the loss of its activity under the oxidative environment. b Co-expression of AcrIIC1-SS can restore the inhibition ring under various redox environments because its activity is resistant to redox fluctuation. Assay panels are in the same order as a. c Statistical analysis of the inhibition zone diameters from a, b. The left part shows the size of the inhibition ring varies in response to environmental redox change when wtAcrIIC1 is co-expressed with Cas9. The right panel shows the size of the inhibition ring remains constant and resistant to environmental redox changes when AcrIIC1-SS is co-expressed with Cas9. Lines in the plots indicate means, dots indicate individual data points from three independent experiments, and every dot is calculated by averaging three rings’ diameters from one plate. Error bars represent SD. Unpaired two-sided t-test, **P < 0.01, ***P < 0.001. The exact P values for wtAcrIIC1 panels 1 with 2, 2 with 4, and 5 with 6 are 0.0005, 0.0019, and 0.0002, respectively. The P values for AcrIIC1-SS panels 1 with 2 is <0.0001, and panels 2 with 4 is 0.0003. Source data are provided as a Source Data file.