Fig. 4

Further validation of CRISPRi-based repression tool through targeting of hipO. Six target sgRNA locations were identified in the hipO gene in M1Cam and 81–176, distributed from the 5’ to 3’ end of the gene (A). Assessment of HipO activity in M1Cam and 81–176 CRISPRi strains targeting hipO in a range of locations (B). All target locations in 81–176 CRISPRi strains showed significantly decreased HipO activity (**** = p < 0.0001, *** = p < 0.001, one way ANOVA). All target locations except HipO_883 showed significantly decreased HipO activity in M1Cam CRISPRi strains (††† = p < 0.001, †† = p < 0.01, † = p < 0.05, one way ANOVA), with a trend of decreased activity from 5’ to 3’, although no significant difference was found between sgRNA target locations. 81–176 – N = 3 biological replicates, M1Cam – N = 5 biological replicates, error bars = SD. Analysis of relative percentage decrease in HipO activity for each strain (C) revealed that the sgRNAs HipO_676, HipO_883 and HipO_976 showed a significantly greater decrease in HipO activity in 81–176 than in M1Cam. Statistics calculated using two-way ANOVA, *** = p < 0.001, ** = p < 0.01 and * = p < 0.05, error bars = SD