Fig. 2

Analysis of guides producing unexpected fitness defects. a Fitness effect of guides targeting the non-essential lpoB and hisI gene. Guides T-lpoB, T-lpoB2, C-lpoB, T-hisI, and C-hisI are highlighted. b The highlighted guides were cloned in plasmid psgRNA and introduced in strain LC-E18 carrying dCas9 under the control of a Ptet promoter in the chromosome. Cells were grown overnight and plated on LB agar with 1 nM of aTc. Representative figure from p = 4 independent experiments. c Expression levels of lpoB and hisI when repressed by the T-lpoB, C-lpoB, T-hisI, and C-hisI guide RNAs, as measured by RT-qPCR. Points show biological replicates (n = 4), the black bar is the median. d Effect of the T-lpoB, T-lpoB2, and control guides on the plating efficiency of LC-E18 and LC-E18ΔlpoB. Cells were grown overnight and plated on LB agar with 1 nM of aTc. Representative figure from n = 4 independent experiments