Figure 4:

HeLa cells were infected with Ct transformed with the pBOMBL-12CRia plasmid, which constitutively expresses a ftsK crRNA or pbp2 crRNA and encodes dCas12 under the control of an aTc-inducible promoter. dCas12 was induced at 17hpi by adding 5nM aTc to the media. In a control infection, the expression of dCas12 was not induced. Cells were harvested at 24hpi and the morphology of Ct in induced and uninduced control cells was assessed in 250 cells (A and B). 3 replicates were performed, and the values shown are the averages of the 3 replicates. The localization of endogenous FtsK, endogenous PBP2, and endogenous PBP3 was assessed in cells transformed with the pBOMBL-12CRia plasmid that targets ftsK or pbp2. The localization is shown in coccoid cells where dCas12 expression was (C) uninduced or (D) induced. White bars are 1μm. The localization profiles of FtsK, PBP2, and PBP3 were quantified in (E) uninduced and (F) induced cells. 3 replicates were performed, and the values shown are the averages of the 3 replicates.