Plasmid curing

To assess Card1’s ability to promote plasmid curing under low transcription conditions, a plasmid curing assay was performed, similarly to previously described⁵. Briefly, overnight cultures of S. aureus cells harbouring pTarget and a pCRISPR containing either Card1, dCard1, or no spacer (Δspc) were diluted to exactly OD 0.15 in tryptic soy broth with 10 ug/ml chloramphenicol. After removing a cell aliquot for the 0 timepoint, aTc was added to a concentration of 9.3 ng/ml (Fig. 3c) or 125 ng/ml (Fig. 3d) and the cells were incubated at 37 °C, with further aliquots taken at the indicated times. The cells were then lysed, and the plasmid DNA isolated using a QIAprep Miniprep kit (Qiagen) according to the manufacturer’s protocol (Qiagen). 400 ng of plasmid was then linearised using BamHI-HF (NEB), which cuts both pTarget and pCRISPR once, followed by visualisation by gel electrophoresis.