Fig. 2

a Selection of gRNA-targets based on conserved regions of popular replicon-families. Selected replicon sequences representing the RNAI and RNAII encoding part of ColE1 and the repA encoding part of pSC101 replicon groups, were aligned and gRNA was selected based on the degree of conservation (illustrated as the color intensity). The center part of repA was fully conserved and omitted in the depiction. The plasmid names in grey boxes are examples of vectors belonging to each replicon family. Two gRNAs were selected for each replicon group, and all four gRNAs were combined into a CRISPR-array (crArray). b Plasmid map of pFREE. The pFREE plasmid was constructed by inserting the crArray targeting the ColE1 and pSC101 replicons into a colA vector encoding Cas9 along with other essential modules for CRISPR-Cas9 activity such as trans-activating CRISPR RNA (trcrRNA). The gRNA array and Cas9 nuclease are controlled by the inducible rhamnose (PrhaBAD) and tetracycline (Ptet) promoters to ensure tight regulation of curing functionality. c One-step curing workflow using the pFREE system. The pFREE plasmid is transformed into a strain harboring the target plasmids for curing. After transformation recovery, cells from the recovered culture are transferred into medium with pFREE selection, 0.2% rhamnose and 200 ng/mL anhydrotetracycline (aTc) added. The system is induced overnight (O/N) to allow the cleavage of target plasmids (red and green respectively) by Cas9 (blue), guided by the gRNA expression from pFREE (black plasmid). The culture is plated on non-selective agar and cured cells can be identified by replicon PCR (Additional file 1: info S2) or by phenotypic screening e.g. antibiotic sensitivity