A versatile one-step CRISPR-Cas9 based approach to plasmid-curing
The pFREE system is a CRISPR-Cas9 based platform that offers plasmid curing of all major classes of vectors used for cloning and expression purposes in bacteria. The temperature-sensitive and broad host-range version of pFREE (pFREE-RK2) was also developed to provide a curing solution for broad range of Gram-negative bacteria.

The curing workflow is performed in a fast one-step protocol that only requires transformation of pFREE into the strain harboring the target plasmids for curing and the addition of the pFREE inducers, followed by overnight incubation. The pFREE plasmid holds a self-curing feature that allows for efficient and complete plasmid-curing.

All the different versions of pFREE and pFREE-RK2 are available from Addgene.

Reference: Lauritsen, I*., Porse, A*., Sommer, M. O. A., & N rholm, M. H. H. (2017).
A versatile one-step CRISPR-Cas9 based approach to plasmid-curing. Microbial
Cell Factories, 16, [135].


Morten Nørholm
Senior Researcher
DTU Biosustain
+45 45 25 80 26