Abstract
The editing of plasmids and construction of plasmid libraries is paramount to the engineering of desired functionalities in synthetic biology. Typically, plasmids with targeted mutations are produced through time- and resource-consuming DNA amplification and/or cloning steps. In this study, we establish MOSAIC, a highly efficient protocol for the editing of plasmids and generation of combinatorial plasmid libraries. This quick protocol employs the efficient single-stranded DNA annealing protein (SSAP) CspRecT to incorporate (libraries of) DNA oligos harboring the desired mutations into a target plasmid in Escherichia coli. In addition to up to 90% single-target plasmid editing efficiency, we demonstrate that MOSAIC enables the generation of a combinatorial plasmid library spanning four different target regions on a plasmid, in a single transformation. Lastly, we integrated a user-friendly validation pipeline using Nanopore sequencing reads, requiring minimal computational experience. We anticipate that MOSAIC will provide researchers with a simple, rapid and resource-effective method to edit plasmids or generate large, diverse plasmid libraries for a wide range of in vivo or in vitro applications in molecular and synthetic biology.