MOSAIC: A Highly Efficient, One-Step Recombineering Approach to Plasmid Editing and Diversification

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.