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 one-step protocol employs efficient single-stranded DNA annealing proteins (SSAP) to incorporate (libraries of) DNA oligos harboring the desired mutations into a target plasmid in E. coli . MOSAIC can be used to modify virtually any plasmid and is integrated with a validation pipeline based on Nanopore sequencing. In addition to up to 90% single-target plasmid editing efficiency, MOSAIC is demonstrated to enable the generation of a combinatorial plasmid library spanning four different target regions on a plasmid, in a single transformation. 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.