Gap-repair recombineering for efficient retrieval of large DNA fragments from BAC clones and manipulation of large high-copy number plasmids

Bacterial high-copy number plasmids are the preferred DNA source for reporter constructs used in cell transfection experiments and making transgenic invertebrates to study gene expression, develop gene therapies and biotechnological application. They can be quickly validated from small cultures and easily generated in large quantities. However, manipulating plasmids above 10 kb can become very tedious. Here, we devised simple and highly efficient gap-repair recombineering methodology in E. coli to manipulate high-copy number plasmids up to 20 kb with up to 100% efficiency. This method utilises rare cutting restriction enzymes to introduce a gap, which is then subsequently repaired through homologous recombination from a provided template. Unlike traditional cloning methods, large concentration differences among fragments are tolerated. Moreover, CRISPR-Cas9-mediated in vitro DNA scission can be sufficiently efficient to overcome limitations from finding rare cutting restriction enzymes. Gap-repair recombineering provides a significant advancement in generating recombinant high-copy number DNA plasmids through enhancing efficiency, speed, and robustness. We validated this technology by generating reporter transgenes of the highly repetitive Drosophila Down Syndrome Cell Adhesion Molecule (Dscam) gene to analyse alternative splicing.