Assembly of large mobilizable genetic cargo by double recombinase operated insertion of DNA (DROID).

There is an important need to develop new therapeutic tools to modulate the gene content of microbiomes. A potential strategy for microbiome engineering relies on the delivery of genetic payloads by conjugative plasmids. Yet, the introduction of large DNA molecules in conjugative plasmids can be challenging. Here, we describe the Double Recombinase Operated Insertion of DNA (DROID), an efficient method to assemble large DNA molecules without introducing antibiotic resistance genes or other unwanted sequences in the final construct. We exemplify this method by demonstrating that the Bxb1 integrase and FLP recombinase can be used successively to stably insert a relatively large DNA cargo consisting of a CRISPR-Cas9 system in a conjugative plasmid. We further show that the resulting CRISPR-Cas9 mobilization system was able to cure a multi-copy antibiotic resistance plasmid in a target bacterium. In addition to its utility for DNA payload integration in conjugative plasmids, the DROID method could readily be adapted to a multitude of other applications that require the manipulation of large DNA molecules.