CRISPR-Cas9-mediated Large Cluster Deletion and Multiplex Genome Editing in Paenibacillus polymyxa .

The use of molecular tools based on the clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems has rapidly advanced genetic engineering. These molecular biological tools have been applied for different genetic engineering purposes in multiple organisms, including the quite rarely explored Paenibacillus polymyxa . However, only limited studies on large cluster deletion and multiplex genome editing have been described for this highly interesting and versatile bacterium. Here, we demonstrate the utilization of a Cas9-based system to realize targeted deletions of four biosynthetic gene clusters in the range of 12-41 kb by the use of a single targeting sgRNA. Furthermore, we also harnessed the system for multiplex editing of genes and large genomic regions. Multiplex deletion was achieved with more than 80% efficiency, while simultaneous integration at two distantly located sites was obtained with 58% efficiency. The findings reported in this study are anticipated to accelerate future research in P. polymyxa and related species.