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.