Harnessing the Endogenous Type I–F CRISPR/Cas System for Efficient Genome Engineering and Gene Repression in <i>Pseudomonas chlororaphis</i> LX24

<i>Pseudomonas chlororaphis</i>, a nonpathogenic plant growth-promoting rhizobacterium, holds immense potential for agricultural and industrial applications due to its ability to biosynthesize bioactive metabolites. However, the lack of efficient genetic tools has hindered its metabolic engineering. In this study, we first characterized an endogenous type I-F CRISPR/Cas system in <i>P. chlororaphis</i> LX24 and established a programmable genome editing toolkit based on this system. Concurrently, the plasmid transformation efficiency of <i>P. chlororaphis</i> LX24 was enhanced by identifying and deleting the restriction-modification systems. We further demonstrated the DNA interference capability with different PAM sequences of the type I-F CRISPR/Cas system, which also exhibited various editing efficiencies ranging from 22 to 87% in <i>P. chlororaphis</i> LX24. By introducing the λ-Red recombination system, the knockout efficiency of the phenazine cluster (8.3 kb) increased by over 9-fold. Next, introducing the <i>sacB</i>-based counterselection marker achieved a 100% plasmid curing success within 36 h. The optimized toolkit was further applied to single-step gene insertion and replacement with 100% success rates. Additionally, we established a CRISPR interference (CRISPRi) system for transcriptional repression in <i>P. chlororaphis</i> LX24 by knocking out nuclease Cas3. Through modulating the induction time and concentration of IPTG, the production of phenazines was reduced to 21-89% within 24 h in <i>P. chlororaphis</i> LX24. Overall, our work developed a convenient and precise genetic tool for the <i>P. chlororaphis</i> LX24, and the methods may also provide a reference for repurposing endogenous CRISPR systems in non-model prokaryotes.