A Broad-Host-Range CRISPRi Toolkit for Silencing Gene Expression in .

Genetic tools are critical to dissecting the mechanisms governing cellular processes, from fundamental physiology to pathogenesis. Members of the genus have potential for biotechnological applications but can also cause disease in humans with a debilitated immune system. The lack of suitable genetic tools to edit GC-rich genomes has hampered the exploration of useful capacities and the understanding of pathogenic features. To address this, we have developed CRISPR interference (CRISPRi) technology for gene silencing in , testing it in , , and . Tunable expression was provided by placing a codon-optimized from under control of a rhamnose-inducible promoter. As a proof of concept, the operon controlling genes necessary for phenylacetic acid degradation was targeted by plasmid-borne gRNAs, resulting in near complete inhibition of growth on phenylacetic acid as the sole carbon source. This was supported by reductions in mRNA expression. The utility of CRISPRi to probe other functions at the single cell level was demonstrated by knocking down and , which dramatically reduces polyhydroxybutyrate granule accumulation and motility, respectively. As a hallmark of the mini-CTX system is the broad host-range of integration, we putatively identified 67 genera of Proteobacteria that might be amenable to modification with our CRISPRi toolkit. Our CRISPRi toolkit provides a simple and rapid way to silence gene expression to produce an observable phenotype. Linking genes to functions with CRISPRi will facilitate genome editing with the goal of enhancing biotechnological capabilities while reducing 's pathogenic arsenal.