A broad-host-range CRISPRi toolkit for silencing gene expression in Burkholderia

Genetic tools are critical to dissecting the mechanisms governing cellular processes, from fundamental physiology to pathogenesis. Species of Burkholderia have emerged over recent decades as both important opportunistic pathogens in immunocompromised patients and as a rich source of secondary metabolites for biotechnology. With large, high GC content genomes, there are limited tools available for genetic manipulation of Burkholderia species. To address this, we have developed CRISPR interference (CRISPRi) technology for gene silencing in Burkholderia. Tunable expression was provided by placing a codon-optimized dcas9 from Streptococcus pyogenes under control of a rhamnose-inducible promoter, which was validated by immunoblot. Expression from a multicopy plasmid caused a growth defect; therefore, the mini-CTX single-copy insertion system was used to deliver dcas9 to the chromosome, resulting in wild-type growth. As a proof of concept, the paaABCDE operon controlling genes necessary for phenylacetic acid degradation was targeted by plasmid-borne sgRNAs, resulting in near complete inhibition of growth on phenylacetic acid as a sole carbon source. The utility of CRISPRi to probe other functions at the single cell level was demonstrated knocking down phbC and fliF, 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.