CRISPRi System as an Efficient, Simple Platform for Rapid Identification of Genes Involved in Pollutant Transformation by .

species are indigenous in diverse aquatic environments and play important roles in environmental remediation. However, the pollutant transformation mechanisms of these bacteria remain elusive, and their potential in pollution control is largely unexploited so far. In this work, we report an efficient and simple genome regulation tool to edit and identify its biomolecular pathways for pollutant transformation. The genome regulation system, which is based on the type II clustered regularly interspaced short palindromic repeat interference (CRISPRi) system from , can serve as a reversible and multiplexible platform for gene knockdown in . A single-plasmid CRISPRi system harboring both dCas9 and the sgRNA was constructed in and used to silence diverse genes with varied sizes and expression levels. With this system, up to 467-fold repression of expression was achieved, and the function of the essential gene- was identified quickly and accurately. Furthermore, simultaneous transcriptional repression of multiple targeted genes was realized. We discovered that the operon played an essential role in arsenic detoxification, and the extracellular electron transfer (EET) pathway was involved in methyl orange reduction, but not in vanadium reduction by . Our method allows better insights and effective genetic manipulation of the pollutant transformation processes in , which might facilitate more efficient utilization of the species and other microbial species for environmental remediation applications.