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

• DOI: 10.1021/acs.est.9b07191
• Publication Date: 2020-02-28T22:03:25Z
• Authors: Jie Wu; Zhou-Hua Cheng; Di Min; Lei Cheng; Ru-Li He; Dong-Feng Liu; Wen-Wei Li
• Keywords: 0301 basic medicine; 0303 health sciences; 03 medical and health sciences; Bacterial Proteins; 13. Climate action; Gene Knockdown Techniques; Gene Expression; Clustered Regularly Interspaced Short Palindromic Repeats; Environmental Pollutants; 01 natural sciences; 0105 earth and related environmental sciences; Aeromonas hydrophila

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