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

Abstract

Aeromonas 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 Aeromonas hydrophila 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 Streptococcus pyogenes, can serve as a reversible and multiplexible platform for gene knockdown in A. hydrophila. A single-plasmid CRISPRi system harboring both dCas9 and the sgRNA was constructed in A. hydrophila and used to silence diverse genes with varied sizes and expression levels. With this system, up to 467-fold repression of gfp expression was achieved, and the function of the essential gene-ftsZ was identified quickly and accurately. Furthermore, simultaneous transcriptional repression of multiple targeted genes was realized. We discovered that the ars 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 A. hydrophila. Our method allows better insights and effective genetic manipulation of the pollutant transformation processes in Aeromonas, which might facilitate more efficient utilization of the Aeromonas species and other microbial species for environmental remediation applications.

SynBioStrainFinder: A microbial strain database of manually curated CRISPR/Cas genetic manipulation system information for biomanufacturing

“…1 c). (2) The most frequently used promoters for Cas9 expression are the tef [ 28 – 32 ] and teto promoters [ 33 – 36 ] in fungi and bacteria, respectively. For sgRNA expression, the commonly used promoters are the SNR52 [ 30 , 37 ] and U6 promoters [ 28 , 38 ] in fungi and the J23119 promoter [ 39 , 40 ] in bacteria (Fig.…”

Recent advances in the roles of minerals for enhanced microbial extracellular electron transfer

“…(2) To promote the conversion of value-added chemicals, exploiting the enzymatic machineries imbedded in native microbial cells might ensure products with exquisite selectivity and circumvent energy/product losses resulting from secondary biomass proliferation [157]. 3To maximize electron flow in clean fuel synthesis, enhancing the electron flux through engineered microbial strains produced by manipulating genetic materials could greatly enhance efficacy (e.g., employing CRISPR [clustered regularly interspaced short palindromic repeats] and CRISPRi [CRISPR interference] technologies) [158,159]. Following the steady improvements of the past decade, the most salient components responsible for electron transfer are primed for further exploitation in bioelectrochemical system to greatly enhance future applications and operational efficiencies.…”

Cadmium sulfide nanoparticles-assisted intimate coupling of microbial and photoelectrochemical processes: Mechanisms and environmental applications

“…(3) To promote targeted environmental applications of biohybrids, genetically modified microbes may improve biohybrid performance. For instance, the electron flux could be re-diverted in engineered strains by manipulating genetic circuits (e.g., employing CRISPR [clustered regularly interspaced short palindromic repeats] and CRISPRi [CRISPR interference] technologies) to strengthen the biocompatibility of the biomaterials (Li et al, 2020a;Wu et al, 2020). (4) Further research is warranted to optimize reaction conditions in terms of specific microorganisms, food substrates and the type/amount of immobilized CdS NPs to maximize operational efficiency.…”