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CRISPRi System as an Efficient, Simple Platform for Rapid Identification of Genes Involved in Pollutant Transformation by Aeromonas hydrophila
journal contribution
posted on 2020-03-05, 17:35 authored by Jie Wu, Zhou-Hua Cheng, Di Min, Lei Cheng, Ru-Li He, Dong-Feng Liu, Wen-Wei LiAeromonas 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.
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biomolecular pathwaysAeromonas hydrophila Aeromonas speciesextracellular electron transfervanadium reductionRapid IdentificationSimple PlatformEETars operonAeromonas speciesgfp expressionAeromonas hydrophilatype IIpollutant transformationpollutant transformation mechanismsdCas 9CRISPRi Systempollutant transformation processessingle-plasmid CRISPRi systemmultiplexible platformStreptococcus pyogenestranscriptional repressionexpression levelsgene knockdownremediation applicationsgenome regulation toolgenome regulation systemrolearsenic detoxificationPollutant Transformationpollution control
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