Dataset Information

CRISPR Interference (CRISPRi) Inhibition of luxS Gene Expression in E. coli: An Approach to Inhibit Biofilm.

ABSTRACT: Biofilm is a sessile bacterial accretion embedded in self-producing matrix. It is the root cause of about 80% microbial infections in human. Among them, E. coli biofilms are most prevalent in medical devices associated nosocomial infections. The objective of this study was to inhibit biofilm formation by targeting gene involved in quorum sensing, one of the main mechanisms of biofilm formation. Hence we have introduced the CRISPRi, first time to target luxS gene. luxS is a synthase, involved in the synthesis of Autoinducer-2(AI-2), which in turn guides the initial stage of biofilm formation. To implement CRISPRi system for luxS gene suppression, we have synthesized complementary sgRNA to target gene sequence and co-expressed with dCas9, a mutated form of an endonuclease. Suppression of luxS expression was confirmed through qRT-PCR. The effect of luxS gene on biofilm inhibition was studied through crystal violet assay, XTT reduction assay and scanning electron microscopy. We conclude that CRISPRi system could be a potential strategy to inhibit bacterial biofilm through mechanism base approach.

SUBMITTER: Zuberi A

PROVIDER: S-EPMC5445563 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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CRISPR Interference (CRISPRi) Inhibition of luxS Gene Expression in <i>E. coli</i>: An Approach to Inhibit Biofilm.

Zuberi Azna A Misba Lama L Khan Asad U AU

Frontiers in cellular and infection microbiology 20170526

Biofilm is a sessile bacterial accretion embedded in self-producing matrix. It is the root cause of about 80% microbial infections in human. Among them, <i>E. coli</i> biofilms are most prevalent in medical devices associated nosocomial infections. The objective of this study was to inhibit biofilm formation by targeting gene involved in quorum sensing, one of the main mechanisms of biofilm formation. Hence we have introduced the CRISPRi, first time to target luxS gene. luxS is a synthase, invol ...[more]

PMID: 28603699

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Project description:BackgroundThe type II based CRISPR-Cas system remains restrictedly utilized in archaea, a featured domain of life that ranks parallelly with Bacteria and Eukaryotes. Methanococcus maripaludis, known for rapid growth and genetic tractability, serves as an exemplary model for studying archaeal biology and exploring CO2-based biotechnological applications. However, tools for controlled gene regulation remain deficient and CRISPR-Cas tools still need improved in this archaeon, limiting its application as an archaeal model cellular factory.ResultsThis study not only improved the CRISPR-Cas9 system for optimizing multiplex genome editing and CRISPR plasmid construction efficiencies but also pioneered an effective CRISPR interference (CRISPRi) system for controlled gene regulation in M. maripaludis. We developed two novel strategies for balanced expression of multiple sgRNAs, facilitating efficient multiplex genome editing. We also engineered a strain expressing Cas9 genomically, which simplified the CRISPR plasmid construction and facilitated more efficient genome modifications, including markerless and scarless gene knock-in. Importantly, we established a CRISPRi system using catalytic inactive dCas9, achieving up to 100-fold repression on target gene. Here, sgRNAs targeting near and downstream regions of the transcription start site and the 5'end ORF achieved the highest repression efficacy. Furthermore, we developed an inducible CRISPRi-dCas9 system based on TetR/tetO platform. This facilitated the inducible gene repression, especially for essential genes.ConclusionsTherefore, these advancements not only expand the toolkit for genetic manipulation but also bridge methodological gaps for controlled gene regulation, especially for essential genes, in M. maripaludis. The robust toolkit developed here paves the way for applying M. maripaludis as a vital model archaeal cell factory, facilitating fundamental biological studies and applied biotechnology development of archaea.

Dataset Information

CRISPR Interference (CRISPRi) Inhibition of luxS Gene Expression in E. coli: An Approach to Inhibit Biofilm.

Publications

CRISPR Interference (CRISPRi) Inhibition of luxS Gene Expression in <i>E. coli</i>: An Approach to Inhibit Biofilm.

Similar Datasets

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