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首页> 外文期刊>Environmental Science & Technology >CRISPRi System as an Efficient, Simple Platform for Rapid Identification of Genes Involved in Pollutant Transformation by Aeromonas hydrophila
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CRISPRi System as an Efficient, Simple Platform for Rapid Identification of Genes Involved in Pollutant Transformation by Aeromonas hydrophila

机译：CRISPRi系统是一种高效，简单的平台，用于快速鉴定涉及嗜水气单胞菌污染物转化的基因

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摘要

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.
机译：气单胞菌物种是多种水生环境中的本土物种，并在环境修复中发挥重要作用。但是，这些细菌的污染物转化机制仍然难以捉摸，到目前为止，它们在污染控制中的潜力尚未得到充分挖掘。在这项工作中，我们报告了一种有效而简单的基因组调控工具，可编辑嗜水气单胞菌并确定其污染物转化的生物分子途径。基因组调控系统基于化脓性链球菌的II型成簇规则间隔的短回文重复干扰（CRISPRi）系统，可作为嗜水链球菌基因敲除的可逆和多重平台。在亲水性链球菌中构建了一个同时包含dCas9和sgRNA的单质粒CRISPRi系统，该系统用于沉默具有不同大小和表达水平的多种基因。使用该系统，可以抑制gfp的表达达到467倍，并且可以快速，准确地识别必需基因ftsZ的功能。此外，实现了多个靶基因的同时转录抑制。我们发现，ars操纵子在砷的解毒中起着至关重要的作用，而胞外电子转移（EET）途径与甲基橙的还原有关，而与嗜水杆菌​​的钒还原无关。我们的方法为气单胞菌中的污染物转化过程提供了更好的见识和有效的遗传操作，这可能有助于更有效地利用气单胞菌物种和其他微生物物种进行环境修复。

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  • 来源
    《Environmental Science & Technology》 |2020年第6期|3306-3315|共10页
  • 作者

    Jie Wu; Zhou-Hua Cheng; Di Min; Lei Cheng; Ru-Li He; Dong-Feng Liu; Wen-Wei Li;

  • 作者单位

    CAS Key Laboratory oj Urban Pollutant Conversion Department of Applied Chemistry University oj Science & Technology of China Hefei 230026 P. R. China;

    School of Life Sciences University of Science & Technology of China Hefei 230026 P. R. China;

    CAS Key Laboratory of Urban Pollutant Conversion Department of Applied Chemistry University of Science & Technology of China Hefei 230026 P. R. China;

    CAS Key Laboratory of Urban Pollutant Conversion Department of Applied Chemistry University of Science & Technology of China Hefei 230026 P. R China;

    CAS Key Laboratory of Urban Pollutant Conversion Department of Applied Chemistry University of Science & Technology of China Hefei 230026 P. R China;

  • 收录信息 美国《科学引文索引》(SCI);美国《工程索引》(EI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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