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Reversible Gene Expression Control in Yersinia pestis by Using an Optimized CRISPR Interference System.

Tong Wang1, Min Wang1, Qingwen Zhang2

  • 1State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China.

Applied and Environmental Microbiology
|April 14, 2019
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Summary

Researchers optimized the clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for gene knockdown in the plague pathogen Yersinia pestis. This efficient and reversible CRISPRi tool aids in understanding Y. pestis genes and developing new plague control strategies.

Keywords:
CRISPRiYersinia pestisgene knockdown

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Area of Science:

  • Microbiology
  • Genetics
  • Molecular Biology

Background:

  • Many genes in *Yersinia pestis*, the plague pathogen, remain uncharacterized, hindering plague prevention and control.
  • Clustered regularly interspaced short palindromic repeat interference (CRISPRi) is an effective gene knockdown tool in bacteria but was not previously reported for *Y. pestis*.

Purpose of the Study:

  • To optimize and validate a CRISPRi system for efficient and reversible gene knockdown in *Yersinia pestis*.
  • To demonstrate the utility of this CRISPRi system for functional genomic studies and phenotypic screening of *Y. pestis*.

Main Methods:

  • An optimized CRISPRi system using *Streptococcus pyogenes* Cas9-derived dCas9 was developed for *Y. pestis*.
  • The system was used for inducible gene knockdown of chromosomal and plasmid-borne genes.
  • Phenotypic assays included *in vitro* biofilm formation, cold tolerance, and virulence studies in HeLa cells and mice.

Main Results:

  • Efficient gene knockdown (up to 380-fold) was achieved in an anhydrotetracycline-inducible manner.
  • Knockdown of *hmsH* and *cspB* genes resulted in decreased biofilm formation and impaired cold tolerance, respectively.
  • Silencing of virulence genes *yscB* and *ail* led to attenuated virulence in cell and mouse models.

Conclusions:

  • The optimized CRISPRi system provides an efficient and reversible method for gene knockdown in *Y. pestis*.
  • This tool facilitates high-throughput phenotypic screening of uncharacterized *Y. pestis* genes.
  • The system is valuable for advancing plague prevention, control, and biodefense strategies.