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Forward Genetic Approaches in Chlamydia trachomatis
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Nonspecific toxicities of Streptococcus pyogenes and Staphylococcus aureus dCas9 in Chlamydia trachomatis

Wurihan Wurihan1, Yehong Huang1,2, Alec M Weber1

  • 1Department of Pharmacology, Rutgers University Robert Wood Johnson Medical School, 675 Hoes Lane West, Piscataway, New Jersey 08854, USA.

Pathogens and Disease
|February 4, 2020
PubMed

Insights

CRISPR interference (CRISPRi) using dCas9 proteins from Streptococcus pyogenes and Staphylococcus aureus showed unexpected toxicity in Chlamydia trachomatis. These dCas9 tools are currently unsuitable for gene knockdown in chlamydial research.

Area of Science:

  • Microbiology
  • Genetics
  • Molecular Biology

Background:

  • Chlamydiae are significant human and animal pathogens.
  • Efficient genetic tools for gene knockout or knockdown in Chlamydia are needed.

Purpose of the Study:

  • To evaluate the utility of dCas9-based CRISPR interference (CRISPRi) for conditional gene knockdown in Chlamydia trachomatis.
  • To assess the feasibility of using an anhydrotetracycline (ATC)-inducible system for CRISPRi in Chlamydia.

Main Methods:

  • Implementation of Streptococcus pyogenes and Staphylococcus aureus dCas9 systems with an anhydrotetracycline (ATC)-inducible promoter.
  • Testing dCas9 expression with and without guide RNA (gRNA) in Chlamydia trachomatis.
  • Monitoring bacterial growth and toxicity in response to varying ATC concentrations.

Main Results:

  • Streptococcus pyogenes dCas9 induced significant growth inhibition even without a guide RNA.
  • Staphylococcus aureus dCas9 exhibited toxicity when expressed with an empty guide RNA scaffold.
  • Toxicity was observed at low anhydrotetracycline concentrations (0.2 nM for S. pyogenes dCas9, 1.0 nM for S. aureus dCas9).
  • Control experiments with methionine-tRNA ligase overexpression showed no adverse effects on Chlamydia trachomatis growth.

Conclusions:

  • Current forms of Streptococcus pyogenes and Staphylococcus aureus dCas9 proteins demonstrate significant toxicity in Chlamydia trachomatis.
  • These dCas9 systems, as implemented, have limited applicability for gene knockdown in chlamydial research.
  • Further strategies are required to adapt dCas9-based CRISPRi technology for effective use in Chlamydia.
Keywords:
CRISPRCRISPRiChlamydiaconditional gene knock-downdCas9essential gene knock-downtoxicity

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