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Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
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A programmable, selection-free CRISPR interference system in Staphylococcus aureus for long-term host interaction

Roni Miah1, Mona Johannessen1, Morten Kjos2

  • 1Department of Medical Biology and Centre for New Antibacterial Strategies (CANS), UiT- The Arctic University of Norway, 9019 Tromsø, Norway.

Iscience
|September 22, 2025
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Summary
This summary is machine-generated.

Researchers developed a selection-free CRISPR interference (CRISPRi) system for Staphylococcus aureus. This novel system enables gene silencing without antibiotics or inducers, advancing bacterial pathogenesis studies.

Keywords:
Biological sciencesBiotechnologyMicrobiologySynthetic biology

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Area of Science:

  • Microbiology and Molecular Biology
  • Bacterial Genetics and Pathogenesis
  • CRISPR-based Gene Regulation

Background:

  • Conventional dCas9-based CRISPR interference (CRISPRi) systems necessitate antibiotic selection and external inducers, restricting their use in infection models.
  • The bacterium *Staphylococcus aureus* is a significant human pathogen, and precise tools are needed to study its virulence factors and pathogenesis.
  • Developing antibiotic-free and inducer-free gene manipulation tools is crucial for advancing studies in complex biological systems.

Purpose of the Study:

  • To engineer a novel, selection-free CRISPR interference (CRISPRi) system for *Staphylococcus aureus*.
  • To enable programmable gene silencing in *S. aureus* without the need for antibiotic resistance markers or exogenous inducer molecules.
  • To validate the system's efficacy for long-term gene function studies in bacterial pathogenesis.

Main Methods:

  • Development of a CRISPRi system utilizing the stable pCM29 plasmid in *S. aureus*.
  • Integration of dCas9 expression under an endogenous promoter and sgRNA expression under a constitutive promoter for selection-free and inducer-free operation.
  • Programming the system to target coagulase and autolysin genes, followed by validation using qPCR and phenotypic assays (in vitro and in vivo infection models).

Main Results:

  • Successful establishment of a selection-free CRISPRi system in *S. aureus*.
  • Demonstrated stable gene silencing for over 27 generations without antibiotic pressure.
  • Confirmed target gene knockdown via qPCR and observed corresponding phenotypic changes in plasma coagulation and infection models (THP-1 cells, *Galleria mellonella*).

Conclusions:

  • The developed CRISPRi system provides a powerful, versatile tool for genetic manipulation in *Staphylococcus aureus*.
  • This selection-free and inducer-free system significantly enhances the study of bacterial gene function, particularly in long-term pathogenesis research.
  • The system serves as a foundational blueprint for creating similar CRISPRi tools in other bacterial species.