Development of an inducer-free, virulence gene promoter-controlled, and fluorescent reporter-labeled CRISPR interference system in Staphylococcus aureus.
Microbiol Spectr
; 12(10): e0060224, 2024 Oct 03.
Article
de En
| MEDLINE
| ID: mdl-39162514
ABSTRACT
The dCas9-based Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi) gene regulation technique requires two components a catalytically inactive Cas9 protein (dCas9) and a single-guide RNA that targets the gene of interest. This system is commonly activated by expressing dCas9 through an inducible gene promoter, but these inducers may affect cellular physiology, and accessibility and permeability of the inducer are limited in relevant model systems. Here, we have developed an alternative approach for CRISPRi activation in the clinical isolate Staphylococcus aureus USA300 LAC, where dCas9 was expressed through endogenous virulence gene promoters (vgp); coagulase, autolysin, or fibronectin-binding protein A. Additionally, we integrated a fluorescent reporter gene into the vgp-CRISPRi system to monitor the activity of the dcas9-controlling promoter. Testing the efficacy of vgp-CRISPRi by inducing growth arrest (when targeting penicillin-binding protein 1), downregulating target gene expression, or blocking coagulase-dependent coagulation of blood plasma, we provide a proof-of-concept demonstration that the virulence gene promoter-driven CRISPRi system is functional in S. aureus.IMPORTANCEThe presented inducer-free, endogenous virulence gene promoter-induced, dCas9-based Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi system addresses several shortcomings related to the use of inducer-dependent systems such as effects on cell physiology or limitations in permeability, and it avoids the high, putatively toxic levels of dCas9 in CRISPRi systems controlled by strong, constitutive promoters.
Mots clés
Texte intégral:
1
Collection:
01-internacional
Base de données:
MEDLINE
Sujet principal:
Staphylococcus aureus
/
Protéines bactériennes
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Régulation de l'expression des gènes bactériens
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Régions promotrices (génétique)
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Gènes rapporteurs
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Facteurs de virulence
/
Systèmes CRISPR-Cas
Limites:
Humans
Langue:
En
Journal:
Microbiol Spectr
/
Microbiol. spectr
/
Microbiology spectrum
Année:
2024
Type de document:
Article
Pays d'affiliation:
Norvège
Pays de publication:
États-Unis d'Amérique