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CRISPR-Cas9 enables efficient genome engineering of the strictly lytic, broad-host-range staphylococcal bacteriophage K

Summary

Scientists have developed a new method to genetically engineer bacteriophages—viruses that infect bacteria—to fight antibiotic-resistant Staphylococcus aureus infections. Using CRISPR technology, they created a special phage that glows when it infects S. aureus cells, allowing doctors to quickly detect this dangerous pathogen in patient blood samples and other clinical samples. This engineered phage works against most S. aureus strains tested, regardless of their resistance to vancomycin, and could lead to new diagnostic tools and treatments for drug-resistant bacterial infections.

Background

Staphylococcus aureus is a major opportunistic pathogen with rising resistance to methicillin and vancomycin. Bacteriophages offer promising alternatives to conventional antibiotics, particularly members of the Twortvirinae subfamily which are strictly lytic with broad host range. However, engineering large phage genomes has been notoriously difficult.

Objective

To develop an efficient phage engineering platform for Twortvirus K using homologous recombination and CRISPR-Cas9-assisted counterselection. The goal was to construct a nanoluciferase-encoding reporter phage for viable S. aureus detection.

Results

The CRISPR-Cas9 counterselection system efficiently isolated recombinant phages at approximately 10^-4 frequency. K::nluc successfully detected all 71 tested S. aureus strains via bioluminescence, including 20 strains showing no plaque formation. Detection limits were 707-2,617 CFU/mL in growth medium and remained effective in complex matrices like human whole blood and bovine raw milk.

Conclusion

A novel CRISPR-Cas9-assisted engineering platform enables efficient modification of large Twortvirinae phage genomes. The resulting reporter phage demonstrates rapid and sensitive S. aureus detection across diverse clinical isolates and complex biological matrices, with potential applications in diagnostics and therapeutic antimicrobial protein delivery.
  • Published in:Applied and Environmental Microbiology,
  • Study Type:Experimental Research,
  • Source: PMID: 40757862, DOI: 10.1128/aem.02014-24
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