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

Staphylococcus aureus is a major opportunistic pathogen, increasingly difficult to treat due to rising resistance to methicillin, vancomycin, and other antimicrobials. Bacteriophages offer a promising alternative, particularly when conventional therapies fail and their efficacy can be enhanced through genetic engineering. Among S. aureus phages, the strictly lytic, broad-host-range members of the Twortvirinae subfamily are among the most promising therapeutic candidates. However, their large genome sizes make them notoriously difficult to engineer. In this study, we utilized Twortvirus K as a model to develop an efficient phage engineering platform, leveraging homologous recombination and CRISPR-Cas9-assisted counterselection. As proof of principle, this platform was utilized to construct a nanoluciferase (nluc)-encoding reporter phage (K::nluc) and tested as a bioluminescence-based approach for identifying viable Staphylococcus cells. Independent of their phage-resistance profile, 100% of tested clinical S. aureus isolates emitted bioluminescence upon K::nluc challenge. This diagnostic assay was further adapted to complex matrices such as human whole blood and bovine raw milk, simulating S. aureus detection scenarios in bacteremia and bovine mastitis. Beyond reporter phage-based diagnostics, our engineering technology opens avenues for the design and engineering of therapeutic Twortvirinae phages to combat drug-resistant S. aureus strains.IMPORTANCEPhage engineering, the process of modifying bacteriophages to enhance or customize their properties, offers significant potential for advancing precision antimicrobial therapies and diagnostics. While methods for engineering small Staphylococcus phage genomes are well-established, larger Staphylococcus phages have historically been challenging to modify. In this study, we present a novel method that enables the engineering of Twortvirinae, a subfamily of Staphylococcus phages known for their broad host range and strictly lytic lifestyle, making them highly relevant for diagnostic and therapeutic applications. Using this method, we successfully developed a phage-based diagnostic tool capable of rapid and sensitive detection of S. aureus cells across various matrices. This approach has the potential to extend beyond diagnostics, enabling applications such as phage-mediated delivery of antimicrobial effector proteins in the future.