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

<i>Staphylococcus aureus</i> 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 <i>S. aureus</i> phages, the strictly lytic, broad-host-range members of the <i>Twortvirinae</i> subfamily are among the most promising therapeutic candidates. However, their large genome sizes make them notoriously difficult to engineer. In this study, we utilized <i>Twortvirus</i> 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 (<i>nluc</i>)-encoding reporter phage (K::<i>nluc</i>) and tested as a bioluminescence-based approach for identifying viable <i>Staphylococcus</i> cells. Independent of their phage-resistance profile, 100% of tested clinical <i>S. aureus</i> isolates emitted bioluminescence upon K::<i>nluc</i> challenge. This diagnostic assay was further adapted to complex matrices such as human whole blood and bovine raw milk, simulating <i>S. aureus</i> detection scenarios in bacteremia and bovine mastitis. Beyond reporter phage-based diagnostics, our engineering technology opens avenues for the design and engineering of therapeutic <i>Twortvirinae</i> phages to combat drug-resistant <i>S. aureus</i> 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 <i>Staphylococcus</i> phage genomes are well-established, larger <i>Staphylococcus</i> phages have historically been challenging to modify. In this study, we present a novel method that enables the engineering of <i>Twortvirinae</i>, a subfamily of <i>Staphylococcus</i> 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 <i>S. aureus</i> 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.