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Bacteriophages offer a promising alternative, particularly when conventional therapies fail and their efficacy can be enhanced through genetic engineering. Among\n                      <jats:italic toggle=\"yes\">S. aureus<\/jats:italic>\n                      phages, the strictly lytic, broad-host-range members of the\n                      <jats:italic toggle=\"yes\">Twortvirinae<\/jats:italic>\n                      subfamily are among the most promising therapeutic candidates. However, their large genome sizes make them notoriously difficult to engineer. In this study, we utilized\n                      <jats:italic toggle=\"yes\">Twortvirus<\/jats:italic>\n                      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 (\n                      <jats:italic toggle=\"yes\">nluc<\/jats:italic>\n                      )-encoding reporter phage (K::\n                      <jats:italic toggle=\"yes\">nluc<\/jats:italic>\n                      ) and tested as a bioluminescence-based approach for identifying viable\n                      <jats:italic toggle=\"yes\">Staphylococcus<\/jats:italic>\n                      cells. Independent of their phage-resistance profile, 100% of tested clinical\n                      <jats:italic toggle=\"yes\">S. aureus<\/jats:italic>\n                      isolates emitted bioluminescence upon K::\n                      <jats:italic toggle=\"yes\">nluc<\/jats:italic>\n                      challenge. This diagnostic assay was further adapted to complex matrices such as human whole blood and bovine raw milk, simulating\n                      <jats:italic toggle=\"yes\">S. aureus<\/jats:italic>\n                      detection scenarios in bacteremia and bovine mastitis. Beyond reporter phage-based diagnostics, our engineering technology opens avenues for the design and engineering of therapeutic\n                      <jats:italic toggle=\"yes\">Twortvirinae<\/jats:italic>\n                      phages to combat drug-resistant\n                      <jats:italic toggle=\"yes\">S. aureus<\/jats:italic>\n                      strains.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Phage 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\n                        <jats:italic toggle=\"yes\">Staphylococcus<\/jats:italic>\n                        phage genomes are well-established, larger\n                        <jats:italic toggle=\"yes\">Staphylococcus<\/jats:italic>\n                        phages have historically been challenging to modify. In this study, we present a novel method that enables the engineering of\n                        <jats:italic toggle=\"yes\">Twortvirinae<\/jats:italic>\n                        , a subfamily of\n                        <jats:italic toggle=\"yes\">Staphylococcus<\/jats:italic>\n                        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\n                        <jats:italic toggle=\"yes\">S. aureus<\/jats:italic>\n                        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.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/aem.02014-24","type":"journal-article","created":{"date-parts":[[2025,8,4]],"date-time":"2025-08-04T13:00:28Z","timestamp":1754312428000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":8,"title":["CRISPR-Cas9 enables efficient genome engineering of the strictly lytic, broad-host-range staphylococcal bacteriophage K"],"prefix":"10.1128","volume":"91","author":[{"given":"Jonas","family":"Fernbach","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05a28rw58","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Health Science and Technology, ETH Z\u00fcrich","place":["Z\u00fcrich, Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jasmin","family":"Baggenstos","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05a28rw58","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Health Science and Technology, ETH Z\u00fcrich","place":["Z\u00fcrich, Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6333-1550","authenticated-orcid":false,"given":"Ellen-Aleksandra","family":"Svorjova","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05a28rw58","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Health Science and Technology, ETH Z\u00fcrich","place":["Z\u00fcrich, Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jeannine","family":"Riedo","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05a28rw58","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Health Science and Technology, ETH Z\u00fcrich","place":["Z\u00fcrich, Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4277-7753","authenticated-orcid":true,"given":"Shawna","family":"McCallin","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02crff812","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Neuro-Urology, Balgrist University Hospital, University of Z\u00fcrich","place":["Z\u00fcrich, Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8162-2631","authenticated-orcid":true,"given":"Martin J.","family":"Loessner","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05a28rw58","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Health Science and Technology, ETH Z\u00fcrich","place":["Z\u00fcrich, Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1162-6549","authenticated-orcid":true,"given":"Samuel","family":"Kilcher","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/05a28rw58","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Health Science and Technology, ETH Z\u00fcrich","place":["Z\u00fcrich, Switzerland"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_4_2_2","doi-asserted-by":"publisher","DOI":"10.2147\/IDR.S173867"},{"key":"e_1_3_4_3_2","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.00181-19"},{"key":"e_1_3_4_4_2","doi-asserted-by":"publisher","DOI":"10.1179\/2047773215Y.0000000030"},{"key":"e_1_3_4_5_2","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2021.771510"},{"key":"e_1_3_4_6_2","volume-title":"Antimicrobial resistance: tackling a crisis for the health and wealth of nations","author":"O\u2019neill J","year":"2014","unstructured":"O\u2019neill J. 2014. 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