Recent advances in genome editing techniques based on CRISPR-Cas have opened up new possibilities in bacteriophage engineering and, thus, enabled key developments in medicine, nanotechnology, and synthetic biology. Although staphylococcal phage genomes have already been edited, the modification of their structural proteins has not yet been reported. Here, the structure of Staphylococcus phage 812h1 of the Kayvirus genus was modified by inserting a poly histidine tag into an exposed loop of the tail sheath protein. A two-strain editing strategy was applied, utilizing homologous recombination followed by CRISPR-Cas10-assisted counter-selection of the recombinant phages. The His-tagged phage particles can be recognized by specific antibodies, enabling the modified bacteriophages to be employed in numerous techniques. The attachment of the engineered phage to bacteria was visualized by fluorescence microscopy, and its functionality was confirmed using biolayer interferometry biosensing, enzyme-linked immunosorbent assay, and flow cytometry, demonstrating that the genetic modification did not impair its biological activity.

Central European Institute of Technology Masaryk University Brno 625 00 Czech Republic

Department of Biochemistry Faculty of Science Masaryk University Brno 625 00 Czech Republic

Department of Chemistry York Structural Biology Laboratory University of York Heslington York YO10 5DD United Kingdom

Department of Experimental Biology Faculty of Science Masaryk University Brno 611 37 Czech Republic

National Centre for Biomolecular Research Faculty of Science Masaryk University Brno 625 00 Czech Republic

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