Šimečková Hana, Bárdy Pavol, Kuntová Lucie, Macháčová Eliška, Botka Tibor, Bíňovský Ján, Houser Josef, Farka Zdeněk, Plevka Pavel, Pantůček Roman, Mašlaňová Ivana
Department of Experimental Biology, Faculty of Science, Masaryk University, Brno 611 37, Czech Republic.
Department of Chemistry, York Structural Biology Laboratory, University of York, Heslington, York YO10 5DD, United Kingdom.
ACS Synth Biol. 2025 Aug 15;14(8):2979-2986. doi: 10.1021/acssynbio.5c00387. Epub 2025 Jul 28.
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 phage 812h1 of the 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.
基于CRISPR-Cas的基因组编辑技术的最新进展为噬菌体工程开辟了新的可能性，从而推动了医学、纳米技术和合成生物学的关键发展。尽管葡萄球菌噬菌体基因组已经被编辑，但它们结构蛋白的修饰尚未见报道。在此，通过将多组氨酸标签插入尾鞘蛋白的一个暴露环中，对该属噬菌体812h1的结构进行了修饰。应用了一种双菌株编辑策略，利用同源重组，随后通过CRISPR-Cas10辅助对重组噬菌体进行反选择。带有His标签的噬菌体颗粒可以被特异性抗体识别，使得修饰后的噬菌体能够应用于多种技术。通过荧光显微镜观察了工程噬菌体与细菌的结合情况，并使用生物层干涉术生物传感、酶联免疫吸附测定和流式细胞术证实了其功能，表明基因修饰并未损害其生物活性。
