CRISPR-Cas10-Assisted Structural Modification of Staphylococcal Kayvirus for Imaging and Biosensing Applications

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• CRISPR-Cas genome editing advances bacteriophage engineering for medicine, nanotechnology, and synthetic biology.
• While staphylococcal phage genomes have been edited, structural protein modification remains unexplored.

Purpose of the Study:

• To genetically modify the structural proteins of Staphylococcus phage 812h1 (Kayvirus genus).
• To introduce a poly histidine tag into the tail sheath protein for enhanced detection and application.

Main Methods:

• A two-strain editing strategy involving homologous recombination.
• CRISPR-Cas10-assisted counter-selection to isolate recombinant phages.
• Insertion of a poly histidine tag into an exposed loop of the tail sheath protein.

Main Results:

• Successfully engineered Staphylococcus phage 812h1 with a His-tagged tail sheath protein.
• His-tagged phage particles were recognized by specific antibodies.
• Engineered phage attachment to bacteria was visualized via fluorescence microscopy.
• Functionality confirmed through biolayer interferometry, ELISA, and flow cytometry, showing no impairment.

Conclusions:

• Genetic modification of bacteriophage structural proteins is feasible using CRISPR-Cas technology.
• The His-tagged phage retains biological activity and can be detected using antibodies.
• This engineered phage offers new possibilities for applications in various scientific fields.