CRISPR/Cas9-Mediated\nGenome Editing of T4 Bacteriophage\nfor High-Throughput Antimicrobial Susceptibility Testing

The accurate and effective determination of antimicrobial\nresistance\nis essential to limiting the spread of infectious diseases and ensuring\nhuman health. Herein, a simple, accurate, and high-throughput phage-based\ncolorimetric sensing strategy was developed for antimicrobial susceptibility\ntesting (AST). Taking advantage of the CRISPR/Cas9 system, the genome\nof the T4 phage was modularly engineered to carry lacZ-α (lacZa),\na marker gene encoding the α-fragment of β-galactosidase\n(β-gal). T4<sub>lacZa</sub> phages were identified by blue-white\nselection and then used for a biosensing application. In this strategy,\nthe bacterial solution is exposed to the T4<sub>lacZa</sub> phage,\ncausing target bacteria to overexpress β-gal. Upon the addition\nof a colorimetric substrate, the β-gal initiates an enzymatic\nreaction, resulting in a solution color change from yellow to red.\nThis sensing strategy offers a visual way to monitor bacterial growth\nin the presence of antibiotics, enabling the determination of bacterial\nantimicrobial susceptibility. As a proof of concept, our developed\nsensing strategy was successfully applied to identify 9 different\nmultidrug-resistant Escherichia coli (E. coli) in urine samples with 100%\nspecificity. Compared with conventional disk diffusion susceptibility\ntests, the engineered phage-based sensing strategy can shorten the\ndetection time by at least half without losing detection sensitivity,\nproviding an alternative high-throughput method for AST in clinical\ndiagnosis.