GENETIC REMOVAL OF TOXIN AND INTEGRASE GENES FROM A STAPHYLOCOCCAL BACTERIOPHAGE

Abstract

Bacteriophages, or phages, are viruses that specifically infect and kill bacterial cells. Phages are the most numerous biological entities on Earth, with an estimated population size of 1031 (1). Discovery, purification, and characterization of phages illuminate the composition of the microbial world and provide potential applications in medicine. To combat antibiotic-resistant bacteria, phages are being investigated as supplements or alternatives to antibiotics. Methicillin-resistant Staphylococcus aureus (MRSA) is a commonly acquired infection in hospitals that has become difficult to treat due to its resistance to antibiotics commonly used to treat ordinary S. aureus infections (20). Bacteriophage, JB, isolated from dairy cow hair samples by Tyler Nygaard at Montana State University, has the ability to infect MRSA USA300 strain LAC. Genome annotation of this phage characterized it as a 42,683 bp circularly permuted genome with 68 putative protein coding genes, and a G/C content of 35.2%. Through bioinformatic analysis it was discovered that in a cluster of reverse transcribed (genes 30-33), putative integrase and toxin genes were present (21). In order for this phage to be used therapeutically in mammalian organisms, the deletion of these genes is essential to prevent the possibility for the phage to produce viable lysogen bacterial cells with enhanced pathogenicity. Genetic engineering to remove genes 30-33 was performed using type the type II CRISPR-Cas9 system to generate a strictly lytic deletion mutant JB bacteriophage, JBΔ30-33. The creation of the lytic derivative of JB bacteriophage, JBΔ30-33, was confirmed by clear plaque morphology, polymerase chain reaction, and DNA sequencing