Genetic engineering of a temperate phage-based delivery system for CRISPR/Cas9 antimicrobials against Staphylococcus aureus.
Discovery of clustered, regularly interspaced, short palindromic repeats and the Cas9 RNA-guided nuclease (CRISPR/Cas9) system provides a new opportunity to create programmable gene-specific antimicrobials that are far less likely to drive resistance than conventional antibiotics. However, the practical therapeutic use of CRISPR/Cas9 is still questionable due to current shortcomings in phage-based delivery systems such as inefficient delivery, narrow host range, and potential transfer of virulence genes by generalized transduction. In this study, we demonstrate genetic engineering strategies to overcome these shortcomings by integrating CRISPR/Cas9 system into a temperate phage genome, removing major virulence genes from the host chromosome, and expanding host specificity of the phage by complementing tail fiber protein. This significantly improved the efficacy and safety of CRISPR/Cas9 antimicrobials to therapeutic levels in both in vitro and in vivo assays. The genetic engineering tools and resources established in this study are expected to provide an efficacious and safe CRISPR/Cas9 antimicrobial, broadly applicable to Staphylococcus aureus.
Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762, United States.
Joo Youn
JY
Park
Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762, United States.|Department of Microbiology, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, Seoul 151-742, South Korea.
Bo Youn
BY
Moon
Department of Computer Engineering and Computer Science, KBRIN, University of Louisville, KY 40208, United States.
Juw Won
JW
Park
Department of Biological Sciences, Mississippi State University, Mississippi State, MS 39762, United States.
Justin A
JA
Thornton
Department of Microbiology, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, Seoul 151-742, South Korea.
Yong Ho
YH
Park
Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS 39762, United States.
Keun Seok
KS
Seo
D001427:Bacterial Toxins; D001435:Bacteriophages / Q000502:physiology*; D064113:CRISPR-Cas Systems; D018014:Gene Transfer Techniques; D005818:Genetic Engineering; D016679:Genome, Viral; D058507:Host Specificity; D006801:Humans; D010957:Plasmids / Q000235:genetics; D013211:Staphylococcus aureus / Q000235:genetics* / Q000821:virology*
D001427:Bacterial Toxins