Genetic Engineering of Therapeutic Phages Using Type III CRISPR-Cas Systems

The functional characterization of “hypothetical” phage genes is a major bottleneck in basic and applied phage research. To compound this issue, the most suitable phages for therapeutic applications—the strictly lytic variety—are largely recalcitrant to classical genetic techniques due to low recombination rates and lack of selectable markers. Here we describe methods for fast and effective phage engineering that rely upon a Type III-A CRISPR-Cas system. In these methods, the CRISPR-Cas system is used as a powerful counterselection tool to isolate rare phage recombinants.