Project summary/Abstract Staphylococcus aureus, causes a wide variety of life-threatening infections, many of which cannot be treated effectively, owing to antibiotic resistance. Consequently, there is an urgent need for new ways to treat these infections, which annually cause some 18,000 deaths in the US. We have developed a novel non-antibiotic method, for treating staphylococcal infections based on the highly mobile staphylococcal pathogenicity islands (SaPIs). The SaPIs are ~15 kb genetic elements that are stably inserted in the staph chromosome but can be induced by “helper” phages to excise and replicate. The replicated SaPI DNA is packaged in infectious phage- like particles which are released upon phage-induced lysis. The SaPIs carry and disseminate genes encoding superantigen toxins and other virulence factors. Instead of working on the prevention of SaPI spread, we hit upon the idea of exploiting SaPI spread by converting these agents of disease into agents of therapy – antibacterial drones (ABDs). To create the ABDs, we have re-engineered the SaPIs, deleting their natural cargo (toxin genes), increasing their packaging capacity from 15 to >40 kb, and inserting antibacterial modules. We have also modified the helper phage so that ABD particles are produced in the absence of functional phage. The ABD particles are administered to an infected animal (or plant), where they attach to the infecting bacteria, insert their DNA, express their antibacterial cargo genes and thus abrogate the infection. As proof of principle, we have begun by incorporating into ABDs either CRISPR/cas9 or CRISPR/dcas9 modules with spacers targeting a chromosomal gene or the promoter region of a global virulence regulator, respectively. Preliminary studies have shown that the CRISPR/cas9-containing ABD kills S. aureus in vitro by DNA cleavage, blocks the development of a subcutaneous S. aureus abscess, and rescues mice given a lethal dose of S. aureus intraperitoneally. The CRISPR/dcas9 containing ABD blocks the expression of staphylococcal virulence in vitro and blocks the formation of a subcutaneous abscess in vivo. This proposal outlines our program to develop the ABD system and validate our underlying hypothesis that SaPIs can be converted to versatile and fully effective anti-staphylococcal therapeutic agents. There are 3 specific aims. In Aim I we will construct and test ABDs designed to treat the wide array of infections caused by S. aureus. In Aim II, we will focus on expanding the host range of the ABDs to target diverse S. aureus strains. In Aim III, we will encapsidate the ABD particles for increased efficacy, test for ABD resistance and immunogenicity, and refine our procedures for producing and preserving high titer ABD preparations.