The emergence of antibiotic resistance combined with a paucity of new antibiotics under development have sparked a renewed interest in phage therapy as a treatment strategy for infectious diseases caused by common pathogens like Staphylococcus aureus and Staphylococcus epidermidis. However, several challenges remain before phage therapy can become a viable treatment option, including the narrow host range of the phages and concerns about genetic mobilization. Most current therapeutic strategies rely on cocktails of poorly characterized phages with uncertain interaction with the human microbiome. There is therefore a need for a more rational approach to phage therapy based on well-characterized components with defined and programmable host specificities. The bacteriophages of the Picovirinae subfamily of the Podoviridae (picoviruses) are attractive candidates for therapeutic applications due to their small (<20 kbp) genomes and strictly lytic lifestyle. The overall objectives of this exploratory/developmental (R21) project are (1) to understand the determinants for host range and specificity in staphylococcal picoviruses, and (2) to uncover the rules for manipulating this specificity to enable the rational design of therapeutic phages with tunable host range. In this proposal we will test our central hypothesis that picovirus receptor binding protein structures correlate with host cell wall teichoic acid composition. This will be accomplished through a combination of phage discovery and sequence analysis, cryo-electron microscopy, and CRISPR- based genome editing, via three specific aims: (1) Determine the genetic basis for host attachment by staphylococcal picoviruses; (2) Define the structural determinants for picovirus host range and specificity; and (3) Engineer phages with altered host ranges. This work will establish a predictive framework for determining the sensitivity of a pathogen to a specific set of picoviruses. In doing so, this research will provide a versatile toolkit for the rational design of therapeutic phages with pre-determined host specificities against pathogenic staphylococci. This novel approach can also be broadly applied to target other Gram-positive pathogens.