PROJECT SUMMARY/ABSTRACT Bacteriophage therapy could be a promising solution to the antibiotic resistance crisis as evidenced by many recent success stories. However, the use of natural phages has fundamental limitations in efficacy, reliability, scalability and speed. Natural phages have lower efficacy due evolutionary constraints, give inconsistent results in unwieldy cocktails, and discovery new phages when bacterial resistance arises is slow and laborious. We propose a new framework by high-throughput precision genome engineering of natural phages (as chassis) to create potent phage variants suitable for therapeutic applications. By combining pooled selection experiments with deep sequencing, our approach samples the sequence space of targeted phage genes via systematic mutational profiling and mines the rich diversity of metagenomic sequences to identify new functional variants. The sequence-function knowledgebase from these experiments enhance our basic understanding of how mutations affect phage function, and enable a design-build-test-learn platform for rapid design of new phages against new and resistant bacterial strains. To implement this idea, we developed what we term as ORACLE technology for generating large libraries of phage variants with pre-defined sequences at a target locus on the phage genome using high-throughput recombinase-mediated genome editing and Cas9-guided enrichment. ORACLE can be applied to diversify any phage gene. In this R21 application, we will characterize and engineer receptor binding proteins (RBP) of T7 phage to elucidate sequence-function relationship and to eliminate pathogenic E. coli known to cause urinary tract infection. RBP is the primary determinant of host range as it mediates interaction between phage and host receptors. In Aim 1, we will use ORACLE to systematically dissect the functional role of individual amino acids of T7 RBP (10,507 variants) to understand which residues are critical for specificity, virulence and stability. Ig-like domains found at the distal tip of RBP play a key role in phage adsorption and specificity, and are rampantly exchanged among Caudovirales phages. In Aim 2, we will functionally screen ~25,000 Ig-like domains mined from viral metagenomes by replacing native T7 Ig-like domain to investigate gain-of-function against new hosts. We will assay both libraries (point mutants and metagenomic variants) against a panel of 82 clinical E. coli isolates found in patients with urinary tract infection to find T7 variants for potential therapeutic use. Our initial screens show T7 gain-of-function variants capable of infecting and killing a spontaneously resistant clinical E. coli isolate from a patient with UTI that could not be killed by wildtype. We envision the ORACLE technology platform as a standard tool for development and optimization of chassis phages to target different bacterial clades, strain variants, and to rapidly develop countermeasures against resistant strains.