# Understanding molecular rules governing bacteriophage specificity and virulence by high-throughput mutational and metagenomic scanning

> **NIH NIH R21** · UNIVERSITY OF WISCONSIN-MADISON · 2021 · $232,813

## Abstract

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.

## Key facts

- **NIH application ID:** 10128864
- **Project number:** 1R21AI156785-01
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** Srivatsan Raman
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $232,813
- **Award type:** 1
- **Project period:** 2020-12-10 → 2022-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10128864

## Citation

> US National Institutes of Health, RePORTER application 10128864, Understanding molecular rules governing bacteriophage specificity and virulence by high-throughput mutational and metagenomic scanning (1R21AI156785-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10128864. Licensed CC0.

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