# Enhancing E. coli Nissle colonization with CRISPR-Cas immunity

> **NIH NIH R41** · ANCILIA, INC. · 2024 · $274,401

## Abstract

Project Summary
 Inflammatory bowel disease (IBD) prevalence is rising globally and live biotherapeutic products (LBPs)
have shown some success in relieving the symptoms of IBD. However, LBP efficacy has been limited in part due
to inadequate engraftment and colonization. Novel technologies are needed to identify and address the factors
limiting the engraftment and efficacy of LBPs in the clinic. Lytic and lysogenic phage predation has the potential
to inhibit the colonization of LBP strains and obstruct their engraftment. Bacterial CRISPR-Cas systems, while
extensively used for genome engineering, provide adaptive immunity against phage predation by degrading
foreign DNA in a programmable and sequence specific manner. Thus, the addition of a rationally designed
CRISPR-Cas system to an LBP can provide phage immunity and support bacterial survival in the presence of
phage predation. Escherichia coli Nissle 1917 (EcN) is an LBP with a long history of human use for a variety of
applications, including treatment of UC and as a chassis for the delivery of therapeutic payloads. However, like
most LBPs, EcN variably engrafts in patients, which can limit its utility in the clinic. We have identified and isolated
EcN phages from stool samples of patients with UC, and further validated the existence of these phages in in
vitro experiments. This work suggests that these phages could directly prevent the survival and efficacy
of this therapeutically significant strain in the clinic.
 Ancilia Bioscience’s long-term goal is to harness CRISPR’s natural function to develop a new class of
LBPs with enhanced engraftment and therapeutic efficacy. The overall objective of this proposal is to improve
EcN colonization in the presence of phage. Our central hypothesis is that phages can directly prevent the
therapeutic efficacy of LBPs. By strategically engineering CRISPR, we can immunize EcN to robustly colonize
the intestinal tract and increase bacterial engraftment. This proposal aims to investigate phage immunity as a
method to augment EcN therapeutic efficacy and fitness in the context of IBD. These studies will be executed
by Ancilia Biosciences through collaborations with Dr. Rodolphe Barrangou, Todd R. Klaenhammer
Distinguished Professor at NC State University, and Dr. Casey Theriot, Associate Professor in Infectious
Disease at NC State University. Furthermore, we expect these results to establish the feasibility of engineering
CRISPR-Cas immunity for improved bacterial engraftment to enable the therapeutic efficacy and
commercialization of LBPs for a range of applications.

## Key facts

- **NIH application ID:** 11007627
- **Project number:** 1R41DK141344-01
- **Recipient organization:** ANCILIA, INC.
- **Principal Investigator:** RODOLPHE BARRANGOU
- **Activity code:** R41 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $274,401
- **Award type:** 1
- **Project period:** 2024-09-19 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11007627

## Citation

> US National Institutes of Health, RePORTER application 11007627, Enhancing E. coli Nissle colonization with CRISPR-Cas immunity (1R41DK141344-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/11007627. Licensed CC0.

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