# CRISPR interference-enabled phenotyping of essential genes in C. difficile to aid in discovery of antibiotic targets

> **NIH NIH R21** · UNIVERSITY OF IOWA · 2022 · $208,575

## Abstract

Project Summary
Clostridioides (Clostridium) difficile infections strike close to 500,000 people a year in the United States, leading
to nearly 30,000 deaths. The CDC has declared this organism an “urgent” threat to public health, the highest
threat category. New treatments are sorely needed. Most antibiotics inhibit proteins that are essential for viability,
so a better understanding of these proteins and the genes that encode them can advance efforts to develop new
antibiotics. Although essential genes are difficult to study, new techniques for manipulating bacterial gene
expression are overcoming this barrier. One emerging technology for studying essential genes in bacteria is
called CRISPR interference (CRISPRi). CRISPRi uses a short RNA to guide a protein called dCas9 to a gene,
thereby creating a roadblock that prevents RNA polymerase from transcribing that gene. CRISPRi is well-suited
for screening large sets of genes because repressing a new target is simply a matter of cloning a synthetic DNA
sequence encoding the right guide RNA into the plasmid, a process that is easily scaled-up. Another key feature
of our CRISPRi system is that dCas9 is expressed from a xylose-inducible promoter, which allows us to control
the timing and extent of gene repression. The specific objective of this proposal is to deploy CRISPRi to
characterize 187 putatively essential genes in C. difficile. In Aim 1 we will construct a CRISPRi library and use it
to vet the essentiality of the genes on our target list. In Aim 2 we will determine whether gene knockdown results
in morphological defects or increases sensitivity to a panel of antibiotics chosen to target a variety of cellular
processes. The information from these assays will enable us to assign many of these genes to functional
pathways. It will also provide foundational knowledge and tools for developing new antibiotics to treat C. difficile
infections.

## Key facts

- **NIH application ID:** 10369416
- **Project number:** 1R21AI159071-01A1
- **Recipient organization:** UNIVERSITY OF IOWA
- **Principal Investigator:** DAVID S WEISS
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $208,575
- **Award type:** 1
- **Project period:** 2021-11-02 → 2023-10-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10369416

## Citation

> US National Institutes of Health, RePORTER application 10369416, CRISPR interference-enabled phenotyping of essential genes in C. difficile to aid in discovery of antibiotic targets (1R21AI159071-01A1). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10369416. Licensed CC0.

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