# Non-canonical functions of Cas9 andthe cell biology of CRISPR-Cas immunity

> **NIH NIH R35** · JOHNS HOPKINS UNIVERSITY · 2022 · $409,375

## Abstract

SUMMARY
CRISPR-Cas loci provide bacteria with adaptive immunity against phages and plasmids. By
remembering and destroying foreign genetic elements, CRISPR-Cas systems also restrict
horizontal gene transfer, a frequent route for the dissemination of antibiotic resistance genes
and toxins. To balance defense with genetic exchange, CRISPR-Cas systems likely undergo
spatiotemporal regulation; however, little is known about the ways in which CRISPR-Cas loci
interact with their bacterial hosts or changing environments.
My laboratory studies type II CRISPR-Cas systems, which encode the gene-editing tool cas9.
CRISPR-Cas9 systems are frequently found in human pathogens like S. pyogenes, and we are
focused on understanding how CRISPR-Cas9 activities are intertwined with and often defined
by the biology of their bacterial host cell. We recently discovered that S. pyogenes Cas9
performs a novel autoregulatory function. A non-canonical guide RNA repurposes Cas9 from a
nuclease into a transcriptional repressor that silences its own promoter. This finding helps
explain how CRISPR-Cas9 systems prevent autoimmunity against the bacterial chromosome,
but it remains unclear how CRISPR-Cas expression can be induced as needed, for instance
during or preceding a phage infection.
In this proposal, we explore two new directions in CRISPR-Cas biology. First, we investigate the
non-canonical regulatory roles of Cas9. Specifically, we characterize the conditions and
mechanisms that allow Cas9 to transiently relieve its repression and induce CRISPR-Cas
expression, and we ask whether Cas9 evolved to regulate other bacterial genes outside the
CRISPR-Cas locus. These studies will establish a new foundation for understanding the role of
Cas9 in the physiology of commensal and pathogenic bacteria. Furthermore, our studies on
non-canonical guide RNAs will lead to new strategies for the development of controllable Cas9
technologies and therapies. In our second project, we establish an innovative imaging platform
to observe live CRISPR-Cas immunization events for the first time. This project will illuminate a
fundamental unanswered question in CRISPR-Cas biology: why are new memories successfully
formed in only one in a million infected cells? The answers will provide new clues into the ways
in which heterogeneity within bacterial populations enables survival. Furthermore, these insights
and tools will be valuable for the development of phage therapies, which are offering promise in
combatting the growing threat of antibiotic-resistant pathogens.

## Key facts

- **NIH application ID:** 10458754
- **Project number:** 5R35GM142731-02
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Joshua Modell
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $409,375
- **Award type:** 5
- **Project period:** 2021-08-01 → 2026-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10458754

## Citation

> US National Institutes of Health, RePORTER application 10458754, Non-canonical functions of Cas9 andthe cell biology of CRISPR-Cas immunity (5R35GM142731-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10458754. Licensed CC0.

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