# Mechanisms and functions of CRISPR-Casautoregulation in bacterial immunity and pathogenesis

> **NIH NIH F31** · JOHNS HOPKINS UNIVERSITY · 2021 · $46,036

## Abstract

Project Summary
The goal of my research is to investigate how bacterial CRISPR-Cas immune systems are regulated and how
this regulation contributes to bacterial immunity and virulence. CRISPR-Cas systems protect bacteria from
bacteriophages and other mobile genetic elements and thereby prevent cell lysis but also limit the potential for
horizontal gene transfer, a major route for the dissemination of antibiotic-resistance genes. Despite all that is
known about CRISPR-Cas biology from a mechanistic standpoint, much remains to be understood about how
these systems function in their native bacterial hosts. In particular, we lack an understanding of the ways by
which bacteria regulate CRISPR-Cas expression to maximize immunity while mitigating autoimmunity and the
metabolic burden of constitutive system expression. Furthermore, Cas9 is itself a virulence factor in many
human pathogens, including S. pyogenes, S. agalactiae, F. novicida, N. meningitidis, and C. jejuni although it
is unclear how Cas9 contributes to pathogenesis. For these reasons, it is critical to understand whether and
how CRISPR-Cas expression is regulated to prepare bacteria for an impending phage infection or for growth in
a human host.
 To address this gap in knowledge, we performed a screen to identify regulators of CRISPR-Cas
immunity. Interestingly, we discovered that trL, a noncoding RNA within the S. pyogenes CRISPR-Cas locus, is
capable of folding into a natural single-guide RNA that directs Cas9 to transcriptionally silence the Cas operon
promoter (Workman et al., 2020). While a trL deletion enhances Cas gene expression by ~50-fold and
stimulates CRISPR-Cas immunity by 3000-fold, it remains unknown how trL de-repression occurs under
physiological conditions. In this proposal, I will identify the conditions and genetic pathways that mediate trL de-
repression and assess the impact of this regulation on bacterial immunity and virulence. We have obtained
preliminary data demonstrating that CRISPR RNAs (crRNAs) and growth-phase specific cues modulate Cas9
expression; however, the mechanisms and consequences of Cas9 induction remain to be tested. In Aim 1, we
test the hypothesis that crRNAs and trL form an integrated genetic circuit that controls CRISPR-Cas
expression, providing a novel mechanism through which spacers, the molecular “memories” of infection,
differentially affect immunity. In Aim 2, we investigate the physiological cues and genetic networks that cause
Cas9 to accumulate in late stationary phase, and we probe whether this regulation affects immunity and
virulence in S. pyogenes. The proposed studies will help us understand how pathogenic bacteria regulate
CRISPR-Cas expression in order to survive in hostile environments. Finally, our work will inform the
development of regulatable Cas9 tools and new therapeutic targets and strategies for human pathogens.

## Key facts

- **NIH application ID:** 10235985
- **Project number:** 1F31GM142275-01
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Rachael Workman Sparklin
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $46,036
- **Award type:** 1
- **Project period:** 2021-05-15 → 2024-05-14

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10235985

## Citation

> US National Institutes of Health, RePORTER application 10235985, Mechanisms and functions of CRISPR-Casautoregulation in bacterial immunity and pathogenesis (1F31GM142275-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10235985. Licensed CC0.

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