# The Escherichia coli Type I-E CRISPR-Cas immunity system

> **NIH NIH R01** · WADSWORTH CENTER · 2020 · $362,803

## Abstract

SUMMARY
CRISPR-Cas systems are diverse adaptive immunity modules, widespread across bacteria and archaea. We
study Type I CRISPR-Cas systems, which represent the majority of all systems. We use the Escherichia coli
Type I-E system as a model. Importantly, many aspects of Type I CRISPR systems are mechanistically distinct
from the well-studied Cas9 Type II system. Our long-term goal is to develop a detailed mechanistic understanding
of all steps of E. coli CRISPR-Cas function, including CRISPR RNA expression, targeting of invading DNA
molecules, and acquisition of new immunity elements (“spacers”). A complete understanding of CRISPR systems
is a highly significant goal, since they have huge potential to be developed as research tools for genome
engineering and high-throughput functional genomic analyses, and as sequence-specific anti-microbials.
The two major aspects of Type I CRISPR-Cas biology are (i) targeting of invading DNA molecules and (ii)
acquisition of new spacers. The Cascade protein complex binds CRISPR RNAs and targets DNA sequences
that are complementary to the CRISPR RNA sequence. This results in destruction of the target DNA by the Cas3
nuclease. The major mechanism by which new spacers are acquired is known as “priming”. Priming requires
that Cascade (bound to a CRISPR RNA) targets an invading DNA molecule, although not necessarily with high
affinity. This results in acquisition of a spacer from a different position in the targeted DNA molecule. Key
questions about Type I CRISPR-Cas biology remain unanswered. First, it is unclear how the processes of
Cascade binding to target DNA, Cas3-mediated destruction of target DNA, and priming, are connected. Second,
it is unclear which sequence elements are required for each of these processes. Third, the process of priming is
poorly understood. We do not know the composition of the priming complex, nor do we know how new spacers
are acquired. We will leverage our expertise in genetics, genomics, and high-throughput techniques to address
these critical, unanswered questions by pursuing the following specific aims:
Specific Aim 1 – Determine the spacer/protospacer sequence requirements for Cascade binding, priming
and target destruction.
Specific Aim 2 – Determine the molecular details of priming.
Specific Aim 3 – Determine the role of Nus factors in CRISPR RNA expression.

## Key facts

- **NIH application ID:** 9858367
- **Project number:** 5R01GM122836-03
- **Recipient organization:** WADSWORTH CENTER
- **Principal Investigator:** Joseph Thomas Wade
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $362,803
- **Award type:** 5
- **Project period:** 2018-02-01 → 2022-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9858367

## Citation

> US National Institutes of Health, RePORTER application 9858367, The Escherichia coli Type I-E CRISPR-Cas immunity system (5R01GM122836-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9858367. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*