# Molecular mechanisms of RNA-targeting CRISPR-Cas systems and their membrane-associated accessory proteins

> **NIH NIH R35** · UNIVERSITY OF ROCHESTER · 2024 · $452,437

## Abstract

Microbes employ diverse defense systems to provide protection from viruses, and studying these systems is
important for understanding immune system evolution, phage therapy, and the discovery of new molecular tools
such as CRISPR-Cas systems. While it’s broadly thought that CRISPR-Cas systems work exclusively by
degrading nucleic acids, we’ve recently shown that membrane perturbation by accessory proteins (e.g., Csx28)
are an unappreciated aspect of CRISPR-Cas’s mechanism of action. However, the molecular mechanisms of
how proteins like Csx28 function are unknown. The goal of my research is to understand how CRISPR-Cas
systems and their membrane-associated proteins function together to enhance anti-viral defense with an eye
towards the discovery of new tools. This proposal focuses on addressing the following gaps: 1) What is the
mechanistic and structural details of Csx28 activation by Cas13? We recently discovered that the RNA-targeting
Cas enzyme Cas13 is communicating with membrane pore protein Csx28, to mount a robust anti-viral immune
response, however we still don’t understand what the precise molecular mechanism involved. Our preliminary
data indicate that Cas13 generated RNA cleavage products are acting as ligands to gate Csx28 channel activity.
We will use RNA sequencing and membrane biophysics approaches to determine the precise RNA ligands
modulating Csx28 activity and how these ligands affect gating of the Csx28 pore, respectively. These
experiments will shed light on the molecular details of fascinating anti-phage synergy between Cas13 and Csx28
and provide a clear path for technology development. 2) Do Csx28-like proteins have roles in anti-viral defense
outside of CRISPR systems? With our structural studies of Csx28 coupled with the recent advances in structure
prediction databases, we discovered that Csx28 shares very close structural similarity to a family of hypothetical
proteins that reside in potentially new immune systems. We hypothesize that these Csx28-like proteins are
employed to modulate membrane function during viral infection, however no experimental studies have been
done. We will use similar genetic, biochemical, and structural approaches to our previous work on Csx28 to
study these putative membrane proteins. 3) How does Cas13 associated protein Csx27 modulate antiphage
defense? Csx27 is a predicted transmembrane CRISPR accessory gene that resides within a subset of Cas13b
containing loci. Csx27 has recently been shown to work with Cas13b from to modulate anti-phage defense,
however the mechanism is not understood. Our preliminary data suggest that Csx27, while bearing no similarity
to Csx28, also acts as a membrane pore and activation by Cas13b results in a similar loss of membrane potential,
and a muted metabolism, however more work needs to be done to understand the details these of effects. We
will use a similar set of genetic, biochemical, and structural approaches that were successful previously for Csx28
with...

## Key facts

- **NIH application ID:** 10842493
- **Project number:** 2R35GM133462-06
- **Recipient organization:** UNIVERSITY OF ROCHESTER
- **Principal Investigator:** Mitchell O'Connell
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $452,437
- **Award type:** 2
- **Project period:** 2019-08-01 → 2029-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10842493

## Citation

> US National Institutes of Health, RePORTER application 10842493, Molecular mechanisms of RNA-targeting CRISPR-Cas systems and their membrane-associated accessory proteins (2R35GM133462-06). Retrieved via AI Analytics 2026-06-25 from https://api.ai-analytics.org/grant/nih/10842493. Licensed CC0.

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