# Determining the specificity and regulation of Type III CRISPR-Cas interference

> **NIH NIH R35** · UNIVERSITY OF ALABAMA IN TUSCALOOSA · 2021 · $368,258

## Abstract

Project Summary
CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins
are a group of diverse crRNA guided nucleases providing prokaryotes adaptive immunity to foreign genetic
elements. Cas10-Csm is an approximately 300 kDa multiprotein complex that upon detecting foreign RNA
transcripts initiates a DNA and RNA degradation response. Distinct CRISPR-Cas types use the information in
crRNA to detect either double-stranded DNA or single-stranded RNA. While much is known concerning the
specificity mechanisms of dsDNA detection by Type I (Cascade) and Type II (Cas9) systems, little is known
concerning the specificity mechanisms of ssRNA detection by Type III (Cas10) systems. This is significant
because evidence exists that the mechanisms for enforcing specificity during dsDNA detection and ssRNA
detection are fundamentally different: competition between rehybridization of the non-base paired DNA strand
to re-form dsDNA or binding to a Cas protein is essential to the dsDNA detecting specificity mechanism. This
mechanism is not possible during specific detection of ssRNA. The hypothesis will be tested that in the Cas10-
Csm system mismatches in specific, sensitive locations within the crRNA-target RNA duplex disrupt
interference. Cas10-Csm structural models suggest the Csm2 component of Cas10-Csm directly contacts
bound target RNA. We will test the hypothesis that Csm2 plays an integral role in detecting cognate RNA
binding to Cas10-Csm and relays this signal to Cas10 via structural changes. We will use cryo-EM to
determine the `structural mechanism' for Cas10 activation. The research proposed will impact several
emerging biotechnologies such as the deployment of Cas10 as a point-of-care RNA virus diagnostic.

## Key facts

- **NIH application ID:** 10276334
- **Project number:** 1R35GM142966-01
- **Recipient organization:** UNIVERSITY OF ALABAMA IN TUSCALOOSA
- **Principal Investigator:** Jack Albert Dunkle
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $368,258
- **Award type:** 1
- **Project period:** 2021-09-01 → 2026-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10276334

## Citation

> US National Institutes of Health, RePORTER application 10276334, Determining the specificity and regulation of Type III CRISPR-Cas interference (1R35GM142966-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10276334. Licensed CC0.

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