# 'Class I and III Multi-subunit CRISPR-Cas Surveillance Complexes: Recognition, Cleavage, Autoimmunity and Inhibition’

> **NIH NIH R01** · SLOAN-KETTERING INST CAN RESEARCH · 2020 · $359,200

## Abstract

Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign mobile
genetic elements, such as invading phages and viruses. A central feature of this immune system is RNA-
guided surveillance complexes capable of targeting non-self DNA or RNA for degradation in a sequence- and
site-specific manner. The effector proteins are composed of either single-subunit Cas nucleases or the more
prevalent multi-subunit CRISPR surveillance ribonucleoprotein complexes, together with either their intrinsic
cis-acting or associated trans-acting helicase-nucleases. This application focuses on cryo-EM structural and
biochemical (structure-guided interfacial mutational) studies to elucidate mechanistic insights related to dsDNA
targeting by type I-F and ssRNA/ssDNA targeting by type III-A multi-subunit CRISPR systems, together with
insights into cleavage mechanisms, as well as cleavage inhibition by phage-evolved anti-CRISPR proteins.
Currently, the type III-A Csm is much less well characterized relative to its type-IIIB Cmr counterpart. We have
recently solved cryo-EM based structures of crRNA-bound type III-A Csm (labeled CsmcrRNA) from T.
onnurneus and its complexes with target RNA. In Aim 1 we propose to extend these studies to address
structure-guided mechanistic issues related to the origins of autoimmunity suppression given that type III
systems unlike type I lack a PAM sequence, to decipher the principles underlying target RNA-mediated
transcription-coupled activation of ssDNA activity, as well as the generation of second messenger cyclic
oligoadenyates, which in turn activate the nonspecific RNA degradation activity of trans-acting nuclease Csm6.
We have recently solved cryo-EM based structures of crRNA-bound type I-F Csy complex (labeled CsycrRNA)
from P. aeruginosa in the absence and presence of partial R-loop dsDNA and identified recognition principles
and associated conformational transitions on ternary complex formation. Aim 2 focuses on extending this
research to structures and conformational transitions of CsycrRNA on binding full R-loop dsDNA in the absence
and presence of trans-acting helicase-nuclease Cas3. These efforts should address the principles underlying
non-target DNA strand displacement and R-loop positioning for recognition and cleavage by Cas3.
We have recently solved cryo-EM based structures of type I-F CsycrRNA with bound anti-CRISPR AcrF proteins
1, 2 and 10, thereby identifying alternate strategies utilized by AcrF suppressors for targeting and blocking
different features of the dsDNA recognition machinery. Aim 3 focuses on a structure-based mechanistic
understanding of the function of additional anti-CRISPR AcrF proteins 6, 7, 8 and 9 targeted to CsycrRNA, with
the potential for identifying alternate anti-CRISPR approaches, including allosteric inhibition, for dsDNA
cleavage suppression. To date, there have been no reports of anti-CRISPRs that target type III CRISPR-Cas
systems, but should these b...

## Key facts

- **NIH application ID:** 9906243
- **Project number:** 5R01GM129430-02
- **Recipient organization:** SLOAN-KETTERING INST CAN RESEARCH
- **Principal Investigator:** DINSHAW J PATEL
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $359,200
- **Award type:** 5
- **Project period:** 2019-04-08 → 2023-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9906243

## Citation

> US National Institutes of Health, RePORTER application 9906243, 'Class I and III Multi-subunit CRISPR-Cas Surveillance Complexes: Recognition, Cleavage, Autoimmunity and Inhibition’ (5R01GM129430-02). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9906243. Licensed CC0.

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