Molecular mechanism of active Cas7-11 in processing CRISPR RNA and interfering target RNA

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Abstract

Cas7-11 is a Type III-E CRISPR Cas effector that confers programmable RNA cleavage and has potential applications in RNA interference. Cas7-11 encodes a single polypeptide containing four Cas7- and one Cas11-like segments that obscures the distinction between the multi-subunit Class 1 and the single-subunit Class-2 CRISPR-Cas systems. We report a cryo-EM structure of the active Cas7-11 from Desulfonema ishimotonii (DiCas7-11) that reveals the molecular basis for RNA processing and interference activities. DiCas7-11 arranges its Cas7- and Cas11-like domains in an extended form that resembles the backbone made up by four Cas7 and one Cas11 subunits in the multi-subunit enzymes. Unlike the multi-subunit enzymes, however, the backbone of DiCas7-11 contains evolutionarily different Cas7 and Cas11 domains, giving rise to their unique functionality. The first Cas7-like domain nearly engulfs the last 15 direct repeat nucleotides in processing and recognition of the CRISPR RNA, and its free-standing fragment retains most of the activity. Both the second and the third Cas7-like domains mediate target RNA cleavage in a metal-dependent manner. The structure and mutational data indicate that the long variable insertion to the fourth Cas7 domain has little impact to RNA processing or targeting, suggesting the possibility for engineering a compact and programmable RNA interference tool.

Data availability

Structure model generated from this study is deposited to Protein Data Bank under the accession code 8D1V. The cryoEM map is deposited to EMDB under the accession code EMD-27138.

The following data sets were generated

(2022) Structure of Desulfonema ishimotonii Cas7-11
PDB, 8D1V.

https://www.rcsb.org/structure/8D1V
(2022) EM map of Desulfonema ishimotonii Cas7-11
EMDB, EMD-27138.

https://www.ebi.ac.uk/emdb/EMD-27138

Article and author information

Author details

Hemant N Goswami

Institute of Molecular Biophysics, Florida State University, Tallahassee, United States

Competing interests
The authors declare that no competing interests exist.
Jay Rai

Institute of Molecular Biophysics, Florida State University, Tallahassee, United States

Competing interests
The authors declare that no competing interests exist.
Anuska Das

Institute of Molecular Biophysics, Florida State University, Tallahassee, United States

Competing interests
The authors declare that no competing interests exist.
Hong Li

Department of Chemistry and Biochemistry, Florida State University, Tallahassee, United States

For correspondence
hong.li@fsu.edu

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0003-2046-9861

Funding

National Institutes of Health (GM101343)

Hong Li

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.