Craspase is a CRISPR RNA-guided, RNA-activated protease

Hu, Chunyi; van Beljouw, Sam P.  B.; Nam, Ki Hyun; Schuler, Gabriel; Ding, Fran; Cui, Yanru; Rodríguez-Molina, Alicia; Haagsma, Anna C.; Valk, Menno; Pabst, Martin; Brouns, Stan J.  J.; Ke, Ailong

doi:10.1126/science.add5064

Craspase is a CRISPR RNA-guided, RNA-activated protease

Journal Article · 16 September 2022 · Science

DOI:https://doi.org/10.1126/science.add5064· OSTI ID:1982963

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Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
Department of Bionanoscience, Delft University of Technology, 2629 HZ Delft, Netherlands.; Kavli Institute of Nanoscience, 2629 HZ Delft, Netherlands.
Department of Life Sciences, Pohang University of Science and Technology, Pohang, Gyeongbuk 37673, Republic of Korea.
Department of Environmental Biotechnology, Delft University of Technology, 2629 HZ Delft, Netherlands.

The CRISPR-Cas type III-E RNA-targeting effector complex gRAMP/Cas7-11 is associated with a caspase-like protein (TPR-CHAT/Csx29) to form Craspase (CRISPR-guided caspase). Here, we use cryo–electron microscopy snapshots of Craspase to explain its target RNA cleavage and protease activation mechanisms. Target-guide pairing extending into the 5' region of the guide RNA displaces a gating loop in gRAMP, which triggers an extensive conformational relay that allosterically aligns the protease catalytic dyad and opens an amino acid side-chain–binding pocket. We further define Csx30 as the endogenous protein substrate that is site-specifically proteolyzed by RNA-activated Craspase. This protease activity is switched off by target RNA cleavage by gRAMP and is not activated by RNA targets containing a matching protospacer flanking sequence. We thus conclude that Craspase is a target RNA–activated protease with self-regulatory capacity.

Research Organization:: Brookhaven National Laboratory (BNL), Upton, NY (United States). Laboratory for BioMolecular Structure (LBMS)

Sponsoring Organization:: USDOE Office of Science (SC)

DOE Contract Number:: SC0012704

OSTI ID:: 1982963

Journal Information:: Science, Journal Name: Science Journal Issue: 6612 Vol. 377; ISSN 0036-8075

Publisher:: AAAS

Country of Publication:: United States

Language:: English

References (35)

Basic local alignment search tool Altschul, Stephen F.; Gish, Warren; Miller, Webb Journal of Molecular Biology, Vol. 215, Issue 3, p. 403-410 https://doi.org/10.1016/S0022-2836(05)80360-2	journal	October 1990
Features and development of Coot Emsley, P.; Lohkamp, B.; Scott, W. G. Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4 https://doi.org/10.1107/S0907444910007493	journal	March 2010
Programmable RNA targeting with the single-protein CRISPR effector Cas7-11 Özcan, Ahsen; Krajeski, Rohan; Ioannidi, Eleonora Nature, Vol. 597, Issue 7878 https://doi.org/10.1038/s41586-021-03886-5	journal	September 2021
A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems Kazlauskiene, Migle; Kostiuk, Georgij; Venclovas, Česlovas Science, Vol. 357, Issue 6351 https://doi.org/10.1126/science.aao0100	journal	June 2017
Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death Shi, Jianjin; Zhao, Yue; Wang, Kun Nature, Vol. 526, Issue 7575 https://doi.org/10.1038/nature15514	journal	September 2015
SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation Steens, Jurre A.; Zhu, Yifan; Taylor, David W. Nature Communications, Vol. 12, Issue 1 https://doi.org/10.1038/s41467-021-25337-5	journal	August 2021
Caspase Activation Shi, Yigong Cell, Vol. 117, Issue 7 https://doi.org/10.1016/j.cell.2004.06.007	journal	June 2004
CRISPR meets caspase Hochstrasser, Megan L.; Nuñez, James K. Nature Microbiology, Vol. 6, Issue 12 https://doi.org/10.1038/s41564-021-01001-y	journal	November 2021
Systematic discovery of antiphage defense systems in the microbial pangenome Doron, Shany; Melamed, Sarah; Ofir, Gal Science, Vol. 359, Issue 6379 https://doi.org/10.1126/science.aar4120	journal	January 2018
The gRAMP CRISPR-Cas effector is an RNA endonuclease complexed with a caspase-like peptidase van Beljouw, Sam P. B.; Haagsma, Anna C.; Rodríguez-Molina, Alicia Science, Vol. 373, Issue 6561 https://doi.org/10.1126/science.abk2718	journal	September 2021
Physiological Characterization of an Anaerobic Ammonium-Oxidizing Bacterium Belonging to the “Candidatus Scalindua” Group Awata, Takanori; Oshiki, Mamoru; Kindaichi, Tomonori Applied and Environmental Microbiology, Vol. 79, Issue 13 https://doi.org/10.1128/AEM.00056-13	journal	July 2013
Type III-A CRISPR-Cas Csm Complexes: Assembly, Periodic RNA Cleavage, DNase Activity Regulation, and Autoimmunity Jia, Ning; Mo, Charlie Y.; Wang, Chongyuan Molecular Cell, Vol. 73, Issue 2 https://doi.org/10.1016/j.molcel.2018.11.007	journal	January 2019
Structure and engineering of the type III-E CRISPR-Cas7-11 effector complex Kato, Kazuki; Zhou, Wenyuan; Okazaki, Sae Cell, Vol. 185, Issue 13 https://doi.org/10.1016/j.cell.2022.05.003	journal	June 2022
Pore-forming activity and structural autoinhibition of the gasdermin family Ding, Jingjin; Wang, Kun; Liu, Wang Nature, Vol. 535, Issue 7610 https://doi.org/10.1038/nature18590	journal	June 2016
RELION: Implementation of a Bayesian approach to cryo-EM structure determination Scheres, Sjors H. W. Journal of Structural Biology, Vol. 180, Issue 3, p. 519-530 https://doi.org/10.1016/j.jsb.2012.09.006	journal	December 2012
Structural basis of cohesin cleavage by separase Lin, Zhonghui; Luo, Xuelian; Yu, Hongtao Nature, Vol. 532, Issue 7597 https://doi.org/10.1038/nature17402	journal	March 2016
Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants Makarova, Kira S.; Wolf, Yuri I.; Iranzo, Jaime Nature Reviews Microbiology, Vol. 18, Issue 2 https://doi.org/10.1038/s41579-019-0299-x	journal	December 2019
Evolutionary and functional classification of the CARF domain superfamily, key sensors in prokaryotic antivirus defense Makarova, Kira S.; Timinskas, Albertas; Wolf, Yuri I. Nucleic Acids Research, Vol. 48, Issue 16 https://doi.org/10.1093/nar/gkaa635	journal	July 2020
Comprehensive search for accessory proteins encoded with archaeal and bacterial type III CRISPR- cas gene cassettes reveals 39 new cas gene families Shah, Shiraz A.; Alkhnbashi, Omer S.; Behler, Juliane RNA Biology, Vol. 16, Issue 4 https://doi.org/10.1080/15476286.2018.1483685	journal	June 2018
Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix Liebschner, Dorothee; Afonine, Pavel V.; Baker, Matthew L. Acta Crystallographica Section D Structural Biology, Vol. 75, Issue 10 https://doi.org/10.1107/S2059798319011471	journal	October 2019
Coupling of ssRNA cleavage with DNase activity in type III-A CRISPR-Csm revealed by cryo-EM and biochemistry Guo, Minghui; Zhang, Kaiming; Zhu, Yuwei Cell Research, Vol. 29, Issue 4 https://doi.org/10.1038/s41422-019-0151-x	journal	February 2019
Structural Mechanism for GSDMD Targeting by Autoprocessed Caspases in Pyroptosis Wang, Kun; Sun, Qi; Zhong, Xiu Cell, Vol. 180, Issue 5 https://doi.org/10.1016/j.cell.2020.02.002	journal	March 2020
Highly accurate protein structure prediction with AlphaFold Jumper, John; Evans, Richard; Pritzel, Alexander Nature https://doi.org/10.1038/s41586-021-03819-2	journal	July 2021
Type III CRISPR–Cas systems produce cyclic oligoadenylate second messengers Niewoehner, Ole; Garcia-Doval, Carmela; Rostøl, Jakob T. Nature, Vol. 548, Issue 7669 https://doi.org/10.1038/nature23467	journal	July 2017
Cryo-EM structure of a metazoan separase–securin complex at near-atomic resolution Boland, Andreas; Martin, Thomas G.; Zhang, Ziguo Nature Structural & Molecular Biology, Vol. 24, Issue 4 https://doi.org/10.1038/nsmb.3386	journal	March 2017
Structural and biochemical characterization of in vivo assembled Lactococcus lactis CRISPR-Csm complex Sridhara, Sagar; Rai, Jay; Whyms, Charlisa Communications Biology, Vol. 5, Issue 1 https://doi.org/10.1038/s42003-022-03187-1	journal	March 2022
Structure Studies of the CRISPR-Csm Complex Reveal Mechanism of Co-transcriptional Interference You, Lilan; Ma, Jun; Wang, Jiuyu Cell, Vol. 176, Issue 1-2 https://doi.org/10.1016/j.cell.2018.10.052	journal	January 2019
The tetratricopeptide repeat: a structural motif mediating protein-protein interactions Blatch, Gregory L.; Lässle, Michael BioEssays, Vol. 21, Issue 11 https://doi.org/10.1002/(SICI)1521-1878(199911)21:11<932::AID-BIES5>3.0.CO;2-N	journal	October 1999
cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination Punjani, Ali; Rubinstein, John L.; Fleet, David J. Nature Methods, Vol. 14, Issue 3 https://doi.org/10.1038/nmeth.4169	journal	February 2017
Bacterial gasdermins reveal an ancient mechanism of cell death Johnson, Alex G.; Wein, Tanita; Mayer, Megan L. Science, Vol. 375, Issue 6577 https://doi.org/10.1126/science.abj8432	journal	January 2022
Structural basis of CRISPR-Cas Type III prokaryotic defence systems Molina, Rafael; Sofos, Nicholas; Montoya, Guillermo Current Opinion in Structural Biology, Vol. 65 https://doi.org/10.1016/j.sbi.2020.06.010	journal	December 2020
UCSF Chimera?A visualization system for exploratory research and analysis Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C. Journal of Computational Chemistry, Vol. 25, Issue 13 https://doi.org/10.1002/jcc.20084	journal	January 2004
MolProbity: More and better reference data for improved all-atom structure validation: PROTEIN SCIENCE.ORG Williams, Christopher J.; Headd, Jeffrey J.; Moriarty, Nigel W. Protein Science, Vol. 27, Issue 1 https://doi.org/10.1002/pro.3330	journal	November 2017
New Type III CRISPR variant and programmable RNA targeting tool: Oh, thank heaven for Cas7-11 Catchpole, Ryan J.; Terns, Michael P. Molecular Cell, Vol. 81, Issue 21 https://doi.org/10.1016/j.molcel.2021.10.014	journal	November 2021
Caspase-1 Engages Full-Length Gasdermin D through Two Distinct Interfaces That Mediate Caspase Recruitment and Substrate Cleavage Liu, Zhonghua; Wang, Chuanping; Yang, Jie Immunity, Vol. 53, Issue 1 https://doi.org/10.1016/j.immuni.2020.06.007	journal	July 2020

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