scholarly journals Control of cyclic oligoadenylate synthesis in a type III CRISPR system

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Christophe Rouillon ◽  
Januka S Athukoralage ◽  
Shirley Graham ◽  
Sabine Grüschow ◽  
Malcolm F White
Keyword(s):  
Transcription Factors ◽  
Rna Binding ◽  
Rna Cleavage ◽  
Type Iii ◽  
Rna Transcripts ◽  
Crispr System ◽  
Cyclase Domain ◽  
Activated State ◽  
Target Rna ◽  
Oligoadenylate Synthesis

The CRISPR system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. When viral RNA transcripts are detected, type III systems adopt an activated state that licenses DNA interference and synthesis of cyclic oligoadenylate (cOA). cOA activates nucleases and transcription factors that orchestrate the antiviral response. We demonstrate that cOA synthesis is subject to tight temporal control, commencing on target RNA binding, and is deactivated rapidly as target RNA is cleaved and dissociates. Mismatches in the target RNA are well tolerated and still activate the cyclase domain, except when located close to the 3’ end of the target. Phosphorothioate modification reduces target RNA cleavage and stimulates cOA production. The ‘RNA shredding’ activity originally ascribed to type III systems may thus be a reflection of an exquisite mechanism for control of the Cas10 subunit, rather than a direct antiviral defence.

scholarly journals Type III CRISPR-Cas systems generate cyclic oligoadenylate second messengers to activate Csm6 RNases

2017 ◽  
Author(s):  
Ole Niewoehner ◽  
Carmela Garcia-Doval ◽  
Jakob T. Rostøl ◽  
Christian Berk ◽  
Frank Schwede ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Rna Binding ◽  
Second Messengers ◽  
Allosteric Activation ◽  
Conceptual Similarity ◽  
Type Iii ◽  
Rna Transcripts ◽  
Rna Targeting ◽  
Target Rna

ABSTRACTIn many prokaryotes, type III CRISPR–Cas systems detect and degrade invasive genetic elements by an RNA-guided, RNA-targeting multisubunit interference complex that possesses dual RNase and DNase activities. The CRISPR-associated protein Csm6 additionally contributes to interference by functioning as a standalone ribonuclease that degrades invader RNA transcripts, but the mechanism linking invader sensing to Csm6 activity is not understood. Here we show that Csm6 proteins are activated through a second messenger generated by the type III interference complex. Upon target RNA binding by the type III interference complex, the Cas10 subunit converts ATP into a cyclic oligoadenylate product, which allosterically activates Csm6 by binding to its CARF domain. CARF domain mutations that abolish allosteric activation inhibit Csm6 activity in vivo, and mutations in the Cas10 Palm domain phenocopy loss of Csm6. Together, these results point to a hitherto unprecedented mechanism for regulation of CRISPR interference that bears striking conceptual similarity to oligoadenylate signalling in mammalian innate immunity.

scholarly journals SCOPE enables type III CRISPR-Cas diagnostics using flexible targeting and stringent CARF ribonuclease activation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jurre A. Steens ◽  
Yifan Zhu ◽  
David W. Taylor ◽  
Jack P. K. Bravo ◽  
Stijn H. P. Prinsen ◽  
...  
Keyword(s):  
Immune Response ◽  
Diagnostic Tool ◽  
Nasal Swab ◽  
Rna Binding ◽  
Seed Region ◽  
Base Pairing ◽  
Specific Detection ◽  
Type Iii ◽  
Single Host ◽  
Target Rna

AbstractCharacteristic properties of type III CRISPR-Cas systems include recognition of target RNA and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of the target RNA and production of cyclic oligoadenylate (cOA) molecules. Here we report that an exposed seed region at the 3′ end of the crRNA is essential for target RNA binding and cleavage, whereas cOA production requires base pairing at the 5′ end of the crRNA. Moreover, we uncover that the variation in the size and composition of type III complexes within a single host results in variable seed regions. This may prevent escape by invading genetic elements, while controlling cOA production tightly to prevent unnecessary damage to the host. Lastly, we use these findings to develop a new diagnostic tool, SCOPE, for the specific detection of SARS-CoV-2 from human nasal swab samples, revealing sensitivities in the atto-molar range.

scholarly journals Specificity and sensitivity of an RNA targeting type III CRISPR complex coupled with a NucC endonuclease effector

2021 ◽  
Author(s):  
Sabine Gruschow ◽  
Catherine S Adamson ◽  
Malcolm F White
Keyword(s):  
Rna Binding ◽  
Limit Of Detection ◽  
Flanking Sequence ◽  
Guide Rna ◽  
Type Iii ◽  
Specificity And Sensitivity ◽  
Virus Rna ◽  
Messenger Molecules ◽  
Target Rna

Type III CRISPR systems detect invading RNA, resulting in the activation of the enzymatic Cas10 subunit. The Cas10 cyclase domain generates cyclic oligoadenylate (cOA) second messenger molecules, activating a variety of effector nucleases that degrade nucleic acids to provide immunity. The prophage-encoded Vibrio metoecus type III-B (VmeCmr) locus is uncharacterised, lacks the HD nuclease domain in Cas10 and encodes a NucC DNA nuclease effector that is also found associated with Cyclic-oligonucleotide-based anti-phage signalling systems (CBASS). Here we demonstrate that VmeCmr is activated by target RNA binding, generating cyclic-triadenylate (cA3) to stimulate a robust NucC-mediated DNase activity. The specificity of VmeCmr is probed, revealing the importance of specific nucleotide positions in segment 1 of the RNA duplex and the protospacer flanking sequence (PFS). We harness this programmable system to demonstrate the potential for a highly specific and sensitive assay for detection of the SARS-CoV-2 virus RNA with a limit of detection (LoD) of 2 fM using a commercial plate reader without any extrinsic amplification step. The sensitivity is highly dependent on the guide RNA used, suggesting that target RNA secondary structure plays an important role that may also be relevant in vivo.

scholarly journals Regulation of the RNA and DNA nuclease activities required for Pyrococcus furiosus Type III-B CRISPR–Cas immunity

2020 ◽  
Vol 48 (8) ◽  
pp. 4418-4434 ◽  
Author(s):  
Kawanda Foster ◽  
Sabine Grüschow ◽  
Scott Bailey ◽  
Malcolm F White ◽  
Michael P Terns
Keyword(s):  
Rna Binding ◽  
Pyrococcus Furiosus ◽  
Type Iii ◽  
Second Messenger Signaling ◽  
Biochemical Analyses ◽  
Rna Interaction ◽  
Dual Mechanism ◽  
Target Rna ◽  
Rna And Dna

Abstract Type III CRISPR–Cas prokaryotic immune systems provide anti-viral and anti-plasmid immunity via a dual mechanism of RNA and DNA destruction. Upon target RNA interaction, Type III crRNP effector complexes become activated to cleave both target RNA (via Cas7) and target DNA (via Cas10). Moreover, trans-acting endoribonucleases, Csx1 or Csm6, can promote the Type III immune response by destroying both invader and host RNAs. Here, we characterize how the RNase and DNase activities associated with Type III-B immunity in Pyrococcus furiosus (Pfu) are regulated by target RNA features and second messenger signaling events. In vivo mutational analyses reveal that either the DNase activity of Cas10 or the RNase activity of Csx1 can effectively direct successful anti-plasmid immunity. Biochemical analyses confirmed that the Cas10 Palm domains convert ATP into cyclic oligoadenylate (cOA) compounds that activate the ribonuclease activity of Pfu Csx1. Furthermore, we show that the HEPN domain of the adenosine-specific endoribonuclease, Pfu Csx1, degrades cOA signaling molecules to provide an auto-inhibitory off-switch of Csx1 activation. Activation of both the DNase and cOA generation activities require target RNA binding and recognition of distinct target RNA 3′ protospacer flanking sequences. Our results highlight the complex regulatory mechanisms controlling Type III CRISPR immunity.

scholarly journals Author response: Control of cyclic oligoadenylate synthesis in a type III CRISPR system

2018 ◽  
Author(s):  
Christophe Rouillon ◽  
Januka S Athukoralage ◽  
Shirley Graham ◽  
Sabine Grüschow ◽  
Malcolm F White
Keyword(s):  
Author Response ◽  
Response Control ◽  
Type Iii ◽  
Crispr System ◽  
Oligoadenylate Synthesis

scholarly journals Ring nucleases deactivate Type III CRISPR ribonucleases by degrading cyclic oligoadenylate

2018 ◽  
Author(s):  
Januka S Athukoralage ◽  
Christophe Rouillon ◽  
Shirley Graham ◽  
Sabine Grüschow ◽  
Malcolm F White
Keyword(s):  
Transcription Factors ◽  
Adaptive Immunity ◽  
Effector Proteins ◽  
Antiviral State ◽  
Type Iii ◽  
Downstream Effector ◽  
Ring Molecules ◽  
Family Protein ◽  
Independent Mechanism ◽  
Target Rna

AbstractThe CRISPR-Cas system provides adaptive immunity against mobile genetic elements in prokaryotes, utilising small CRISPR RNAs which direct effector complexes to degrade invading entities. Type III effector complexes were recently demonstrated to synthesise a novel second messenger, cyclic oligoadenylate (cOA), on binding target RNA. cOA in turn binds to and activates a range of downstream effector proteins including ribonucleases (Csm6/Csx1) and transcription factors via a CARF (CRISPR associated Rossman Fold) domain, inducing an antiviral state in the cell that is important for immunity. The mechanism of the “off-switch” that resets the system is not understood. Here, we report the identification of the nuclease that degrades these cOA ring molecules. The “Ring nuclease” is itself a CARF family protein with a metal independent mechanism, which cleaves cOA4 rings to generate linear di-adenylate species and switches off the antiviral state. The identification of Ring nucleases adds an important insight to the CRISPR-Cas system.

scholarly journals SCOPE: Flexible targeting and stringent CARF activation enables type III CRISPR-Cas diagnostics

2021 ◽  
Author(s):  
Jurre A. Steens ◽  
Yifan Zhu ◽  
David W. Taylor ◽  
Jack P.K. Bravo ◽  
Stijn H.P Prinsen ◽  
...  
Keyword(s):  
Natural Variation ◽  
Nasal Swab ◽  
Rna Binding ◽  
Seed Region ◽  
Specific Detection ◽  
Unique Region ◽  
Type Iii ◽  
Single Host ◽  
Messenger Molecules ◽  
Target Rna

SummaryCharacteristic properties of type III CRISPR-Cas systems include recognition of target RNA (rather than DNA) and the subsequent induction of a multifaceted immune response. This involves sequence-specific cleavage of a target RNA and production of cyclic oligoadenylate (cOA) second messenger molecules that may trigger dormancy or cell death. In this study, we discovered that a largely exposed seed region at the 3’ end of the crRNA is essential for target RNA binding and cleavage, whereas base pairing at a unique region at the 5’ end of the guide is required to trigger cOA production. Moreover, we uncovered that the natural variation in the composition of type III complexes within a single host results in different guide lengths, and hence variable seed regions. This shifting seed may prevent escape by invading genetic elements, while controlling cOA production very tightly to prevent unnecessary damage to the host. Lastly, we used these findings to develop a new diagnostic tool, named SCOPE, which was used for the specific detection of SARS-CoV-2 from human nasal swab samples, showing sensitivities in the atto-molar range.

scholarly journals Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence

2019 ◽  
Vol 47 (17) ◽  
pp. 9259-9270 ◽  
Author(s):  
Sabine Grüschow ◽  
Januka S Athukoralage ◽  
Shirley Graham ◽  
Tess Hoogeboom ◽  
Malcolm F White
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dna Cleavage ◽  
Type Iii ◽  
Crispr System ◽  
Interference System ◽  
First Time ◽  
Fully Functioning ◽  
Target Rna

Abstract The CRISPR system provides adaptive immunity against mobile genetic elements (MGE) in prokaryotes. In type III CRISPR systems, an effector complex programmed by CRISPR RNA detects invading RNA, triggering a multi-layered defence that includes target RNA cleavage, licencing of an HD DNA nuclease domain and synthesis of cyclic oligoadenylate (cOA) molecules. cOA activates the Csx1/Csm6 family of effectors, which degrade RNA non-specifically to enhance immunity. Type III systems are found in diverse archaea and bacteria, including the human pathogen Mycobacterium tuberculosis. Here, we report a comprehensive analysis of the in vitro and in vivo activities of the type III-A M. tuberculosis CRISPR system. We demonstrate that immunity against MGE may be achieved predominantly via a cyclic hexa-adenylate (cA6) signalling pathway and the ribonuclease Csm6, rather than through DNA cleavage by the HD domain. Furthermore, we show for the first time that a type III CRISPR system can be reprogrammed by replacing the effector protein, which may be relevant for maintenance of immunity in response to pressure from viral anti-CRISPRs. These observations demonstrate that M. tuberculosis has a fully-functioning CRISPR interference system that generates a range of cyclic and linear oligonucleotides of known and unknown functions, potentiating fundamental and applied studies.

scholarly journals Cyclic oligoadenylate signalling mediatesMycobacterium tuberculosisCRISPR defence

2019 ◽  
Author(s):  
Sabine Grüschow ◽  
Januka S. Athukoralage ◽  
Shirley Graham ◽  
Tess Hoogeboom ◽  
Malcolm F. White
Keyword(s):  
Dna Cleavage ◽  
Effector Protein ◽  
Type Iii ◽  
Nuclease Domain ◽  
Crispr System ◽  
Interference System ◽  
Fully Functioning ◽  
Target Rna

ABSTRACTThe CRISPR system provides adaptive immunity against mobile genetic elements (MGE) in prokaryotes. In type III CRISPR systems, an effector complex programmed by CRISPR RNA detects invading RNA, triggering a multi-layered defence that includes target RNA cleavage, licencing of an HD DNA nuclease domain and synthesis of cyclic oligoadenylate (cOA) molecules. cOA activates the Csx1/Csm6 family of effectors, which degrade RNA non-specifically to enhance immunity. Type III systems are found in diverse archaea and bacteria, including the human pathogenMycobacterium tuberculosis. Here, we report a comprehensive analysis of thein vitroandin vivoactivities of the type III-AM. tuberculosisCRISPR system. We demonstrate that immunity against MGE is achieved predominantly via a cyclic hexa-adenylate (cA6) signalling pathway and the ribonuclease Csm6, rather than through DNA cleavage by the HD domain. Furthermore, we show that the mechanism can be reprogrammed to operate as a cyclic tetra-adenylate (cA4) system by replacing the effector protein. These observations demonstrate thatM. tuberculosishas a fully-functioning CRISPR interference system that generates a range of cyclic and linear oligonucleotides of known and unknown functions, potentiating fundamental and applied studies.

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