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

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.

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

10.1101/2021.02.01.429135 ◽

2021 ◽

Cited By ~ 1

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.

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

10.1101/2021.09.13.460032 ◽

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.

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

Nucleic Acids Research ◽

10.1093/nar/gkaa176 ◽

2020 ◽

Vol 48 (8) ◽

pp. 4418-4434 ◽

Cited By ~ 7

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.

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

10.1101/153262 ◽

2017 ◽

Cited By ~ 1

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.

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Control of cyclic oligoadenylate synthesis in a type III CRISPR system

eLife ◽

10.7554/elife.36734 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 55

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.

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RNA-binding and HEPN-nuclease activation are decoupled in CRISPRCas13a

10.1101/190603 ◽

2017 ◽

Author(s):

Akshay Tambe ◽

Alexandra East-Seletsky ◽

Gavin J. Knott ◽

Jennifer A. Doudna ◽

Mitchell R. O’Connell

Keyword(s):

Rna Binding ◽

Tight Binding ◽

Nuclease Activity ◽

Base Pairing ◽

Rna Regulation ◽

Rna Detection ◽

Rna Targeting ◽

Target Binding ◽

Target Rna

SUMMARYCRISPR-Cas13a enzymes are RNA-guided, RNA-activated ribonucleases. Their properties have been exploited as powerful tools for RNA detection, RNA imaging and RNA regulation. However, the relationship between target RNA binding and HEPN (higher-eukaryotes-and-prokaryotes nucleotide-binding)- domain nuclease activation is not well understood. Using sequencing experiments coupled with in vitro biochemistry, we find that Cas13a’s target RNA binding affinity and HEPN-nuclease activity are differentially affected by the number of and position of mismatches between the guide and target. We identify a central ‘binding seed’ where perfect base pairing is absolutely required for target binding, and a separate ‘nuclease switch’ where imperfect base-pairing results in tight binding but no HEPN-nuclease activation. These results demonstrate that the binding and cleavage activities of Cas13a are decoupled, highlighting a complex specificity landscape. Our findings underscore a need to consider the range of effects off-target recognition has on Cas13a’s RNA binding and cleavage behavior for RNA-targeting tool development.

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Structures of the Cmr-β Complex Reveal the Regulation of the Immunity Mechanism of Type III-B CRISPR-Cas

10.1101/2020.06.24.163345 ◽

2020 ◽

Author(s):

Nicholas Sofos ◽

Mingxia Feng ◽

Stefano Stella ◽

Tillmann Pape ◽

Anders Fuglsang ◽

...

Keyword(s):

Immune Response ◽

Rna Recognition ◽

Defense Strategies ◽

Type Iii ◽

Self Recognition ◽

Function Relationship ◽

Immunity Mechanism ◽

Relationship Of ◽

Target Rna ◽

Oligoadenylate Synthesis

AbstractCmr-β is a Type III-B CRISPR-Cas complex that upon target RNA recognition unleashes a multifaceted immune response against invading genetic elements, including ssDNA cleavage, cyclic oligoadenylate synthesis, and also a unique UA-specific ssRNA hydrolysis by the Cmr2 subunit. Here, we present the structure-function relationship of Cmr-β unveiling how binding of the target RNA regulates the Cmr2 activities. CryoEM analysis revealed the unique subunit architecture of Cmr-β and captured the complex in different conformational stages of the immune response, including the non-cognate and cognate target-RNA bound complexes. The binding of the target RNA induces a conformational change of Cmr2, which together with the complementation between the 5’-handle in the crRNA and the 3’-antitag of the target RNA, activate different configurations in a unique loop of the Cmr3 subunit, which acts as an allosteric sensor signaling the self vs. non-self recognition. These findings highlight the diverse defense strategies of Type III complexes.

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Structural Organization of a Type III-A CRISPR Effector Subcomplex Determined by X-ray Crystallography and Cryo-EM

10.1101/452599 ◽

2018 ◽

Author(s):

Bryan W. Dorsey ◽

Lei Huang ◽

Alfonso Mondragón

Keyword(s):

Staphylococcus Epidermidis ◽

Rna Binding ◽

Guide Rna ◽

Type Iii ◽

X Ray Crystallography ◽

Cryo Electron Microscopy ◽

Class 1 ◽

Target Rna ◽

Resolution Single ◽

Cas Proteins

AbstractClustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas proteins provide an immune-like response in many prokaryotes against extraneous nucleic acids. CRISPR-Cas systems are classified into different classes and types. Class 1 CRISPR-Cas systems form multi-protein effector complexes that includes a guide RNA (crRNA) used to identify the target for destruction. Here we present crystal structures of Staphylococcus epidermidis Type III-A CRISPR subunits Csm2 and Csm3 and a 5.2 Å resolution single-particle cryo-electron microscopy (cryo-EM) reconstruction of an effector subcomplex including the crRNA. The structures help to clarify the quaternary architecture of Type III-A effector complexes, as well as to provide details on crRNA binding, target RNA binding and cleavage, and intermolecular interactions essential for effector complex assembly. The structures allow a better understanding of the organization of Type III-A CRISPR effector complexes as well as highlighting the overall similarities and differences with other Class 1 effector complexes.

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