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.

The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes, using small CRISPR RNAs that direct effector complexes to degrade invading nucleic acids. Type III effector complexes were recently demonstrated to synthesize a novel second messenger, cyclic oligoadenylate, on binding target RNA. Cyclic oligoadenylate, in turn, binds to and activates ribonucleases and other factors-via a CRISPR-associated Rossman-fold domain-and thereby induces in the cell an antiviral state that is important for immunity. The mechanism of the 'off-switch' that resets the system is not understood. Here we identify the nuclease that degrades these cyclic oligoadenylate ring molecules. This 'ring nuclease' is itself a protein of the CRISPR-associated Rossman-fold family, and has a metal-independent mechanism that cleaves cyclic tetraadenylate rings to generate linear diadenylate species and switches off the antiviral state. The identification of ring nucleases adds an important insight to the CRISPR system.

The CRISPR system provides adaptive immunity against mobile genetic elements in bacteria and archaea. Type III CRISPR systems detect viral RNA, resulting in activation of a HD nuclease domain for DNA degradation 1,2 and a Cyclase domain that synthesises cyclic oligoadenylate (cOA) from ATP 3-5. cOA activates defence enzymes with a CARF (CRISPR Associated Rossmann Fold) domain 6 , sculpting a powerful antiviral response 7-10 that can drive viruses to extinction 7,8. Cyclic nucleotides are increasingly implicated as playing an important role in host-pathogen interactions 11-13. Here, we identify a new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA 4). The viral ring nuclease (AcrIII-1) is widely distributed in archaeal and bacterial viruses, and proviruses. The enzyme uses a novel fold to bind cA 4 specifically and utilizes a conserved active site to rapidly cleave the signalling molecule, allowing viruses to neutralise the type III CRISPR defence system. The AcrIII-1 family has a broad host range as it targets cA 4 signalling molecules rather than specific CRISPR effector proteins. This study highlights the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

The CRISPR system provides adaptive immunity against mobile genetic elements in bacteria and archaea. On detection of viral RNA, type III CRISPR systems generate a cyclic oligoadenylate (cOA) second messenger 1-3 , activating defence enzymes and sculpting a powerful antiviral response that can drive viruses to extinction 4,5 . Cyclic nucleotides are increasingly implicated as playing an important role in host-pathogen interactions 6,7 . Here, we identify a widespread new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease (AcrIII-1) is the first Acr described for type III CRISPR systems and is widely distributed in archaeal and bacterial viruses, and proviruses. The enzyme uses a novel fold to bind cA4 specifically and utilizes a conserved active site to rapidly cleave the signalling molecule, allowing viruses to neutralise the type III CRISPR defence system. The AcrIII-1 family has a broad host range as it targets cA4 signalling molecules rather than specific CRISPR effector proteins. This study highlights the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.Type III CRISPR-Cas systems synthesise the signalling molecule cyclic oligoadenylate (cOA) from ATP 1,2 when they detect viral RNA. cOA molecules are synthesised with a range of ring sizes with 3-6 AMP subunits (denoted cA3, cA4 etc.) by the cyclase domain of the Cas10 protein [1][2][3]9,10 . cOA binds to a specific protein domain, known as a CARF (CRISPR Associated Rossman Fold) domain. CARF domains are found fused to a variety of effector domains that are known or predicted to cleave RNA, DNA, or function as transcription factors 11 . The best characterised CARF protein family is the Csx1/Csm6 family of HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) ribonucleases, which are activated by cOA binding and cleave RNA with minimal sequence dependence 1-3 . A number of studies have demonstrated that the cOA signalling component of type III systems is crucial for effective immunity against viruses 4,12-15 , highlighting the importance of this facet of CRISPR immunity.Recently, we identified a cellular enzyme in Sulfolobus solfataricus, hereafter referred to as the Crn1 family (for "CRISPR associated ring nuclease 1"), that degrades cA4 molecules and thus deactivates the Csx1 ribonuclease in vitro 16 . These enzymes exhibit very slow kinetics, and are thought to act by mopping up cA4 molecules in the cell without compromising the immunity provided by the type III CRISPR system. Unsurprisingly, viruses have responded to the threat of the CRISPR system by evolving a range of anti-CRISPR (Acr) proteins, which are used to inhibit and overcome the cell's CRISPR defences (reviewed in 17 ). Acr's have been identified for the type I-D 18 , I-F , II-A and V-A effector complexes (reviewed in 17,19,20 ), numbering over 40 families 21 , but importantly not for type III systems. We focussed on one of the protein families, DUF1874, conserved and widespread ...

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.