Bacteria SAVED from Viruses.

Increasingly, cyclic nucleotide second messengers are implicated in antiviral defense systems in bacteria and archaea as well as in eukaryotes. In this issue of Cell, Lowey et al. describe SAVED-a widespread, uncharacterized cyclic nucleotide sensor protein domain that activates cell defense systems. The structure of SAVED reveals links to the CRISPR system, which also generates cyclic nucleotides in response to viral infection.

Antiviral type III CRISPR signalling via conjugation of ATP and SAM.

CRISPR systems are widespread in the prokaryotic world, providing adaptive immunity against mobile genetic elements1,2. Type III CRISPR systems, with the signature gene cas10, use CRISPR RNA to detect non-self RNA, activating the enzymatic Cas10 subunit to defend the cell against mobile genetic elements either directly, via the integral histidine-aspartate (HD) nuclease domain3-5 or indirectly, via synthesis of cyclic oligoadenylate second messengers to activate diverse ancillary effectors6-9. A subset of type III CRISPR systems encode an uncharacterized CorA-family membrane protein and an associated NrN family phosphodiesterase that are predicted to function in antiviral defence. Here we demonstrate that the CorA-associated type III-B (Cmr) CRISPR system from Bacteroides fragilis provides immunity against mobile genetic elements when expressed in Escherichia coli. However, B. fragilis Cmr does not synthesize cyclic oligoadenylate species on activation, instead generating S-adenosyl methionine (SAM)-AMP (SAM is also known as AdoMet) by conjugating ATP to SAM via a phosphodiester bond. Once synthesized, SAM-AMP binds to the CorA effector, presumably leading to cell dormancy or death by disruption of the membrane integrity. SAM-AMP is degraded by CRISPR-associated phosphodiesterases or a SAM-AMP lyase, potentially providing an 'off switch' analogous to cyclic oligoadenylate-specific ring nucleases10. SAM-AMP thus represents a new class of second messenger for antiviral signalling, which may function in different roles in diverse cellular contexts.

Antiviral signalling by a cyclic nucleotide activated CRISPR protease.

CRISPR defence systems such as the well-known DNA-targeting Cas9 and the RNA-targeting type III systems are widespread in prokaryotes1,2. The latter orchestrates a complex antiviral response that is initiated through the synthesis of cyclic oligoadenylates after recognition of foreign RNA3-5. Among the large set of proteins that are linked to type III systems and predicted to bind cyclic oligoadenylates6,7, a CRISPR-associated Lon protease (CalpL) stood out to us. CalpL contains a sensor domain of the SAVED family7 fused to a Lon protease effector domain. However, the mode of action of this effector is unknown. Here we report the structure and function of CalpL and show that this soluble protein forms a stable tripartite complex with two other proteins, CalpT and CalpS, that are encoded on the same operon. After activation by cyclic tetra-adenylate (cA4), CalpL oligomerizes and specifically cleaves the MazF homologue CalpT, which releases the extracytoplasmic function σ factor CalpS from the complex. Our data provide a direct connection between CRISPR-based detection of foreign nucleic acids and transcriptional regulation. Furthermore, the presence of a SAVED domain that binds cyclic tetra-adenylate in a CRISPR effector reveals a link to the cyclic-oligonucleotide-based antiphage signalling system.

Cyclic nucleotide-induced helical structure activates a TIR immune effector.

Cyclic nucleotide signalling is a key component of antiviral defence in all domains of life. Viral detection activates a nucleotide cyclase to generate a second messenger, resulting in activation of effector proteins. This is exemplified by the metazoan cGAS-STING innate immunity pathway1, which originated in bacteria2. These defence systems require a sensor domain to bind the cyclic nucleotide and are often coupled with an effector domain that, when activated, causes cell death by destroying essential biomolecules3. One example is the Toll/interleukin-1 receptor (TIR) domain, which degrades the essential cofactor NAD+ when activated in response to infection in plants and bacteria2,4,5 or during programmed nerve cell death6. Here we show that a bacterial antiviral defence system generates a cyclic tri-adenylate that binds to a TIR-SAVED effector, acting as the 'glue' to allow assembly of an extended superhelical solenoid structure. Adjacent TIR subunits interact to organize and complete a composite active site, allowing NAD+ degradation. Activation requires extended filament formation, both in vitro and in vivo. Our study highlights an example of large-scale molecular assembly controlled by cyclic nucleotides and reveals key details of the mechanism of TIR enzyme activation.

An anti-CRISPR viral ring nuclease subverts type III CRISPR immunity.

The CRISPR system in bacteria and archaea provides adaptive immunity against mobile genetic elements. Type III CRISPR systems detect viral RNA, resulting in the activation of two regions of the Cas10 protein: an HD nuclease domain (which degrades viral DNA)1,2 and a cyclase domain (which synthesizes cyclic oligoadenylates from ATP)3-5. Cyclic oligoadenylates in turn activate defence enzymes with a CRISPR-associated Rossmann fold domain6, sculpting a powerful antiviral response7-10 that can drive viruses to extinction7,8. Cyclic nucleotides are increasingly implicated in host-pathogen interactions11-13. Here we identify a new family of viral anti-CRISPR (Acr) enzymes that rapidly degrade cyclic tetra-adenylate (cA4). The viral ring nuclease AcrIII-1 is widely distributed in archaeal and bacterial viruses and in proviruses. The enzyme uses a previously unknown fold to bind cA4 specifically, and a conserved active site to rapidly cleave this signalling molecule, allowing viruses to neutralize 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. Our findings highlight the crucial role of cyclic nucleotide signalling in the conflict between viruses and their hosts.

Bioinformatic analysis of type III CRISPR systems reveals key properties and new effector families.

Recognition of RNA from invading mobile genetic elements (MGE) prompts type III CRISPR systems to activate an HD nuclease domain and/or a nucleotide cyclase domain in the Cas10 subunit, eliciting an immune response. The cyclase domain can generate a range of nucleotide second messengers, which in turn activate a diverse family of ancillary effector proteins. These provide immunity by non-specific degradation of host and MGE nucleic acids or proteins, perturbation of membrane potentials, transcriptional responses, or the arrest of translation. The wide range of nucleotide activators and downstream effectors generates a complex picture that is gradually being resolved. Here, we carry out a global bioinformatic analysis of type III CRISPR loci in prokaryotic genomes, defining the relationships of Cas10 proteins and their ancillary effectors. Our study reveals that cyclic tetra-adenylate is by far the most common signalling molecule used and that many loci have multiple effectors. These typically share the same activator and may work synergistically to combat MGE. We propose four new candidate effector protein families and confirm experimentally that the Csm6-2 protein, a highly diverged, fused Csm6 effector, is a ribonuclease activated by cyclic hexa-adenylate.

CRISPR antiphage defence mediated by the cyclic nucleotide-binding membrane protein Csx23.

CRISPR-Cas provides adaptive immunity in prokaryotes. Type III CRISPR systems detect invading RNA and activate the catalytic Cas10 subunit, which generates a range of nucleotide second messengers to signal infection. These molecules bind and activate a diverse range of effector proteins that provide immunity by degrading viral components and/or by disturbing key aspects of cellular metabolism to slow down viral replication. Here, we focus on the uncharacterised effector Csx23, which is widespread in Vibrio cholerae. Csx23 provides immunity against plasmids and phage when expressed in Escherichia coli along with its cognate type III CRISPR system. The Csx23 protein localises in the membrane using an N-terminal transmembrane α-helical domain and has a cytoplasmic C-terminal domain that binds cyclic tetra-adenylate (cA4), activating its defence function. Structural studies reveal a tetrameric structure with a novel fold that binds cA4 specifically. Using pulse EPR, we demonstrate that cA4 binding to the cytoplasmic domain of Csx23 results in a major perturbation of the transmembrane domain, consistent with the opening of a pore and/or disruption of membrane integrity. This work reveals a new class of cyclic nucleotide binding protein and provides key mechanistic detail on a membrane-associated CRISPR effector.

Many anti-viral defence systems generate a cyclic nucleotide signal that activates cellular defences in response to infection. Type III CRISPR systems use a specialised polymerase to make cyclic oligoadenylate (cOA) molecules from ATP. These can bind and activate a range of effector proteins that slow down viral replication. In this study, we focussed on the Csx23 effector from the human pathogen Vibrio cholerae ­ a trans-membrane protein that binds a cOA molecule, leading to anti-viral immunity. Structural studies revealed a new class of nucleotide recognition domain, where cOA binding is transmitted to changes in the trans-membrane domain, most likely resulting in membrane depolarisation. This study highlights the diversity of mechanisms for anti-viral defence via nucleotide signalling.

RNA processing by the CRISPR-associated NYN ribonuclease.

CRISPR-Cas systems confer adaptive immunity in prokaryotes, facilitating the recognition and destruction of invasive nucleic acids. Type III CRISPR systems comprise large, multisubunit ribonucleoprotein complexes with a catalytic Cas10 subunit. When activated by the detection of foreign RNA, Cas10 generates nucleotide signalling molecules that elicit an immune response by activating ancillary effector proteins. Among these systems, the Bacteroides fragilis type III CRISPR system was recently shown to produce a novel signal molecule, SAM-AMP, by conjugating ATP and SAM. SAM-AMP regulates a membrane effector of the CorA family to provide immunity. Here, we focus on NYN, a ribonuclease encoded within this system, probing its potential involvement in crRNA maturation. Structural modelling and in vitro ribonuclease assays reveal that NYN displays robust sequence-nonspecific, Mn2+-dependent ssRNA-cleavage activity. Our findings suggest a role for NYN in trimming crRNA intermediates into mature crRNAs, which is necessary for type III CRISPR antiviral defence. This study sheds light on the functional relevance of CRISPR-associated NYN proteins and highlights the complexity of CRISPR-mediated defence strategies in bacteria.

Activation of Csm6 ribonuclease by cyclic nucleotide binding: in an emergency, twist to open.

Type III CRISPR systems synthesize cyclic oligoadenylate (cOA) second messengers as part of a multi-faceted immune response against invading mobile genetic elements (MGEs). cOA activates non-specific CRISPR ancillary defence nucleases to create a hostile environment for MGE replication. Csm6 ribonucleases bind cOA using a CARF (CRISPR-associated Rossmann Fold) domain, resulting in activation of a fused HEPN (Higher Eukaryotes and Prokaryotes Nucleotide binding) ribonuclease domain. Csm6 enzymes are widely used in a new generation of diagnostic assays for the detection of specific nucleic acid species. However, the activation mechanism is not fully understood. Here we characterised the cyclic hexa-adenylate (cA6) activated Csm6' ribonuclease from the industrially important bacterium Streptococcus thermophilus. Crystal structures of Csm6' in the inactive and cA6 bound active states illuminate the conformational changes which trigger mRNA destruction. Upon binding of cA6, there is a close to 60° rotation between the CARF and HEPN domains, which causes the 'jaws' of the HEPN domain to open and reposition active site residues. Key to this transition is the 6H domain, a right-handed solenoid domain connecting the CARF and HEPN domains, which transmits the conformational changes for activation.

Ring nucleases deactivate type III CRISPR ribonucleases by degrading cyclic oligoadenylate.

The CRISPR system provides adaptive immunity against mobile genetic elements in prokaryotes, using small CRISPR RNAs that direct effector complexes to degrade invading nucleic acids1-3. Type III effector complexes were recently demonstrated to synthesize a novel second messenger, cyclic oligoadenylate, on binding target RNA4,5. 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.

Structure and mechanism of the type I-G CRISPR effector.

Type I CRISPR systems are the most common CRISPR type found in bacteria. They use a multisubunit effector, guided by crRNA, to detect and bind dsDNA targets, forming an R-loop and recruiting the Cas3 enzyme to facilitate target DNA destruction, thus providing immunity against mobile genetic elements. Subtypes have been classified into families A-G, with type I-G being the least well understood. Here, we report the composition, structure and function of the type I-G Cascade CRISPR effector from Thioalkalivibrio sulfidiphilus, revealing key new molecular details. The unique Csb2 subunit processes pre-crRNA, remaining bound to the 3' end of the mature crRNA, and seven Cas7 subunits form the backbone of the effector. Cas3 associates stably with the effector complex via the Cas8g subunit and is important for target DNA recognition. Structural analysis by cryo-Electron Microscopy reveals a strikingly curved backbone conformation with Cas8g spanning the belly of the structure. These biochemical and structural insights shed new light on the diversity of type I systems and open the way to applications in genome engineering.

Repurposing the atypical type I-G CRISPR system for bacterial genome engineering.

The CRISPR-Cas system functions as a prokaryotic immune system and is highly diverse, with six major types and numerous sub-types. The most abundant are type I CRISPR systems, which utilize a multi-subunit effector, Cascade, and a CRISPR RNA (crRNA) to detect invading DNA species. Detection leads to DNA loading of the Cas3 helicase-nuclease, leading to long-range deletions in the targeted DNA, thus providing immunity against mobile genetic elements (MGE). Here, we focus on the type I-G system, a streamlined, 4-subunit complex with an atypical Cas3 enzyme. We demonstrate that Cas3 helicase activity is not essential for immunity against MGE in vivo and explore applications of the Thioalkalivibrio sulfidiphilus Cascade effector for genome engineering in Escherichia coli. Long-range, bidirectional deletions were observed when the lacZ gene was targeted. Deactivation of the Cas3 helicase activity dramatically altered the types of deletions observed, with small deletions flanked by direct repeats that are suggestive of microhomology mediated end joining. When donor DNA templates were present, both the wild-type and helicase-deficient systems promoted homology-directed repair (HDR), with the latter system providing improvements in editing efficiency, suggesting that a single nick in the target site may promote HDR in E. coli using the type I-G system. These findings open the way for further application of the type I-G CRISPR systems in genome engineering.

Cyclic oligoadenylate signalling and regulation by ring nucleases during type III CRISPR defence.

In prokaryotes, CRISPR-Cas immune systems recognise and cleave foreign nucleic acids to defend against Mobile Genetic Elements (MGEs). Type III CRISPR-Cas complexes also synthesise cyclic oligoadenylate (cOA) second messengers, which activate CRISPR ancillary proteins involved in antiviral defence. In particular, cOA-stimulated nucleases degrade RNA and DNA non-specifically, which slows MGE replication but also impedes cell growth, necessitating mechanisms to eliminate cOA in order to mitigate collateral damage. Extant cOA is degraded by a new class of enzyme termed a 'ring nuclease', which cleaves cOA specifically and switches off CRISPR ancillary enzymes. Several ring nuclease families have been characterised to date, including a family used by MGEs to circumvent CRISPR immunity, and encompass diverse protein folds and distinct cOA cleavage mechanisms. In this review we outline cOA signalling, discuss how different ring nucleases regulate the cOA signalling pathway, and reflect on parallels between cyclic nucleotide-based immune systems to reveal new areas for exploration.

Structure of the DNA repair helicase XPD.

The XPD helicase (Rad3 in Saccharomyces cerevisiae) is a component of transcription factor IIH (TFIIH), which functions in transcription initiation and Nucleotide Excision Repair in eukaryotes, catalyzing DNA duplex opening localized to the transcription start site or site of DNA damage, respectively. XPD has a 5' to 3' polarity and the helicase activity is dependent on an iron-sulfur cluster binding domain, a feature that is conserved in related helicases such as FancJ. The xpd gene is the target of mutation in patients with xeroderma pigmentosum, trichothiodystrophy, and Cockayne's syndrome, characterized by a wide spectrum of symptoms ranging from cancer susceptibility to neurological and developmental defects. The 2.25 A crystal structure of XPD from the crenarchaeon Sulfolobus tokodaii, presented here together with detailed biochemical analyses, allows a molecular understanding of the structural basis for helicase activity and explains the phenotypes of xpd mutations in humans.

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

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.

The CRISPR ancillary effector Can2 is a dual-specificity nuclease potentiating type III CRISPR defence.

Cells and organisms have a wide range of mechanisms to defend against infection by viruses and other mobile genetic elements (MGE). Type III CRISPR systems detect foreign RNA and typically generate cyclic oligoadenylate (cOA) second messengers that bind to ancillary proteins with CARF (CRISPR associated Rossman fold) domains. This results in the activation of fused effector domains for antiviral defence. The best characterised CARF family effectors are the Csm6/Csx1 ribonucleases and DNA nickase Can1. Here we investigate a widely distributed CARF family effector with a nuclease domain, which we name Can2 (CRISPR ancillary nuclease 2). Can2 is activated by cyclic tetra-adenylate (cA4) and displays both DNase and RNase activity, providing effective immunity against plasmid transformation and bacteriophage infection in Escherichia coli. The structure of Can2 in complex with cA4 suggests a mechanism for the cA4-mediated activation of the enzyme, whereby an active site cleft is exposed on binding the activator. These findings extend our understanding of type III CRISPR cOA signalling and effector function.

Fuse to defuse: a self-limiting ribonuclease-ring nuclease fusion for type III CRISPR defence.

Type III CRISPR systems synthesise cyclic oligoadenylate (cOA) second messengers in response to viral infection of bacteria and archaea, potentiating an immune response by binding and activating ancillary effector nucleases such as Csx1. As these effectors are not specific for invading nucleic acids, a prolonged activation can result in cell dormancy or death. Some archaeal species encode a specialised ring nuclease enzyme (Crn1) to degrade cyclic tetra-adenylate (cA4) and deactivate the ancillary nucleases. Some archaeal viruses and bacteriophage encode a potent ring nuclease anti-CRISPR, AcrIII-1, to rapidly degrade cA4 and neutralise immunity. Homologues of this enzyme (named Crn2) exist in type III CRISPR systems but are uncharacterised. Here we describe an unusual fusion between cA4-activated CRISPR ribonuclease (Csx1) and a cA4-degrading ring nuclease (Crn2) from Marinitoga piezophila. The protein has two binding sites that compete for the cA4 ligand, a canonical cA4-activated ribonuclease activity in the Csx1 domain and a potent cA4 ring nuclease activity in the C-terminal Crn2 domain. The cA4 binding affinities and activities of the two constituent enzymes in the fusion protein may have evolved to ensure a robust but time-limited cOA-activated ribonuclease activity that is finely tuned to cA4 levels as a second messenger of infection.

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

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.

Asymmetric base-pair opening drives helicase unwinding dynamics.

The opening of a Watson-Crick double helix is required for crucial cellular processes, including replication, repair, and transcription. It has long been assumed that RNA or DNA base pairs are broken by the concerted symmetric movement of complementary nucleobases. By analyzing thousands of base-pair opening and closing events from molecular simulations, here, we uncover a systematic stepwise process driven by the asymmetric flipping-out probability of paired nucleobases. We demonstrate experimentally that such asymmetry strongly biases the unwinding efficiency of DNA helicases toward substrates that bear highly dynamic nucleobases, such as pyrimidines, on the displaced strand. Duplex substrates with identical thermodynamic stability are thus shown to be more easily unwound from one side than the other, in a quantifiable and predictable manner. Our results indicate a possible layer of gene regulation coded in the direction-dependent unwindability of the double helix.