Critical factors for precise and efficient RNA cleavage by RNase Y in Staphylococcus aureus.

Cellular processes require precise and specific gene regulation, in which continuous mRNA degradation is a major element. The mRNA degradation mechanisms should be able to degrade a wide range of different RNA substrates with high efficiency, but should at the same time be limited, to avoid killing the cell by elimination of all cellular RNA. RNase Y is a major endoribonuclease found in most Firmicutes, including Bacillus subtilis and Staphylococcus aureus. However, the molecular interactions that direct RNase Y to cleave the correct RNA molecules at the correct position remain unknown. In this work we have identified transcripts that are homologs in S. aureus and B. subtilis, and are RNase Y targets in both bacteria. Two such transcript pairs were used as models to show a functional overlap between the S. aureus and the B. subtilis RNase Y, which highlighted the importance of the nucleotide sequence of the RNA molecule itself in the RNase Y targeting process. Cleavage efficiency is driven by the primary nucleotide sequence immediately downstream of the cleavage site and base-pairing in a secondary structure a few nucleotides downstream. Cleavage positioning is roughly localised by the downstream secondary structure and fine-tuned by the nucleotide immediately upstream of the cleavage. The identified elements were sufficient for RNase Y-dependent cleavage, since the sequence elements from one of the model transcripts were able to convert an exogenous non-target transcript into a target for RNase Y.

Structural basis for the activity of the type VII CRISPR-Cas system.

The newly identified type VII CRISPR-Cas candidate system uses a CRISPR RNA-guided ribonucleoprotein complex formed by Cas5 and Cas7 proteins to target RNA1. However, the RNA cleavage is executed by a dedicated Cas14 nuclease, which is distinct from the effector nucleases of the other CRISPR-Cas systems. Here we report seven cryo-electron microscopy structures of the Cas14-bound interference complex at different functional states. Cas14, a tetrameric protein in solution, is recruited to the Cas5-Cas7 complex in a target RNA-dependent manner. The N-terminal catalytic domain of Cas14 binds a stretch of the substrate RNA for cleavage, whereas the C-terminal domain is primarily responsible for tethering Cas14 to the Cas5-Cas7 complex. The biochemical cleavage assays corroborate the captured functional conformations, revealing that Cas14 binds to different sites on the Cas5-Cas7 complex to execute individual cleavage events. Notably, a plugged-in arginine of Cas7 sandwiched by a C-shaped clamp of C-terminal domain precisely modulates Cas14 binding. More interestingly, target RNA cleavage is altered by a complementary protospacer flanking sequence at the 5' end, but not at the 3' end. Altogether, our study elucidates critical molecular details underlying the assembly of the interference complex and substrate cleavage in the type VII CRISPR-Cas system, which may help rational engineering of the type VII CRISPR-Cas system for biotechnological applications.

Assembly mechanism of Integrator's RNA cleavage module.

The modular Integrator complex is a transcription regulator that is essential for embryonic development. It attenuates coding gene expression via premature transcription termination and performs 3'-processing of non-coding RNAs. For both activities, Integrator requires endonuclease activity that is harbored by an RNA cleavage module consisting of INTS4-9-11. How correct assembly of Integrator modules is achieved remains unknown. Here, we show that BRAT1 and WDR73 are critical biogenesis factors for the human cleavage module. They maintain INTS9-11 inactive during maturation by physically blocking the endonuclease active site and prevent premature INTS4 association. Furthermore, BRAT1 facilitates import of INTS9-11 into the nucleus, where it is joined by INTS4. Final BRAT1 release requires locking of the mature cleavage module conformation by inositol hexaphosphate (IP6). Our data explain several neurodevelopmental disorders caused by BRAT1, WDR73, and INTS11 mutations as Integrator assembly defects and reveal that IP6 is an essential co-factor for cleavage module maturation.

The kinase Rio1 and a ribosome collision-dependent decay pathway survey the integrity of 18S rRNA cleavage.

The 18S rRNA sequence is highly conserved, particularly at its 3'-end, which is formed by the endonuclease Nob1. How Nob1 identifies its target sequence is not known, and in vitro experiments have shown Nob1 to be error-prone. Moreover, the sequence around the 3'-end is degenerate with similar sites nearby. Here, we used yeast genetics, biochemistry, and next-generation sequencing to investigate a role for the ATPase Rio1 in monitoring the accuracy of the 18S rRNA 3'-end. We demonstrate that Nob1 can miscleave its rRNA substrate and that miscleaved rRNA accumulates upon bypassing the Rio1-mediated quality control (QC) step, but not in healthy cells with intact QC mechanisms. Mechanistically, we show that Rio1 binding to miscleaved rRNA is weaker than its binding to accurately processed 18S rRNA. Accordingly, excess Rio1 results in accumulation of miscleaved rRNA. Ribosomes containing miscleaved rRNA can translate, albeit more slowly, thereby inviting collisions with trailing ribosomes. These collisions result in degradation of the defective ribosomes utilizing parts of the machinery for mRNA QC. Altogether, the data support a model in which Rio1 inspects the 3'-end of the nascent 18S rRNA to prevent miscleaved 18S rRNA-containing ribosomes from erroneously engaging in translation, where they induce ribosome collisions. The data also demonstrate how ribosome collisions purify cells of altered ribosomes with different functionalities, with important implications for the concept of ribosome heterogeneity.

TaqTth-hpRNA: a novel compact RNA-targeting tool for specific silencing of pathogenic mRNA.

Pathogenic allele silencing is a promising treatment for genetic hereditary diseases. Here, we develop an RNA-cleaving tool, TaqTth-hpRNA, consisting of a small, chimeric TaqTth, and a hairpin RNA guiding probe. With a minimal flanking sequence-motif requirement, in vitro and in vivo studies show TaqTth-hpRNA cleaves RNA efficiently and specifically. In an Alzheimer's disease model, we demonstrate silencing of mutant APPswe mRNA without altering the wild-type APP mRNA. Notably, due to the compact size of TaqTth, we are able to combine with APOE2 overexpression in a single AAV vector, which results in stronger inhibition of pathologies.

Endonucleolytic RNA cleavage drives changes in gene expression during the innate immune response.

Viral infection triggers several double-stranded RNA (dsRNA) sensors that lead to changes in gene expression in the cell. One of these sensors activates an endonuclease, ribonuclease L (RNase L), that cleaves single-stranded RNA. However, how the resultant widespread RNA fragmentation affects gene expression is not fully understood. Here, we show that this fragmentation induces the ribotoxic stress response via ZAKα, potentially through stalled ribosomes and/or ribosome collisions. The p38 and JNK pathways that are activated as part of this response promote outcomes that inhibit the virus, such as programmed cell death. We also show that RNase L limits the translation of stress-responsive genes. Intriguingly, we found that the activity of the generic endonuclease, RNase A, recapitulates many of the same molecular phenotypes as activated RNase L, demonstrating how widespread RNA cleavage can evoke an antiviral program.

Structural mechanism of angiogenin activation by the ribosome.

Angiogenin, an RNase-A-family protein, promotes angiogenesis and has been implicated in cancer, neurodegenerative diseases and epigenetic inheritance1-10. After activation during cellular stress, angiogenin cleaves tRNAs at the anticodon loop, resulting in translation repression11-15. However, the catalytic activity of isolated angiogenin is very low, and the mechanisms of the enzyme activation and tRNA specificity have remained a puzzle3,16-23. Here we identify these mechanisms using biochemical assays and cryogenic electron microscopy (cryo-EM). Our study reveals that the cytosolic ribosome is the activator of angiogenin. A cryo-EM structure features angiogenin bound in the A site of the 80S ribosome. The C-terminal tail of angiogenin is rearranged by interactions with the ribosome to activate the RNase catalytic centre, making the enzyme several orders of magnitude more efficient in tRNA cleavage. Additional 80S-angiogenin structures capture how tRNA substrate is directed by the ribosome into angiogenin's active site, demonstrating that the ribosome acts as the specificity factor. Our findings therefore suggest that angiogenin is activated by ribosomes with a vacant A site, the abundance of which increases during cellular stress24-27. These results may facilitate the development of therapeutics to treat cancer and neurodegenerative diseases.

RNA targeting and cleavage by the type III-Dv CRISPR effector complex.

CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction1-5. Target RNA cleavage at regular intervals is characteristic of type III effector complexes6-8. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors9,10, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.

Molecular mechanism for target RNA recognition and cleavage of Cas13h.

RNA-targeting type VI CRISPR-Cas effectors are widely used in RNA applications. Cas13h is a recently identified subtype of Cas13 ribonuclease, with strong RNA cleavage activity and robust in vivo RNA knockdown efficiency. However, little is known regarding its biochemical properties and working mechanisms. Biochemical characterization of Cas13h1 indicated that it lacks in vitro pre-crRNA processing activity and adopts a central seed. The cleavage activity of Cas13h1 is enhanced by a R(G/A) 5'-PFS, and inhibited by tag:anti-tag RNA pairing. We determined the structures of Cas13h1-crRNA binary complex at 3.1 Å and Cas13h1-crRNA-target RNA ternary complex at 3.0 Å. The ternary complex adopts an elongated architecture, and encodes a nucleotide-binding pocket within Helical-2 domain to recognize the guanosine at the 5'-end of the target RNA. Base pairing between crRNA guide and target RNA disrupts Cas13h1-guide interactions, leading to dramatic movement of HEPN domains. Upon target RNA engagement, Cas13h1 adopts a complicated activation mechanism, including separation of HEPN catalytic residues and destabilization of the active site loop and NTD domain, to get activated. Collectively, these insights expand our understanding into Cas13 effectors.

In-vitro Clinical Diagnostics using RNA-Cleaving DNAzymes.

Over the last three decades, significant advancements have been made in the development of biosensors and bioassays that use RNA-cleaving DNAzymes (RCDs) as molecular recognition elements. While early examples of RCDs were primarily responsive to metal ions, the past decade has seen numerous RCDs reported for more clinically relevant targets such as bacteria, cancer cells, small metabolites, and protein biomarkers. Over the past 5 years several RCD-based biosensors have also been evaluated using either spiked biological matrixes or patient samples, including blood, serum, saliva, nasal mucus, sputum, urine, and faeces, which is a critical step toward regulatory approval and commercialization of such sensors. In this review, an overview of the methods used to generate RCDs and the properties of key RCDs that have been utilized for in vitro testing is first provided. Examples of RCD-based assays and sensors that have been used to test either spiked biological samples or patient samples are then presented, highlighting assay performance in different biological matrixes. A summary of current prospects and challenges for development of in vitro diagnostic tests incorporating RCDs and an overview of future directions of the field is also provided.

Multivalent DNAzyme agents for cleaving folded RNA.

Multivalent recognition and binding of biological molecules is a natural phenomenon that increases the binding stability (avidity) without decreasing the recognition specificity. In this study, we took advantage of this phenomenon to increase the efficiency and maintain high specificity of RNA cleavage by DNAzymes (Dz). We designed a series of DNA constructs containing two Dz agents, named here bivalent Dz devices (BDD). One BDD increased the cleavage efficiency of a folded RNA fragment up to 17-fold in comparison with the Dz of a conventional design. Such an increase was achieved due to both the improved RNA binding and the increased probability of RNA cleavage by the two catalytic cores. By moderating the degree of Dz agent association in BDD, we achieved excellent selectivity in differentiating single-base mismatched RNA, while maintaining relatively high cleavage rates. Furthermore, a trivalent Dz demonstrated an even greater efficiency than the BDD in cleaving folded RNA. The data suggests that the cooperative action of several RNA-cleaving units can significantly improve the efficiency and maintain high specificity of RNA cleavage, which is important for the development of Dz-based gene knockdown agents.

Identifying human pre-mRNA cleavage and polyadenylation factors by genome-wide CRISPR screens using a dual fluorescence readthrough reporter.

Messenger RNA precursors (pre-mRNA) generally undergo 3' end processing by cleavage and polyadenylation (CPA), which is specified by a polyadenylation site (PAS) and adjacent RNA sequences and regulated by a large variety of core and auxiliary CPA factors. To date, most of the human CPA factors have been discovered through biochemical and proteomic studies. However, genetic identification of the human CPA factors has been hampered by the lack of a reliable genome-wide screening method. We describe here a dual fluorescence readthrough reporter system with a PAS inserted between two fluorescent reporters. This system enables measurement of the efficiency of 3' end processing in living cells. Using this system in combination with a human genome-wide CRISPR/Cas9 library, we conducted a screen for CPA factors. The screens identified most components of the known core CPA complexes and other known CPA factors. The screens also identified CCNK/CDK12 as a potential core CPA factor, and RPRD1B as a CPA factor that binds RNA and regulates the release of RNA polymerase II at the 3' ends of genes. Thus, this dual fluorescence reporter coupled with CRISPR/Cas9 screens reliably identifies bona fide CPA factors and provides a platform for investigating the requirements for CPA in various contexts.

tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector.

Type VI CRISPR-Cas systems are among the few CRISPR varieties that target exclusively RNA. The CRISPR RNA-guided, sequence-specific binding of target RNAs, such as phage transcripts, activates the type VI effector, Cas13. Once activated, Cas13 causes collateral RNA cleavage, which induces bacterial cell dormancy, thus protecting the host population from the phage spread. We show here that the principal form of collateral RNA degradation elicited by Leptotrichia shahii Cas13a expressed in Escherichia coli cells is the cleavage of anticodons in a subset of transfer RNAs (tRNAs) with uridine-rich anticodons. This tRNA cleavage is accompanied by inhibition of protein synthesis, thus providing defense from the phages. In addition, Cas13a-mediated tRNA cleavage indirectly activates the RNases of bacterial toxin-antitoxin modules cleaving messenger RNA, which could provide a backup defense. The mechanism of Cas13a-induced antiphage defense resembles that of bacterial anticodon nucleases, which is compatible with the hypothesis that type VI effectors evolved from an abortive infection module encompassing an anticodon nuclease.

Retention of the RNA ends provides the molecular memory for maintaining the activation of the Csm complex.

The type III CRISPR-Cas effector complex Csm functions as a molecular Swiss army knife that provides multilevel defense against foreign nucleic acids. The coordinated action of three catalytic activities of the Csm complex enables simultaneous degradation of the invader's RNA transcripts, destruction of the template DNA and synthesis of signaling molecules (cyclic oligoadenylates cAn) that activate auxiliary proteins to reinforce CRISPR-Cas defense. Here, we employed single-molecule techniques to connect the kinetics of RNA binding, dissociation, and DNA hydrolysis by the Csm complex from Streptococcus thermophilus. Although single-stranded RNA is cleaved rapidly (within seconds), dual-color FCS experiments and single-molecule TIRF microscopy revealed that Csm remains bound to terminal RNA cleavage products with a half-life of over 1 hour while releasing the internal RNA fragments quickly. Using a continuous fluorescent DNA degradation assay, we observed that RNA-regulated single-stranded DNase activity decreases on a similar timescale. These findings suggest that after fast target RNA cleavage the terminal RNA cleavage products stay bound within the Csm complex, keeping the Cas10 subunit activated for DNA destruction. Additionally, we demonstrate that during Cas10 activation, the complex remains capable of RNA turnover, i.e. of ongoing degradation of target RNA.

A novel "on-off-on" electrochemiluminescence strategy based on RNA cleavage propelled signal amplification and resonance energy transfer for Pb2+ detection.

BACKGROUND: Lead (Pb) is one of the most toxic heavy-metal pollutants. Additionally, lead ions (Pb2+) can accumulate in the human body through the food chain, causing irreversible damage through organ damage and system disorders. In the past few years, the detection of Pb2+ has mainly relied on instrumental methods such as atomic absorption spectroscopy (AAS) and inductively coupled plasma mass spectrometry (ICP-MS). Nonetheless, these techniques are complicated in terms of equipment and procedures, along with being time-intensive and expensive in terms of detection. These drawbacks have limited their wide application. Hence, there is a pressing need to develop detection techniques for Pb2+ that are not only cost-efficient but also highly sensitive and specific. RESULTS: A novel "on-off-on" electrochemiluminescence (ECL) sensor for detecting Pb2+ was developed based on the resonance energy transfer (RET) effect between AuNPs and boron nitride quantum dots (BN QDs) and the recognition of Pb2+ by DNAzyme along with the cleavage reaction of the substrate chain. Poly(6-carboxyindole)/stannic sulfide (P6ICA/SnS2) nanocomposite was employed as a co-reaction accelerator to consequently facilitate the production of intermediate SO4•-. This effective enhancement of the reaction led to an improved ECL intensity of BN QDs and enabled the sensor platform to exhibit a higher original ECL response. Benefiting from the combination of the DNAzyme signal amplification strategy with the "on-off-on" design, the ECL sensor showed satisfactory selectivity, good stability, and high sensitivity. This ECL sensor exhibited a linear detection range (LDR) of 10-12-10-5 M and a limit of detection (LOD) of 2.6 × 10-13 M. SIGNIFICANCE: In the present work, an "on-off-on" ECL sensor is constructed based on RET effect for ultrasensitive detection of Pb2+. P6ICA/SnS2 was investigated as the co-reaction accelerator in this sensor. Moreover, this ECL sensor exhibited excellent analytical capability for detecting Pb2+ in actual water samples, providing a method for detecting other heavy metal ions as well.

CRISPR dual enzyme cleavage triggers DNA and RNA substrate cleavage for SARS-CoV-2 dual gene detection.

The widespread dissemination of coronavirus 2019 imposes a significant burden on society. Therefore, rapid detection facilitates the reduction of transmission risk. In this study, we proposed a multiplex diagnostic platform for the rapid, ultrasensitive, visual, and simultaneous detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frame 1ab (ORF1ab) and N genes. A visual diagnostic method was developed using a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a/Cas13a dual-enzyme digestion system integrated with multiplex reverse transcriptase-recombinase polymerase amplification (RT-RPA). Two CRISPR-Cas proteins (Cas12a and Cas13a) were introduced into the system to recognize and cleave the N gene and ORF1ab gene, respectively. We used fluorescent or CRISPR double digestion test strips to detect the digested products, with the N gene corresponding to the FAM channel in the PCR instrument or the T1 line on the test strip, and the ORF1ab gene corresponding to the ROX channel in the PCR instrument or the T2 line on the test strip. The analysis can be completed in less than 20 min. Meanwhile, we assessed the application of the platform and determined a sensitivity of up to 200 copies/mL. Additionally, dual gene validation in 105 clinical nasopharyngeal swab samples showed a 100% positive predictive value agreement and a 95.7% negative predictive value agreement between our method and quantitative reverse transcription-polymerase chain reaction. Overall, our method offered a novel insight into the rapid diagnosis of SARS-CoV-2.

Structural transitions upon guide RNA binding and their importance in Cas12g-mediated RNA cleavage.

Cas12g is an endonuclease belonging to the type V RNA-guided CRISPR-Cas family. It is known for its ability to cleave RNA substrates using a conserved endonuclease active site located in the RuvC domain. In this study, we determined the crystal structure of apo-Cas12g, the cryo-EM structure of the Cas12g-sgRNA binary complex and investigated conformational changes that occur during the transition from the apo state to the Cas12g-sgRNA binary complex. The conserved zinc finger motifs in Cas12g undergo an ordered-to-disordered transition from the apo to the sgRNA-bound state and their mutations negatively impact on target RNA cleavage. Moreover, we identified a lid motif in the RuvC domain that undergoes transformation from a helix to loop to regulate the access to the RuvC active site and subsequent cleavage of the RNA substrate. Overall, our study provides valuable insights into the mechanisms by which Cas12g recognizes sgRNA and the conformational changes it undergoes from sgRNA binding to the activation of the RNase active site, thereby laying a foundation for the potential repurposing of Cas12g as a tool for RNA-editing.

A ratiometric fluorescence platform for lead ion detection via RNA cleavage-inhibited self-assembly of three-arm branched junction.

Heavy metal pollution can pose a threat to food safety and human health, and accurate quantification of heavy metal ions is a vital requirement. Emerging DNA nanostructures-based biosensors offer attractive tools toward ultra-sensitive or rapid analysis of heavy metal ions. However, the problems including complex design, severe reaction conditions and undesirable reliability are inevitable obstacle in advancing their extension and application. Herein, a ratiometric fluorescent platform was established for monitoring lead ion (Pb2+) in food based on dual Förster resonance energy transfer (FRET) and RNA cleavage-inhibited self-assembly of three-arm branched junction (TBJ). GR-5 DNAzyme was employed for Pb2+ recognition, and enzyme-free amplification technique catalytic hairpin assembly (CHA) served to form FRET probes-carried TBJ. The substrate strand (S) of DNAzyme triggered the generation of CHA-TBJ, and Pb2+-responsive cleavage of S hindered the assembly of CHA-TBJ, causing opposite changes in the FRET states of FAM/BHQ1 and ROX/BHQ2 pairs. The fluorescence responses were recorded through synchronous fluorescence spectrometry to indicate Pb2+ concentration, allowing sensitive and reliable identification of Pb2+ in the linear range of 0.05-5 ng mL-1 with the detection limit of 0.03 ng mL-1. The Pb2+ detection can be achieved under conventional reaction conditions, simple mixing procedures and one-step measurement operation. The approach can afford excellent specificity for Pb2+ against competing metal ions, and can be applied to analyze Pb2+ in tea samples with satisfactory results. This facile fluorescence platform shows a capable method for Pb2+ detection, and provides new avenue in the development of ratiometric approaches and DNAzyme strategies for monitoring heavy metal pollution, facilitating the transformation of DNAzyme-based biosensors for food safety control.

Kinetic analysis of RNA cleavage by coronavirus Nsp15 endonuclease: Evidence for acid-base catalysis and substrate-dependent metal ion activation.

Understanding the functional properties of severe acute respiratory syndrome coronavirus 2 nonstructural proteins is essential for defining their roles in the viral life cycle, developing improved therapeutics and diagnostics, and countering future variants. Coronavirus nonstructural protein Nsp15 is a hexameric U-specific endonuclease whose functions, substrate specificity, mechanism, and dynamics are not fully defined. Previous studies report that Nsp15 requires Mn2+ ions for optimal activity; however, the effects of divalent ions on Nsp15 reaction kinetics have not been investigated in detail. Here, we analyzed the single- and multiple-turnover kinetics for model ssRNA substrates. Our data confirm that divalent ions are dispensable for catalysis and show that Mn2+ activates Nsp15 cleavage of two different ssRNA oligonucleotide substrates but not a dinucleotide. Biphasic kinetics of ssRNA substrates demonstrates that Mn2+ stabilizes alternative enzyme states that have faster substrate cleavage on the enzyme. However, we did not detect Mn2+-induced conformational changes using CD and fluorescence spectroscopy. The pH-rate profiles in the presence and absence of Mn2+ reveal active-site ionizable groups with similar pKas of ca. 4.8 to 5.2. An Rp stereoisomer phosphorothioate modification at the scissile phosphate had minimal effect on catalysis supporting a mechanism involving an anionic transition state. However, the Sp stereoisomer is inactive because of weak binding, consistent with models that position the nonbridging phosphoryl oxygen deep in the active site. Together, these data demonstrate that Nsp15 employs a conventional acid-base catalytic mechanism passing through an anionic transition state, and that divalent ion activation is substrate dependent.

Csx28 is a membrane pore that enhances CRISPR-Cas13b-dependent antiphage defense.

Type VI CRISPR-Cas systems use RNA-guided ribonuclease (RNase) Cas13 to defend bacteria against viruses, and some of these systems encode putative membrane proteins that have unclear roles in Cas13-mediated defense. We show that Csx28, of type VI-B2 systems, is a transmembrane protein that assists to slow cellular metabolism upon viral infection, increasing antiviral defense. High-resolution cryo-electron microscopy reveals that Csx28 forms an octameric pore-like structure. These Csx28 pores localize to the inner membrane in vivo. Csx28's antiviral activity in vivo requires sequence-specific cleavage of viral messenger RNAs by Cas13b, which subsequently results in membrane depolarization, slowed metabolism, and inhibition of sustained viral infection. Our work suggests a mechanism by which Csx28 acts as a downstream, Cas13b-dependent effector protein that uses membrane perturbation as an antiviral defense strategy.