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

VanderWal, Arica R.; Park, Jung-Un; Polevoda, Bogdan; Nicosia, Julia K; Vargas, Adrian M. Molina; Kellogg, Elizabeth H.; O’Connell, Mitchell R.

doi:10.1126/science.abm1184

Cited by 14 publications

(8 citation statements)

References 56 publications

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“…The two known variants of subtype VI-B systems, VI-B1 and VI-B2, can be differentiated on the basis of the presence of additional small accessory proteins containing predicted transmembrane (TM) helices, known as Csx27 (VI-B1) and Csx28 (VI-B2). These accessory proteins regulate Cas13b-mediated RNA interference (Smargon et al in 2017; VanderWal et al 2023). These variants are believed to have diverged during the course of evolution, leading to different architectural features associated with these unique accessory transmembrane proteins (Shmakov et al, 2017).…”

Section: Resultsmentioning

confidence: 99%

“…On the other hand, it has been suggested that the VI-B1-associated ancillary Csx27 protein, which downregulates the activity of Cas13b, may form membrane channels for ssDNA uptake, and that its nascent transcripts are degraded by the VI-B1 interference machinery (Makarova et al, 2019). Additionally, the Csx28 protein associated with VI-B2 systems forms pores, and its antiviral activity requires sequence-specific cleavage of viral mRNAs by Cas13b leading to membrane depolarization (VanderWal et al, 2023). The presumed role of CorA in the interference step in type VI-B/RT-associated systems needs to be determined, but we are more inclined towards a role similar to that shown by Csx28.…”

Section: Discussionmentioning

confidence: 99%

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Spacer acquisition in type VI CRISPR-Cas systems associated with reverse transcriptase-Cas1 fusion proteins

Molina-Sanchez,

Martinez-Abarca,

Millan

et al. 2024

Preprint

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In prokaryotes, CRISPR-Cas systems store memories of past infections in the form of spacers integrated into CRISPR arrays. When associated with type III CRISPR-Cas systems, Reverse transcriptase-Cas1 fusion proteins (RT-Cas1) enable these defense systems to acquire spacers from RNA sources. However, despite the specific targeting of RNA by the Cas13-containing type VI CRISPR-Cas systems, there is no evidence of RNA-origin spacer acquisition. Using computational analyses, we recently reported the association of RT-Cas1 fusion proteins with type VI-A systems. In this study, we found that RT-Cas1 fusion proteins were also associated with complete type VI-B systems in bacteria from gut metagenomes, constituting a variant system that harbors a linked CorA-encoding locus in addition to the CRISPR array and adaptation RT-Cas1/Cas2 module. By combining in vitro and in vivo experiments, we demonstrated that type VI RT-CRISPR systems are functional for spacer acquisition and CRISPR array processing, and that the associated RT enables spacer acquisition from RNA molecules, thus demonstrating that the system is capable of functioning independently of other in-trans systems. These findings highlight the importance of RTs in RNA-targeting CRISPR-Cas systems, suggesting a potential defense mechanism against RNA-based invaders in specific environments.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Spacer acquisition in type VI CRISPR-Cas systems associated with reverse transcriptase-Cas1 fusion proteins

Molina-Sanchez,

Martinez-Abarca,

Millan

et al. 2024

Preprint

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“…In addition, the tRNA fragments or nicked tRNAs produced by the Cas13a cleavage might act as signaling or inhibitory molecules. Recently, the signaling function of cleavage products, presumed to be tRNA fragments, generated by Cas13b ortholog, was proposed for the CRISPR-Cas13b–assisted mechanism of antiphage defense through membrane pore activation ( 24 ).…”

Section: Discussionmentioning

confidence: 99%

tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector

Jain,

Kolesnik,

Kuznedelov

et al. 2024

Sci. Adv.

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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.

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“…Membrane-associated CRISPR effectors have been detected bioinformatically but characterised examples are still scarce. In addition to the aforementioned B. fragilis CorA effector [28], some type V CRISPR systems have an associated Csx28 protein that forms an octameric membrane pore, resulting in membrane depolarisation, although the mechanism of activation remains unclear [45]. More broadly, membrane associated effectors function in many other prokaryotic defence pathways that are presumed to function via programmed cell death.…”

Section: Discussionmentioning

confidence: 99%

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

Grüschow,

McQuarrie,

Ackermann

et al. 2023

Preprint

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CRISPR 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 inVibrio cholerae. Csx23 provides immunity against plasmids and phage when expressed inEscherichia colialong with its cognate type III CRISPR system. The Csx23 protein localises in the membrane using a 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 cA4specifically. Using pulse EPR, we demonstrate that cA4binding 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.

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

Cited by 14 publications

References 56 publications

Spacer acquisition in type VI CRISPR-Cas systems associated with reverse transcriptase-Cas1 fusion proteins

Spacer acquisition in type VI CRISPR-Cas systems associated with reverse transcriptase-Cas1 fusion proteins

tRNA anticodon cleavage by target-activated CRISPR-Cas13a effector

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

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