CRISPR-Csx28 forms a Cas13b-activated membrane pore required for robust CRISPR-Cas adaptive immunity

Abstract

Type VI CRISPR-Cas systems use the RNA-guided RNase Cas13 to defend bacteria against viruses, and some of these systems encode putative membrane proteins that have unclear roles in Cas13-mediated defense. Here we show that Csx28, of Type VI-B2 systems, forms membrane pore structures to slow cellular metabolism upon viral infection, and this activity drastically increases anti-viral defense. High- resolution cryo-EM reveals that Csx28 exists unexpectedly as a detergent-encapsulated octameric pore, and we then show these Csx28 pores are membrane localized in vivo. Activation of Csx28 in vivo strictly requires sequence-specific recognition of viral mRNAs by Cas13b, and this activation results in Csx28-mediated membrane depolarization, slowed metabolism, and inhibition of sustained viral infection. Together, our work reveals an unprecedented mechanism by which Csx28 acts as a downstream, Cas13b-activated, effector protein that uses membrane perturbation as an anti-viral defense strategy.

A target expression threshold dictates invader defense and prevents autoimmunity by CRISPR-Cas13

“…Furthermore, the existence of cOA-degrading Acrs could offer a means to artificially raise the target expression threshold to the point where this mode of immunity is never activated over the course of the infection. Similar behaviors may be expected for type VI CRISPR-Cas systems encoding Cas13b and the accessory protein Csx28 that appears to amplify the immune response (Smargon et al, 2017;VanderWal et al, 2021). Exploring the extent to which a target expression threshold exists for different type III and VI-B CRISPR-Cas systems could broaden our understanding of the target expression threshold, particularly when back-up defenses exist that enhance the immune response.…”

RNA-targeting CRISPR-Cas13 Provides Broad-spectrum Phage Immunity

“…Two parsimonious explanations for the phylogenetic distribution of Cas13 effectors are that (1) Cas13 effectors are relatively ineffective anti-phage systems, limiting their phylogenetic spread from evolutionary pressure or (2) Cas13 effectors are potent anti-phage systems, but the fitness cost of their abortive-infection- (abi-) like effects 30, 33 is selected against. To explore these possibilities, we tested the anti-phage activity of the most- and least-widely dispersed Cas13 effectors based on our analysis of bacterial phylogeny, Cas13a and Cas13d, respectively (Fig.…”

“…Possibly, the highly potent anti-phage activity observed by Cas13a explains the relative scarcity of Type-VI CRISPR-Cas systems. All known Type-VI systems are thought to facilitate anti-phage activity through mechanisms similar to abortive infection 30, 33 . Although the use of crRNA confers specificity for the activation of Cas13, we noticed substantial activation and toxicity in the absence of phage for RfxCas13d.…”

“…Upon target RNA binding, Cas13 unleashes general, non-specific RNA degradation that arrests growth of the virocell to block infection progression, thereby limiting infection of neighboring cells 30 . Since all known viruses produce RNA 31 , Cas13 is capable of inhibiting dsDNA phages, primarily shown through studies investigating temperate 30, 32, 33 and nucleus forming 19, 34 phages. However, other Class 2 CRISPR effectors have encountered serious limitations in overcoming the diversity of genetic content encoded in phages 12, 19, 20, 35 .…”

Cas12a2 elicits abortive infection via RNA-triggered destruction of double-stranded DNA

“…In bacteria and archaea, this strategy is called Abortive infection (Abi), and it is used by a vast variety of bacterial defense systems 1,2 . Recently, it was shown that CRISPR RNA (crRNA)-guided adaptive immune systems that target RNA cause Abi phenotypes [3][4][5][6] . In type III systems, target RNA binding triggers production of cyclic oligoadenylate secondary messengers that in turn activate indiscriminate accessory RNases (e.g., Cms6/Csx1) 4,5,[7][8][9] .…”