Toxin-antitoxin RNA pairs safeguard CRISPR-Cas systems

Small RNAs guard CRISPR-Cas The microbial adaptive immunity system CRISPR-Cas benefits microbes by warding off genetic invaders, but it also inflicts a fitness cost because of occasional autoimmune reactions, rendering CRISPR loci evolutionarily unstable. Li et al. identified previously unnoticed toxin-antitoxin RNA pairs embedded within diverse CRISPR-Cas loci. The antitoxin RNA mimics a CRISPR RNA and repurposes the CRISPR immunity effector to transcriptionally repress a toxin RNA that would otherwise arrest cell growth by sequestering a rare transfer RNA. These small RNAs thus form a symbiosis with CRISPR, rendering CRISPR addictive to the host despite its fitness cost. These findings reveal how CRISPR-Cas can operate as a selfish genetic element. Science, this issue p. eabe5601 CRISPR effectors can regulate expression of RNA toxin genes, thereby preventing loss of CRISPR-Cas systems. INTRODUCTION CRISPR-Cas systems efficiently protect bacteria and archaea from viruses and other types of foreign DNA, but, characteristically of defense systems, they also impart non-negligible fitness costs on the host, for example, the risk of autoimmunity and the repulsion to exogenous beneficial genes. Presumably, these costs result in frequent loss of CRISPR-Cas in bacteria, which is reflected in its patchy distribution, even among closely related bacterial strains. Nevertheless, in the current genome sequence databases, ~40% of bacterial and ~90% of archaeal genomes carry CRISPR-cas loci, suggesting the possibility that in addition to the direct benefits of adaptive immunity, mechanisms might exist that mitigate the costs of CRISPR systems and prevent their loss. RATIONALE We specifically looked into an archaeal type I-B CRISPR-Cas, where the genes encoding the subunits of the CRISPR effector Cascade cannot be deleted individually but can be readily deleted as a whole, including a 311–base pair intergenic region. These observations suggest that the Cascade gene cassette (cas6-cas8-cas7-cas5) includes a toxic component that makes it addictive to the host (elicits cellular toxicity once any of the cascade genes is deleted). We cloned and extensively analyzed the intergenic region between cas6 and cas8, which allowed us to identify the Cascade-repressed toxin gene creT, along with an associating CRISPR repeat–like sequence that appears to be required for transcriptional repression of creT. We hypothesized that the repeat-like sequence is part of a CRISPR RNA–resembling antitoxin (CreA) RNA, which represses the toxin jointly with Cascade. We reasoned that CreTA would make the cascade genes addictive for the host. RESULTS The intergenic sequence between cas6 and cas8 caused toxicity in cells lacking one or more cascade genes. By extensive mutational analysis, we identified the RNA toxin gene creT and its critical elements, namely, a combination of a strong Shine-Dalgarno motif, an efficient start codon, two minor arginine codons (AGA) located immediately downstream, and a stable stem-loop structure. Overexpression of tRNAUCU relieved the toxicity of CreT, supporting a mechanism whereby this RNA toxin arrests cellular growth by sequestering the rare arginine tRNAUCU. Mutational analysis of creT and its neighboring sequences revealed an adjacent CRISPR repeat–like sequence that is required to suppress the toxicity of CreT. This repeat-like sequence is immediately followed by a spacer-like sequence and a transcription terminator. By Northern blotting and RNA sequencing, we validated the expression of CreA RNA, a CRISPR RNA variant that lacks a 3′ handle. The spacer of CreA partially matches the promoter of creT (PcreT), and using a reporter gene, we confirmed that CreA, as a complex with Cascade, represses PcreT. Similar to CRISPR interference, repression of creT requires a protospacer adjacent motif (PAM) and the PAM-proximal base pairing. In cells lacking CreTA, the cascade genes become susceptible to disruption by transposable elements. Our bioinformatic analysis identified several CreTA analogs associated with diverse archaeal and bacterial CRISPR-cas loci and containing PAMs corresponding to those of the respective CRISPR systems. Notably, these CreTA analogs hold little conservation in nucleic acid sequence, suggesting that they have highly divergently evolved and, conceivably, exploited different toxicity mechanisms. CONCLUSION Our data unearth previously unnoticed toxin-antitoxin RNA pairs that prevent the loss of CRISPR-cas loci by making them addictive to the host cell. The naturally occurring reprogramming of CRISPR effectors for gene regulation highlights the multifunctionality of CRISPR-Cas in bacteria and archaea and illuminates the emerging topic of the evolution of antiviral defense and gene regulation. Toxin-antitoxin RNA pair CreTA safeguards CRISPR-Cas. CRISPR effector (Cascade) is not only guided by CRISPR RNA to inactivate full-matching foreign nucleic acids but is also co-opted by CreA RNA to transcriptionally repress the toxin gene creT through partial complementarity between CreA and the creT promoter. When Cascade is inactivated, the derepressed CreT RNA sequesters the rare tRNAUCU that decodes a minor arginine codon and arrests cellular growth, thus making the CRISPR effector addictive to the host cell. CRISPR-Cas systems provide RNA-guided adaptive immunity in prokaryotes. We report that the multisubunit CRISPR effector Cascade transcriptionally regulates a toxin-antitoxin RNA pair, CreTA. CreT (Cascade-repressed toxin) is a bacteriostatic RNA that sequesters the rare arginine tRNAUCU (transfer RNA with anticodon UCU). CreA is a CRISPR RNA–resembling antitoxin RNA, which requires Cas6 for maturation. The partial complementarity between CreA and the creT promoter directs Cascade to repress toxin transcription. Thus, CreA becomes antitoxic only in the presence of Cascade. In CreTA-deleted cells, cascade genes become susceptible to disruption by transposable elements. We uncover several CreTA analogs associated with diverse archaeal and bacterial CRISPR-cas loci. Thus, toxin-antitoxin RNA pairs can safeguard CRISPR immunity by making cells addicted to CRISPR-Cas, which highlights the multifunctionality of Cas proteins and the intricate mechanisms of CRISPR-Cas regulation.