Phage CRISPR-like regulatory RNAs silence bacterial adaptive and innate immunity

In prokaryotes, arrayed CRISPR RNAs (crRNAs) guide Cas proteins to destroy phage DNA/RNA, while solitary crRNA-like RNAs (crlRNAs) program Cas proteins for auto-regulation or to control abortive infection (Abi)-inducing toxins that activate when CRISPR-Cas fails. Here, we report that phages exploit crlRNA mimics to hijack these multi-layered host defenses. Pseudomonas aeruginosa phages use crlRNAs to thwart CRISPR-Cas immunity by inhibiting Cas expression, or to block Abi by silencing an unprecedented RNA toxin that features consecutive proline codons. Remarkably, the anti-CRISPR protein AcrIF24 selectively inhibits Cas proteins loaded with host crRNAs/crlRNAs, while allowing those complexed with viral crlRNAs to synergistically block Abi responses, as viral crlRNAs have co-adaptively evolved shorter spacer sequences. Furthermore, viral crlRNAs frequently organize as multiplexed arrays, mirroring the architecture of bacterial CRISPRs. Our findings showcase how phage RNAs hijack Cas proteins to silence multi-layered bacterial defenses, and highlight the delicate synergy between RNA and protein-based anti-CRISPR strategies.