Phage CRISPR-like regulatory RNAs silence multilayered bacterial immune systems

Abstract

CRISPR RNAs (crRNAs) encoded by CRISPR arrays direct Cas nucleases against viruses. Some CRISPR-Cas loci also contain mini-arrays encoding crRNA-like RNAs (crlRNAs), which modulate transcription of cas or associated toxin genes that safeguard CRISPR-Cas activity. Here, we report that phages hijack these host crlRNA regulatory circuits to suppress bacterial immunity. Viral crlRNAs compete with host crRNAs for Cas protein binding and reprogram Cas proteins to silence their own expression or suppress transcription of CRISPR-safeguarding toxins. These anti-defense crlRNAs are found across diverse phages and plasmids, frequently organized into multiplexed transcriptional units resembling CRISPR arrays, and often synergize with co-encoded anti-CRISPR proteins to disable hierarchical host immune responses. Furthermore, we demonstrate that these compact viral crlRNA architectures can be harnessed for efficient mammalian genome editing. Our findings uncover widespread viral hijacking of host Cas proteins via crlRNAs, highlighting the recurrent exaptation of CRISPR-Cas regulatory functions to counter multilayered bacterial immune defenses.