A leader-repeat hairpin blocks extraneous CRISPR RNA production in diverse CRISPR-Cas13 systems

Abstract CRISPR RNAs (crRNAs) guide recognition and targeting of intracellular invaders as part of adaptive immunity by CRISPR-Cas systems. crRNAs are transcribed from CRISPR arrays of conserved repeats interlaced with invader-derived spacers. While crRNA production is essential for immunity, its optimization for defense remains poorly understood. Here, we show that, in diverse RNA-targeting type VI CRISPR-Cas systems, the leader RNA encoded upstream of the CRISPR array prevents formation of an invader-independent extraneous crRNA (ecrRNA) by blocking processing of the first repeat. Using the VI-B2 system from Porphyromonas gingivalis as a model, we demonstrate that the leader RNA and first repeat form a conserved inhibitory hairpin that precludes binding and processing by the system’s Cas13b nuclease. Disrupting this hairpin enables ecrRNA production, which in turn can deplete invader-derived crRNAs and reduce Cas13b-mediated phage defense. Structure prediction indicates that these leader-repeat hairpins are widespread across diverse type VI subtypes, highlighting a conserved regulatory mechanism. Our findings reveal how a prevalent branch of CRISPR-Cas systems suppresses ecrRNA formation to promote RNA-guided immunity.