Spacer prioritization in CRISPR-Cas9 immunity is enabled by the leader RNA

CRISPR-Cas systems store fragments of foreign DNA called spacers as immunological recordings used to combat future infections. Of the many spacers stored in a CRISPR array, the newest spacers are known to be prioritized for immune defense. However, the underlying mechanism remains unclear. Here we show that the leader region upstream of CRISPR arrays in CRISPR-Cas9 systems enhances CRISPR RNA (crRNA) processing from the newest spacer, prioritizing defense against the matching invader. Using the CRISPR-Cas9 system from Streptococcus pyogenes as a model, we found that the transcribed leader interacts with the conserved repeats bordering the newest spacer. The resulting interaction promotes tracrRNA hybridization with the second repeat, accelerating crRNA processing. Accordingly, disrupting this structure reduces the abundance of the associated crRNA and immune defense against targeted plasmids and bacteriophages. Beyond the S. pyogenes system, bioinformatics analyses revealed that leader-repeat structures appear across CRISPR-Cas9 systems. CRISPR-Cas systems thus possess an RNA-based mechanism to prioritize defense against the most recently encountered invaders.