A novel pathway for the maturation of CRISPR-derived small guide RNAs (crRNAs) has been identified in the human pathogen Streptococcus pyogenes. This process involves a trans-encoded small RNA called tracrRNA, which pairs with the CRISPR precursor transcript (pre-crRNA) to facilitate its processing by the host-encoded RNase III and the CRISPR-associated Csn1 protein. These components are essential for the bacteria's defense against prophage-derived DNA. The study reveals that tracrRNA directs the maturation of crRNAs by forming a duplex with pre-crRNA, which is then cleaved by RNase III to generate mature crRNAs. The Csn1 protein, a Cas protein, acts as a molecular anchor to facilitate the base-pairing of tracrRNA with pre-crRNA, enabling RNase III to cleave the pre-crRNA. This process is crucial for the bacteria's immunity against lysogenic phages and limits horizontal virulence gene transfer among pathogenic streptococcal species. The findings highlight the diversity and complexity of CRISPR/Cas systems and suggest that host factors like RNase III can contribute to the evolutionary diversification of these systems. The study also indicates that the requirement of a trans-encoded small RNA for pre-crRNA processing into active crRNAs is a general RNA maturation mechanism shared by type II CRISPR/Cas systems that lack the cse3, cas6, or csy4 proteins but possess csn1. The research underscores the importance of RNA-based immunity mechanisms in bacteria and the role of host factors in CRISPR activity.A novel pathway for the maturation of CRISPR-derived small guide RNAs (crRNAs) has been identified in the human pathogen Streptococcus pyogenes. This process involves a trans-encoded small RNA called tracrRNA, which pairs with the CRISPR precursor transcript (pre-crRNA) to facilitate its processing by the host-encoded RNase III and the CRISPR-associated Csn1 protein. These components are essential for the bacteria's defense against prophage-derived DNA. The study reveals that tracrRNA directs the maturation of crRNAs by forming a duplex with pre-crRNA, which is then cleaved by RNase III to generate mature crRNAs. The Csn1 protein, a Cas protein, acts as a molecular anchor to facilitate the base-pairing of tracrRNA with pre-crRNA, enabling RNase III to cleave the pre-crRNA. This process is crucial for the bacteria's immunity against lysogenic phages and limits horizontal virulence gene transfer among pathogenic streptococcal species. The findings highlight the diversity and complexity of CRISPR/Cas systems and suggest that host factors like RNase III can contribute to the evolutionary diversification of these systems. The study also indicates that the requirement of a trans-encoded small RNA for pre-crRNA processing into active crRNAs is a general RNA maturation mechanism shared by type II CRISPR/Cas systems that lack the cse3, cas6, or csy4 proteins but possess csn1. The research underscores the importance of RNA-based immunity mechanisms in bacteria and the role of host factors in CRISPR activity.