RNA-guided immunity: a novel RNA maturation pathway for the production of CRISPR RNAs targeting genome invaders

To protect themselves against infection by the enemy (phages, plasmids), bacteria and archaea have invented a sophisticated adaptive RNA-based defence mechanism, CRISPR/Cas. A key event in the activation of this immune system is the maturation of the short spacer/repeat-derived CRISPR RNAs (crRNAs) that silence invading foreign nucleic acids in a sequence-specific manner leading to their destruction. Certain CRISPR/Cas subtypes use Cas endoribonucleases (Cse3 (CasE), Cas6, Csy4) to maturate the crRNAs. We have analyzed the CRISPR/Cas system II (Nmeni/CASS4 subtype) in our model organism Streptococcus pyogenes. Our study revealed a fundamentally novel crRNA maturation pathway. A new trans-acting small RNA, tracrRNA, directs the processing of CRISPR pre-crRNA into mature crRNAs by the activities of the widely conserved endogenous RNase III and the CRISPR-associated protein, Csn1. All these components are essential to protect S. pyogenes against lysogenic phage-derived DNA. Furthermore, we show evidence that tracrRNA and pre-crRNA of the CRISPR/Cas system II from other bacterial species (Listeria innocua, Neisseria meningitidis, Streptococcus mutans and Streptococcus thermophilus) are expressed and co-processed. Thus, the type II CRISPR/Cas system including tracrRNA and the host RNase III seems to be constitutively activated to target and affect the maintenance of invader genomes to protect bacteria against cell death (lytic phages, e.g. S. thermophilus) or the acquisition of virulence genes (lysogenic phages, S. pyogenes). Taken together, our work uncovered a novel pathway of small guide RNA maturation and the first example of a host factor (RNase III) required for bacterial RNA-mediated immunity against invaders.