CRISPR Processing Many bacteria and archaea recognize invading viruses and plasmids. Foreign DNA is integrated into so-called clustered regularly interspaced short palindromic repeat (CRISPR) loci, and transcripts from these loci are processed into RNAs that can target the invading DNA or RNA for destruction. To investigate the molecular basis for this processing, Haurwitz et al. (p. 1355) screened CRISPR-associated (Cas) proteins in the opportunistic pathogen Pseudomonas aeruginosa and found they were capable of cleaving the CRISPR transcripts. The crystal structure of Cas4 with the CRISPR RNA transcript revealed how the protein specifically recognized RNA repeats, as well as the mechanism of endonucleolytic cleavage. How a prokaryotic immune system makes small RNAs that target invading nucleic acids. Many bacteria and archaea contain clustered regularly interspaced short palindromic repeats (CRISPRs) that confer resistance to invasive genetic elements. Central to this immune system is the production of CRISPR-derived RNAs (crRNAs) after transcription of the CRISPR locus. Here, we identify the endoribonuclease (Csy4) responsible for CRISPR transcript (pre-crRNA) processing in Pseudomonas aeruginosa. A 1.8 angstrom crystal structure of Csy4 bound to its cognate RNA reveals that Csy4 makes sequence-specific interactions in the major groove of the crRNA repeat stem-loop. Together with electrostatic contacts to the phosphate backbone, these enable Csy4 to bind selectively and cleave pre-crRNAs using phylogenetically conserved serine and histidine residues in the active site. The RNA recognition mechanism identified here explains sequence- and structure-specific processing by a large family of CRISPR-specific endoribonucleases.
