Many prokaryotes contain genomic 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) following transcription of the CRISPR locus. Here we identify the endoribonuclease (Csy4) responsible for pre-crRNA processing in Pseudomonas aeruginosa. A 1.8 Å crystal structure of Csy4 in complex with its cognate RNA reveals an unexpected recognition mechanism whereby Csy4 makes sequence-specific interactions in the major groove of the CRISPR repeat stem-loop. Together with electrostatic contacts to the phosphate backbone, these enable Csy4 to selectively bind and cleave pre-crRNAs. The active site of Csy4 comprises two invariant residues, a serine and a histidine. The RNA recognition mechanism identified here explains sequence- and structure-specific processing by a large family of CRISPR-specific endoribonucleases.

The RNA-guided Cas9 endonuclease specifically targets and cleaves DNA in a sequence-dependent manner and has been widely used for programmable genome editing. Cas9 activity is dependent on interactions with guide RNAs, and evolutionarily divergent Cas9 nucleases have been shown to work orthogonally. However, the molecular basis of selective Cas9:guide-RNA interactions is poorly understood. Here, we identify and characterize six conserved modules within native crRNA:tracrRNA duplexes and single guide RNAs (sgRNAs) that direct Cas9 endonuclease activity. We show the bulge and nexus are necessary for DNA cleavage and demonstrate that the nexus and hairpins are instrumental in defining orthogonality between systems. In contrast, the crRNA:tracrRNA complementary region can be modified or partially removed. Collectively, our results establish guide RNA features that drive DNA targeting by Cas9 and open new design and engineering avenues for CRISPR technologies.

Complex interactions among genetic components often result in variable systemic performance in designed multigene systems. Using the bacterial clustered regularly interspaced short palindromic repeat (CRISPR) pathway we develop a synthetic RNA-processing platform, and show that efficient and specific cleavage of precursor mRNA enables reliable and predictable regulation of multigene operons. Physical separation of linked genetic elements by CRISPR-mediated cleavage is an effective strategy to achieve assembly of promoters, ribosome binding sites, cis-regulatory elements, and riboregulators into single- and multigene operons with predictable functions in bacteria. We also demonstrate that CRISPR-based RNA cleavage is effective for regulation in bacteria, archaea and eukaryotes. Programmable RNA processing using CRISPR offers a general approach for creating context-free genetic elements and can be readily used in the bottom-up construction of increasingly complex biological systems in a plug-and-play manner.