Rachel E. Haurwitz

Sequence- and Structure-Specific RNA Processing by a CRISPR Endonuclease

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.

RNA processing enables predictable programming of gene expression

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.

Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality

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.

Csy4 relies on an unusual catalytic dyad to position and cleave CRISPR RNA.

CRISPR‐Cas adaptive immune systems protect prokaryotes against foreign genetic elements. crRNAs derived from CRISPR loci base pair with complementary nucleic acids, leading to their destruction. In Pseudomonas aeruginosa, crRNA biogenesis requires the endoribonuclease Csy4, which binds and cleaves the repetitive sequence of the CRISPR transcript. Biochemical assays and three co‐crystal structures of wild‐type and mutant Csy4/RNA complexes reveal a substrate positioning and cleavage mechanism in which a histidine deprotonates the ribosyl 2′‐hydroxyl pinned in place by a serine, leading to nucleophilic attack on the scissile phosphate. The active site catalytic dyad lacks a general acid to protonate the leaving group and positively charged residues to stabilize the transition state, explaining why the observed catalytic rate constant is ∼104‐fold slower than that of RNase A. We show that this RNA cleavage step is essential for assembly of the Csy protein‐crRNA complex that facilitates target recognition. Considering that Csy4 recognizes a single cellular substrate and sequesters the cleavage product, evolutionary pressure has likely selected for substrate specificity and high‐affinity crRNA interactions at the expense of rapid cleavage kinetics.

RNA–protein analysis using a conditional CRISPR nuclease

RNA-binding proteins control the fate and function of the transcriptome in all cells. Here we present technology for isolating RNA–protein partners efficiently and accurately using an engineered clustered regularly interspaced short palindromic repeats (CRISPR) endoribonuclease. An inactive version of the Csy4 nuclease binds irreversibly to transcripts engineered with a 16-nt hairpin sequence at their 5′ ends. Once immobilized by Csy4 on a solid support, contaminating proteins and other molecules can be removed by extensive washing. Upon addition of imidazole, Csy4 is activated to cleave the RNA, removing the hairpin tag and releasing the native transcript along with its specifically bound protein partners. This conditional Csy4 enzyme enables recovery of specific RNA-binding partners with minimal false-positive contamination. We use this method, coupled with quantitative MS, to identify cell type-specific human pre-microRNA-binding proteins. We also show that this technology is suitable for analyzing diverse size transcripts, and that it is suitable for adaptation to a high-throughput discovery format.