Primed CRISPR DNA uptake in Pyrococcus furiosus.

CRISPR-Cas adaptive immune systems are used by prokaryotes to defend against invaders like viruses and other mobile genetic elements. Immune memories are stored in the form of 'spacers' which are short DNA sequences that are captured from invaders and added to the CRISPR array during a process called 'adaptation'. Spacers are transcribed and the resulting CRISPR (cr)RNAs assemble with different Cas proteins to form effector complexes that recognize matching nucleic acid and destroy it ('interference'). Adaptation can be 'naïve', i.e. independent of any existing spacer matches, or it can be 'primed', i.e. spurred by the crRNA-mediated detection of a complete or partial match to an invader sequence. Here we show that primed adaptation occurs in Pyrococcus furiosus. Although P. furiosus has three distinct CRISPR-Cas interference systems (I-B, I-A and III-B), only the I-B system and Cas3 were necessary for priming. Cas4, which is important for selection and processing of new spacers in naïve adaptation, was also essential for priming. Loss of either the I-B effector proteins or Cas3 reduced naïve adaptation. However, when Cas3 and all crRNP genes were deleted, uptake of correctly processed spacers was observed, indicating that none of these interference proteins are necessary for naïve adaptation.

Regulation of the RNA and DNA nuclease activities required for Pyrococcus furiosus Type III-B CRISPR-Cas immunity.

Type III CRISPR-Cas prokaryotic immune systems provide anti-viral and anti-plasmid immunity via a dual mechanism of RNA and DNA destruction. Upon target RNA interaction, Type III crRNP effector complexes become activated to cleave both target RNA (via Cas7) and target DNA (via Cas10). Moreover, trans-acting endoribonucleases, Csx1 or Csm6, can promote the Type III immune response by destroying both invader and host RNAs. Here, we characterize how the RNase and DNase activities associated with Type III-B immunity in Pyrococcus furiosus (Pfu) are regulated by target RNA features and second messenger signaling events. In vivo mutational analyses reveal that either the DNase activity of Cas10 or the RNase activity of Csx1 can effectively direct successful anti-plasmid immunity. Biochemical analyses confirmed that the Cas10 Palm domains convert ATP into cyclic oligoadenylate (cOA) compounds that activate the ribonuclease activity of Pfu Csx1. Furthermore, we show that the HEPN domain of the adenosine-specific endoribonuclease, Pfu Csx1, degrades cOA signaling molecules to provide an auto-inhibitory off-switch of Csx1 activation. Activation of both the DNase and cOA generation activities require target RNA binding and recognition of distinct target RNA 3' protospacer flanking sequences. Our results highlight the complex regulatory mechanisms controlling Type III CRISPR immunity.

Cas4 Nucleases Define the PAM, Length, and Orientation of DNA Fragments Integrated at CRISPR Loci.

To achieve adaptive and heritable immunity against viruses and other mobile genetic elements, CRISPR-Cas systems must capture and store short DNA fragments (spacers) from these foreign elements into host genomic CRISPR arrays. This process is catalyzed by conserved Cas1/Cas2 integration complexes, but the specific roles of another highly conserved protein linked to spacer acquisition, the Cas4 nuclease, are just now emerging. Here, we show that two Cas4 nucleases (Cas4-1 and Cas4-2) play critical roles in CRISPR spacer acquisition in Pyrococcus furiosus. The nuclease activities of both Cas4 proteins are required to process protospacers to the correct size. Cas4-1 specifies the upstream PAM (protospacer adjacent motif), while Cas4-2 specifies the conserved downstream motif. Both Cas4 proteins ensure CRISPR spacer integration in a defined orientation leading to CRISPR immunity. Collectively, these findings provide in vivo evidence for critical roles of Cas4 nucleases in protospacer generation and functional spacer integration at CRISPR arrays.

The CRISPR-associated Csx1 protein of Pyrococcus furiosus is an adenosine-specific endoribonuclease.

Prokaryotes are frequently exposed to potentially harmful invasive nucleic acids from phages, plasmids, and transposons. One method of defense is the CRISPR-Cas adaptive immune system. Diverse CRISPR-Cas systems form distinct ribonucleoprotein effector complexes that target and cleave invasive nucleic acids to provide immunity. The Type III-B Cmr effector complex has been found to target the RNA and DNA of the invader in the various bacterial and archaeal organisms where it has been characterized. Interestingly, the gene encoding the Csx1 protein is frequently located in close proximity to the Cmr1-6 genes in many genomes, implicating a role for Csx1 in Cmr function. However, evidence suggests that Csx1 is not a stably associated component of the Cmr effector complex, but is necessary for DNA silencing by the Cmr system in Sulfolobus islandicus. To investigate the function of the Csx1 protein, we characterized the activity of recombinant Pyrococcus furiosus Csx1 against various nucleic acid substrates. We show that Csx1 is a metal-independent, endoribonuclease that acts selectively on single-stranded RNA and cleaves specifically after adenosines. The RNA cleavage activity of Csx1 is dependent upon a conserved HEPN motif located within the C-terminal domain of the protein. This motif is also key for activity in other known ribonucleases. Collectively, the findings indicate that invader silencing by Type III-B CRISPR-Cas systems relies both on RNA and DNA nuclease activities from the Cmr effector complex as well as on the affiliated, trans-acting Csx1 endoribonuclease.

The RNA- and DNA-targeting CRISPR-Cas immune systems of Pyrococcus furiosus.

Using the hyperthermophile Pyrococcus furiosus, we have delineated several key steps in CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) invader defence pathways. P. furiosus has seven transcriptionally active CRISPR loci that together encode a total of 200 crRNAs (CRISPR RNAs). The 27 Cas proteins in this organism represent three distinct pathways and are primarily encoded in two large gene clusters. The Cas6 protein dices CRISPR locus transcripts to generate individual invader-targeting crRNAs. The mature crRNAs include a signature sequence element (the 5' tag) derived from the CRISPR locus repeat sequence that is important for function. crRNAs are tailored into distinct species and integrated into three distinct crRNA-Cas protein complexes that are all candidate effector complexes. The complex formed by the Cmr [Cas module RAMP (repeat-associated mysterious proteins)] (subtype III-B) proteins cleaves complementary target RNAs and can be programmed to cleave novel target RNAs in a prokaryotic RNAi-like manner. Evidence suggests that the other two CRISPR-Cas systems in P. furiosus, Csa (Cas subtype Apern) (subtype I-A) and Cst (Cas subtype Tneap) (subtype I-B), target invaders at the DNA level. Studies of the CRISPR-Cas systems from P. furiosus are yielding fundamental knowledge of mechanisms of crRNA biogenesis and silencing for three of the diverse CRISPR-Cas pathways, and reveal that organisms such as P. furiosus possess an arsenal of multiple RNA-guided mechanisms to resist diverse invaders. Our knowledge of the fascinating CRISPR-Cas pathways is leading in turn to our ability to co-opt these systems for exciting new biomedical and biotechnological applications.

Structure of an RNA silencing complex of the CRISPR-Cas immune system.

Bacterial and archaeal clustered regularly interspaced short palindromic repeat (CRISPR) loci capture virus and plasmid sequences and use them to recognize and eliminate these invaders. CRISPR RNAs (crRNAs) containing the acquired sequences are incorporated into effector complexes that destroy matching invader nucleic acids. The multicomponent Cmr effector complex cleaves RNA targets complementary to the crRNAs. Here, we report cryoelectron microscopy reconstruction of a functional Cmr complex bound with a target RNA at ~12 Å. Pairs of the Cmr4 and Cmr5 proteins form a helical core that is asymmetrically capped on each end by distinct pairs of the four remaining subunits: Cmr2 and Cmr3 at the conserved 5' crRNA tag sequence and Cmr1 and Cmr6 near the 3' end of the crRNA. The shape and organization of the RNA-targeting Cmr complex is strikingly similar to the DNA-targeting Cascade complex. Our results reveal a remarkably conserved architecture among very distantly related CRISPR-Cas complexes.

Structure of the Cmr2-Cmr3 subcomplex of the Cmr RNA silencing complex.

The Cmr complex is an RNA-guided effector complex that cleaves invader RNA in the prokaryotic immune response mediated by the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas system. Here, we report the crystal structure of a Cmr subcomplex containing Cmr2 (Cas10) and Cmr3 subunits at 2.8 Å resolution. The structure revealed a dual ferredoxin fold and glycine-rich loops characteristic of previously known repeat-associated mysterious proteins and two unique insertion elements in Cmr3 that mediate its interaction with Cmr2. Surprisingly, while mutation of both insertion elements significantly weakened Cmr3-Cmr2 interaction, they exhibit differential effects on Cmr-mediated RNA cleavage by the Cmr complex, suggesting stabilization of Cmr2-Cmr3 interactions by other subunits. Further mutational analysis of the two conserved (but non-Cmr2-binding) glycine-rich loops of Cmr3 identified a region that is likely involved in assembly or the RNA cleavage function of the Cmr complex.

Crystal structure of Cmr5 from Pyrococcus furiosus and its functional implications.

The bacterial acquired immune system consists of clustered regularly interspaced short palindromic repeats (CRISPRs) and CRIPSR-associated (Cas) genes, which include Cas-module repeat-associated mysterious proteins (Cmr). The six Cmr proteins of Pyrococcus furiosus (pfCmr1-pfCmr6) form a Cmr effector complex that functions against exogenous nucleic acid. Among the Cmr proteins, the role of pfCmr5 and its involvement in the complex's cleavage activity have been obscure. The elucidated pfCmr5 structure has two inserted α-helices compared with the other trimeric Cmr5 structure. However, pfCmr5 exists as a monomeric protein both in the crystalline state and in solution. In vitro assays indicate that pfCmr5 interacts with pfCmr4. These structural and biophysical data might help in understanding the complicated and ill-characterized Cmr effector complex.

The impact of CRISPR repeat sequence on structures of a Cas6 protein-RNA complex.

The repeat-associated mysterious proteins (RAMPs) comprise the most abundant family of proteins involved in prokaryotic immunity against invading genetic elements conferred by the clustered regularly interspaced short palindromic repeat (CRISPR) system. Cas6 is one of the first characterized RAMP proteins and is a key enzyme required for CRISPR RNA maturation. Despite a strong structural homology with other RAMP proteins that bind hairpin RNA, Cas6 distinctly recognizes single-stranded RNA. Previous structural and biochemical studies show that Cas6 captures the 5' end while cleaving the 3' end of the CRISPR RNA. Here, we describe three structures and complementary biochemical analysis of a noncatalytic Cas6 homolog from Pyrococcus horikoshii bound to CRISPR repeat RNA of different sequences. Our study confirms the specificity of the Cas6 protein for single-stranded RNA and further reveals the importance of the bases at Positions 5-7 in Cas6-RNA interactions. Substitutions of these bases result in structural changes in the protein-RNA complex including its oligomerization state.

RNAi: prokaryotes get in on the act.

The small CRISPR-derived RNAs of bacteria and archaea provide adaptive immunity by targeting the DNA of invading viruses and plasmids. Hale et al. (2009) now report on a new variant CRISPR/Cas complex in the archaeon Pyrococcus furiosus that uses guide RNAs to specifically target and cleave RNA not DNA.

RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex.

Compelling evidence indicates that the CRISPR-Cas system protects prokaryotes from viruses and other potential genome invaders. This adaptive prokaryotic immune system arises from the clustered regularly interspaced short palindromic repeats (CRISPRs) found in prokaryotic genomes, which harbor short invader-derived sequences, and the CRISPR-associated (Cas) protein-coding genes. Here, we have identified a CRISPR-Cas effector complex that is comprised of small invader-targeting RNAs from the CRISPR loci (termed prokaryotic silencing (psi)RNAs) and the RAMP module (or Cmr) Cas proteins. The psiRNA-Cmr protein complexes cleave complementary target RNAs at a fixed distance from the 3' end of the integral psiRNAs. In Pyrococcus furiosus, psiRNAs occur in two size forms that share a common 5' sequence tag but have distinct 3' ends that direct cleavage of a given target RNA at two distinct sites. Our results indicate that prokaryotes possess a unique RNA silencing system that functions by homology-dependent cleavage of invader RNAs.