Three CRISPR-Cas immune effector complexes coexist in <i>Pyrococcus furiosus</i>

Majumdar, Sonali; Zhao, Peng; Pfister, Neil T.; Compton, Mark M.; Olson, Sara; Glover, Claiborne V.C.; Wells, Lance; Graveley, Brenton R.; Terns, Rebecca M.; Terns, Michael P.

doi:10.1261/rna.049130.114

Cited by 51 publications

(73 citation statements)

References 76 publications

(137 reference statements)

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“…S1). The effector complexes in P. furiosus use crRNAs produced from seven shared CRISPR loci (Majumdar et al 2015). Using deletion strains containing a single CRISPRCas system, we found that the P. furiosus Type I-A Csa and Type I-G Cst systems silence plasmid DNA invaders in a PAM-dependent manner (Elmore et al 2015).…”

Section: Resultsmentioning

confidence: 97%

Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system

et al. 2016

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CRISPR-Cas systems eliminate nucleic acid invaders in bacteria and archaea. The effector complex of the Type III-B Cmr system cleaves invader RNAs recognized by the CRISPR RNA (crRNA ) of the complex. Here we show that invader RNAs also activate the Cmr complex to cleave DNA. As has been observed for other Type III systems, Cmr eliminates plasmid invaders in Pyrococcus furiosus by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Notably, we found that the target RNA per se induces DNA cleavage by the Cmr complex in vitro. DNA cleavage activity does not depend on cleavage of the target RNA but notably does require the presence of a short sequence adjacent to the target sequence within the activating target RNA (rPAM [RNA protospacer-adjacent motif]). The activated complex does not require a target sequence (or a PAM) in the DNA substrate. Plasmid elimination by the P. furiosus Cmr system also does not require the Csx1 (

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Section: Resultsmentioning

confidence: 97%

Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system

et al. 2016

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“…Some systems co-existing in the same species have been demonstrated to share the same set of guides, e.g. Type III-A (Csm) and Type III-B (Cmr) of Thermus thermophilus [83], Type III-B (Cmr), Type I-A (Csa) and Type I-G (Cst) of Pyrococcus furiosus [84]. Given that the Type III loci usually lack cas6 genes, a single stand-alone Cas6 nuclease is likely to be responsible for the supply of crRNAs to the Type III complexes in T. thermophilus [83].…”

Section: Biogenesis Of Crrnasmentioning

confidence: 99%

“…Given that the Type III loci usually lack cas6 genes, a single stand-alone Cas6 nuclease is likely to be responsible for the supply of crRNAs to the Type III complexes in T. thermophilus [83]. In P. furiosus, Cas6 nuclease of Type I generates the crRNAs from all CRISPR loci for the different co-existing complexes [84]. Cas6-based processing of pre-crRNA in Type III systems is typically followed by a sequence-unspecific trimming at the 3' end (by yet to be identified RNases) to yield mature crRNAs with a defined 8-nt 5' end and a variable 3' end [34,85,86].…”

Section: Biogenesis Of Crrnasmentioning

confidence: 99%

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

Mohanraju

Makarova

Zetsche

et al. 2016

Science

545

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Background: Prokaryotes have evolved multiple systems to combat invaders such as viruses and plasmids. Examples of such defence systems include receptor masking, restriction-modification (R-M systems), DNA interference (Argonaute), bacteriophage exclusion (BREX or PGL) and abortive infection, all of which act in an innate, non-specific manner. In addition, prokaryotes have evolved adaptive, heritable immune systems, i.e. clustered regularly interspaced palindromic repeats (CRISPR) and the CRISPR-associated proteins (CRISPR-Cas). Adaptive immunity is conferred by the integration of DNA sequences from an invading element into the CRISPR array (adaptation), which is transcribed into long pre-CRISPR (pre-cr) RNAs and processed into short crRNAs (expression), which guide Cas proteins to specifically degrade the cognate DNA on subsequent exposures (interference). Advances:A plethora of distinct CRISPR-Cas systems are represented in genomes of most archaea and almost half of the bacteria. The latest CRISPR-Cas classification scheme delineates two classes that are each subdivided into three types. Integration of biochemistry and molecular genetics has contributed significantly to revealing many of the unique features of the variant CRISPR-Cas types. Additionally, structural analysis and single molecule studies have further advanced our understanding of the molecular basis of CRISPR-Cas functionality. Recent progress includes relevant steps in the adaptation stage, when fragments of foreign DNA are processed and incorporated as new spacers into the CRISPR array. In addition, three novel CRISPR-Cas types (IV, V, and VI) have been identified, and in particular, the type V interference complexes have been experimentally characterized. Moreover, the ability to easily program sequence-specific DNA targeting and cleavage by CRISPRCas components, as demonstrated for Cas9 and Cpf1, allows for the application of CRISPR-Cas components as highly effective tools for genetic engineering and gene regulation in a wide range of eukaryotes and prokaryotes.The pressing issue of off-target cleavage by the Cas9 nuclease is being actively addressed using structure-guided engineering.Outlook: Although our understanding of the CRISPR-Cas system has increased tremendously over the past few years, much remains to be done. About the evolution of CRISPR-Cas systems, the continuing discovery of novel CRISPR-Cas variants will provide direct tests of the recently proposed modular scenario. The recent discovery and characterization of new CRISPR-Cas types with many unique features implies that our current knowledge has relatively limited power for predicting the functional details of distantly related variants. Hence, newly discovered CRISPR-Cas systems need to be thoroughly dissected with the aforementioned multi-disciplinary approaches to gain insight in their biological role, to unravel their molecular mechanism, and to harness their potential for biotechnology. As to the biology, key outstanding questions include the ecological roles of microbial ...

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“…The hyperthermophilic archaeon Pyrococcus furiosus (Pfu) harbors three coexisting immune effector crRNP complexes: Type I-A (Csa), Type I-G (Cst), and Type III-B (Cmr), along with seven functional CRISPR loci (Hale et al 2008;Terns and Terns 2013;Majumdar et al 2015). There is evidence that the Pfu Csa and Cst effector complexes target DNA (Elmore et al 2015), while the Cmr complex has been shown to target DNA and RNA in vitro and in vivo (Hale et al 2009(Hale et al , 2012Deng et al 2013;Spilman et al 2013;Benda et al 2014;Ramia et al 2014;J Elmore, N Sheppard, R Terns, and M Terns, unpubl.…”

Section: Introductionmentioning

confidence: 99%

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

Sheppard

Glover

Terns

et al. 2015

RNA

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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.

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Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus

Cited by 51 publications

References 76 publications

Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system

Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system

Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems

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

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