Characterization of the CRISPR/Cas Subtype I-A System of the Hyperthermophilic Crenarchaeon Thermoproteus tenax

Plagens, André; Tjaden, Britta; Hagemann, Anna; Randau, Lennart; Hensel, Reinhard

doi:10.1128/jb.00206-12

Cited by 103 publications

(115 citation statements)

References 86 publications

(111 reference statements)

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“…The first experimental evidence for the existence of native Csa crRNPs came from work with the thermophilic archaeaon Sulfolobus solfataricus, where affinity purifications performed with tagged Csa2 (Cas7) provided evidence for a stable interaction of Csa2 (Cas7) with Cas5a (Cas5) and crRNA with possible additional (less stable) interactions with Csa5 (Cas11 or small subunit protein), Csa4 (Cas8), and Cas6 (Lintner et al 2011b). Similar to our findings, Type I-A (Csa) crRNPs reconstituted with recombinant proteins in Thermoproteus tenax contained Cas3 ′′ and Cas3 ′ as stable components of the complex (Plagens et al 2012(Plagens et al , 2014. The recombinant T. tenax Csa crRNP targets DNA, and Cas3 ′′ (HD-nuclease protein) is the effector nuclease (Plagens et al 2014).…”

Section: Cas Protein Composition and Functional Roles Of Csa And Cst supporting

confidence: 66%

“…1A). While the Csa and Cst complexes have not been extensively studied, Cas5, Cas7, Cas8 (large subunit), and Cas11 (small subunit) superfamily proteins comprise other characterized Type I crRNPs (the latter sometimes fused to the large subunit as a domain) (Brouns et al 2008;Lintner et al 2011b;Wiedenheft et al 2011a,b;Nam et al 2012;Plagens et al 2012;van Duijn et al 2012;Brendel et al 2014). The signature Cas3 (HD nuclease-DExH helicase) proteins of Type I systems interact with the crRNPs and cleave invader DNA (Cady and O'Toole 2011;Westra et al 2012a;Mulepati and Bailey 2013;Sinkunas et al 2013;Hochstrasser et al 2014;Plagens et al 2014).…”

Section: Introductionmentioning

confidence: 99%

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Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus

Majumdar

Zhao

Pfister

et al. 2015

RNA

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CRISPR-Cas immune systems function to defend prokaryotes against potentially harmful mobile genetic elements including viruses and plasmids. The multiple CRISPR-Cas systems (Types I, II, and III) each target destruction of foreign nucleic acids via structurally and functionally diverse effector complexes (crRNPs). CRISPR-Cas effector complexes are comprised of CRISPR RNAs (crRNAs) that contain sequences homologous to the invading nucleic acids and Cas proteins specific to each immune system type. We have previously characterized a crRNP in Pyrococcus furiosus (Pfu) that contains Cmr (Type III-B) Cas proteins associated with one of two size classes of crRNAs and cleaves complementary target RNAs. Here, we have isolated and characterized two additional native Pfu crRNPs containing either Csa (Type I-A) or Cst (Type I-G) Cas proteins and distinct profiles of associated crRNAs. For each complex, the Cas proteins were identified by mass spectrometry and immunoblotting and the crRNAs by RNA sequencing and Northern blot analysis. The crRNAs associated with both the Csa and Cst complexes originate from all seven Pfu CRISPR loci and contain identical 5 ′ ends (8-nt repeat-derived 5 ′ tag sequences) but heterogeneous 3 ′ ends (containing variable amounts of downstream repeat sequences). These crRNA forms are distinct from Cmr-associated crRNAs, indicating different 3 ′ end processing pathways following primary cleavage of common pre-crRNAs. Like other previously characterized Type I CRISPR-Cas effector complexes, we predict that the newly identified Pfu Csa and Cst crRNPs each function to target invading DNA, adding an additional layer of protection beyond that afforded by the previously characterized RNA targeting Cmr complex.

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Section: Cas Protein Composition and Functional Roles Of Csa And Cst supporting

confidence: 66%

Section: Introductionmentioning

confidence: 99%

Three CRISPR-Cas immune effector complexes coexist in Pyrococcus furiosus

Majumdar

Zhao

Pfister

et al. 2015

RNA

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“…In type II systems, the csn2 gene has been shown to be required for spacer acquisition from phages and plasmids (Barrangou et al, 2007;Garneau et al, 2010). Cas4 is also thought to be involved in spacer acquisition (Plagens et al, 2012;Zhang et al, 2012). Nucleotide (BLASTN) and protein (BLASTP) BLAST analyses of the genomes for the presence of an orthologous csn2 or cas4 returned no significant hit, confirming the absence of both of these genes in C. diphtheriae.…”

Section: Crispr-cas Organization In C Diphtheriaementioning

confidence: 77%

“…2a). Csn2 was required for spacer acquisition (Barrangou et al, 2007) and Cas4 is predicted to participate in adaptation (Plagens et al, 2012;Zhang et al, 2012), suggesting that the ability of these strains to acquire new spacers may be compromised. Alternatively, it is possible in theory that this system might still be acquiring new spacers via Cas1-and Cas2-mediated adaptation (Yosef et al, 2012).…”

Section: Discussionmentioning

confidence: 99%

Novel configurations of type I and II CRISPR–Cas systems in Corynebacterium diphtheriae

2013

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Clustered regularly interspaced short palindromic repeats (CRISPRs) are major barriers to recombination through recognition of invading nucleic acids, such as phage and plasmids, and promoting their degredation through the action of CRISPR associated (Cas) proteins. The genomic comparison of 17 Corynebacterium diphtheriae strains led to the identification of three novel CRISPR-Cas system variants, based on the Type II (Type II-C) or type I-E systems. The type II-C system was the most common (11/17 isolates) but it lacked the csn2 and cas4 genes that are involved in spacer acquisition. We also identified that this variant type II-C CRISPR-Cas system is present in other bacteria, and the first system was recently characterized in Neisseria meningitidis. In the remaining isolates, the type II-C system was replaced by a variant of type I-E (I-E-a), where the repeat arrays are inserted between the cas3 and cse1 genes. Three isolates with the type II-C system also possess an additional variant of type I-E (I-E-b), elsewhere in the genome, that exhibits a novel divergent gene organization within the cas operon. The nucleotide sequences of the palindromic repeats and the cas1 gene were phylogenetically incongruent to the core genome. The G+C content of the systems is lower (46.0-49.5 mol%) than the overall DNA G+C content (53 mol%), and they are flanked by mobile genetic elements, providing evidence that they were acquired in three independent horizontal gene transfer events. The majority of spacers lack identity with known phage or plasmid sequences, indicating that there is an unexplored reservoir of corynebacteriophages and plasmids. These novel CRISPR-Cas systems may represent a unique mechanism for spacer acquisitions and defence against invading DNA.

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“…CRISPR and Cas expression is often under the control of cellular regulators [38][39][40] and seems to be inducible by abiotic and/or biotic stress. 38,41,42 Upon transcription, the long CRISPR pre-RNA is processed by other Cas proteins (i.e., Cas6) or by endogenous proteins, to the short mature crRNAs. 11,43,44 A mature crRNA is composed of a spacer derived from the extra-chromosomal genetic element, an 8 nt 5'tag derived from the preceding repeat and a 3'end handle of variable length that stems from the downstream repeat.…”

Section: Introductionmentioning

confidence: 99%