Article

Small CRISPR RNAs guide antiviral defense in prokaryotes. Science

Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Dreijenplein 10, 6703 HB Wageningen, Netherlands.
Science (Impact Factor: 31.48). 09/2008; 321(5891):960-4. DOI: 10.1126/science.1159689
Source: PubMed

ABSTRACT Prokaryotes acquire virus resistance by integrating short fragments of viral nucleic acid into clusters of regularly interspaced short palindromic repeats (CRISPRs). Here we show how virus-derived sequences contained in CRISPRs are used by CRISPR-associated (Cas) proteins from the host to mediate an antiviral response that counteracts infection. After transcription of the CRISPR, a complex of Cas proteins termed Cascade cleaves a CRISPR RNA precursor in each repeat and retains the cleavage products containing the virus-derived sequence. Assisted by the helicase Cas3, these mature CRISPR RNAs then serve as small guide RNAs that enable Cascade to interfere with virus proliferation. Our results demonstrate that the formation of mature guide RNAs by the CRISPR RNA endonuclease subunit of Cascade is a mechanistic requirement for antiviral defense.

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Available from: Bram Snijders, Nov 25, 2014
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    • "Following transcription and subsequent cleavage of the pre-crRNA within the repeats, short crRNAs are produced, each carrying a single spacer (Brouns et al., 2008; Carte et al., 2008). crRNAs together with Cas proteins are then combined into effector complexes that are multimeric for type I and III CRISPR-Cas systems (Brouns et al., 2008; Sinkunas et al., 2013; Zhang et al., 2012; Tamulaitis et al., 2014) or monomeric for type II systems (Gasiunas et al., 2012). Type I and II effector complexes recognize putative DNA targets by establishing base-specific pairing between the crRNA guide and the target sequence (called the ''protospacer''), forming a so-called R-loop where the crRNA forms a heteroduplex with the complementary DNA strand while the non-targeting strand is displaced. "
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    ABSTRACT: CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against foreign nucleic acids. In type I CRISPR-Cas systems, invading DNA is detected by a large ribonucleoprotein surveillance complex called Cascade. The crRNA component of Cascade is used to recognize target sites in foreign DNA (protospacers) by formation of an R-loop driven by base-pairing complementarity. Using single-molecule supercoiling experiments with near base-pair resolution, we probe here the mechanism of R-loop formation and detect short-lived R-loop intermediates on off-target sites bearing single mismatches. We show that R-loops propagate directionally starting from the protospacer-adjacent motif (PAM). Upon reaching a mismatch, R-loop propagation stalls and collapses in a length-dependent manner. This unambiguously demonstrates that directional zipping of the R-loop accomplishes efficient target recognition by rapidly rejecting binding to off-target sites with PAM-proximal mutations. R-loops that reach the protospacer end become locked to license DNA degradation by the auxiliary Cas3 nuclease/helicase without further target verification. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 03/2015; 10(9). DOI:10.1016/j.celrep.2015.01.067 · 8.36 Impact Factor
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    • "Two years later, CRISPR arrays were confirmed to provide protection against invading viruses when combined with Cas genes (Barrangou et al., 2007). The mechanism of this immune system based on RNA-mediated DNA targeting was demonstrated shortly thereafter (Brouns et al., 2008; Deltcheva et al., 2011; Garneau et al., 2010; Marraffini and Sontheimer, 2008). "
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    ABSTRACT: Targeted genome editing using artificial nucleases has the potential to accelerate basic research as well as plant breeding by providing the means to modify genomes rapidly in a precise and predictable manner. Here we describe the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, a recently-developed tool for the introduction of site-specific double stranded DNA breaks. We highlight the strengths and weaknesses of this technology compared with two well-established genome editing platforms: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). We summarize recent results obtained in plants using CRISPR/Cas9 technology, discuss possible applications in plant breeding and consider potential future developments. Copyright © 2014. Published by Elsevier Inc.
    Biotechnology Advances 12/2014; 33(1). DOI:10.1016/j.biotechadv.2014.12.006 · 8.91 Impact Factor
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    • "deviation of 3.13 Å (190 aligned C α atoms) (Fig. 2d). The structural similarity of these proteins is consistent with the fact that both Csm4 and Cmr3 are orthologs of the Cas5 protein [32], a constituent of the Cascade complex in the type I CRISPR–Cas system [9]. MjCsm3 is a single domain protein that structurally resembles a ferredoxin-like fold, with a four-stranded antiparallel β-sheet buttressed on one side by several α-helices (Figs. "
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    ABSTRACT: Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci play a pivotal role in the prokaryotic host defense system against invading genetic materials. The CRISPR loci are transcribed to produce CRISPR RNAs (crRNAs), which form interference complexes with CRISPR-associated (Cas) proteins to target the invading nucleic acid for degradation. The interference complex of the type III-A CRISPR-Cas system is composed of five Cas proteins (Csm1-Csm5) and a crRNA, and targets invading DNA. Here, we show that the Csm1, Csm3, and Csm4 proteins from Methanocaldococcus jannaschii form a stable subcomplex. We also report the crystal structure of the M. jannaschii Csm3-Csm4 subcomplex at 3.1 Å resolution. The complex structure revealed the presence of a basic concave surface around their interface, suggesting the RNA and/or DNA binding ability of the complex. A gel retardation analysis showed that the Csm3-Csm4 complex binds single-stranded RNA in a non-sequence-specific manner. Csm4 structurally resembles Cmr3, a component of the type III-B CRISPR-Cas interference complex. Based on bioinformatics, we constructed a model structure of the Csm1-Csm4-Csm3 ternary complex, which provides insights into its role in the Csm interference complex.
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