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: 33.61). 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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    • "In type I CRISPR–Cas systems, multiple Cas proteins assemble with a mature crRNA in a large multisubunit complex , termed Cascade, that facilitates recognition of doublestranded DNA (dsDNA) targets (Brouns et al. 2008). Upon target binding, the Cascade complex recruits the type I-specific helicase/exonuclease Cas3 that degrades the target DNA in a processive manner (Brouns et al. 2008; Beloglazova et al. 2011; Sinkunas et al. 2011; Westra et al. 2012). In contrast , type II and type V systems target dsDNA by means of single effector proteins Cas9 and Cpf1, respectively, that function as RNA-guided DNA endonucleases (Deltcheva et al. 2011; Zetsche et al. 2015). "
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    ABSTRACT: Prokaryotic CRISPR-Cas systems provide an RNA-guided mechanism for genome defense against mobile genetic elements such as viruses and plasmids. In type III-A CRISPR-Cas systems, the RNA-guided multisubunit Csm effector complex targets both single-stranded RNAs and double-stranded DNAs. In addition to the Csm complex, efficient anti-plasmid immunity mediated by type III-A systems also requires the CRISPR-associated protein Csm6. Here we report the crystal structure of Csm6 from Thermus thermophilus and show that the protein is a ssRNA-specific endoribonuclease. The structure reveals a dimeric architecture generated by interactions involving the N-terminal CARF and C-terminal HEPN domains. HEPN domain dimerization leads to the formation of a composite ribonuclease active site. Consistently, mutations of invariant active site residues impair catalytic activity in vitro. We further show that the ribonuclease activity of Csm6 is conserved across orthologs, suggesting that it plays an important functional role in CRISPR-Cas systems. The dimer interface of the CARF domains features a conserved electropositive pocket that may function as a ligand-binding site for allosteric control of ribonuclease activity. Altogether, our work suggests that Csm6 proteins provide an auxiliary RNA-targeting interference mechanism in type III-A CRISPR-Cas systems that operates in conjunction with the RNA- and DNA-targeting endonuclease activities of the Csm effector complex.
    Preview · Article · Jan 2016 · RNA
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    • "When foreign DNA is introduced, either by phage infection or plasmid uptake, small fragments of the invasive DNA become integrated within the CRISPR locus as a spacer (Fineran and Charpentier 2012; Nuñez et al. 2014). The primary transcript of the CRISPR locus is processed into multiple unit CRISPR RNAs (crRNAs) (Brouns et al. 2008; Carte et al. 2008). Mature crRNAs each form ribonucleoprotein complexes with associated Cas (CRISPR-associated) proteins , and these complexes then recognize and cleave the foreign nucleic acid that is complementary to the crRNA guide element (Terns and Terns 2011; Westra et al. 2012; Sorek et al. 2013; van der Oost et al. 2014; Jackson and Wiedenheft 2015). "
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    ABSTRACT: 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.
    Full-text · Article · Dec 2015 · RNA
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    • "Type I and type III systems encode Cascade, Csy, and Csm proteins that constitute the multi-subunit effector complexes responsible for target nucleic acid recognition (Brouns and others 2008). The signature gene of type I systems is cas3, a single stranded nickase with 3'-5' exonuclease activity, which is recruited to the target via the CRISPR-associated complex for antiviral defense (Brouns and others 2008; Sinkunas and others 2011). In contrast to other CRISPR system types, type III systems target either or both DNA and RNA, and the signature gene is cas10 (Makarova and others 2011) Estimates indicate that 46% of bacterial and 84% of archaeal genomes contain at least one CRISPR-Cas system (Makarova and others 2011). "
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    ABSTRACT: The on-going CRISPR craze is focused on the use of Cas9-based technologies for genome editing applications in eukaryotes, with high potential for translational medicine and next-generation gene therapy. Nevertheless, CRISPR-Cas systems actually provide adaptive immunity in bacteria, and have much promise for various applications in food bacteria that include high-resolution typing of pathogens, vaccination of starter cultures against phages, and the genesis of programmable and specific antibiotics that can selectively modulate bacterial population composition. Indeed, the molecular machinery from these DNA-encoded, RNA-mediated, DNA-targeting systems can be harnessed in native hosts, or repurposed in engineered systems for a plethora of applications that can be implemented in all organisms relevant to the food chain, including agricultural crops trait-enhancement, livestock breeding, and fermentation-based manufacturing, and for the genesis of next-generation food products with enhanced quality and health-promoting functionalities. CRISPR-based applications are now poised to revolutionize many fields within food science, from farm to fork. In this review, we describe CRISPR-Cas systems and highlight their potential for the development of enhanced foods.
    Full-text · Article · Oct 2015 · Journal of Food Science
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