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


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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    • "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.
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    • "Acquisition occurs via molecular 'sampling' of foreign DNA, from which short sequences termed spacers are integrated in a polarized manner at the leader end of the CRISPR array (Barrangou et al., 2007). CRISPR arrays are transcribed constitutively and inducibly, directed by promoter elements in the preceding leader sequence during expression (Brouns et al., 2008; Young et al., 2012). The transcript is processed selectively at each repeat sequence, forming small interfering CRISPR RNAs (crRNAs) that function to guide Cas proteins. "
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    ABSTRACT: Bacteria encode clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes, which collectively form an RNA-guided adaptive immune system against invasive genetic elements. In silico surveys have revealed that lactic acid bacteria harbor a prolific and diverse set of CRISPR-Cas systems. Thus, the natural evolutionary role of CRISPR-Cas systems may be investigated in these ecologically, industrially, scientifically, and medically important microbes. In this study, 17 L. gasseri strains were investigated and 6 harbored a Type II-A CRISPR-Cas system, with considerable diversity in array size and spacer content. Several of the spacers showed similarity to phage and plasmid sequences, which are typical targets of CRISPR-Cas immune systems. Aligning the protospacers facilitated inference of the Protospacer Adjacent Motif (PAM) sequence, determined to be 5'-NTAA-3' flanking the 3'end of protospacer. The system in L. gasseri JV-V03 and NCK1342, interfered with transforming plasmids containing sequences matching the most recently acquired CRISPR spacers in each strain. We report the distribution and function of a native Type II-A CRISPR-Cas system in the commensal species, L. gasseri. Collectively, these results open avenues for applications for bacteriophage protection and genome modification in L. gasseri and contribute to the fundamental understanding of CRISPR-Cas systems in bacteria.
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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.
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