Sequence- and structure-specific RNA processing by a CRISPR endonuclease

Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
Science (Impact Factor: 31.48). 09/2010; 329(5997):1355-8. DOI: 10.1126/science.1192272
Source: PubMed

ABSTRACT Many bacteria and archaea contain clustered regularly interspaced short palindromic repeats (CRISPRs) that confer resistance to invasive genetic elements. Central to this immune system is the production of CRISPR-derived RNAs (crRNAs) after transcription of the CRISPR locus. Here, we identify the endoribonuclease (Csy4) responsible for CRISPR transcript (pre-crRNA) processing in Pseudomonas aeruginosa. A 1.8 angstrom crystal structure of Csy4 bound to its cognate RNA reveals that Csy4 makes sequence-specific interactions in the major groove of the crRNA repeat stem-loop. Together with electrostatic contacts to the phosphate backbone, these enable Csy4 to bind selectively and cleave pre-crRNAs using phylogenetically conserved serine and histidine residues in the active site. The RNA recognition mechanism identified here explains sequence- and structure-specific processing by a large family of CRISPR-specific endoribonucleases.

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    • "Some of the RAMPs have been shown to possess sequence-or structure-specific RNAse activity that is involved in the processing of precrRNA transcripts (Brouns et al., 2008; Hale et al., 2009). The crystal structures of several RAMPs have been solved and indicate that they contain one or two domains which display distinct versions of the RNA recognition motif (RRM) or ferredoxin fold (Lintner et al., 2011; Wang et al., 2011; Haurwitz et al., 2010). The RNA-binding RAMP domain is present in the Cas5, Cas6, Cas7 and Cmr3 protein families and RAMP-like domains are found in Cas2 and Cas10 (Reeks et al., 2013; Makarova et al., 2011b). "
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    ABSTRACT: Approximately all sequenced archaeal and half of eubacterial genomes have some sort of adaptive immune system, which enables them to target and cleave invading foreign genetic elements by an RNAi-like pathway. CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) systems consist of the CRISPR loci with multiple copies of a short repeat sequence separated by variable sequences with similar size that are derived from invaders and cas genes encode proteins involved in RNA binding, endo-and exo-nucleases, helicases, and polymerases activities. There are three main types (I, II and III) of CRISPR/Cas systems. All systems function in three distinct stages: (1) adaptation, (2) crRNA biogenesis, and (3) interference. This review focuses on the features and mechanisms of the CRISPR-Cas systems and current finding about them.
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    • "This technology has been successfully used in many plant species to generate targeted mutants (Osakabe et al., 2010; Shan et al., 2013a; Shukla et al., 2009). Similarly, a recent method called clustered regularly interspaced short palindromic repeats (CRISPRs) generate DNA mutation using precursor RNA guided mechanism to edit genomic sequences (Haurwitz et al., 2010; Liu et al., 2013). The CRISPR-Cas9 system is best characterized between the different types of CRISPRs, and successful examples have been reported in tobacco, Arabidopsis and rice (Chen and Gao, 2014; Li et al., 2013; Shan et al., 2013b). "
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    ABSTRACT: Fine-tuning plant cell wall properties to render plant biomass more amenable to biofuel conversion is a colossal challenge. A deep knowledge of the biosynthesis and regulation of plant cell wall and a high-precision genome engineering toolset are the two essential pillars of efforts to alter plant cell walls and reduce biomass recalcitrance. The past decade has seen a meteoric rise in use of transcriptomics and high-resolution imaging methods resulting in fresh insights into composition, structure, formation and deconstruction of plant cell walls. Subsequent gene manipulation approaches, however, commonly include ubiquitous mis-expression of a single candidate gene in a host that carries an intact copy of the native gene. The challenges posed by pleiotropic and unintended changes resulting from such an approach are moving the field towards synthetic biology approaches. Synthetic biology builds on a systems biology knowledge base and leverages high-precision tools for high-throughput assembly of multigene constructs and pathways, precision genome editing and site-specific gene stacking, silencing and/or removal. Here, we summarize the recent breakthroughs in biosynthesis and remodelling of major secondary cell wall components, assess the impediments in obtaining a systems-level understanding and explore the potential opportunities in leveraging synthetic biology approaches to reduce biomass recalcitrance.
    Plant Biotechnology Journal 11/2014; 12(9). DOI:10.1111/pbi.12283 · 5.68 Impact Factor
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    • "Therefore, prokaryotes harboring the CRISPR–Cas system memorize the previous attacks of foreign genetic elements, by incorporating invader-derived sequences into CRISPR loci for protection against subsequent invasions [8]. Upon infection, the CRISPR arrays are transcribed into long precursor CRISPR RNAs (crRNAs), which are then processed within each repeat sequence to generate crRNAs by the dedicated Cas endoribonucleases [9] [10] [11] [12]. Subsequently , in a process known as interference, the mature crRNAs associate with Cas proteins to form ribonucleoprotein interference complexes that degrade the foreign genetic elements [9] [13] [14]. "
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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.
    Journal of Molecular Biology 10/2014; 427(2). DOI:10.1016/j.jmb.2014.09.029 · 4.33 Impact Factor
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