Article

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: 33.61). 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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    • "These RNA-guided genome defense systems typically consist of an array of short repeats intercalated with invader-derived spacer sequences, and an operon containing several CRISPR-associated (cas) genes encoding the molecular machinery involved in spacer acquisition, guide RNA processing, and target interference. Transcription of CRISPR spacer-repeat arrays and subsequent processing of the precursor transcripts yields individual CRISPR RNAs (crRNAs) (Brouns et al. 2008; Carte et al. 2008; Hale et al. 2008; Haurwitz et al. 2010). These crRNA guides in turn associate with Cas proteins in effector complexes in which they mediate target detection by Watson–Crick base-pairing interactions (Brouns et al. 2008; Hale et al. 2009; Jore et al. 2011). "
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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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    • ". This " memory system " can destroy DNA or RNA if reinfection occurs in the same bacteria or in its descendants [14] [15] [16] [17] [18] [19]. Three types of CRISPR loci exist, all of which acquire short pieces of DNA called spacers from foreign DNA elements [20]. "
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    ABSTRACT: Genetic manipulation is a powerful tool to establish the causal relationship between a genetic lesion and a particular pathological phenotype. The rise of CRISPR/Cas9 genome-engineering tools overcame the traditional technical bottleneck for routine site-specific genetic manipulation in cells. To create the perfect in vitro cell model, there is significant interest from the stem cell research community to adopt this fast evolving technology. This review addresses this need directly by providing both the up-to-date biochemical rationale of CRISPR-mediated genome engineering and detailed practical guidelines for the design and execution of CRISPR experiments in cell models. Ultimately, this review will serve as a timely and comprehensive guide for this fast developing technology.
    Full-text · Article · Dec 2015 · Stem cell International
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    • "A critical aspect of designing multiplex CRISPR/Cas9 experiments is deciding which strategy to 392 use for expressing multiple gRNAs simultaneously. Different strategies have been explored 393 such as a self-processing ribozyme system (Gao and Zhao, 2014), a tRNA-processing system 394 (Xie et al., 2015) or the Csy4 RNase system (Haurwitz et al., 2010; Tsai et al., 2014). However, 395 the most popular approach uses small RNA promoters such as U6 or U3. "
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    ABSTRACT: The relative ease, speed and biological scope of CRISPR/Cas9-based reagents for genomic manipulations are revolutionizing virtually all areas of molecular biosciences, including functional genomics, genetics, applied biomedical research and agricultural biotechnology. In plant systems, however, a number of hurdles currently exist that limit this technology from reaching its full potential. For example, significant plant molecular biology expertise and effort is still required to generate functional expression constructs that allow simultaneous editing, and especially transcriptional regulation, of multiple different genomic loci or "multiplexing", which is a significant advantage of CRISPR/Cas9 versus other genome editing systems. In order to streamline and facilitate rapid and wide-scale use of CRISPR/Cas9-based technologies for plant research, we developed and implemented a comprehensive molecular toolbox for multifaceted CRISPR/Cas9 applications in plants. This toolbox provides researchers with a protocol and reagents to quickly and efficiently assemble functional CRISPR/Cas9 T-DNA constructs for monocots and dicots using Golden Gate and Gateway cloning methods. It comes with a full suite of capabilities, including multiplexed gene editing and transcriptional activation or repression of plant endogenous genes. We report the functionality and effectiveness of this toolbox in model plants such as tobacco, Arabidopsis and rice, demonstrating its utility for basic and applied plant research. Copyright © 2015, Plant Physiology.
    Full-text · Article · Aug 2015 · Plant physiology
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