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


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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    • "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.
    Plant physiology 08/2015; DOI:10.1104/pp.15.00636 · 6.84 Impact Factor
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    • "In the Cascade context, the I-E Cas6 is delivering the mature crRNA and tightly caps the 3 handle (Sashital, Jinek and Doudna 2011; Jackson et al. 2014a). This protection results in crRNAs with a complete 3 handle, which was also shown for type I-F (Haurwitz et al. 2010). In contrast, crRNAs from other type I (I-A, I-B, I-D) systems and all type III systems were shown to harbor trimmed 3 ends. "
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    ABSTRACT: The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) adaptive immune systems use small guide RNAs, the CRISPR RNAs (crRNAs), to mark foreign genetic material, e.g. viral nucleic acids, for degradation. Archaea and bacteria encode a large variety of Cas proteins that bind crRNA molecules and build active ribonucleoprotein surveillance complexes. The evolution of CRISPR-Cas systems has resulted in a diversification of cas genes and a classification of the systems into three types and additional subtypes characterized by distinct surveillance and interfering complexes. Recent crystallographic and biochemical advances have revealed detailed insights into the assembly and DNA/RNA targeting mechanisms of the various complexes. Here, we review our knowledge on the molecular mechanism involved in the DNA and RNA interference stages of type I (Cascade: CRISPR-associated complex for antiviral defense), type II (Cas9) and type III (Csm, Cmr) CRISPR-Cas systems. We further highlight recently reported structural and mechanistic themes shared among these systems. © FEMS 2015.
    FEMS microbiology reviews 04/2015; 39(3). DOI:10.1093/femsre/fuv019 · 13.24 Impact Factor
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    • "One or several spacer arrays in a prokaryotic cell can be transcribed and processed into small CRISPR RNA (crRNA) molecules by a complex of several Cas proteins known as Cascade. The Cascade complex then mediates the formation of a duplex between the crRNA and the cognate protospacer sequence in an invading nucleic acid molecule triggering degradation of the latter (Haurwitz et al., 2010; Semenova et al., 2011). The fact that the CRISPR–Cas systems are able to continuously acquire new spacers enables partial reconstruction of the history of past selfish-element infections (Tyson and Banfield, 2008; Denef et al., 2010; Held et al., 2010; Stern et al., 2012; Weinberger et al., 2012a). "
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    ABSTRACT: The CRISPR (clustered, regularly, interspaced, short, palindromic repeats)-Cas (CRISPR-associated genes) systems of archaea and bacteria provide adaptive immunity against viruses and other selfish elements and are believed to curtail horizontal gene transfer (HGT). Limiting acquisition of new genetic material could be one of the sources of the fitness cost of CRISPR-Cas maintenance and one of the causes of the patchy distribution of CRISPR-Cas among bacteria, and across environments. We sought to test the hypothesis that the activity of CRISPR-Cas in microbes is negatively correlated with the extent of recent HGT. Using three independent measures of HGT, we found no significant dependence between the length of CRISPR arrays, which reflects the activity of the immune system, and the estimated number of recent HGT events. In contrast, we observed a significant negative dependence between the estimated extent of HGT and growth temperature of microbes, which could be explained by the lower genetic diversity in hotter environments. We hypothesize that the relevant events in the evolution of resistance to mobile elements and proclivity for HGT, to which CRISPR-Cas systems seem to substantially contribute, occur on the population scale rather than on the timescale of species evolution.The ISME Journal advance online publication, 24 February 2015; doi:10.1038/ismej.2015.20.
    The ISME Journal 02/2015; 9(9). DOI:10.1038/ismej.2015.20 · 9.30 Impact Factor
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