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  1. Article: Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes

    Ma, Enbo / Jennifer A. Doudna / Kaihong Zhou / Lucas B. Harrington / Mitchell R. O’Connell

    Molecular cell. 2015 Nov. 05, v. 60

    2015  

    Abstract: Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9-guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome ... ...

    Abstract Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9-guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family.
    Keywords bacteria ; DNA damage ; enzymes ; genetic engineering ; genome ; immune system ; nucleotide sequences ; proteins ; RNA ; single-stranded DNA
    Language English
    Dates of publication 2015-1105
    Size p. 398-407.
    Publishing place Elsevier Inc.
    Document type Article
    ZDB-ID 1415236-8
    ISSN 1097-4164 ; 1097-2765
    ISSN (online) 1097-4164
    ISSN 1097-2765
    DOI 10.1016/j.molcel.2015.10.030
    Database NAL-Catalogue (AGRICOLA)

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    In stock of ZB MED Bonn / Germany

    Z 2005/501: Show issues

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  2. Article: Synthesis of the Bacteriocin Glycopeptide Sublancin 168 and S-Glycosylated Variants

    Hsieh, Yves S. Y / Wilkinson Brendan L / O’Connell Mitchell R / Mackay Joel P / Matthews Jacqueline M / Payne Richard J

    Organic letters. 2012 Apr. 06, v. 14, no. 7

    2012  

    Abstract: The synthesis of sublancin 168, a unique S-glucosylated bacteriocin antibiotic, is described. The natural product and two S-glycosylated variants were successfully prepared via native chemical ligation followed by folding. The synthetic glycopeptides ... ...

    Abstract The synthesis of sublancin 168, a unique S-glucosylated bacteriocin antibiotic, is described. The natural product and two S-glycosylated variants were successfully prepared via native chemical ligation followed by folding. The synthetic glycopeptides were shown to possess primarily an α-helical secondary structure by CD and NMR studies.
    Keywords antibiotics ; bacteriocins ; chemical reactions ; chemical structure ; glycopeptides ; nuclear magnetic resonance spectroscopy ; organic compounds
    Language English
    Dates of publication 2012-0406
    Size p. 1910-1913.
    Publishing place American Chemical Society
    Document type Article
    ISSN 1523-7052
    DOI 10.1021%2Fol300557g
    Database NAL-Catalogue (AGRICOLA)

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  3. Article: Programmable RNA Tracking in Live Cells with CRISPR/Cas9

    Nelles, David A / Mark Y. Fang / Mitchell R. O’Connell / Jia L. Xu / Sebastian J. Markmiller / Jennifer A. Doudna / Gene W. Yeo

    Cell. 2016 Apr. 07, v. 165

    2016  

    Abstract: RNA-programmed genome editing using CRISPR/Cas9 from Streptococcus pyogenes has enabled rapid and accessible alteration of specific genomic loci in many organisms. A flexible means to target RNA would allow alteration and imaging of endogenous RNA ... ...

    Abstract RNA-programmed genome editing using CRISPR/Cas9 from Streptococcus pyogenes has enabled rapid and accessible alteration of specific genomic loci in many organisms. A flexible means to target RNA would allow alteration and imaging of endogenous RNA transcripts analogous to CRISPR/Cas-based genomic tools, but most RNA targeting methods rely on incorporation of exogenous tags. Here, we demonstrate that nuclease-inactive S. pyogenes CRISPR/Cas9 can bind RNA in a nucleic-acid-programmed manner and allow endogenous RNA tracking in living cells. We show that nuclear-localized RNA-targeting Cas9 (RCas9) is exported to the cytoplasm only in the presence of sgRNAs targeting mRNA and observe accumulation of ACTB, CCNA2, and TFRC mRNAs in RNA granules that correlate with fluorescence in situ hybridization. We also demonstrate time-resolved measurements of ACTB mRNA trafficking to stress granules. Our results establish RCas9 as a means to track RNA in living cells in a programmable manner without genetically encoded tags.[Display omitted]
    Keywords Streptococcus pyogenes ; cytoplasmic granules ; fluorescence in situ hybridization ; genome ; genomics ; granules ; image analysis ; loci ; messenger RNA
    Language English
    Dates of publication 2016-0407
    Size p. 488-496.
    Publishing place Elsevier Inc.
    Document type Article
    ZDB-ID 187009-9
    ISSN 1097-4172 ; 0092-8674
    ISSN (online) 1097-4172
    ISSN 0092-8674
    DOI 10.1016/j.cell.2016.02.054
    Database NAL-Catalogue (AGRICOLA)

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    In stock of ZB MED Bonn / Germany

    Z 75/702: Show issues
    Z 93: Show issues

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    This service is chargeable due to the Delivery terms set by subito. Orders including an article and supplementary material will be classified as separate orders. In these cases, fees will be demanded for each order.

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