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    Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering

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    Type
    Article
    Authors
    Aouida, Mustapha cc
    Eid, Ayman
    Ali, Zahir cc
    Cradick, Thomas
    Lee, Ciaran
    Deshmukh, Harshavardhan
    Ahmed, Atef
    Abu Samra, Dina Bashir Kamil cc
    Gadhoum, Samah
    Merzaban, Jasmeen cc
    Bao, Gang
    Mahfouz, Magdy M. cc
    KAUST Department
    Biological and Environmental Sciences and Engineering (BESE) Division
    Bioscience Program
    Desert Agriculture Initiative
    KAUST Supercomputing Laboratory (KSL)
    Laboratory for Genome Engineering
    Laboratory of Cell Signaling and Migration
    Plant Science
    Plant Science Program
    Date
    2015-07-30
    Permanent link to this record
    http://hdl.handle.net/10754/579491
    
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    Abstract
    The Cas9 endonuclease is used for genome editing applications in diverse eukaryotic species. A high frequency of off-target activity has been reported in many cell types, limiting its applications to genome engineering, especially in genomic medicine. Here, we generated a synthetic chimeric protein between the catalytic domain of the FokI endonuclease and the catalytically inactive Cas9 protein (fdCas9). A pair of guide RNAs (gRNAs) that bind to sense and antisense strands with a defined spacer sequence range can be used to form a catalytically active dimeric fdCas9 protein and generate double-strand breaks (DSBs) within the spacer sequence. Our data demonstrate an improved catalytic activity of the fdCas9 endonuclease, with a spacer range of 15–39 nucleotides, on surrogate reporters and genomic targets. Furthermore, we observed no detectable fdCas9 activity at known Cas9 off-target sites. Taken together, our data suggest that the fdCas9 endonuclease variant is a superior platform for genome editing applications in eukaryotic systems including mammalian cells.
    Citation
    Efficient fdCas9 Synthetic Endonuclease with Improved Specificity for Precise Genome Engineering 2015, 10 (7):e0133373 PLOS ONE
    Publisher
    Public Library of Science (PLoS)
    Journal
    PLoS ONE
    DOI
    10.1371/journal.pone.0133373
    PubMed ID
    26225561
    Additional Links
    http://dx.plos.org/10.1371/journal.pone.0133373
    ae974a485f413a2113503eed53cd6c53
    10.1371/journal.pone.0133373
    Scopus Count
    Collections
    Articles; Biological and Environmental Sciences and Engineering (BESE) Division; Bioscience Program; KAUST Supercomputing Laboratory (KSL); Plant Science Program; Desert Agriculture Initiative

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