Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification View Full Text


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Article Info

DATE

2014-06

AUTHORS

John P Guilinger, David B Thompson, David R Liu

ABSTRACT

Genome editing by Cas9, which cleaves double-stranded DNA at a sequence programmed by a short single-guide RNA (sgRNA), can result in off-target DNA modification that may be detrimental in some applications. To improve DNA cleavage specificity, we generated fusions of catalytically inactive Cas9 and FokI nuclease (fCas9). DNA cleavage by fCas9 requires association of two fCas9 monomers that simultaneously bind target sites ∼15 or 25 base pairs apart. In human cells, fCas9 modified target DNA sites with >140-fold higher specificity than wild-type Cas9 and with an efficiency similar to that of paired Cas9 'nickases', recently engineered variants that cleave only one DNA strand per monomer. The specificity of fCas9 was at least fourfold higher than that of paired nickases at loci with highly similar off-target sites. Target sites that conform to the substrate requirements of fCas9 occur on average every 34 bp in the human genome, suggesting the versatility of this approach for highly specific genome-wide editing. More... »

PAGES

577-582

References to SciGraph publications

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  • 2008-07. Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases in NATURE BIOTECHNOLOGY
  • 2014-03. Improving CRISPR-Cas nuclease specificity using truncated guide RNAs in NATURE BIOTECHNOLOGY
  • 2013-11. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing in NATURE METHODS
  • 2011-01. Enhancing zinc-finger-nuclease activity with improved obligate heterodimeric architectures in NATURE METHODS
  • 2011-09. Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection in NATURE METHODS
  • 2009-12. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner in NATURE BIOTECHNOLOGY
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  • Journal

    TITLE

    Nature Biotechnology

    ISSUE

    6

    VOLUME

    32

    Author Affiliations

    From Grant

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  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1038/nbt.2909

    DOI

    http://dx.doi.org/10.1038/nbt.2909

    DIMENSIONS

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    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/24770324


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