CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2013-09

AUTHORS

Prashant Mali, John Aach, P Benjamin Stranges, Kevin M Esvelt, Mark Moosburner, Sriram Kosuri, Luhan Yang, George M Church

ABSTRACT

Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a transcriptional activation-based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator-like (TALs) effectors. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1-3 and 1-2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity. More... »

PAGES

833

References to SciGraph publications

  • 2013-03. Synergistic and tunable human gene activation by combinations of synthetic transcription factors in NATURE METHODS
  • 2011-09. Revealing off-target cleavage specificities of zinc-finger nucleases by in vitro selection in NATURE METHODS
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  • 2012-05. FLASH assembly of TALENs for high-throughput genome editing in NATURE BIOTECHNOLOGY
  • 2012-01. A transcription activator-like effector toolbox for genome engineering in NATURE PROTOCOLS
  • 2005-08. Gene targeting using zinc finger nucleases in NATURE BIOTECHNOLOGY
  • 2011-03. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III in NATURE
  • 2012-03. Massively parallel functional dissection of mammalian enhancers in vivo in NATURE BIOTECHNOLOGY
  • 2011-02. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription in NATURE BIOTECHNOLOGY
  • 2013-03. Efficient genome editing in zebrafish using a CRISPR-Cas system in NATURE BIOTECHNOLOGY
  • 2011-09. An unbiased genome-wide analysis of zinc-finger nuclease specificity in NATURE BIOTECHNOLOGY
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  • 2011-08. Tracking genome engineering outcome at individual DNA breakpoints in NATURE METHODS
  • Journal

    TITLE

    Nature Biotechnology

    ISSUE

    9

    VOLUME

    31

    Author Affiliations

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  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1038/nbt.2675

    DOI

    http://dx.doi.org/10.1038/nbt.2675

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1041659759

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/23907171


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