High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2013-09

AUTHORS

Vikram Pattanayak, Steven Lin, John P Guilinger, Enbo Ma, Jennifer A Doudna, David R Liu

ABSTRACT

The RNA-programmable Cas9 endonuclease cleaves double-stranded DNA at sites complementary to a 20-base-pair guide RNA. The Cas9 system has been used to modify genomes in multiple cells and organisms, demonstrating its potential as a facile genome-engineering tool. We used in vitro selection and high-throughput sequencing to determine the propensity of eight guide-RNA:Cas9 complexes to cleave each of 10(12) potential off-target DNA sequences. The selection results predicted five off-target sites in the human genome that were confirmed to undergo genome cleavage in HEK293T cells upon expression of one of two guide-RNA:Cas9 complexes. In contrast to previous models, our results show that guide-RNA:Cas9 specificity extends past a 7- to 12-base-pair seed sequence. Our results also suggest a tradeoff between activity and specificity both in vitro and in cells as a shorter, less-active guide RNA is more specific than a longer, more-active guide RNA. High concentrations of guide-RNA:Cas9 complexes can cleave off-target sites containing mutations near or within the PAM that are not cleaved when enzyme concentrations are limiting. More... »

PAGES

839-843

Journal

TITLE

Nature Biotechnology

ISSUE

9

VOLUME

31

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  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1038/nbt.2673

    DOI

    http://dx.doi.org/10.1038/nbt.2673

    DIMENSIONS

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    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/23934178


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