An unbiased genome-wide analysis of zinc-finger nuclease specificity View Full Text


Ontology type: schema:ScholarlyArticle     


Article Info

DATE

2011-09

AUTHORS

Richard Gabriel, Angelo Lombardo, Anne Arens, Jeffrey C Miller, Pietro Genovese, Christine Kaeppel, Ali Nowrouzi, Cynthia C Bartholomae, Jianbin Wang, Geoffrey Friedman, Michael C Holmes, Philip D Gregory, Hanno Glimm, Manfred Schmidt, Luigi Naldini, Christof von Kalle

ABSTRACT

Zinc-finger nucleases (ZFNs) allow gene editing in live cells by inducing a targeted DNA double-strand break (DSB) at a specific genomic locus. However, strategies for characterizing the genome-wide specificity of ZFNs remain limited. We show that nonhomologous end-joining captures integrase-defective lentiviral vectors at DSBs, tagging these transient events. Genome-wide integration site analysis mapped the actual in vivo cleavage activity of four ZFN pairs targeting CCR5 or IL2RG. Ranking loci with repeatedly detectable nuclease activity by deep-sequencing allowed us to monitor the degree of ZFN specificity in vivo at these positions. Cleavage required binding of ZFNs in specific spatial arrangements on DNA bearing high homology to the intended target site and only tolerated mismatches at individual positions of the ZFN binding sites. Whereas the consensus binding sequence derived in vivo closely matched that obtained in biochemical experiments, the ranking of in vivo cleavage sites could not be predicted in silico. Comprehensive mapping of ZFN activity in vivo will facilitate the broad application of these reagents in translational research. More... »

PAGES

816

Journal

TITLE

Nature Biotechnology

ISSUE

9

VOLUME

29

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  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1038/nbt.1948

    DOI

    http://dx.doi.org/10.1038/nbt.1948

    DIMENSIONS

    https://app.dimensions.ai/details/publication/pub.1033698278

    PUBMED

    https://www.ncbi.nlm.nih.gov/pubmed/21822255


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