An unbiased genome-wide analysis of zinc-finger nuclease specificity

An unbiased genome-wide analysis of zinc-finger nuclease specificity 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Biotechnology Springer Journals

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Publisher
Springer Journals
Copyright
Copyright © 2011 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.
ISSN
1087-0156
eISSN
1546-1696
DOI
10.1038/nbt.1948
Publisher site
See Article on Publisher Site

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.

Journal

Nature BiotechnologySpringer Journals

Published: Aug 7, 2011

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