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

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

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)816-823
Number of pages8
JournalNature Biotechnology
Volume29
Issue number9
DOIs
Publication statusPublished - Sep 2011

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Biotechnology
  • Molecular Medicine
  • Bioengineering
  • Biomedical Engineering

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