title: An unbiased genome-wide analysis of zinc-finger nuclease specificity.
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.
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors.forename: Richard
authors.initials: R
authors.lastname: Gabriel
authors.forename: Angelo
authors.initials: A
authors.lastname: Lombardo
authors.forename: Anne
authors.initials: A
authors.lastname: Arens
authors.forename: Jeffrey C
authors.initials: JC
authors.lastname: Miller
authors.forename: Pietro
authors.initials: P
authors.lastname: Genovese
authors.forename: Christine
authors.initials: C
authors.lastname: Kaeppel
authors.forename: Ali
authors.initials: A
authors.lastname: Nowrouzi
authors.forename: Cynthia C
authors.initials: CC
authors.lastname: Bartholomae
authors.forename: Jianbin
authors.initials: J
authors.lastname: Wang
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Friedman
authors.forename: Michael C
authors.initials: MC
authors.lastname: Holmes
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.forename: Hanno
authors.initials: H
authors.lastname: Glimm
authors.forename: Manfred
authors.initials: M
authors.lastname: Schmidt
authors.forename: Luigi
authors.initials: L
authors.lastname: Naldini
authors.forename: Christof
authors.initials: C
authors.lastname: von Kalle
mesh_terms: D001665:Binding Sites; D045744:Cell Line, Tumor; D016000:Cluster Analysis; D053903:DNA Breaks, Double-Stranded; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D018390:Gene Targeting; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics; D019427:HIV Integrase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D011485:Protein Binding; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D019427:HIV Integrase; D004706:Endodeoxyribonucleases; C058608:endonuclease-integrase