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

被引：396

作者：

Gabriel, Richard ^{[2
,3
]}

Lombardo, Angelo ^{[1
]}

Arens, Anne ^{[2
,3
]}

Miller, Jeffrey C. ^{[4
]}

Genovese, Pietro ^{[1
]}

Kaeppel, Christine ^{[2
,3
]}

Nowrouzi, Ali ^{[2
,3
]}

Bartholomae, Cynthia C. ^{[2
,3
]}

Wang, Jianbin ^{[4
]}

Friedman, Geoffrey ^{[4
]}

Holmes, Michael C. ^{[4
]}

Gregory, Philip D. ^{[4
]}

Glimm, Hanno ^{[2
,3
]}

Schmidt, Manfred ^{[2
,3
]}

Naldini, Luigi ^{[1
]}

von Kalle, Christof ^{[2
,3
]}

机构：

[1] Univ Vita Salute San Raffaele, San Raffaele Telethon Inst Gene Therapy HSR TIGET, Milan, Italy

[2] Natl Ctr Tumor Dis NCT, Dept Translat Oncol, Heidelberg, Germany

[3] German Canc Res Ctr, Heidelberg, Germany

[4] Sangamo BioSci Inc, Richmond, CA USA

来源：

NATURE BIOTECHNOLOGY | 2011年 / 29卷 / 09期

基金：

欧洲研究理事会;

关键词：

DOUBLE-STRAND BREAKS; MAMMALIAN-CELLS; RESTRICTION ENZYMES; VECTOR INTEGRATION; LENTIVIRAL VECTORS; GENE MODIFICATION; DNA CLEAVAGE; IN-VIVO; KNOCKOUT; SITES;

D O I：

10.1038/nbt.1948

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

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.

引用

页码：816 / U72

页数：9

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